Novel polyglycerol-conjugated lipids and lipid nanoparticle compositions containing the same

Abstract

Lipid-based nanoparticles have played a key role in the success of many other nanomedicines and are therefore considered a leading candidate among nanoscale drug delivery systems. However, effective targeted delivery of biologically active substances, such as therapeutic nucleic acids, remains a continuing medical challenge. The present disclosure provides novel polymer-conjugated lipids, including, for example, DODA conjugated to polyglycerol or polyglycerol derivatives. The present disclosure also provides lipid nanoparticle (LNP) formulations using the polymer-conjugated lipids and methods for treating diseases by administering the LNP formulations.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 429,267, filed December 1, 2022, U.S. Provisional Patent Application No. 63 / 429,226, filed December 1, 2022, U.S. Provisional Patent Application No. 63 / 449,610, filed March 3, 2023, U.S. Provisional Patent Application No. 63 / 449,617, filed March 3, 2023, U.S. Provisional Patent Application No. 63 / 452,077, filed March 14, 2023, U.S. Provisional Patent Application No. 63 / 467,045, filed May 17, 2023, U.S. Provisional Patent Application No. 63 / 467,116, filed May 17, 2023, and U.S. Provisional Patent Application No. 63 / 592,852, filed October 24, 2023. The entire contents of each of the aforementioned applications are expressly incorporated herein by reference in their entirety. [Background technology]

[0002] Lipid-based nanoparticles have played a key role in the success of COVID-19 vaccines and many other nanomedicines, such as Doxil® and Onpattro®, and are therefore considered a leading candidate among nanoscale drug delivery systems. However, the effective targeted delivery of biologically active substances, such as therapeutic nucleic acids, remains a continuing medical challenge. This significantly limits the widespread application of nucleic acids, such as mRNA and DNA, in protein replacement therapy, gene therapy, gene editing, and vaccination.

[0003] The lack of effective methods and vehicles for intracellular delivery is a major barrier to the widespread use of nucleic acid therapeutics. Generally, intracellular delivery of mRNA or DNA is more difficult than intracellular delivery of small oligonucleotides, partly due to the fact that mRNA and DNA molecules (typically ranging from 300 kDa to 5,000 kDa or about 1 to 15 kb) are significantly larger than other types of RNA, such as small interfering RNA (siRNA, typically about 14 kDa) or antisense oligonucleotides (ASO, typically ranging from 4 kDa to 10 kDa).

[0004] Furthermore, intracellular delivery of nucleic acid therapeutics to target cells is hindered by activation of the innate and / or adaptive immune response. While it is possible to avoid RNA sensing by myeloid dendritic cells (MDCs) by chemically modifying RNA cargo (e.g., with 1mΨ, 2'OMe, etc.), there are no known chemical modifications to DNA cargo that can limit pattern recognition receptor (PRR) sensing and still maintain transcriptional activity.

[0005] An alternative approach to gene therapy is the recombinant adeno-associated virus (rAAV) vector platform, which packages heterologous DNA into viral capsids. However, there are several major disadvantages to using rAAV vectors as gene delivery vectors. One major drawback associated with rAAV is the limited viral packaging capacity of approximately 4.5 kb of heterologous DNA. Another major drawback is capsid immunogenicity, which prevents re-administration to patients.

[0006] Thus, there remains a need for effective delivery vehicles that allow for the safe and effective delivery of nucleic acid therapeutics to desired cell populations. Summary of the Invention

[0007] Thus, in some aspects, the present disclosure provides a polymer-conjugated lipid comprising: (i) polyglycerol (PG) or a PG derivative; (ii) Formula (I) [ka] (I) (In the formula, R 1 is absent, hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10 to 30 carbon atoms; R 2 is absent, hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10 to 30 carbon atoms; R 1 and R 2 are each hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10 to 30 carbon atoms, N is positively charged, R 3 is a hydrophobic tail containing 10 to 30 carbon atoms), or a pharmaceutically acceptable salt thereof; (iii) a linker that conjugates the PG or PG derivative to the lipid moiety, In formula (I), [ka] is a bond conjugating the lipid moiety and the linker.

[0008] In some embodiments, the PG derivative is carboxylated PG. In some embodiments, the carboxylated PG is glutarylated PG. In one embodiment, the glutarylated PG is 3-methylglutarylated PG. In one embodiment, the carboxylated PG is 2-carboxycyclohexane-1-carboxylated PG.

[0009] In some embodiments, the PG or PG derivative is linear or branched. In some embodiments, in the lipid moiety represented by formula (I), R 1 is absent, and R 2 and R 3are each independently a hydrophobic tail containing 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon atoms. 2 and R 3 are each independently a hydrophobic tail containing 16, 17, 18, 19, 20, 21, or 22 carbon atoms. 2 and R 3 are each independently a hydrophobic tail containing 18 carbon atoms, and the lipid moiety is dioctadecylamine (DODA).

[0010] In some embodiments, the lipid moiety conjugated to the linker has the following structure: [ka] is expressed by

[0011] In some embodiments, the PG or PG derivative comprises about 5-100 monomeric units, or an average of 5-100 monomeric units.

[0012] In some embodiments, the PG or PG derivative comprises about 5, 6, 7, 8, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 monomer units, or an average of 5, 6, 7, 8, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 monomer units.

[0013] In some embodiments, PG or a PG derivative comprises about 8, 34, 45, 46, or 58 monomer units, or an average of 8, 34, 45, 46, or 58 monomer units. In some embodiments, PG or a PG derivative comprises about 8 monomer units, or an average of 8 monomer units. In some embodiments, PG or a PG derivative comprises about 34 monomer units, or an average of 34 monomer units. In some embodiments, PG or a PG derivative comprises about 45 monomer units, or an average of 45 monomer units. In some embodiments, PG or a PG derivative comprises about 46 monomer units, or an average of 46 monomer units. In some embodiments, PG or a PG derivative comprises about 58 monomer units, or an average of 58 monomer units.

[0014] In some embodiments, the linker is an alkyl linker, a glycerol linker, a phosphate linker, a phosphate ester linker, an ether linker, an ester linker, a diester linker, an amide linker, a diamide linker, an amine linker, a peptide linker, a phosphoethanolamine linker, a phosphocholine linker, a carbamate linker, a diamide alkyl linker, a cleavable linker, or any combination thereof. In some embodiments, the linker is -(CH) n -, -C(O)(CH2) n -, -C(O)O(CH2) n , -OC(O)(CH2) n C(O)O- and -NH(CH2) n In some embodiments, the linker is selected from the group consisting of -C(O)O-, where n is an integer ranging from 1 to 20. In some embodiments, the linker is a glutaryl linker or a succinyl linker. In some embodiments, the linker is -C(O)(CH) n -, where n is 2, 3, 4, 5, or 6. In some embodiments, n is 4.

[0015] In some aspects, the present disclosure provides a compound having the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0016] In some aspects, the present disclosure provides a compound having the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0017] In some aspects, the present disclosure provides a compound having the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0018] In some aspects, the present disclosure provides a compound having the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0019] In some aspects, the present disclosure provides a compound having the following structure: [ka] (Wherein R is [ka] or [ka] a polymer-conjugated lipid represented by or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0020] In some embodiments, the polymer-conjugated lipid of the present disclosure further comprises a reactive species conjugated to PG or a PG derivative, wherein the reactive species is functionalized for conjugation to a targeting moiety. In some embodiments, the reactive species is a click chemistry reagent or a maleimide. In some embodiments, the click chemistry reagent is selected from the group consisting of a dibenzocyclooctyne (DBCO) reagent, a transcyclooctene (TCO) reagent, a tetrazine (Tz) reagent, an alkyne reagent, and an azide reagent.

[0021] In some embodiments, the polymer-conjugated lipid of the present disclosure further comprises a targeting moiety conjugated to PG or a PG derivative via a reactive species. In some embodiments, the targeting moiety is conjugated to PG or a PG derivative via dibenzocyclooctyne (DBCO)-azide conjugation, azide-alkyne conjugation, TCO-Tz conjugation, or thiol-maleimide conjugation. In some embodiments, the targeting moiety is capable of binding to liver cells. In some embodiments, the liver cells are hepatocytes. In some embodiments, the targeting moiety is N-acetylgalactosamine (GalNAc) or a GalNAc derivative. In some embodiments, the targeting moiety is a triantennary GalNAc conjugate or a tetraantennary GalNAc conjugate.

[0022] In some embodiments, the targeting moiety is selected from the group consisting of an ApoE protein, an ApoE polypeptide, an ApoB protein, an ApoB polypeptide, and fragments or variants thereof. In some embodiments, the targeting moiety is an antibody or antibody fragment, wherein the antibody or antibody fragment is capable of specifically binding to an antigen present on the surface of a cell. In some embodiments, the antibody or antibody fragment is a monoclonal antibody (mAb), a single chain variable fragment (scF), or a combination thereof. v), heavy chain antibodies (hcAb), nanobodies (Nb), heavy chain-only immunoglobulins (HC1g), immunoglobulin neoantigen receptors (IgNAR), variable domains of immunoglobulin neoantigen receptors (V NAR ), single domain antibodies, or antibodies with only variable heavy chains (V HH )

[0023] In some aspects, the present disclosure provides a lipid nanoparticle (LNP), comprising: (i) a therapeutic nucleic acid (TNA); (ii) an ionizable lipid; and (iii) a sterol; (iv) a first lipid-anchored polymer, wherein the first lipid-anchored polymer comprises a polymer-conjugated lipid of the present disclosure.

[0024] In some embodiments, the LNP further comprises a helper lipid. In some embodiments, the helper lipid comprises a phospholipid or phosphatidylcholine (PC). In some embodiments, the helper lipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), hydrogenated soy PC (HSPC), phosphatidylserine (PS), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1-margalloyl-2-oleoyl-sn-glycero-3-phosphocholine (MO ...-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC The helper lipid is selected from the group consisting of mytoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the helper lipid is DSPC.

[0025] In some embodiments, the helper lipid has the formula (II): [ka] (II) (In the formula, [ka] is a single bond or a double bond, R 1 But C1-C 17 Alkyl or C2-C 17 is alkenyl, R 2 But C1-C 22Alkyl or C2-C 22 is alkenyl, R 3 is hydrogen or C1-C2 alkyl, R 4 is hydrogen or C1-C2 alkyl), or a pharmaceutically acceptable salt or ester thereof.

[0026] In some embodiments, [ka] is a double bond.

[0027] In some embodiments, R 1 is C 10 -C 20 alkenyl, and R 2 is C 10 -C 20 alkyl, and R 3 is hydrogen.

[0028] In some embodiments, the helper lipid represented by formula (II) is [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0029] In some embodiments, the helper lipid represented by formula (II) is [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0030] In some embodiments, the helper lipid represented by formula (II) is [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0031] In some embodiments, the helper lipid represented by formula (II) is [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0032] In some embodiments, the helper lipid represented by formula (II) is [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0033] In some embodiments, the helper lipid represented by formula (II) is [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0034] In some embodiments, the sterol is selected from the group consisting of cholesterol, beta-sitosterol, stigmasterol, beta-sitostanol, campesterol, brassicasterol, and derivatives thereof, hi one embodiment, the sterol is cholesterol.

[0035] In some embodiments, the ionizable lipid is a) Formula (A): [ka] Formula (A) (In the formula, R 1 and R 1’each independently represents an optionally substituted linear or branched C 1-3 is alkylene, R 2 and R 2’ each independently represents an optionally substituted linear or branched C 1-6 is alkylene, R 3 and R 3’ each independently represents an optionally substituted linear or branched C 1-6 Is it alkyl? Alternatively, R 2 optionally substituted branched C 1-6 When R is alkylene, 2 and R 3 together with the intervening N atom to form a 4- to 8-membered heterocyclyl, or Alternatively, R 2’ optionally substituted branched C 1-6 When R is alkylene, 2’ and R 3’ together with the intervening N atom to form a 4- to 8-membered heterocyclyl; R 4 and R 4’ are each independently -CR a , -C(R a )2CR a , or -[C(R a )2]2CR a and R a each occurrence independently represents H or C 1-3 Is it alkyl? Alternatively, R 4 -C(R a )2CR a or -[C(R a )2]2CR a and R a C 1-3 If it is alkyl, R 3 and R 4 together with the intervening N atom to form a 4- to 8-membered heterocyclyl, or Alternatively, R 4’ -C(Ra )2CR a or -[C(R a )2]2CR a and R a C 1-3 If it is alkyl, R 3’ and R 4’ together with the intervening N atom to form a 4- to 8-membered heterocyclyl; R 5 and R 5’ are each independently hydrogen, C 1-20 Alkylene or C 2-20 is alkenylene, R 6 and R 6’ But for each occurrence, independently, C 1-20 Alkylene, C 3-20 Cycloalkylene or C 2-20 is alkenylene, m and n are each independently an integer selected from 1, 2, 3, 4, and 5, or a pharmaceutically acceptable salt thereof; b) Formula (B): [ka] Formula (B) (In the formula, a is an integer ranging from 1 to 20; b is an integer ranging from 2 to 10; R 1 is absent or (C2-C 20 ) alkenyl, -C(O)O(C2-C 20 ) alkyl, and (C2-C 20 ) cyclopropyl substituted with alkyl; R 2 However, (C2-C 20 ) alkyl) or a pharmaceutically acceptable salt thereof; or c) Formula (C): [ka] Formula (C) (In the formula, R 1 and R 1’ are each independently R a and (C1-C6) alkylene optionally substituted with one or more groups selected from R 2 and R 2’ are each independently (C1-C2) alkylene; R 3 and R 3’ are each independently R b or (C1-C6) alkyl optionally substituted with one or more groups selected from Alternatively, R 2 and R 3 and / or R 2’ and R 3’ together with the intervening N atom to form a 4- to 7-membered heterocyclyl; R 4 and R 4 each ' is (C2-C6)alkylene interrupted by -C(O)O-; R 5 and R 5 each independently being optionally interrupted by —C(O)O— or (C3-C6)cycloalkyl, (C2-C 30 ) alkyl or (C2-C 30 ) alkenyl, R a and R b wherein each is halo or cyano, or a pharmaceutically acceptable salt thereof; d) Formula (D): [ka] Formula (D) (In the formula, R' is absent, hydrogen, or C1-C6 alkyl, provided that when R' is hydrogen or C1-C6 alkyl, R', R 1 , and R 2 are all positively charged nitrogen atoms, R 1 and R 2are each independently hydrogen, C1-C6 alkyl, or C2-C6 alkenyl; R 3 But C1-C 12 Alkylene or C2-C 12 is alkenylene, R 4 But C1-C 18 Unbranched alkyl, C2-C 18 unbranched alkenyl, or [ka] where: R 4a and R 4b are each independently, C1-C 16 Unbranched alkyl or C2-C 16 is an unbranched alkenyl, R 5 is absent, C1-C8 alkylene, or C2-C8 alkenylene; R 6a and R 6b are each independently, C7-C 16 Alkyl or C7-C 16 alkenyl, provided that R 6a and R 6b provided that the total number of carbon atoms in X 1 and X 2 each independently represents -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -SS-, -C(R a )=N-, -N=C(R a )-, -C(R a )=NO-, -ON=C(R a )-, -C(=O)NR a -, -NR a C(=O)-, -NR a C(=O)NR a -, -OC(=O)O-, -OSi(R a )2O-, -C(=O)(CR a 2) C(=O)O- or OC(=O)(CRa 2) C(=O)-, wherein: R a is independently at each occurrence hydrogen or C1-C6 alkyl; n is an integer selected from 1, 2, 3, 4, 5, and 6; In some embodiments, R 4 But C1-C 16 Unbranched alkyl, C2-C 16 unbranched alkenyl, or [ka] where R 4a and R 4b wherein R is as defined above, or a pharmaceutically acceptable salt thereof; e) Formula (E): [ka] Formula (E) (In the formula, R' is absent, hydrogen, or C1-C3 alkyl, provided that when R' is hydrogen or C1-C3 alkyl, R', R 1 , and R 2 provided that all nitrogen atoms to which they are attached are positively charged, R 1 and R 2 are each independently hydrogen or C1-C3 alkyl; R 3 But C3-C 10 Alkylene or C3-C 10 is alkenylene, R 4 But C1-C 16 Unbranched alkyl, C2-C 16 unbranched alkenyl, or [ka] where: R 4a and R 4b are each independently, C1-C 16Unbranched alkyl or C2-C 16 is an unbranched alkenyl, R 5 is absent, C1-C6 alkylene, or C2-C6 alkenylene; R 6a and R 6b are each independently, C7-C 14 Alkyl or C7-C 14 is alkenyl, X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -SS-, -C(R a )=N-, -N=C(R a )-, -C(R a )=NO-, -ON=C(R a )-, -C(=O)NR a -, -NR a C(=O)-, -NR a C(=O)NR a -, -OC(=O)O-, -OSi(R a )2O-, -C(=O)(CR a )2C(=O)O- or OC(=O)(CR a 2) C(=O)-, wherein: R a is independently at each occurrence hydrogen or C1-C6 alkyl; n is an integer selected from 1, 2, 3, 4, 5, and 6, or a pharmaceutically acceptable salt thereof; f) An ionizable lipid selected from the group consisting of any of the ionizable lipids of Table 1, Table 4, Table 5, Table 6, or Table 7.

[0036] In some embodiments, the ionizable lipid has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0037] In some embodiments, the ionizable lipid has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0038] In some embodiments, the ionizable lipid has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0039] In some embodiments, the ionizable lipid has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0040] In some embodiments, the ionizable lipid has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0041] In some embodiments, the ionizable lipid has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0042] In some embodiments, the ionizable lipid has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0043] In some embodiments, the ionizable lipid has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0044] In some embodiments, the ionizable lipid has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0045] In some embodiments, the ionizable lipid has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0046] In some embodiments, the ionizable lipid has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0047] In some embodiments, the ionizable lipid has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0048] In some embodiments, the ionizable lipid has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0049] In some embodiments, the ionizable lipid has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0050] In some embodiments, the ionizable lipid has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0051] In some embodiments, the ionizable lipid has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0052] In some embodiments, the LNPs of the present disclosure further comprise a second lipid-anchored polymer, wherein the second lipid-anchored polymer is (i) a lipid moiety comprising at least one hydrophobic tail; (ii) a polymer; and (iii) a linker, wherein the polymer is conjugated to the lipid moiety via the linker; and (iv) a reactive species conjugated to a polymer, wherein the reactive species is functionalized to be conjugated to a targeting moiety.

[0053] In some embodiments, the polymer of the second lipid-anchored polymer is selected from the group consisting of polyethylene (PE), polypropylene (PP), polyethylene glycol (PEG), polyvinyl alcohol (PVOH), polysarcosine (pSar), poly(2-methacryloyloxyethylphosphorylcholine) (PMPC), polyglycerol (PG), and derivatives of any of the foregoing. In some embodiments, the PG derivative is carboxylated PG. In one embodiment, the carboxylated PG is glutarylated PG or 2-carboxycyclohexane-1-carboxylated PG. In one embodiment, the glutarylated PG is 3-methylglutarylated PG.

[0054] In some embodiments, the PG or PG derivative is linear or branched.

[0055] In some embodiments, the reactive species is a click chemistry reagent or a maleimide, hi some embodiments, the click chemistry reagent is selected from the group consisting of a dibenzocyclooctyne (DBCO) reagent, a transcyclooctene (TCO) reagent, a tetrazine (Tz) reagent, an alkyne reagent, and an azide reagent.

[0056] In some embodiments, the LNPs of the present disclosure further comprise a targeting moiety conjugated to the polymer via a reactive species. In some embodiments, the targeting moiety is conjugated to the polymer via dibenzocyclooctyne (DBCO)-azide conjugation, azide-alkyne conjugation, TCO-Tz conjugation, or thiol-maleimide conjugation. In some embodiments, the targeting moiety is capable of binding to liver cells. In some embodiments, the liver cells are hepatocytes. In some embodiments, the targeting moiety is N-acetylgalactosamine (GalNAc) or a GalNAc derivative. In some embodiments, the targeting moiety is a triantennary GalNAc conjugate or a tetraantennary GalNAc conjugate.

[0057] In some embodiments, the targeting moiety is selected from the group consisting of an ApoE protein, an ApoE polypeptide, an ApoB protein, an ApoB polypeptide, and fragments or variants thereof.

[0058] In some embodiments, the targeting moiety is an antibody or antibody fragment, and the antibody or antibody fragment is capable of specifically binding to an antigen present on the surface of a cell. In some embodiments, the antibody or antibody fragment is a monoclonal antibody (mAb), a single chain variable fragment (scF), or a combination thereof. v ), heavy chain antibodies (hcAb), nanobodies (Nb), heavy chain-only immunoglobulins (HC1g), immunoglobulin neoantigen receptors (IgNAR), variable domains of immunoglobulin neoantigen receptors (V NAR ), single domain antibodies, or antibodies with only variable heavy chains (V HH )

[0059] In some embodiments, the linker is selected from the group consisting of an alkyl linker, a glycerol linker, a phosphate linker, a phosphate ester linker, an ether linker, an ester linker, a diester linker, an amide linker, a diamide linker, an amine linker, a peptide linker, a phosphoethanolamine linker, a phosphocholine linker, a carbamate linker, a diamide alkyl linker, a cleavable linker, and any combination thereof. In some embodiments, the linker is selected from the group consisting of -(CH2) n -, -C(O)(CH2) n -, -C(O)O(CH2) n -, -OC(O)(CH2) n C(O)O- and -NH(CH2) n In some embodiments, the linker is selected from the group consisting of -C(O)O-, where n is an integer ranging from 1 to 20. In some embodiments, the linker is a glutaryl linker or a succinyl linker. In some embodiments, the linker is -C(O)(CH) n -, where n is 2, 3, 4, 5, or 6. In one embodiment, n is 4.

[0060] In some embodiments, in the LNPs of the present disclosure, the lipid portion of the second lipid-anchored polymer has the formula (I): [ka] (I) (In the formula, R 1 is absent, hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10 to 30 carbon atoms; R 2 is absent, hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10 to 30 carbon atoms; R 1 and R 2 are each hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10 to 30 carbon atoms, N is positively charged, R 3is a hydrophobic tail containing 10 to 30 carbon atoms), or a pharmaceutically acceptable salt thereof.

[0061] In some embodiments, R 1 is absent, and R 2 and R 3 are each independently a hydrophobic tail containing 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon atoms. 2 and R 3 are each independently a hydrophobic tail containing 16, 17, 18, 19, 20, 21, or 22 carbon atoms. 2 and R 3 are each independently a hydrophobic tail containing 18 carbon atoms, and the lipid moiety is dioctadecylamine (DODA).

[0062] In some embodiments, the lipid moiety of the second lipid-anchored polymer is selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielideyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl-2-oleo ...-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1-stearoyl-2-oleoyl-sn-glycero-3- -glycero-3-phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 18-1-trans PE, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), dioctadecylamine (DODA), dihexadecylamine, distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and derivatives thereof. In some embodiments, the lipid portion of the second lipid-anchored polymer comprises a moiety selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, and derivatives of any of the foregoing, hi some embodiments, the lipid portion of the second lipid-anchored polymer comprises DSPE.

[0063] In some embodiments, the polymer of the second lipid-anchored polymer has an average molecular weight of about 500 Da to about 5000 Da. In some embodiments, the polymer has an average molecular weight of about 1500 Da to about 5000 Da. In some embodiments, the polymer has an average molecular weight of about 2000 Da.

[0064] In some embodiments, the second lipid-anchored polymer has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0065] In some embodiments, the second lipid-anchored polymer has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0066] In some embodiments, the ionizable lipids are present in the LNPs in an amount of about 20 mol % to about 60 mol % of the total lipids present in the LNPs, hi some embodiments, the ionizable lipids are present in the LNPs in an amount of about 35 mol % to about 50 mol % of the total lipids present in the LNPs.

[0067] In some embodiments, the sterol is present in the LNP in an amount of about 20 mol % to about 50 mol % of the total lipid present in the LNP, hi some embodiments, the sterol is present in the LNP in an amount of about 30 mol % to about 45 mol % of the total lipid present in the LNP.

[0068] In some embodiments, the helper lipid is present in the LNP in an amount of about 1 mol % to about 40 mol % of the total lipid present in the LNP, hi some embodiments, the helper lipid is present in the LNP in an amount of about 5 mol % to about 15 mol % of the total lipid present in the LNP.

[0069] In some embodiments, the first lipid-anchored polymer is present in the LNP in an amount of about 0.5 mol % to about 5 mol % of the total lipid present in the LNP, hi some embodiments, the first lipid-anchored polymer is present in the LNP in an amount of about 1.5 mol % to about 3 mol % of the total lipid present in the LNP.

[0070] In some embodiments, the second lipid-anchored polymer is present in the LNP in an amount of about 0.05 mol % to about 5 mol % of the total lipid present in the LNP, hi some embodiments, the second lipid-anchored polymer is present in the LNP in an amount of about 0.1 mol % to about 1 mol % of the total lipid present in the LNP.

[0071] In some aspects, the present disclosure provides a lipid nanoparticle (LNP), comprising: (i) a therapeutic nucleic acid (TNA); (ii) an ionizable lipid, wherein the ionizable lipid has the following structure: [ka] an ionizable lipid which is heptadecan-9-yl 9-((4-(dimethylamino)butanoyl)oxy)hexadecanoate having the formula: (iii) a sterol, wherein the sterol is cholesterol; (iv) a helper lipid, wherein the helper lipid is DSPC; (v) a first lipid-anchored polymer, wherein the first lipid-anchored polymer comprises DODA conjugated to a linear PG via a linker; and (vi) a second lipid-anchored polymer, wherein the second lipid-anchored polymer comprises DSPE conjugated to PEG.

[0072] In some embodiments, the liner PG comprises about 30-60 monomer units, or an average of 30-60 monomer units.

[0073] In some embodiments, the linear PG comprises about 34 monomer units or about 45 monomer units, or an average of 34 monomer units or an average of 45 monomer units.

[0074] In some embodiments, the PEG has an average molecular weight of about 1000 Da to about 5000 Da, hi some embodiments, the PEG has an average molecular weight of 2000 Da.

[0075] In some embodiments, in the LNPs of the disclosure, the ionizable lipid is present in the LNP in an amount of about 35 mol % to about 50 mol % of the total lipid present in the LNP; the sterol is present in the LNP in an amount of about 30 mol % to about 45 mol % of the total lipid present in the LNP; the helper lipid is present in the LNP in an amount of about 5 mol % to about 15 mol % of the total lipid present in the LNP; the first lipid-anchored polymer is present in the LNP in an amount of about 1.5 mol % to about 3 mol % of the total lipid present in the LNP; and The second lipid-anchored polymer is present in the LNP in an amount of about 0.1 mol % to about 1 mol % of the total lipid present in the LNP.

[0076] In some embodiments, the TNA included in the LNPs of the present disclosure is selected from the group consisting of a minigene, a plasmid, a minicircle, a small interfering RNA (siRNA), a microRNA (miRNA), a guide RNA (gRNA), an antisense oligonucleotide (ASO), a ribozyme, a deoxyribozyme, a closed-end DNA (ceDNA), a ssDNA, a ministring, a doggybone™, a protelomeric closed-end DNA, a dumbbell-shaped linear DNA, a Dicer substrate dsRNA, a small hairpin RNA (shRNA), an asymmetric interfering RNA (aiRNA), an mRNA, a tRNA, a rRNA, a gRNA, a DNA viral vector, a viral RNA vector, a non-viral vector, and combinations thereof.

[0077] In some embodiments, the TNA is ceDNA.

[0078] In some embodiments, the TNA is a single-stranded nucleic acid or a double-stranded nucleic acid. In some embodiments, the single-stranded nucleic acid is an mRNA. In some embodiments, the single-stranded nucleic acid is a DNA molecule (ssDNA).

[0079] In some embodiments, the ssDNA is linear ssDNA comprising at least one nucleic acid sequence of interest adjacent to at least one stem-loop structure at the 3' end. In some embodiments, the at least one stem-loop structure at the 3' end is sufficient to prime replication and / or transcription. In some embodiments, the stem structure at the 3' end comprises a partial DNA duplex of 4 to 500 nucleotides. In some embodiments, the stem structure at the 3' end comprises a partial DNA duplex of 4 to 50 nucleotides.

[0080] In some embodiments, the loop structure at the 3'-end comprises 3 to 500 non-linked nucleotides, In some embodiments, the loop structure at the 3'-end comprises at least 3 non-linked nucleotides.

[0081] In some embodiments, the ssDNA comprises at least two stem-loop structures at the 3' end. In some embodiments, the ssDNA comprises at least three stem-loop structures at the 3' end. In some embodiments, the ssDNA comprises at least four or more stem-loop structures at the 3' end. In some embodiments, at least one stem-loop structure at the 3' end comprises a hairpin DNA structure. In some embodiments, at least one stem-loop structure at the 3' end comprises a DNA structure selected from the group consisting of a cruciform DNA structure, a hammerhead DNA structure, a quadruplex DNA structure, a bulge DNA structure, and a multi-branched loop structure. In some embodiments, at least one stem-loop structure at the 3' end does not include the A, A', D, and D' regions that would be present in a wild-type AAV ITR. In some embodiments, at least one stem-loop structure at the 3' end does not include the A, A', B, B', C, C', D, and D' regions that would be present in a wild-type AAV ITR. In some embodiments, at least one stem-loop structure at the 3' end does not include a rep-binding element (RBE) that would be present in a wild-type ITR. In some embodiments, at least one stem-loop structure at the 3' end does not include a terminal resolution site (trs) that would be present in a wild-type ITR.

[0082] In some embodiments, the stem structure at the 3' end comprises four or more nucleotides modified to be exonuclease resistant. In some embodiments, the nucleotides are phosphorothioate-modified nucleotides.

[0083] In some embodiments, at least one stem-loop structure at the 3' end further comprises a functional moiety.

[0084] In some embodiments, the ssDNA molecule further comprises a 5' end comprising at least one stem-loop structure. In some embodiments, the ssDNA comprises at least two stem-loop structures at the 5' end. In some embodiments, the ssDNA comprises at least three stem-loop structures at the 5' end. In some embodiments, the ssDNA comprises at least four or more stem-loop structures at the 5' end. In some embodiments, at least one stem-loop structure at the 5' end comprises a hairpin DNA structure. In some embodiments, At least one stem-loop structure at the 5' end comprises a DNA structure selected from the group consisting of a cruciform DNA structure, a hammerhead DNA structure, a quadruplex DNA structure, a bulge DNA structure, and a multi-branched loop structure.

[0085] In some embodiments, at least one stem-loop structure at the 5' end does not include the A, A', D, and D' regions that would be present in a wild-type AAV ITR. In some embodiments, at least one stem-loop structure at the 5' end does not include the A, A', B, B', C, C', D, and D' regions that would be present in a wild-type AAV ITR. In some embodiments, at least one stem-loop structure at the 5' end does not include a rep-binding element (RBE) that would be present in a wild-type ITR. In some embodiments, at least one stem-loop structure at the 5' end does not include a terminal resolution site (trs) that would be present in a wild-type ITR.

[0086] In some embodiments, the stem structure at the 5' end comprises four or more nucleotides modified to be exonuclease resistant, hi some embodiments, the nucleotides are phosphorothioate-modified nucleotides.

[0087] In some embodiments, the 5'-end loop structure further comprises one or more nucleic acids to stabilize the end. In some embodiments, the 5'-end loop structure further comprises one or more chemically modified nucleic acids. In some embodiments, the 5'-end loop structure further comprises one or more aptamers. In some embodiments, the 5'-end loop structure further comprises one or more synthetic ribozymes.

[0088] In some embodiments, the 5'-terminal loop structure further comprises one or more antisense oligonucleotides (ASOs). In some embodiments, the 5'-terminal loop structure further comprises one or more short interfering RNAs (siRNAs). In some embodiments, the 5'-terminal loop structure further comprises one or more antiviral nucleoside analogs (ANAs).

[0089] In some embodiments, the 5'-terminal loop structure further comprises one or more triplex-forming oligonucleotides. In some embodiments, the 5'-terminal loop structure further comprises one or more gRNAs or gDNAs. In some embodiments, the 5'-terminal loop structure further comprises one or more molecular probes.

[0090] In some embodiments, the ssDNA molecule lacks any viral capsid protein coding sequence. In some embodiments, the ssDNA molecule is synthetically produced in vitro. In some embodiments, the ssDNA molecule is synthetically produced in vitro in a cell-free environment.

[0091] In some embodiments, the ssDNA molecule does not activate or only minimally activates an immune pathway. In some embodiments, the immune pathway is an innate immune pathway. In some embodiments, the innate immune pathway is selected from the group consisting of the cGAS / STING pathway, the TLR9 pathway, the inflammasome-mediated pathway, and combinations thereof.

[0092] In some embodiments, the ssDNA molecule is capable of expressing at least one therapeutic protein or therapeutic fragment thereof, hi some embodiments, the at least one therapeutic protein is selected from the group consisting of an antibody, an enzyme, a clotting factor, a transcription factor, a replication factor, a growth factor, a hormone, and a fusion protein. In some embodiments, the at least one therapeutic protein is selected from the group consisting of melanoma, hemophilia A (clotting factor VIII (FVIII) deficiency) and hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited liver metabolic disorders, Lesch-Nyhan syndrome, sickle cell disease, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharidosis (e.g., Hurler syndrome (MPS I), Scheie syndrome (MPS IS), Hurler-Scheie syndrome (MPS I HS), Hunter syndrome (MPS II), Sanfilippo syndrome types A, B, C, and D (MPS III), and others). A, B, C, and D), Morquio syndrome types A and B (MPS IVA and MPS IVB), Maroteaux-Lamy syndrome (MPS VI), Sly syndrome (MPS VII), hyaluronidase deficiency (MPSIX), Niemann-Pick disease types A / B, C1, and C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandhoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis types I, II / III, and IV, sialidosis types I and II, glycogen storage disease types I and II (Pompe disease), Gaucher disease types I, II, and III, Fabry disease, cystinosis, Batten disease, aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidoses, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, The present invention is useful for treating a genetic disorder selected from the group consisting of Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber's congenital amaurosis, Stargardt's macular dystrophy (ABCA4), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC) type I (ATP8B1 deficiency), type II (ABCB11), type III (ABCB4), or type IV (TJP2), and cathepsin A deficiency.

[0093] In some embodiments, the present disclosure also provides pharmaceutical compositions comprising an LNP of the present disclosure and a pharmaceutically acceptable carrier.

[0094] In some aspects, the present disclosure also provides a method of treating a genetic disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of an LNP of the present disclosure or a pharmaceutical composition of the present disclosure.

[0095] In some embodiments, the subject is a human.

[0096] In some embodiments, the genetic disorder is sickle cell anemia, melanoma, hemophilia A (clotting factor VIII (FVIII) deficiency), hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited liver metabolic disorders, Lesch-Nyhan syndrome, thalassemia, xeroderma pigmentosum, Fan et al. Koni anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharidosis, Niemann-Pick disease, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandhoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis (ML), sialidosis type II, glycogen storage disease (GSD), Gaucher disease, cystinosis, Batten disease, aspartylglucosamine Diarrhea, Salla disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (NCL), sphingolipidosis, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy (SMA), Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy ( BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber's congenital amaurosis, Stargardt disease, wet macular degeneration (wet AMD), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC), and cathepsin A deficiency.

[0097] In some aspects, the present disclosure also provides methods of providing anti-tumor immunity to a subject in need thereof, the method comprising administering to the subject an effective amount of an LNP of the present disclosure or a pharmaceutical composition of the present disclosure.

[0098] In some aspects, the present disclosure also provides methods of treating a subject having a disease, disorder, or condition associated with elevated expression of a tumor antigen, the method comprising administering to the subject an effective amount of an LNP of the present disclosure or a pharmaceutical composition of the present disclosure.

[0099] In some aspects, the present disclosure also provides methods of treating a hematological disease, disorder, or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of an LNP of the present disclosure or a pharmaceutical composition of the present disclosure.

[0100] In some aspects, the present disclosure provides a method of synthesizing a polymer-conjugated lipid of the present disclosure, comprising: a) reacting a lipid moiety to be conjugated to a linker with 2,3-epoxy-1-(1-ethoxyethoxy)propane (EEGE) in the presence of a base or an organic catalyst under an argon atmosphere to produce a lipid moiety conjugated to a linker and polymerized EEGE; b) subjecting the lipid moiety conjugated to the linker and the polymerized EEGE to acidic conditions to produce a polymer-conjugated lipid.

[0101] In some embodiments, the base is a phosphazene base. In some embodiments, the phosphazene base is P4-t-Bu.

[0102] In some embodiments, the organic catalyst is an N-heterocyclic carbene (NHC) or an N-heterocyclic olefin (NHO). In some embodiments, the acidic conditions include HCl, Br, HI, HClO, HClO, HSO, or HNO.

[0103] In some embodiments, the lipid moiety comprises DODA.

[0104] In some embodiments, the lipid moiety conjugated to the linker has the following structure: [ka] is expressed by

[0105] In some embodiments, the polymer-conjugated lipid has the following structure: [ka] (wherein n is a number in the range of 10 to 100).

[0106] In some embodiments, n is about 34, 45, 46, or 58. [Brief explanation of the drawings]

[0107] [Figure 1A] 1 shows the MALDI-TOF spectrum of DODA-PG34. [Figure 1B] 1 shows the MALDI-TOF spectrum of DODA-PG45. [Figure 1C] 1 shows the MALDI-TOF spectrum of DODA-PG58. [Figure 1D] "Scheme 1" showing the synthesis of DODA-PG41 and DODA-PG46 is shown. [Figure 1E] "Scheme 2" showing the synthesis of DODA-PG45 and DODA-PG58 is shown. [Figure 2A] 1 shows the total luminous flux measured by total photon counts per region of interest, i.e., liver, measured in mice by an in vivo imaging system (IVIS) on day 4 after administration of an LNP formulation of the present disclosure and a negative control (PBS). [Figure 2B] 1 shows the total luminous flux measured by total photon counts per region of interest, i.e., liver, measured in mice by IVIS on day 7 after administration of an LNP formulation of the present disclosure and a negative control (PBS). [Figure 2C] Shown is the total luminous flux measured by total photon counts per region of interest, i.e., liver, measured in mice by IVIS over two collection days (days 4 and 7) after administration of an LNP formulation of the present disclosure and a negative control (PBS). [Figure 2D]1 shows the percent change in body weight (BW) of mice 1 day after administration of LNP formulations of the present disclosure. [Figure 3] 1 shows the luciferase activity of LNP formulations of the present disclosure containing different lipid-anchored polymers. [Figure 4A] FIG. 1 is a schematic diagram showing a proposed mechanism for opsonization-driven cellular uptake of LNPs. [Figure 4B] FIG. 1 is a schematic diagram showing the assay used to evaluate opsonization-driven cellular uptake of LNPs. [Figure 4C] 1 shows the DiD fluorescence area normalized to the area of ​​viable nuclei measured for LNP formulations of the present disclosure containing different lipid-anchored polymers. [Figure 5] 1 shows the area of ​​DiD fluorescence normalized to the area of ​​living nuclei for LNP formulations of the present disclosure containing different amounts of polyglycerol-conjugated lipids and a control. [Figure 6] 1 shows the amount of endosomal escape measured as the amount of luciferase expression normalized to DiD uptake in mouse hepatocytes treated with LNP formulations of the present disclosure containing different amounts of polyglycerol-conjugated lipids and a control. [Figure 7] 1 shows the whole blood clearance of control LNPs and LNPs of the present disclosure with different lipid Z. [Figure 8A] 1 shows the total luminous flux quantified by total photon counts per region of interest, i.e., liver, measured in mice by IVIS on day 7 after administration of an LNP formulation of the present disclosure and a negative control (DPBS). [Figure 8B] 1 shows the percent change in body weight on day 1 for mice injected with LNP formulations of the present disclosure. [Figure 9A] The levels of various cytokines involved in regulating innate immune responses, namely, IFN-alpha (Figure 9A), IFN-gamma (Figure 9B), IL-6 (Figure 9C), and IL-18 (Figure 9D), were measured in mice after administration of the LNP formulations of the present disclosure. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D]Same as above. [Figure 10] 1 shows the DiD fluorescence area normalized to the area of ​​living nuclei for LNP formulations of the present disclosure containing different amounts of polyglycerol-conjugated lipid and a control, and formulated with DSPE-PEG5K-N3 using 0.5 mol %. DETAILED DESCRIPTION OF THE INVENTION

[0108] The present disclosure provides polymer-conjugated lipids, including polyglycerol (PG) conjugated to dioctadecylamine (DODA), such as DODA-PG34, DODA-PG45, and DODA-PG58, as well as methods for their synthesis. The present disclosure also provides, inter alia, lipid nanoparticles (LNPs) including the polymer-conjugated lipids of the present disclosure, and methods for treating various disorders, including administering the LNPs of the present disclosure to a subject in need thereof. Surprisingly, it has been discovered that LNPs including the polymer-conjugated lipids of the present disclosure, as described herein, are characterized by low levels of undesired opsonization-driven uptake of the LNP into non-target cells, balanced with a desirable level of endosomal escape, thereby achieving a favorable stealth / endosomal escape trade-off.

[0109] I. Definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs. It is understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc., described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition (published by Merck Sharp & Dohme Corp.), 2011 (ISBN 978-0-911910-19-3), Robert S. Porter et al. (eds.), Fields Virology, 6th Edition (published by Lippincott Williams & Wilkins, Philadelphia, PA, USA) (2013), Knipe, DM and Howley, PM (ed.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine (published by Blackwell Science Ltd.), 1999-2012 (ISBN 9783527600908), and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference (VCH Publishers, Inc.), 1995 (ISBN1-56081-569-8), Immunology by Werner Luttmann (published by Elsevier), 2006, Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN0815345305, 9780815345305), Lewin's Genes Harbor, NY, USA (2012) (ISBN1936113414), Davis et al. Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN044460149X), Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN0124199542), Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005, and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBNs 0471142735, 9780471142737), the contents of all of which are incorporated herein by reference in their entireties.

[0110] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0111] As used herein, the term "about," when referring to a measurable value such as an amount, temporal duration, etc., is intended to encompass a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as appropriate for performing the disclosed methods.

[0112] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and fractions thereof, where appropriate (such as integer tenths and hundredths).

[0113] As used herein, "comprise," "comprising," and "comprises" are intended to be synonymous with "include," "including," "includes," or "contain," "containing," "contains," and are, for example, inclusive or open-ended terms specifying the presence of following components, but do not exclude or preclude the presence of additional, unrecited components, features, elements, members, steps that are known in the art or disclosed therein.

[0114] The term "consisting of" refers to compositions, methods, processes, and their respective components described herein, excluding any element not recited in the description of the embodiment.

[0115] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic novel or functional characteristics of that embodiment of the present disclosure.

[0116] As used herein, the terms "administration," "administering," and variations thereof refer to the introduction of a composition or agent (e.g., ceDNA, ssDNA, mRNA, etc.) into a subject, including simultaneous and sequential introduction of one or more compositions or agents. "Administration" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. "Administration" also encompasses in vitro and ex vivo treatments. Introduction of a composition or agent into a subject is by any suitable route, including oral, pulmonary, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intratumoral, or topical. Administration includes self-administration and administration by another. Administration can be by any suitable route. A suitable route of administration enables the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into the subject's vein.

[0117] As used herein, the term "immunogenicity of an LNP" or "immunogenicity of a composition comprising an LNP" refers to the ability of a composition comprising an LNP of the present disclosure to stimulate an unwanted immune response in a subject after the LNP or composition comprising an LNP of the present disclosure is administered to the subject. In some embodiments, the immune response can be measured, for example, by measuring the levels of one or more pro-inflammatory cytokines before and after administration of a composition comprising an LNP of the present disclosure. Examples of pro-inflammatory cytokines that can be used to determine the immunogenicity of LNPs of the present disclosure or compositions comprising LNPs of the present disclosure include, but are not limited to, granulocyte colony-stimulating factor (G-CSF), interleukin 1 alpha (IL-1α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), interleukin 8 (IL-8 or CXCL8), interleukin 11 (IL-11), interleukin 17 (IL-17), interleukin 18 (IL-18), interferon alpha (IFN-α), interferon beta (IFN-β), interferon gamma (IFN-γ), C-X-C motif chemokine ligand 10 (CXCL10 or IP-10), monocyte chemoattractant protein 1 (MCP-1), CD40L, CCL2, CCL3, CCL4, CCL5, CCL11, tumor necrosis factor alpha (TNF-α), and combinations thereof.

[0118] As used herein, the term "off-target delivery" refers to the delivery of LNPs of the present disclosure to non-target cells. For example, LNPs of the present disclosure containing GalNAc target delivery of the LNP to hepatocytes, and off-target delivery of LNPs refers to the delivery of LNPs to random non-target cells, e.g., cells that are not hepatocytes. In some embodiments, the non-target cells can be blood cells, such as leukocytes, neutrophils, eosinophils, basophils, macrophages, or monocytes. In some embodiments, the non-target cells can be immune cells, such as T cells, B cells, or macrophages. In some embodiments, the non-target cells can be liver sinusoidal endothelial cells (LSEC cells), spleen cells, or Kupffer cells.

[0119] After administration to a subject, the LNPs may be delivered to non-target cells, e.g., one or more of the blood cells listed above, resulting in expression of a therapeutic nucleic acid (TNA) in the non-target cells, or may be degraded, e.g., when phagocytosed by macrophages. In some embodiments, a reference LNP that does not contain polyglycerol-conjugated lipids may be characterized by a higher delivery rate to non-target cells, e.g., one or more of the blood cells listed above, compared to an LNP of the present disclosure. In some embodiments, the LNPs of the present disclosure result in an uptake level of TNA (e.g., ceDNA or mRNA) in non-target cells, e.g., blood cells, that is lower than the uptake level of the reference LNPs. In some embodiments, the reference LNPs are LNPs that do not contain polymer-conjugated lipids. In some embodiments, the blood cells are cells selected from the group consisting of red blood cells, white blood cells, neutrophils, macrophages, monocytes, T cells, B cells, macrophages, and peripheral blood mononuclear cells.

[0120] As used herein, the term "aqueous solution" refers to a composition that comprises, in whole or in part, water.

[0121] As used herein, "base" includes purines and pyrimidines, which further include the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and naturally occurring analogues, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0122] As used herein, the terms "carrier" and "excipient" are intended to include any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce toxic, allergic, or similar untoward reactions when administered to a host.

[0123] As used herein, the term "ceDNA" refers to capsid-free, closed-end, linear, double-stranded (ds) duplex DNA, whether synthetic or otherwise, for non-viral gene transfer. According to some embodiments, the ceDNA is closed-end, linear, double-stranded (CELiD) CELiD DNA. According to some embodiments, the ceDNA is a DNA-based minicircle. According to some embodiments, the ceDNA is a minimal immunologically defined gene expression (MIDGE) vector. According to some embodiments, the ceDNA is ministering DNA. According to some embodiments, the ceDNA is a dumbbell-shaped, linear, double-stranded, closed-end DNA containing two hairpin structures of ITRs at the 5' and 3' ends of the expression cassette. According to some embodiments, the ceDNA is doggybone™ DNA. A detailed description of ceDNA is provided in International Patent Application No. PCT / US2017 / 020828, filed March 3, 2017, the entire contents of which are expressly incorporated herein by reference. Certain methods for the generation of ceDNA containing various inverted terminal repeat (ITR) sequences and configurations using cell-based methods are described in Example 1 of International Patent Application No. PCT / US18 / 49996, filed September 7, 2018, and International Patent Application No. PCT / US2018 / 064242, filed December 6, 2018, each of which is incorporated herein by reference in its entirety. Certain methods for the generation of synthetic ceDNA vectors containing various ITR sequences and configurations are described, for example, in International Application No. PCT / US2019 / 14122, filed January 18, 2019, the entire contents of which are incorporated herein by reference.

[0124] As used herein, the term "closed-end DNA vector" refers to a capsid-free DNA vector that has at least one covalently closed end, and at least a portion of the vector has an intramolecular double-stranded structure.

[0125] As used herein, the terms "ceDNA vector" and "ceDNA" are used interchangeably and refer to a closed-end DNA vector containing at least one terminal palindrome. In some embodiments, the ceDNA contains two covalently closed ends.

[0126] As used herein, the term "ceDNA-bacmid" refers to an infectious baculovirus genome containing the ceDNA genome as an intermolecular duplex that can be propagated in E. coli as a plasmid and therefore act as a shuttle vector for baculovirus.

[0127] As used herein, the term "ceDNA-baculovirus" refers to a baculovirus that contains a ceDNA genome as an intermolecular duplex within the baculovirus genome.

[0128] As used herein, the terms "ceDNA-baculovirus-infected insect cells" and "ceDNA-BIIC" are used interchangeably and refer to invertebrate host cells (including, but not limited to, insect cells (e.g., Sf9 cells)) infected with a ceDNA-baculovirus.

[0129] As used herein, the term "ceDNA genome" refers to an expression cassette that further incorporates at least one inverted terminal repeat (ITR) region. The ceDNA genome may further comprise one or more spacer regions. In some embodiments, the ceDNA genome is incorporated into a plasmid or viral genome as an intermolecular double-stranded polynucleotide of DNA.

[0130] As used herein, the terms "DNA regulatory sequence," "control element," and "regulatory element" are used interchangeably herein and refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, proteolysis signals, etc., that provide and / or regulate the transcription of a non-coding sequence (e.g., a DNA-targeting RNA) or a coding sequence (e.g., a site-directed modifying polypeptide or a Cas9 / Csn1 polypeptide) and / or regulate the translation of an encoded polypeptide.

[0131] "ITRs" can be artificially synthesized using a set of oligonucleotides containing one or more desired functional sequences (e.g., palindromic sequences, RBSs). The ITR sequences can be AAV ITRs, artificial non-AAV ITRs, or ITRs physically derived from viral AAV ITRs (e.g., ITR fragments removed from the viral genome). For example, ITRs can be derived from the Parvoviridae family, which includes parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, and human parvovirus B-19), or the SV40 hairpin that functions as the origin of SV40 replication can be used as an ITR, which can be further modified by truncation, substitution, deletion, insertion, and / or addition. The Parvoviridae family of viruses consists of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. Dependoparvoviruses include the adeno-associated virus (AAV) family of viruses that can replicate in vertebrate hosts, including, but not limited to, humans, primates, bovine, canine, equine, and bovine species. Typically, ITR sequences can be derived from AAV, as well as parvoviruses, lentiviruses, goose viruses, and B19, in wild-type, "dogbone," and "dumbbell" configurations, symmetric, or even asymmetric ITR orientations. While ITRs are typically present at both the 5' and 3' ends of AAV vectors, ITRs can be present at only one end of a linear vector. For example, ITRs can be present only at the 5' end. In some other cases, ITRs can be present only at the 3' end of a synthetic AAV vector. For convenience herein, an ITR located 5' to (upstream of) the expression cassette of a synthetic AAV vector is referred to as the "5' ITR" or "left ITR," and an ITR located 3' to (downstream of) the expression cassette of a vector or synthetic AAV is referred to as the "3' ITR" or "right ITR."

[0132] As used herein, "wild-type ITR" or "WT-ITR" refers to the sequence of a naturally occurring ITR sequence in an AAV or other dependovirus that retains, for example, Rep binding activity and Rep nicking ability. The nucleic acid sequence of a WT-ITR from any AAV serotype may differ slightly from the naturally occurring reference sequence due to degeneracy or drift in the genetic code; therefore, WT-ITR sequences encompassed for use herein include WT-ITR sequences that result from naturally occurring variations (e.g., replication errors).

[0133] As used herein, the terms "substantially symmetric WT-ITR" or "substantially symmetric WT-ITR pair" refer to a pair of wild-type ITRs in a synthetic AAV vector, both of which have reverse-complementary sequences across their entire length. For example, an ITR can be considered a wild-type sequence even if it has one or more nucleotides that deviate from a naturally occurring reference sequence, as long as the changes do not affect the sequence's physical and functional properties and overall three-dimensional structure (secondary and tertiary structure). In some embodiments, the deviating nucleotides represent conservative sequence changes. As one non-limiting example, a sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity with a reference sequence (e.g., as measured using BLAST with default settings) and also has a symmetrical three-dimensional spatial configuration relative to the other WT-ITR, such that their three-dimensional structures have the same shape in geometric space. A substantially symmetric WT-ITR has the same A, C-C', and B-B' loops in three-dimensional space. A substantially symmetric WT ITR can be functionally confirmed as WT by determining that it has an operable Rep binding site (RBE or RBE') and terminal resolution site (trs) that pairs with the appropriate Rep protein. One skilled in the art can optionally test other functions, including transgene expression under permissive conditions.

[0134] As used herein, the phrases "modified ITR" or "mod-ITR" or "mutated ITR" are used interchangeably herein and refer to an ITR that has a mutation in at least one or more nucleotides compared to a wild-type ITR from the same serotype. The mutation can result in a change in one or more of the A, C, C', B, and B' regions in the ITR, and can result in a change in the three-dimensional spatial configuration (i.e., its three-dimensional structure in geometric space) compared to the three-dimensional spatial configuration of a wild-type ITR from the same serotype.

[0135] As used herein, the term "asymmetric ITR," also referred to as an "asymmetric ITR pair," refers to a pair of ITRs in a single-stranded synthetic AAV genome that are not reverse complements over their entire length. As one non-limiting example, an asymmetric ITR pair does not have a symmetrical three-dimensional spatial configuration relative to its cognate ITR, such that their three-dimensional structures are different shapes in geometric space. In other words, an asymmetric ITR pair has a different overall geometric structure, i.e., they have a different configuration of their A, C-C', and B-B' loops in three-dimensional space (e.g., one ITR may have a shorter C-C' arm and / or a shorter B-B' arm compared to the cognate ITR). The sequence difference between the two ITRs may be due to one or more nucleotide additions, deletions, truncations, or point mutations. In one embodiment, one ITR of the asymmetric ITR pair may be a wild-type AAV ITR sequence, and the other ITR may be a modified ITR (e.g., a non-wild-type or synthetic ITR sequence) as defined herein. In another embodiment, neither ITR of the asymmetric ITR pair is a wild-type AAV sequence, but rather the two ITRs are modified ITRs with different shapes in geometric space (i.e., different overall geometric structures). In some embodiments, one mod-ITR of the asymmetric ITR pair can have a short C-C' arm, and the other ITR can have a different modification (e.g., a single arm or a short B-B' arm) such that it has a different three-dimensional spatial configuration compared to the cognate asymmetric mod-ITR.

[0136] As used herein, the term "symmetric ITRs" refers to a pair of ITRs in a single-stranded AAV genome that are wild-type or mutant (e.g., modified compared to wild-type) dependovirus ITR sequences and are reverse-complementary throughout their entire length. In one non-limiting example, both ITRs are wild-type ITR sequences from AAV2. In another example, neither ITR is a wild-type ITR AAV2 sequence (i.e., they are modified ITRs, also referred to as mutant ITRs), but may have a sequence that differs from the wild-type ITR due to nucleotide additions, deletions, substitutions, truncations, or point mutations. For convenience herein, the ITR located 5' to (upstream of) the expression cassette of a synthetic AAV vector is referred to as the "5' ITR" or "left ITR," and the ITR located 3' to (downstream of) the expression cassette of a synthetic AAV vector is referred to as the "3' ITR" or "right ITR."

[0137] As used herein, the term "substantially symmetric modified ITR" or "substantially symmetric mod-ITR pair" refers to a pair of modified ITRs in a synthetic AAV that both have reverse-complementary sequences throughout their entire length. For example, modified ITRs can be considered substantially symmetric even if they have some nucleotide sequence deviations from their reverse-complementary sequences, as long as the changes do not affect their properties and overall shape. As one non-limiting example, a sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a reference sequence (as measured using BLAST with default settings) and also has a symmetrical three-dimensional spatial organization relative to its cognate modified ITRs such that their three-dimensional structures have the same shape in geometric space. In other words, a substantially symmetric modified ITR pair has the same A, C-C', and B-B' loops organized in three-dimensional space. In some embodiments, the ITRs from a mod-ITR pair can have different reverse-complementary nucleotide sequences but still have the same symmetrical three-dimensional spatial organization, i.e., both ITRs have mutations that result in the same overall 3D shape. For example, one ITR (e.g., the 5' ITR) of a mod-ITR pair can be from one serotype, and the other ITR (e.g., the 3' ITR) can be from a different serotype, but both can have the same corresponding mutations such that the modified ITR pair has the same symmetrical three-dimensional spatial organization (e.g., if the 5' ITR has a deletion in the C region, the cognate modified 3' ITR from the different serotype has a deletion at a corresponding position in the C' region). In such embodiments, each ITR of a modified ITR pair can be from a different serotype (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), e.g., a combination of AAV2 and AAV6, with modifications in one ITR mirrored at the corresponding position in the cognate ITR from the different serotype. In one embodiment, a substantially symmetric modified ITR pair refers to a pair of modified ITRs (mod-ITRs), so long as the nucleotide sequence differences between the ITRs do not affect the properties or overall shape and they have substantially the same shape in three-dimensional space.Non-limiting examples include mod-ITRs that have at least 95%, 96%, 97%, 98%, or 99% sequence identity with a reference mod-ITR, as determined by standard means known in the art, such as BLAST (Basic Local Alignment Search Tool) or BLASTN in default settings, and also have symmetrical three-dimensional spatial configurations such that their three-dimensional structures are the same shape in geometric space. A substantially symmetric mod-ITR pair will have the same A, C-C', and B-B' loops in three-dimensional space; for example, if the modified ITR of a substantially symmetric mod-ITR pair has a deletion of the C-C' arm, the cognate mod-ITR will have a corresponding deletion of the C-C' loop and also have a similar three-dimensional structure of the remaining A and B-B' loops that are the same shape in the geometric space of the cognate mod-ITR.

[0138] As used herein, the phrase "effective amount" or "therapeutically effective amount" of a therapeutic agent, such as an active agent or therapeutic nucleic acid, is an amount sufficient to produce a desired effect, e.g., inhibition of expression of a target sequence, compared to expression levels detected in the absence of the therapeutic nucleic acid. Suitable assays for measuring expression of a target gene or target sequence include, for example, examination of protein or RNA levels using techniques known to those of skill in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those of skill in the art.

[0139] As used herein, the term "expression" refers to the cellular processes involved in the production of RNA and proteins, and optionally secreted proteins, including, for example, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing, where applicable. As used herein, the phrase "expression product" includes RNA transcribed from a gene (e.g., a transgene) and polypeptides obtained by translation of mRNA transcribed from a gene.

[0140] As used herein, the term "expression vector" refers to a vector that directs the expression of RNA or polypeptides from sequences linked to transcriptional regulatory sequences on the vector. The expressed sequences are often, but not necessarily, heterologous to the host cell. An expression vector may contain additional elements; for example, an expression vector may have two replication systems, thus allowing it to be maintained in two organisms, e.g., human cells in the case of expression and prokaryotic hosts in the case of cloning and amplification. An expression vector may be a recombinant vector.

[0141] As used herein, the term "flanking" refers to the relative position of one nucleic acid sequence with respect to another. Generally, in the sequence ABC, B is flanked by A and C. Similarly for the arrangement AxBxC. Thus, a flanking sequence precedes or follows the flanked sequence, but need not be contiguous with or immediately adjacent to it.

[0142] As used herein, the term "spacer region" refers to an intervening sequence that separates functional elements within a vector or genome. In some embodiments, a spacer region maintains two functional elements at a desired distance for optimal functionality. In some embodiments, a spacer region provides or adds to the genetic stability of a vector or genome. In some embodiments, a spacer region facilitates easy genetic manipulation of the genome by providing convenient locations for cloning sites and gaps of a designed number of nucleotides.

[0143] As used herein, the terms "expression cassette" and "expression unit" are used interchangeably and refer to a heterologous DNA sequence operably linked to a promoter or other DNA regulatory sequence sufficient to direct transcription of a transgene in a DNA vector, e.g., a synthetic AAV vector. Suitable promoters include, for example, tissue-specific promoters. The promoter can also be of AAV origin.

[0144] As used herein, a "vector" or "expression vector" is a replicon, which can be a nucleic acid construct designed to be delivered to a host cell or moved between different host cells. As used herein, a vector can be of viral or non-viral origin in its final form. For the purposes of this disclosure, "vector" generally refers to a synthetic AAV that does not contain a capsid, such as a single-stranded (ss) synthetic vector or a nicked ceDNA vector. Thus, the term "vector" encompasses any genetic element that, when associated with appropriate control elements, can replicate or express and transfer gene sequences into a cell. In some embodiments, a vector can be a recombinant vector or an expression vector. As used herein, it should be understood that the term "single-stranded (ss) synthetic vector" includes a single-stranded AAV-like vector that does not have any viral sequences.

[0145] As used herein, the term "genetic disease" refers to a disease caused, directly or indirectly, in part or in whole by one or more abnormalities in the genome, particularly a condition that is present from birth and that can be treated by the single-stranded (ssDNA) molecules described herein. The abnormality can be a mutation, insertion, or deletion. The abnormality can affect the coding sequence of a gene or its regulatory sequence. Genetic disorders include phenylketonuria (PKU), melanoma, hemophilia A (clotting factor VIII (FVIII) deficiency) and hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis, Huntington's disease, familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, congenital hepatic porphyria, inherited liver metabolic disorders, Lesch-Nyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom's syndrome, retinoblastoma, and mucopolysaccharidosis (e.g., Hurler syndrome (MPS type I), Scheie syndrome (MPS type IS), Hurler-Scheie syndrome (MPS type I HS), Hunter syndrome (MPS type II), Sanfilippo types A, B, C, and D (MPS type III). A, B, C, and D), Morquio syndrome types A and B (MPS IVA and MPS IVB), Maroteaux-Lamy syndrome (MPS VI), Sly syndrome (MPS VII), hyaluronidase deficiency (MPS IX), Niemann-Pick disease types A / B, C1, and C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandhoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis types I, II / III, and IV, sialidosis types I and II, glycogen storage disease types I and II (Pompe disease), Gaucher disease types I, II, and III, Fabry disease, cystinosis, Batten disease, aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidoses, and galactosialidosis.Genetic disorders also include amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber's congenital amaurosis (LCA, e.g., LCA10 [CEP290]), Stargardt macular dystrophy (ABCA4), or cathepsin A deficiency.

[0146] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which may include chemically or biochemically modified or derivatized amino acids and polypeptides having modified peptide backbones.

[0147] In some embodiments, the polypeptide of the present disclosure is an ApoE polypeptide or an ApoB polypeptide. In some embodiments, the ApoE polypeptide is a functional fragment (or functional portion) of a full-length ApoE polypeptide. In some embodiments, the ApoE polypeptide is a functional fragment (or functional portion) of a full-length ApoB polypeptide. In some embodiments, the ApoE polypeptide is 30 amino acids or less in length. In some embodiments, the ApoB polypeptide is 30 amino acids or less in length.

[0148] As used herein, the term "lipid" refers to a group of organic compounds, including, but not limited to, esters of fatty acids, which are characterized by being insoluble in water but soluble in many organic solvents. Lipids are generally divided into at least three classes: (1) "simple lipids," which include fats and oils and waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0149] Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine.Other compounds that lack phosphorus, such as sphingolipids, glycosphingolipid family, diacylglycerol, and β-acyloxyacid, are also included in the group called amphipathic lipid.In addition, the above amphipathic lipids can be mixed with other lipids, including triglycerides and sterols.

[0150] As used herein, the term "polyglycerol" refers to an organic compound that is a polymeric condensation product of glycerol. Polyglycerols obtained from the dehydration of glycerol can have a linear, branched, or cyclic structure. In some embodiments, polyglycerols of the present disclosure are linear or branched. In one embodiment, polyglycerols are linear. In one embodiment, polyglycerols are branched. In some embodiments, the term "polyglycerol" encompasses a population of polyglycerol molecules. A population of polyglycerol molecules can include a distribution of polyglycerol molecules of different lengths, i.e., a distribution of polyglycerol molecules containing different numbers of monomer units. Thus, as used herein with respect to polyglycerol, the term "average molecular weight" refers to the average molecular weight of a population of polyglycerol molecules. The average molecular weight of a polyglycerol can be determined by any method known in the art, such as MALDI-MS or NMR. As used herein with respect to the number of monomer units present in a polyglycerol, the term "average" refers to the average number of monomer units per polyglycerol molecule in a population of polyglycerol molecules. Thus, as used herein with respect to the number of monomer units present in a polyglycerol, language such as "an average of 45 monomer units" refers to an average of 45 monomer units per polyglycerol molecule in a population of polyglycerol molecules. The average number of monomer units per polyglycerol molecule can be calculated based on the average molecular weight of the polyglycerol.

[0151] In some embodiments, the polyglycerol may contain an average of 8 to 100 monomer units, e.g., an average of 8 to 40 monomer units, an average of 15 to 75 monomer units, an average of 20 to 50 monomer units, an average of 30 to 70 monomer units, an average of 40 to 90 monomer units, or an average of 50 to 100 monomer units. In some embodiments, the polyglycerols of the present disclosure may contain an average of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 monomer units.

[0152] As used herein, the term "linear" when referring to a polyglycerol or aliphatic hydrocarbon chain means that the chain is unbranched.

[0153] As used herein, the term "polyglycerol derivative" or "PG derivative" refers to a polyglycerol in which the free alcohol groups have been modified. In some embodiments, the polyglycerol derivatives of the present disclosure are linear or branched. In one embodiment, the polyglycerol derivative is linear. In one embodiment, the polyglycerol derivative is branched.

[0154] In some embodiments, the term "polyglycerol derivative" encompasses a population of polyglycerol derivative molecules. A population of polyglycerol derivative molecules can include a distribution of polyglycerol derivative molecules of different lengths, i.e., a distribution of polyglycerol derivative molecules containing different numbers of monomer units. Thus, when used herein with respect to a polyglycerol derivative, the term "average molecular weight" refers to the average molecular weight of a population of polyglycerol derivative molecules. The average molecular weight of a polyglycerol derivative can be determined by any method known in the art, such as MALDI-MS or NMR. When used herein with respect to the number of monomer units present in a polyglycerol derivative, the term "average" refers to the average number of monomer units per polyglycerol derivative molecule in the population of polyglycerol derivative molecules. Thus, when used herein with respect to the number of monomer units present in a polyglycerol derivative, language such as "an average of 8 monomer units" refers to an average of 8 monomer units per polyglycerol derivative molecule in the population of polyglycerol derivative molecules. The average number of monomer units per polyglycerol derivative molecule can be calculated based on the average molecular weight of the polyglycerol derivative.

[0155] In some embodiments, the polyglycerol derivatives of the present disclosure can include an average of 8 to 100 monomer units, e.g., an average of 8 to 40 monomer units, an average of 15 to 75 monomer units, an average of 20 to 50 monomer units, an average of 30 to 70 monomer units, an average of 40 to 90 monomer units, or an average of 50 to 100 monomer units. In some embodiments, the polyglycerols of the present disclosure have an average of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 monomer units.

[0156] In some embodiments, the polyglycerol derivative can be a carboxylic acid polyglycerol, i.e., a polyglycerol in which free alcohol groups have been modified by converting them to a moiety containing one or more carboxylic acid groups, such as 2-carboxycyclohexane-1-carboxylic acid polyglycerol. In some embodiments, the polyglycerol derivative can be a glutarylated polyglycerol, i.e., a polyglycerol in which free alcohol groups have been modified by converting them to glutarate or glutarate derivatives, such as 3-methylglutarylated polyglycerol. In some embodiments, the polyglycerol derivative can be conjugated to a lipid moiety, such as a lipid moiety represented by formula (I) described herein. In some embodiments, the polyglycerol derivative conjugated to a lipid moiety can be represented by the following structural formula: [ka] , During the ceremony, n is an integer ranging from 8 to 100; R, [ka] and [ka] is selected from the group consisting of:

[0157] As used herein, the term "hydrophobic tail" refers to a hydrocarbon chain, i.e., a chain comprising carbon and hydrogen atoms, which may be saturated or unsaturated. In one embodiment, the hydrophobic tail may contain 10 to 30 carbon atoms, e.g., 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon atoms. In some embodiments, the hydrocarbon chain of the hydrophobic tail is unsaturated, i.e., does not contain double or triple bonds. In other embodiments, the hydrocarbon chain of the hydrophobic tail is unsaturated and contains one or more double bonds and / or one or more triple bonds. In some embodiments, the hydrocarbon chain may be linear. In other embodiments, the hydrocarbon chain may be branched. Non-limiting examples of backbone hydrophobic tails of the present disclosure include the hydrophobic tails present in lauric acid, myristic acid, myristoleic acid, octadecylamine, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, and derivatives thereof.

[0158] As used herein, the term "click chemistry reaction product" refers to the moiety formed by two click chemistry reagents of a "click pair." In some embodiments, the click chemistry reaction product is the product of a reaction between a) a tetrazine reagent (i.e., a reagent comprising a tetrazine moiety) and a transcyclooctene reagent (i.e., a reagent comprising a transcyclooctene moiety), b) a tetrazine reagent and a norbornene reagent (i.e., a reagent comprising a norbornene moiety), or c) an azide reagent (i.e., a reagent comprising an azide moiety) and an alkyne reagent, such as a dibenzocyclooctyne (DBCO) reagent.

[0159] As used herein, the term "lipid-anchored polymer," which may be used interchangeably with the term "lipid conjugate," refers to a molecule comprising a lipid moiety covalently attached to a polymer via a linker. Without wishing to be bound by any particular theory, it is believed that lipid-anchored polymers can inhibit aggregation and provide steric stabilization of LNPs. Exemplary lipid-anchored polymers include the polymer-conjugated lipids described herein, PEGylated lipids, such as PEG coupled to dialkyloxypropyl (e.g., PEG-DAA conjugates), PEG coupled to diacylglycerol (e.g., PEG-DAG conjugates), PEG coupled to dimyristylglycerol (e.g., PEG-DMG), PEG coupled to distearoylglycerol (e.g., PEG-DSG), PEG coupled to poly(2-methacryloyloxyethyl phosphorylcholine) (e.g., PEG-PMPC), PEG coupled to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (e.g., PEG-DSPE), and diacylglycerol-conjugated lipids. Examples of suitable lipid conjugates include, but are not limited to, polyglycerols coupled to tadecylamine (e.g., DODA-PG), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine, and PEG conjugated to ceramide (see, e.g., U.S. Pat. No. 5,885,613), cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates (e.g., POZ-DAA conjugates, see, e.g., U.S. Provisional Patent Application Nos. 61 / 294,828, filed January 13, 2010, and 61 / 295,140, ​​filed January 14, 2010), polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof. Further examples of POZ-lipid conjugates are described in International Patent Application Publication No. 2010 / 006282. The PEG, PG, or POZ can be directly conjugated to the lipid or may be linked to the lipid via a linker moiety.For example, any suitable linker moiety can be used for coupling PEG, PG, or POZ to lipid, including non-ester-containing linker moieties and ester-containing linker moieties.In certain preferred embodiments, non-ester-containing linker moieties such as amides or carbamates are used.The disclosures of each of the above patent documents are incorporated herein by reference in their entirety for all purposes.

[0160] Exemplary linkers that can be used to conjugate lipid moieties to polymers in the lipid-anchored polymers of the present disclosure can be selected from the group consisting of alkyl linkers, glycerol linkers, phosphate linkers, phosphate ester linkers, ether linkers, ester linkers, diester linkers (e.g., glutaryl linkers, succinyl linkers), amide linkers, diamide linkers, amine linkers, peptide linkers, phosphoethanolamine linkers, phosphocholine linkers, carbamate linkers, diamide alkyl linkers, cleavable linkers, click reaction products, and any combination thereof. In some embodiments, the linker is -(CH2) n -, -C(O)(CH2) n -, -C(O)O(CH2) n -, -OC(O)(CH2) n C(O)O- and -NH(CH2) n In some embodiments, the linker may be selected from the group consisting of -C(O)O-, where n is an integer ranging from 1 to 20. In some embodiments, the linker may be selected from the group consisting of -C(O)(CH) n -, where n is 2, 3, 4, 5, or 6. In one particular embodiment, n is 4.

[0161] As used herein, the term "encapsulated lipid" refers to a lipid particle that provides an active or therapeutic agent, such as a nucleic acid (e.g., ceDNA), that is fully encapsulated, partially encapsulated, or both. In preferred embodiments, the nucleic acid is fully encapsulated in the lipid particle (e.g., to form a nucleic acid-containing lipid particle).

[0162] As used herein, the term "lipid particle" or "lipid nanoparticle" or "LNP" refers to a lipid formulation that can be used to deliver a therapeutic agent, such as a nucleic acid therapeutic, to a desired target site (e.g., a cell, tissue, organ, etc.). In one embodiment, the lipid particle of the present disclosure is a nucleic acid-containing lipid particle formed from a cationic lipid, a non-cationic lipid, and optionally conjugated lipids, which typically prevent particle aggregation. In another preferred embodiment, a therapeutic agent, such as a therapeutic nucleic acid, may be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation. In one embodiment, the lipid particle comprises a nucleic acid (e.g., ceDNA, ssDNA, mRNA, etc.) and a lipid comprising one or more tertiary amino groups, one or more phenyl ester bonds, and a disulfide bond.

[0163] According to some embodiments, lipid particles of the present disclosure typically have a size of about 20 nm to about 75 nm, about 20 nm to about 70 nm, about 25 nm to about 75 nm, about 25 nm to about 70 nm, about 30 nm to about 75 nm, about 30 nm to about 70 nm, about 35 nm to about 75 nm, about 35 nm to about 70 nm, about 40 nm to about 75 nm, about 40 nm to about 70 nm, about 45 nm to about 75 nm, about 50 nm to about 5 ... nm to about 75nm, about 50nm to about 70nm, about 60nm to about 75nm, about 60nm to about 70nm, about 65nm to about 75nm, about 65nm to about 70nm, or about 20nm, about 25nm, Approximately 30nm, approximately 35nm, approximately 40nm, approximately 45nm, approximately 50nm, approximately 51nm, approximately 52nm, approximately 53nm, approximately 54nm, approximately 55nm, approximately 56nm, approximately 57nm, approximately 58nm, approximately 59nm having an average diameter of a size of about 60 nm, about 61 nm, about 62 nm, about 63 nm, about 64 nm, about 65 nm, about 66 nm, about 67 nm, about 68 nm, about 69 nm, about 70 nm, about 71 nm, about 72 nm, about 73 nm, about 74 nm, or about 75 nm (±3 nm).

[0164] Generally, the LNPs of the present disclosure have an average diameter selected to produce an intended therapeutic effect. For example, the LNPs of the present disclosure have an average diameter that is compatible with a target organ (e.g., the liver) so that the LNPs of the present disclosure can diffuse through fenestrations in the target organ (e.g., liver) or target cell subpopulation (e.g., hepatocytes).

[0165] According to some embodiments, the lipid particles of the present disclosure typically have an average diameter of less than about 75 nm, less than about 70 nm, less than about 65 nm, less than about 60 nm, less than about 55 nm, less than about 50 nm, less than about 45 nm, less than about 40 nm, less than about 35 nm, less than about 30 nm, less than about 25 nm, or less than about 20 nm in size.

[0166] As used herein, the term "cationic lipid" refers to any lipid that is positively charged at physiological pH.The cationic lipid in lipid particles can include one or more cationic lipids, such as 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linoleyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), "SS-cleavable lipid", or mixtures thereof.In some embodiments, the cationic lipid can also be an ionizable lipid, i.e., an ionizable cationic lipid. That is, the term cationic lipid also encompasses lipids that are positively charged at any pH, such as lipids containing quaternary amine groups, i.e., quaternary lipids. Any cationic lipid described herein that contains a primary, secondary, or tertiary amine group can be converted to the corresponding quaternary lipid, for example, by treatment with a solution of chloromethane (CHCl) in acetonitrile (CHCN) and chloroform (CHCl).

[0167] As used herein, the term "ionizable lipid" refers to a lipid, e.g., a cationic lipid, that has at least one protonatable or deprotonatable group such that the lipid is positively charged at a pH below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably above physiological pH. It will be understood by those skilled in the art that the addition or removal of protons as a function of pH is an equilibrium process, and reference to a charged or neutral lipid refers to the nature of the predominant species and does not require that all lipids exist in a charged or neutral form. Generally, ionizable lipids have a pKa of the protonatable group in the range of about 4 to about 7. In some embodiments, ionizable lipids may include "cleavable lipids" or "SS-cleavable lipids."

[0168] As used herein, the term "neutral lipid" refers to any of several lipid species that exist in either an uncharged form or a neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0169] As used herein, the term "non-cationic lipid" refers to any amphipathic helper lipid, as well as any other neutral or anionic lipid.

[0170] As used herein, the term "cleavable lipid" or "SS-cleavable lipid" refers to an ionizable lipid containing a disulfide bond-cleavable unit. The cleavable lipid may contain a cleavable disulfide bond (SS) containing pH-sensitive amine, e.g., a tertiary amine, and a self-degrading phenyl ester-containing lipid-like material. For example, the SS-cleavable lipid can be ss-OP lipid (COATSOME® SS-OP), ss-M lipid (COATSOME® SS-M), ss-E lipid (COATSOME® SS-E), ss-EC lipid (COATSOME® SS-EC), ss-LC lipid (COATSOME® SS-LC), ss-OC lipid (COATSOME® SS-OC), and ss-PalmE lipid (see, e.g., Formulas I-IV), or lipids described in Togashi et al., (2018) Journal of Controlled Release “A hepatic pDNA delivery system based on an intracellular environment sensitive vitamin E-scaffold lipid-like material with the aid of an anti-inflammatory drug” 279:262-270. Further examples of cleavable lipids are described in U.S. Patent No. 9,708,628 and U.S. Patent No. 10,385,030, the entire contents of which are incorporated herein by reference. In one embodiment, the cleavable lipid comprises a tertiary amine that responds to disulfide bonds, which can be cleaved under a reducing environment, such as an acidic compartment, for example, an endosome or lysosome for membrane destabilization, and the cytoplasm. In one embodiment, the cleavable lipid is a cationic lipid. In one embodiment, the cleavable lipid is an ionizable cationic lipid. Cleavable lipids are described in more detail herein.

[0171] As used herein, the term "organic lipid solution" refers to a composition that comprises, in whole or in part, an organic solvent with a lipid.

[0172] As used herein, the term "liposome" refers to lipid molecules assembled in a spherical configuration that encapsulates an aqueous interior volume separated from an aqueous exterior. Liposomes are vesicles with at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic drug delivery in the context of pharmaceutical development. They function by fusing with cell membranes and rearranging their lipid structure to deliver the drug or active pharmaceutical ingredient (API). Liposomal compositions for such delivery are typically composed of phospholipids, particularly compounds with phosphatidylcholine groups, although these compositions may also contain other lipids.

[0173] As used herein, the term "local delivery" refers to the delivery of an active agent, such as an interfering RNA (e.g., siRNA), to a target site within an organism. For example, the agent can be locally delivered by direct injection into a disease site, such as a tumor, or other target site, such as an inflammation site, or into a target organ, such as the liver, heart, pancreas, or kidney.

[0174] As used herein, the term "nucleic acid" refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form, including DNA, RNA, and hybrids thereof. DNA can be in the form of, for example, an antisense molecule, a plasmid DNA, a DNA-DNA duplex, a precondensed DNA, a PCR product, a vector (P1, PAC, BAC, YAC, artificial chromosome), an expression cassette, a chimeric sequence, chromosomal DNA, or derivatives and combinations thereof. DNA can be in the form of a minicircle, a plasmid, a bacmid, a minigene, a ministring DNA (a covalently closed linear DNA vector), a closed-end linear double-stranded DNA (CELiD or ceDNA), doggybone™ DNA, dumbbell-shaped DNA, a minimal immunologically defined gene expression (MIDGE) vector, a viral vector, or a non-viral vector. RNA can be in the form of small interfering RNA (siRNA), Dicer substrate dsRNA, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), messenger RNA (mRNA), rRNA, tRNA, gRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and have similar binding properties to the reference nucleic acid. Examples of such analogs and / or modified residues include, but are not limited to, phosphorothioates, phosphorodiamidate morpholino oligomers (morpholinos), phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, locked nucleic acids (LNA™), and peptide nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated.

[0175] As used herein, the phrases "nucleic acid therapeutic agent," "therapeutic nucleic acid," and "TNA" are used interchangeably and refer to any therapeutic modality that uses a nucleic acid as the active ingredient of a therapeutic agent to treat a disease or disorder. As used herein, these terms refer to RNA-based therapeutic agents and DNA-based therapeutic agents. Non-limiting examples of RNA-based therapeutic agents include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), Dicer substrate dsRNA, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), or guide RNA (gRNA). Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigenes, viral DNA (e.g., lentivirus or AAV genome) or non-viral synthetic DNA vectors, closed-ended linear double-stranded DNA (ceDNA / CELiD), single-stranded DNA (ssDNA) molecules, plasmids, bacmids, DOGGYBONE™ DNA vectors, minimal immunologically defined gene expression (MIDGE) vectors, non-viral ministring DNA vectors (covalently closed linear DNA vectors), or dumbbell-shaped DNA minimal vectors ("dumbbell DNA").

[0176] As used herein, a "nucleotide" comprises the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate group.

[0177] As used herein, the terms "single-stranded DNA molecule," "ssDNA molecule," or "SSD molecule" refer to a deoxyribonucleic acid (DNA) molecule comprising at least one single-stranded nucleic acid sequence adjacent to at least one stem-loop structure at the 3'-end. In some embodiments, the single-stranded DNA molecule further comprises at least one stem-loop structure at the 5'-end. As used herein, a single-stranded DNA molecule may comprise a region of double-stranded DNA (or partial duplex), e.g., a stem-loop structure, e.g., an inverted terminal repeat sequence or a portion thereof, at an end, e.g., the 3'-end and / or the 5'-end. In some embodiments, the ssDNA molecule is a synthetic ssDNA molecule. In some embodiments, the ssDNA molecule comprises at least one stem-loop structure at the 5'-end and at least one stem-loop structure at the 3'-end.

[0178] As used herein, the terms "single-stranded (ss) synthetic DNA molecule," "single-stranded (ss) synthetic vector," "synthetic production of ssDNA molecule," and "synthetic production of ss vector" refer to single-stranded (ss) synthetic DNA molecules (ssDNA), single-stranded vectors, and methods for their synthetic production in a completely cell-free environment. This production can involve one or more molecules in a manner that does not require replication or other propagation of the molecule by a cell or the interior of a cell or by the use of a cell extract. Synthetic production avoids contamination of the produced molecule with cellular contaminants, such as cellular proteins or nucleic acids, viral proteins or DNA, or insect proteins or DNA, and further minimizes undesired cell-specific modifications of the molecule during the production process, such as methylation or glycosylation, or other post-translational modifications.

[0179] As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions such as oil / water or water / oil, and various types of wetting agents. The term also encompasses any of the agents approved by a U.S. federal regulatory agency or listed in the U.S. Pharmacopoeia for use in animals, including humans, as well as any carrier or diluent that does not cause significant irritation to the subject and does not destroy the biological activity and properties of the administered compound.

[0180] As used herein, the term "gap" refers to an interrupted portion of the synthetic DNA vector of the present disclosure that creates a continuous single-stranded DNA segment in an otherwise double-stranded ceDNA. The gap can be 1 to 100 nucleotides in length in one strand of the duplex DNA. Exemplary gaps designed and generated by the methods described herein, and synthetic vectors generated thereby, can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nt in length. Exemplary gaps in the present disclosure can be 1 nt to 10 nt in length, 1 to 20 nt in length, or 1 to 30 nt in length.

[0181] As used herein, the term "nick" refers to a discontinuity in a double-stranded DNA molecule, typically due to damage or enzymatic action, in which a phosphodiester bond is absent between adjacent nucleotides on one strand. It is understood that one or more nicks allow for twist release within the strand during DNA replication, and that nicks are also thought to play a role in facilitating the binding of the transcription machinery.

[0182] As used herein, the term "ceDNA" refers to closed-ended, linear, double-stranded (ds) duplex DNA, whether synthetic or otherwise, that does not contain a capsid for non-viral gene transfer. A detailed description of ceDNA is provided in International Patent Application No. PCT / US2017 / 020828, filed March 3, 2017, the entire contents of which are expressly incorporated herein by reference. Certain methods for producing ceDNA containing various inverted terminal repeat (ITR) sequences and configurations using cell-based methods are described in Example 1 of International Application No. PCT / US18 / 49996, filed September 7, 2018, and International Application No. PCT / US2018 / 064242, filed December 6, 2018, each of which is incorporated herein by reference in its entirety. Certain methods for generating synthetic ceDNA vectors containing various ITR sequences and configurations are described, for example, in International Application PCT / US2019 / 14122, filed January 18, 2019, the entire contents of which are incorporated herein by reference. As used herein, the terms "ceDNA vector" and "ceDNA" are used interchangeably. According to some embodiments, the ceDNA is a closed-end linear duplex (CELiD) CELiD DNA. According to some embodiments, the ceDNA is a DNA-based minicircle. According to some embodiments, the ceDNA is a minimal immunologically defined gene expression (MIDGE) vector. According to some embodiments, the ceDNA is ministring DNA. According to some embodiments, the ceDNA is doggybone™ DNA. According to some embodiments, the ceDNA comprises one or more phosphorothioate-modified nucleotides. According to some embodiments, the ceDNA does not comprise phosphorothioate-modified nucleotides.

[0183] As used herein, the term "neDNA" or "nicked ceDNA" refers to closed-end DNA that has a nick or gap of 1 to 100 nucleotides in the stem or spacer region upstream of the open reading frame (e.g., the expressed promoter and transgene).

[0184] As used herein, the term "inverted terminal repeat" or "ITR" refers to a nucleic acid sequence located at the 5' and / or 3' end of an ssDNA molecule disclosed herein that comprises at least one stem-loop structure that is partially duplexed and includes at least one loop.

[0185] As used herein, the term "stem-loop structure" refers to a nucleic acid structure comprising at least one double-stranded region (referred to herein as a "stem") and at least one single-stranded region (referred to herein as a "loop"). In some embodiments, the stem-loop structure is a hairpin structure. In some embodiments, the stem-loop structure comprises two or more stems and two or more loops. In some embodiments, a loop is located at the end of a stem (such that a single loop connects two strands of a double-stranded stem, e.g., similar to a hairpin structure). In some embodiments, a loop may be located between two stems (which may be referred to herein as a "bulge" or "bubble"), such that a loop connects two strands of different stems. In some embodiments, as described in more detail herein, the stem-loop structure may comprise a more complex secondary structure comprising multiple stems and multiple loops.

[0186] According to some embodiments, the 5' and / or 3' ends of certain ssDNA molecules contain approximately 145-nucleotide inverted terminal repeats (ITRs) or fragments thereof at both ends. The terminal 125 nucleotides in each ITR form a palindromic double-stranded T-shaped hairpin structure, with the A-A' palindrome forming the stem and two smaller palindromes, B-B' and C-C', forming the cross arms of the T. The other 20 nucleotides in the ITR remain single-stranded and are referred to as the D sequence. The D(-) sequence (also referred to herein as the "ssD(-) sequence") is at the 3' end, and the complementary D(-) sequence (also referred to herein as the "ssD(-) sequence") is at the 5' end. Upon double-stranded DNA synthesis, both the ssD(-) sequence and the ssD(+) sequence become double-stranded (ds)D(±) sequences, each of which contains a D region and a D' region. Ling et al. J Virol. 2015 Jan 15;89(2):952-61, WO2016081927A2 (incorporated herein by reference in its entirety) have reported that ssD(-) and ssD(+) contain one or more transcription factor binding sites and are required for packaging and replication (Ling et al. J Virol. 2015 Jan 15;89(2):952-61, WO2016081927A2 (incorporated herein by reference in its entirety)).

[0187] According to some embodiments, the ITRs can be viral ITRs (e.g., AAV or other dexoviruses), sequences derived from or modified from viral ITRs (e.g., truncations, deletions, substitutions, insertions, and / or additions), or completely artificial sequences (e.g., the ITRs do not contain sequences derived from a virus). The ITRs can further comprise a stem-loop structure (e.g., a "hairpin") or two or more stem-loop structures. For example, the ITRs can comprise two stem-loop structures (e.g., a "hammerhead," "dogbone," or "dumbbell"), three stem-loop structures (e.g., a "cruciform"), or a more complex structure (e.g., a four-stranded stem-loop structure). The ITRs can comprise an aptamer sequence or one or more chemical modifications. The ITRs can be made entirely of an aptamer sequence having at least one stem region and at least one loop region.

[0188] As used herein, the terms "substantially symmetric WT-ITR" or "substantially symmetric WT-ITR pair" refer to a pair of WT-ITRs in a single-stranded DNA (ssDNA) molecule, both of which are wild-type ITRs with reverse-complementary sequences throughout their entire length. For example, an ITR can be considered a wild-type sequence even if it has one or more nucleotides that deviate from a naturally occurring reference sequence, as long as the changes do not affect the physical and functional properties and overall three-dimensional structure (secondary and tertiary) of the sequence. In some embodiments, the deviating nucleotides represent conservative sequence changes. As one non-limiting example, a sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity with a reference sequence (e.g., as determined using BLAST with default settings) and also has a symmetric three-dimensional spatial configuration relative to the other WT-ITR, such that their three-dimensional structures are the same shape in geometric space. A substantially symmetric WT ITR has the same ssD(-) / ssD(+), A-A', C-C', and B-B' loops in three-dimensional space. A substantially symmetric WT ITR can be functionally confirmed as WT by determining that it has an operable Rep binding site (RBE or RBE') and terminal resolution site (TRS) that pairs with the appropriate Rep protein. One skilled in the art can optionally test other functions, including transgene expression under permissive conditions.

[0189] As used herein, the phrases "modified ITR" or "mod-ITR" or "mutated ITR" are used interchangeably herein and refer to an ITR that has a mutation in at least one or more nucleotides compared to a wild-type ITR from the same serotype. The mutation can result in a change in one or more of the ssD(-) or ssD(+), A region, A' region, C region, C' region, B region, or B' region in the ITR, and can result in a change in the three-dimensional spatial configuration (i.e., its three-dimensional structure in geometric space) compared to the three-dimensional spatial configuration of a wild-type ITR from the same serotype.

[0190] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the terms include single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. An "oligonucleotide" generally refers to a polynucleotide of about 5 to about 100 nucleotides, either single- or double-stranded. However, for purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides, also known as "oligomers" or "oligos," can be isolated from genes or chemically synthesized by methods known in the art. The terms "polynucleotide" and "nucleic acid" should be understood to include single-stranded polynucleotides (e.g., sense or antisense) and double-stranded polynucleotides, as applicable to the described embodiments. According to some embodiments, the nucleic acid is a single-stranded DNA (ssDNA) molecule as described herein. The DNA may be in the form of, for example, an antisense molecule, a plasmid DNA, a DNA-DNA duplex, a pre-condensed DNA, a PCR product, a vector (P1, PAC, BAC, YAC, artificial chromosome), an expression cassette, a chimeric sequence, a chromosomal DNA, or derivatives and combinations thereof. The DNA may be in the form of a minicircle, a plasmid, a bacmid, a minigene, a ministring DNA (a covalently closed linear DNA vector), a closed-end linear double-stranded DNA (CELiD or ceDNA), a doggybone (dbDNA™) DNA, a dumbbell-shaped DNA, a minimal immunologically defined gene expression (MIDGE) vector, a viral vector, or a non-viral vector. The RNA may be in the form of a small interfering RNA (siRNA), a Dicer substrate dsRNA, a short hairpin RNA (shRNA), an asymmetric interfering RNA (aiRNA), a microRNA (miRNA), an mRNA, a rRNA, a tRNA, a viral RNA (vRNA), or a combination thereof.Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, both synthetic, naturally occurring, and non-naturally occurring, that have similar binding properties to the reference nucleic acid. Examples of such analogs and / or modified residues include, but are not limited to, phosphorothioates, phosphorodiamidate morpholino oligomers (morpholinos), phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, locked nucleic acids (LNA™), and peptide nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly indicated sequence.

[0191] As used herein, "inhibitory polynucleotide" refers to a DNA or RNA molecule that reduces or prevents the expression (transcription or translation) of a second (target) polynucleotide. Inhibitory polynucleotides include antisense polynucleotides, ribozymes, and external guide sequences. The term "inhibitory polynucleotide" further includes DNA molecules and RNA molecules, e.g., RNAi, that encode the actual inhibitory species, such as a DNA molecule encoding a ribozyme.

[0192] A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate group.

[0193] "Base" includes purines and pyrimidines, which further include the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and naturally occurring analogues, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0194] "Receptor" refers to a polypeptide or portion thereof present on a cell membrane that selectively binds to one or more ligands. As used herein, the term "receptor" is intended to encompass the entire receptor or its ligand-binding portion. These portions of the receptor specifically include a region sufficient for specific binding of the ligand to occur.

[0195] As used herein, the term "subject" refers to a human or animal to which treatment, including prophylactic treatment, using a therapeutic nucleic acid according to the present disclosure is provided. Typically, the animal is a vertebrate, such as, but not limited to, a primate, a rodent, a domestic animal, or a game animal. Primates include, but are not limited to, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include, but are not limited to, cattle, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cats, canine species, e.g., dogs, foxes, wolves, avian species, e.g., chickens, emus, ostriches, and fish, e.g., trout, catfish, and salmon. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate or a human. The subject can be male or female. Additionally, the subject can be an infant or a child. In some embodiments, the subject can be a newborn or unborn subject, e.g., the subject is present in utero. Preferably, the subject is a mammal. The mammal can be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals can be advantageously used as subjects that represent animal models of diseases and disorders. Additionally, the methods and compositions described herein can be used with domestic animals and / or pets. Human subjects can be of any age, sex, race, or ethnic group, e.g., Caucasian (white), Asian, African, Black, African-American, African-European, Hispanic, Middle Eastern, etc. In some embodiments, the subject can be a patient or other subject in a clinical setting. In some embodiments, the subject has already received treatment. In some embodiments, the subject is an embryo, fetus, newborn, infant, child, adolescent, or adult. In some embodiments, the subject is a human fetus, human newborn, human infant, human child, human adolescent, or human adult. In some embodiments, the subject is an animal embryo, or a non-human embryo, or a non-human primate embryo.In some embodiments, the subject is a human embryo.

[0196] As used herein, unless the context and use of the phrase dictate otherwise, the phrase "subject in need" refers to a subject: (i) who is to be administered ceDNA-lipid particles (or a pharmaceutical composition comprising ceDNA-lipid particles) according to the described disclosure, (ii) who has received ceDNA-lipid particles (or a pharmaceutical composition comprising ceDNA-lipid particles) according to the described disclosure, or (iii) who has received ceDNA-lipid particles (or a pharmaceutical composition comprising ceDNA-lipid particles) according to the described disclosure.

[0197] As used herein, the terms "suppress," "reduce," "interfere," "inhibit," and / or "decrease" (and similar terms) generally refer to the act of decreasing, either directly or indirectly, a concentration, level, function, activity, or behavior compared to native, expected, or average conditions, or compared to a control condition.

[0198] As used herein, the term "systemic delivery" refers to the delivery of lipid particles that result in widespread biodistribution of an active agent, such as an interfering RNA (e.g., siRNA), within an organism. Some administration techniques can result in systemic delivery of a particular agent, while others cannot. Systemic delivery means that a useful, preferably therapeutic, amount of the agent is exposed to most parts of the body. To achieve widespread biodistribution, a blood lifetime is generally required such that the agent is not rapidly degraded or eliminated (e.g., by first-pass organs (liver, lung, etc.) or rapid nonspecific cellular binding) before reaching disease sites distal to the administration site. Systemic delivery of LNPs can be by any means known in the art, including, for example, intravenous, subcutaneous, and intraperitoneal. In a preferred embodiment, systemic delivery of LNPs is by intravenous delivery.

[0199] As used herein, the term "effective amount," which may be used interchangeably with the terms "therapeutic amount," "therapeutically effective amount," "effective amount," or "pharmaceutically effective amount" of an active agent (e.g., a ceDNA described herein), refers to an amount sufficient to provide the intended benefit of treatment or effect, e.g., expression or inhibition of expression of a target sequence compared to the expression level detected in the absence of the therapeutic nucleic acid. Suitable assays for measuring the expression of a target gene or target sequence include, for example, testing protein or RNA levels using techniques known to those skilled in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those skilled in the art. Dosage levels are based on a variety of factors, including the type of injury, the patient's age, weight, sex, medical condition, severity of the condition, route of administration, and the particular active agent used. Thus, dosing regimens can vary widely but can be routinely determined by a physician using standard methods. Additionally, the terms "effective amount," "therapeutic amount," "therapeutically effective amount," and "pharmaceutically effective amount" include prophylactic or preventative amounts of the described compositions of the invention. In the described prophylactic or preventative uses of the invention, a sufficient amount is administered to a patient susceptible to or otherwise at risk of a disease, disorder, or condition, including the biochemical, histological, and / or behavioral symptoms of the disease, disorder, or condition, its complications, and intermediate pathological phenotypes manifesting during the development of the disease, disorder, or condition, to eliminate or reduce the risk of, lessen the severity of, or delay the onset of, the disease, disorder, or condition. It is generally preferred to use a maximum dose, i.e., the highest safe dose according to medical judgment. The terms "dose" and "dosage" are used interchangeably herein. In one aspect of any of the aspects or embodiments herein, the terms "therapeutic amount," "therapeutically effective amount," and "pharmaceutically effective amount" refer to non-prophylactic or non-preventative uses.

[0200] As used herein, the term "therapeutic effect" refers to an outcome of treatment, which outcome is deemed desirable and beneficial. A therapeutic effect can include, directly or indirectly, the arrest, reduction, or elimination of disease symptoms. A therapeutic effect can also include, directly or indirectly, the arrest, reduction, or elimination of the progression of disease symptoms.

[0201] For any therapeutic agent described herein, the therapeutically effective amount can be initially determined from preliminary in vitro tests and / or animal models. The therapeutically effective dose can also be determined from human data. The applied dose can be adjusted based on the relative bioavailability and efficacy of the administered compound. Adjusting the dose to achieve maximum efficacy based on the above and other well-known methods is within the ability of a person skilled in the art. The general principles for determining therapeutic efficacy, which can be found in Chapter 1 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001) (incorporated herein by reference), are summarized below.

[0202] Pharmacokinetic principles provide a basis for modifying dosing regimens to achieve a desired degree of therapeutic efficacy with minimal unacceptable adverse effects. Additional guidance for dosage modifications can be obtained in situations where the plasma concentration of the drug is measured and can be related to a therapeutic window.

[0203] As used herein, the terms "treat," "treating," and / or "treatment" include arresting, inhibiting, slowing, or reversing the progression of a condition, ameliorating clinical symptoms of a condition, or preventing the appearance of clinical symptoms to achieve a beneficial or desired clinical result. Treating further refers to achieving one or more of: (a) reducing the severity of the disorder; (b) limiting the onset of symptoms characteristic of the disorder being treated; (c) limiting the worsening of symptoms characteristic of the disorder being treated; (d) limiting the recurrence of the disorder in patients who previously had the disorder; and (e) limiting the recurrence of symptoms in patients who did not previously exhibit symptoms of the disorder. In one aspect of any of the aspects or embodiments herein, the terms "treat," "treating," and / or "treatment" include arresting, inhibiting, slowing, or reversing the progression of a condition, or ameliorating clinical symptoms of a condition.

[0204] Beneficial or desired clinical results, e.g., pharmacological and / or physiological effects, include, but are not limited to, preventing a disease, disorder, or condition from occurring in a subject who may be predisposed to the disease, disorder, or condition but who has not yet experienced or exhibited symptoms of the disease (prophylactic treatment), alleviating the disease, disorder, or condition, reducing the severity of the disease, disorder, or condition, stabilizing (i.e., not worsening) the disease, disorder, or condition, preventing the spread of the disease, disorder, or condition, delaying or slowing the progression of the disease, disorder, or condition, ameliorating or alleviating the disease, disorder, or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.

[0205] As used herein, the term "combination therapy" refers to a treatment regimen for a clinical indication that includes two or more therapeutic agents. Thus, the term refers to a treatment regimen in which a first therapy containing a first composition (e.g., active ingredient) is administered to a patient in conjunction with a second therapy containing a second composition (active ingredient) intended to treat the same or overlapping disease or clinical condition. Both the first and second compositions may act on the same or distinct cellular targets. The phrase "in conjunction with" in the context of combination therapy means that the therapeutic effect of the first therapy overlaps temporally and / or spatially with the therapeutic effect of the second therapy in a subject receiving the combination therapy. Thus, combination therapy can be formulated as a single formulation for simultaneous administration of the therapies or as separate formulations for sequential administration of the therapies.

[0206] As used herein, the term "alkyl" refers to a saturated monovalent hydrocarbon radical of 1 to 20 carbon atoms (i.e., C 1-20 "Monovalent" means that the alkyl has one point of attachment to the rest of the molecule. In one embodiment, an alkyl has 1 to 12 carbon atoms (i.e., C 1-12 alkyl) or 1 to 10 carbon atoms (i.e., C 1-10 In one embodiment, alkyl has 1 to 8 carbon atoms (i.e., C 1-8 alkyl), 1 to 7 carbon atoms (i.e., C 1-7 alkyl), 1 to 6 carbon atoms (i.e., C 1-6 alkyl), 1 to 4 carbon atoms (i.e., C 1-4 alkyl), or 1 to 3 carbon atoms (i.e., C 1-3Examples include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and the like. 1-6 Alkyl," "linear or branched C 1-4 Alkyl" or "Straight or branched C 1-3 Linear or branched alkyl, such as "alkyl," means that the saturated monovalent hydrocarbon radical is straight-chained or branched. As used herein, the term "straight-chained" in reference to an aliphatic hydrocarbon chain means that the chain is unbranched.

[0207] As used herein, the term "alkylene" refers to a saturated divalent hydrocarbon radical of 1 to 20 carbon atoms (i.e., C 1-20 "Alkylene" refers to an alkylene group, examples of which include, but are not limited to, those having the same core structure as the alkyl groups illustrated above. "Divalent" means that the alkylene has two points of attachment to the rest of the molecule. In one embodiment, alkylene is an alkylene group having 1 to 12 carbon atoms (i.e., C 1-12 alkylene) or 1 to 10 carbon atoms (i.e., C 1-10 In one embodiment, the alkylene has 1 to 8 carbon atoms (i.e., C 1-8 alkylene), 1 to 7 carbon atoms (i.e., C 1-7 alkylene), 1 to 6 carbon atoms (i.e., C 1-6 alkylene), 1 to 4 carbon atoms (i.e., C 1-4 alkylene), 1 to 3 carbon atoms (i.e., C 1-3alkylene), ethylene, or methylene. 1-6 alkylene," "linear or branched C 1-4 Alkylene" or "linear or branched C 1-3 Linear or branched alkylene, such as "alkylene," means that the saturated divalent hydrocarbon radical is linear or branched.

[0208] The term "alkenyl" refers to a straight-chain or branched aliphatic hydrocarbon radical having one or more (e.g., 1 or 2) carbon-carbon double bonds, and alkenyl radicals include radicals having "cis" and "trans" configurations, or, alternatively, "E" and "Z" configurations.

[0209] "Alkenylene" means an aliphatic divalent hydrocarbon radical of 2 to 20 carbon atoms having one or two carbon-carbon double bonds (i.e., C 2-20 "Alkenylene" refers to an alkylene group having 2 to 12 carbon atoms (i.e., C 2-12 alkenylene), 2 to 10 carbon atoms (i.e., C 2-10 In one embodiment, the alkenylene has 2 to 4 carbon atoms (C 2-4 Examples include, but are not limited to, ethylene or vinylene (-CH=CH-), allyl (-CHCH=CH-), etc. 2-6 Alkenylene," "linear or branched C 2-4 Alkenylene" or "linear or branched C 2-3 Linear or branched alkenylene, such as "alkenylene," means that the unsaturated divalent hydrocarbon radical is linear or branched.

[0210] As used herein, "cycloalkylene" refers to a divalent saturated carbocyclic ring radical having 3 to 12 carbon atoms as a monocyclic ring or 7 to 12 carbon atoms as a bicyclic ring. "Divalent" means that the cycloalkylene has two points of attachment to the rest of the molecule. In one embodiment, the cycloalkylene is a 3- to 7-membered monocyclic or a 3- to 6-membered monocyclic ring. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, cyclononylene, cyclodecylene, cycloundecylene, cyclododecylene, and the like. In one embodiment, the cycloalkylene is cyclopropylene.

[0211] The terms "heterocycle," "heterocyclyl," heterocyclic, and "heterocyclic ring" are used interchangeably herein and refer to a cyclic group containing at least one N atom, a heteroatom selected from N and S, and optionally 1 to 3 additional heteroatoms, that is non-aromatic (i.e., partially saturated or fully saturated). It can be monocyclic or bicyclic (bridged or fused). Examples of heterocyclic rings include, but are not limited to, aziridinyl, diaziridinyl, thiaaziridinyl, azetidinyl, diazetidinyl, triazetidinyl, thiadiazetidinyl, thiazetidinyl, pyrrolidinyl, pyrazolidinyl, imidazolinyl, isothiazolidinyl, thiazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl, azepanyl, azocanyl, and the like. A heterocycle contains 1 to 4 heteroatoms, which may be the same or different, selected from N and S. In one embodiment, the heterocycle contains 1 to 3 N atoms. In another embodiment, the heterocycle contains 1 or 2 N atoms. In another embodiment, the heterocycle contains 1 N atom. "4- to 8-membered heterocyclyl" refers to a radical having 4 to 8 atoms (including 1 to 4 heteroatoms selected from N and S, or 1 to 3 N atoms, or 1 or 2 N atoms, or 1 N atom) arranged in a monocyclic ring. "5- or 6-membered heterocyclyl" refers to a radical having 5 or 6 atoms (including 1 to 4 heteroatoms selected from N and S, or 1 to 3 N atoms, or 1 or 2 N atoms, or 1 N atom) arranged in a monocyclic ring. The term "heterocycle" is intended to include all possible isomeric forms.Heterocycles are described in Paquette, Leo A., Principles of Modern Heterocyclic Chemistry (WA Benjamin, New York, 1968), especially Chapters 1, 3, 4, 6, 7, and 9; The Chemistry of Heterocyclic Compounds, A Series of Monographs (John Wiley & Sons, New York, 1950 to present), especially Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. Heterocyclyl groups may be carbon (carbon-linked) or nitrogen (nitrogen-linked) attached to the remainder of the molecule, where such is possible.

[0212] When a group is described as "optionally substituted," the group can be either (1) unsubstituted or (2) substituted. When a carbon of a group is described as optionally substituted with one or more of a list of substituents, one or more of the hydrogen atoms on the carbon (to the extent present) can be replaced separately and / or together with any independently selected substituents.

[0213] Suitable substituents for alkyl, alkylene, alkenylene, cycloalkylene, and heterocyclyl are those that do not significantly adversely affect the biological activity of the molecule. Unless otherwise specified, exemplary substituents for these groups include linear, branched, or cyclic alkyl, alkenyl, or alkynyl having 1 to 10 carbon atoms, aryl, heteroaryl, heterocyclyl, halogen, guanidinium [—NH(C═NH)NH], —OR, —O— ... 100 , N.R. 101 R 102 , -NO2, -NR 101 COR 102 , -SR 100 , -SOR 101 sulfoxides, represented by -SO2R 101 Sulfone, sulfonic acid -SO3M, sulfate -OSO3M, -SO2NR 101 R102 Sulfonamide, cyano, azido, -COR 101 , -OCOR 101 , -OCONR 101 R 102 , and polyethylene glycol units (-OCH2CH2) n R 101 wherein M is H or a cation (Na + or K + ) etc.), and R 101 , R 102 , and R 103 are each independently H- a linear, branched, or cyclic alkyl, alkenyl, or alkynyl having 1 to 10 carbon atoms, a polyethylene glycol unit (-OCH2CH2) n -R 104 wherein n is an integer from 1 to 24, an aryl having 6 to 10 carbon atoms, a heterocyclic ring having 3 to 10 carbon atoms, and a heteroaryl having 5 to 10 carbon atoms; R 104 is H or a linear or branched alkyl having 1 to 4 carbon atoms, and R 100 , R 101 , R 102 , R 103 , and R 104 The alkyl, alkenyl, alkynyl, aryl, heteroaryl, and heterocyclyl in the group represented by the formula (I) are optionally substituted with one or more (e.g., 2, 3, 4, 5, 6, or more) substituents independently selected from halogen, —OH, —CN, —NO2, and unsubstituted linear or branched alkyl having 1 to 4 carbon atoms. Preferably, the substituents of the above optionally substituted alkyl, alkylene, alkenylene, cycloalkylene, and heterocyclyl are halogen, —CN, —NR 101 R 102 , -CF3, -OR 100 , aryl, heteroaryl, heterocyclyl, -SR 101 , -SOR 101 , -SO2R 101 Alternatively, suitable substituents are selected from the group consisting of halogen, —OH, —NO, —CN, C1-4 Alkyl, -OR 100 , N.R. 101 R 102 , -NR 101 COR 102 , -SR 100 , -SO2R 101 , -SO2NR 101 R 102 , -COR 101 , -OCOR 101 , and -OCONR 101 R 102 wherein R 100 , R 101 , and R 102 are each independently -H or C 1-4 It is alkyl.

[0214] As used herein, the term "halogen" refers to F, Cl, Br, or I. "Cyano" is --CN.

[0215] The terms "amine" or "amino" are used interchangeably herein and refer to a functional group containing a basic nitrogen atom bearing a lone pair of electrons.

[0216] As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of an ionizable lipid of the present disclosure. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, acid tartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate "mesylate", ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. In cases where multiple charged atoms are part of a pharmaceutically acceptable salt, it may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0217] Grouping of alternative elements or embodiments of the present disclosure disclosed herein should not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. For reasons of convenience and / or patentability, one or more members of a group may be included in, or deleted from, a group. When any such inclusion or deletion occurs, the specification herein is deemed to include the group as modified and, therefore, to satisfy the specification of all Markush groups used in the appended claims.

[0218] In some embodiments, the disclosure described herein does not relate to processes for cloning humans, processes for modifying the germline genetic identity of humans, the use of human embryos for industrial or commercial purposes, or processes for modifying the genetic identity of animals that are likely to cause suffering to humans or animals without providing any substantial medical benefit to them, and similarly processes for modifying the genetic identity of animals resulting from such processes.

[0219] Other terms are defined herein within the description of various aspects of the disclosure.

[0220] All patents and other publications, including literature references, issued patents, published patent applications, and copending patent applications, cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodology described in such publications, which might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicant and do not constitute an admission as to the correctness of the dates or contents of these documents.

[0221] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While certain specific embodiments and examples of the present disclosure are described herein for illustrative purposes, those skilled in the art will recognize that various equivalent modifications are possible within the scope of the present disclosure. For example, while method steps or functions are presented in a given order, alternative embodiments may perform the functions in a different order, or may perform the functions substantially simultaneously. The teachings of the present disclosure provided herein can be applied to other procedures or methods, where appropriate. The various embodiments described herein can be combined to provide further embodiments. Where necessary, aspects of the present disclosure can be modified to employ compositions, functions, and concepts from the above references and applications to provide still further embodiments of the present disclosure. Furthermore, due to considerations of biological functional equivalence, some changes can be made to protein structure without affecting biological or chemical activity in terms of type or amount. These and other modifications can be made to the present disclosure in light of the Detailed Description. All such modifications are intended to be within the scope of the appended claims.

[0222] Specific elements of any of the foregoing embodiments can be combined with or substituted for elements of other embodiments. Furthermore, although advantages associated with certain embodiments of the present disclosure are described in connection with those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily exhibit such advantages to fall within the scope of the present disclosure.

[0223] The technology described herein is further illustrated by the following examples, which should not be construed as further limiting in any way. It is understood that the disclosure is not limited in any way to the particular methodology, protocols, and reagents, etc., described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the disclosure, which is defined solely by the claims.

[0224] II. Polymer-conjugated lipids In some embodiments, the present disclosure provides a polymer-conjugated lipid comprising: (i) polyglycerol (PG) or a PG derivative; (ii) a lipid moiety; and (iii) a linker that conjugates the PG or PG derivative to the lipid moiety.

[0225] Polyglycerol and polyglycerol derivatives The PG or PG derivative contained in the polymer-conjugated lipid of the present disclosure can be linear or branched. In one specific embodiment, the PG or PG derivative is linear. In another embodiment, the PG or PG derivative is branched.

[0226] The polymer-conjugated lipids of the present disclosure can comprise PG or a PG derivative comprising 5 to 100 monomer units, e.g., an average of 10 to 100 monomer units, e.g., an average of 10 to 40 monomer units, an average of 15 to 75 monomer units, an average of 20 to 50 monomer units, an average of 30 to 70 monomer units, an average of 40 to 90 monomer units, or an average of 50 to 100 monomer units. In some embodiments, the polyglycerols of the present disclosure have an average of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 monomer units.

[0227] In one embodiment, the PG or PG derivative of the present disclosure contains an average of 8 monomer units.

[0228] In one embodiment, the PG or PG derivative of the present disclosure contains an average of 34 monomeric units.

[0229] In one embodiment, the PG or PG derivative of the present disclosure contains an average of 45 monomeric units.

[0230] In one embodiment, the PG or PG derivative of the present disclosure contains an average of 46 monomer units.

[0231] In one embodiment, the PG or PG derivative of the present disclosure contains an average of 58 monomer units.

[0232] In one embodiment, the polymer-conjugated lipid of the present disclosure comprises PG.

[0233] In one embodiment, the polymer-conjugated lipid of the present disclosure comprises a PG derivative.

[0234] The PG derivative included in the polymer-conjugated lipid of the present disclosure can be a carboxylated PG, such as 2-carboxycyclohexane-1-carboxylated polyglycerol.

[0235] The PG derivative included in the polymer-conjugated lipid of the present disclosure can also be glutarylated PG, for example, 3-methylglutarylated PG.

[0236] In some embodiments, the PG derivative included in the polymer-conjugated lipid of the present disclosure is represented by the following structural formula: [ka] , During the ceremony, n is an integer ranging from 8 to 100; R, [ka] and [ka] is selected from the group consisting of:

[0237] lipid part In some embodiments, the lipid moiety included in the polymer-conjugated lipid of the present disclosure is a lipid moiety of formula (I): [ka] (I) (In the formula, R 1 is absent, hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10 to 30 carbon atoms; R 2 is absent, hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10 to 30 carbon atoms; R 1 and R 2 are each hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10 to 30 carbon atoms, N is positively charged, R 3 is a hydrophobic tail containing 10 to 30 carbon atoms, In formula (I), [ka] is the bond conjugating the lipid moiety and the linker), or a pharmaceutically acceptable salt thereof.

[0238] In some embodiments, R 1 is absent, and R 2 and R 3 are each independently a hydrophobic tail containing 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon atoms. 2 and R 3 are each independently a hydrophobic tail containing 16, 17, 18, 19, 20, 21, or 22 carbon atoms. In one particular embodiment, R 2 and R 3 are each independently a hydrophobic tail containing 18 carbon atoms, and the lipid moiety is dioctadecylamine (DODA).

[0239] Linker The linker conjugating the polyglycerol or polyglycerol derivative to the lipid moiety in the polymer-conjugated lipid of the present disclosure can be an alkyl linker, a glycerol linker, a phosphate linker, a phosphate ester linker, an ether linker, an ester linker, a diester linker (e.g., a glutaryl linker, a succinyl linker), an amide linker, a diamide linker, an amine linker, a peptide linker, a phosphoethanolamine linker, a phosphocholine linker, a carbamate linker, a diamide alkyl linker, a cleavable linker, or any combination thereof.

[0240] In some embodiments, the linker is -(CH) n -, -C(O)(CH2) n -, -C(O)O(CH2) n , -OC(O)(CH2) n C(O)O- and -NH(CH2) n In some embodiments, the linker may be selected from the group consisting of -C(O)O-, where n is an integer ranging from 1 to 20. In some embodiments, the linker may be selected from the group consisting of -C(O)(CH) n -, where n is 2, 3, 4, 5, or 6. In one embodiment, n is 4.

[0241] In some embodiments, the polymer-conjugated lipid of the present disclosure has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0242] In some embodiments, the polymer-conjugated lipid of the present disclosure has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0243] In some embodiments, the polymer-conjugated lipid of the present disclosure has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0244] In some embodiments, the polymer-conjugated lipid of the present disclosure has the following structure: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0245] In some embodiments, the polymer-conjugated lipid of the present disclosure has the following structure: [ka] (Wherein R is [ka] or [ka] (which is or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0246] The polymer-conjugated lipids of the present disclosure may also include a targeting moiety. Various targeting moieties that may be included in the polymer-conjugated lipids of the present disclosure are described in the "Targeting Moieties" section herein.

[0247] The polymer-conjugated lipid of the present disclosure may also contain a reactive species conjugated to PG or a PG derivative. The reactive species present in the polymer-conjugated lipid of the present disclosure may be used, for example, to conjugate a targeting moiety functionalized with a complementary reactive species, i.e., a reactive species capable of reacting with the reactive species contained in the polymer-conjugated lipid of the present disclosure. In some embodiments, the reactive species conjugated to the polymer-conjugated lipid of the present disclosure may be a reagent selected from the group consisting of a thiol reagent, a maleimide reagent, or a click chemistry reagent, for example, an alkyne reagent such as a dibenzocyclooctyne (DBCO) reagent, a transcyclooctene (TCO) reagent, a tetrazine (TZ) reagent, and an azide (AZ) reagent.

[0248] In some embodiments, the polymer-conjugated lipid of the present disclosure can comprise a targeting moiety conjugated to the polymer-conjugated lipid via a reactive species.For example, the polymer-conjugated lipid of the present disclosure, which comprises an azide reagent as a reactive species, can react with a targeting moiety functionalized with a DBCO reagent as a complementary reactive species to produce a polymer-conjugated lipid comprising a targeting moiety.In another example, the polymer-conjugated lipid of the present disclosure, which comprises a thiol reagent, can react with a targeting moiety functionalized with a maleimide reagent to produce a polymer-conjugated lipid comprising a targeting moiety.Any targeting moiety described herein can be conjugated to the polymer-conjugated lipid of the present disclosure.

[0249] Synthesis of polymer-conjugated lipids The polymer-conjugated lipids of the present disclosure, where the polymer is PG, are (a) reacting a lipid moiety to be conjugated to a linker with 2,3-epoxy-1-(1-ethoxyethoxy)propane (EEGE) in the presence of a base or an organic catalyst under an argon atmosphere to form a lipid moiety conjugated to a linker and polymerized EEGE; (b) subjecting a lipid moiety conjugated to a linker and a polymerized EEGE to acidic conditions to produce a polymer-conjugated lipid.

[0250] In some embodiments, the molar ratio of lipid moieties conjugated to the linker to EEGE can vary from about 1:20 to about 1:100. For example, in step (a), the molar ratio of lipid moieties conjugated to the linker to EEGE can be about 1:20 to about 1:40, about 1:25 to about 1:50, about 1:30 to about 1:60, about 1:50 to about 1:75, or about 1:60 to about 1:100, e.g., about 1:20, about 1:25, about 1:30, about 1:35, about 1:40, about 1:45, about 1:50, about 1:55, about 1:60, about 1:65, about 1:70, about 1:75, about 1:80, about 1:85, about 1:90, or about 1:100. In one embodiment, the ratio of lipid moieties conjugated to a linker to EEGE can be about 1:50. In another embodiment, the ratio of lipid moieties conjugated to a linker to EEGE can be about 1:60.

[0251] In some embodiments, the ratio of lipid moieties conjugated to the linker in step (a) to EEGE determines the average number of monomeric units present in the PG portion of the polymer-conjugated lipid in the final product.

[0252] A base useful for carrying out step (a) of the present method can be a phosphazene base such as P4-t-Bu.

[0253] In some embodiments, an organocatalyst useful for carrying out step (a) of the present method can be an N-heterocyclic carbene (NHC) or an N-heterocyclic olefin (NHO).

[0254] In some embodiments, step (a) may be carried out overnight.

[0255] Useful acidic conditions for carrying out step (b) of the present method include strong acids such as hydrochloric acid (HCl). Other strong acids that can be used in this step include hydrobromic acid (HBr), hydroiodic acid (HI), perchloric acid (HClO), chloric acid (HClO), sulfuric acid (HSO), and nitric acid (HNO).

[0256] In some embodiments, the lipid moiety is DODA, and the lipid moiety conjugated to the linker is represented by the following structure: [ka]

[0257] In some embodiments, the polymer-conjugated lipid is DODA-PG, where PG contains an average of 5 to 100 monomer units. In some embodiments, the polymer-conjugated lipid is DODA-PG34, DODA-PG45, DODA-PG46, or DODA-PG58.

[0258] III. Lipid Nanoparticles (LNPs) The present disclosure also provides a lipid nanoparticle (LNP) comprising (i) a therapeutic nucleic acid (TNA), (ii) an ionizable lipid, (iii) a sterol, and (iv) a first lipid-anchored polymer, optionally further comprising a helper lipid, wherein the first lipid-anchored polymer comprises a polymer-conjugated lipid of the present disclosure. Also provided herein is a LNP consisting essentially of (i) a therapeutic nucleic acid (TNA), (ii) an ionizable lipid, (iii) a sterol, and (iv) a first lipid-anchored polymer, optionally further consisting essentially of a helper lipid, wherein the first lipid-anchored polymer comprises a polymer-conjugated lipid of the present disclosure. Also provided herein is a LNP consisting of (i) a therapeutic nucleic acid (TNA), (ii) an ionizable lipid, (iii) a sterol, and (iv) a first lipid-anchored polymer, optionally further comprising a helper lipid, wherein the first lipid-anchored polymer comprises a polymer-conjugated lipid of the present disclosure.

[0259] A. Ionizable lipids In some embodiments, ionizable lipids are present in LNPs provided by the present disclosure in an amount of about 20 mol% to about 60 mol%, or about 35 mol% to about 50 mol% of the total lipids present in the LNP.

[0260] In some embodiments, the LNPs provided by the present disclosure comprise an ionizable lipid. Exemplary ionizable lipids in the LNPs of the present disclosure are those disclosed in International Patent Application Publication Nos. 2015 / 095340, 2015 / 199952, 2018 / 011633, 2017 / 049245, 2015 / 061467, 2012 / 040184, 2012 / 000104, 2015 / 074085, 2016 / 081029, 2017 / 004143, 2017 / 075531, 2017 / 117528, 2011 / 022460, and 2013 / 148 No. 541, No. 2013 / 116126, No. 2011 / 153120, No. 2012 / 044638, No. 2012 / 05 No. 4365, No. 2011 / 090965, No. 2013 / 016058, No. 2012 / 162210, No. 2008 / 0 No. 42973, No. 2010 / 129709, No. 2010 / 144740, No. 2012 / 099755, No. 2013 / No. 049328, No. 2013 / 086322, No. 2013 / 086373, No. 2011 / 071860, No. 2009 / 132131, 2010 / 048536, 2010 / 088537, 2010 / 054401, 201 No. 0 / 054406, No. 2010 / 054405, No. 2010 / 054384, No. 2012 / 016184, No. 20 09 / 086558, 2010 / 042877, 2011 / 000106, 2011 / 000107, 2 005 / 120152, 2011 / 141705, 2013 / 126803, 2006 / 007712, Nos. 2011 / 038160, 2005 / 121348, 2011 / 066651, 2009 / 127060, 2011 / 141704, 2006 / 069782, 2012 / 031043, 2013 / 006825, 2013 / 033563, 2013 / 089151, 2017 / 099823, 2015 / 095346, 2013 / 086354, and 2021 / 102411, and U.S. Patent Application Publication No. 2016 / 0311759;Same No. 2015 / 0376115, No. 2016 / 0151284, No. 2017 / 0210697, No. 2015 / 0140070, No. 2013 / 0178541, No. 2013 / 0303587, No. 2015 / No. 0141678, No. 2015 / 0239926, No. 2016 / 0376224, No. 2017 / 0119904, No. 2012 / 0149894, No. 2015 / 0057373, No. 2013 / 0090372 , 2013 / 0274523, 2013 / 0274504, 2013 / 0274504, 2009 / 0023673, 2012 / 0128760, 2010 / 0324120, 2014 / 0200257, 2015 / 0203446, 2018 / 0005363, 2014 / 0308304, 2013 / 0338210, 2012 / 0101148, 2012 / 0027796 No. 2012 / 0058144, No. 2013 / 0323269, No. 2011 / 0117125, No. 2011 / 0256175, No. 2012 / 0202871, No. 2011 / 0076335, No. 200 6 / 0083780, 2013 / 0123338, 2015 / 0064242, 2006 / 0051405, 2013 / 0065939, 2006 / 0008910, 2003 / 002264 Nos. 9, 2010 / 0130588, 2013 / 0116307, 2010 / 0062967, 2013 / 0202684, 2014 / 0141070, 2014 / 0255472, 2014 / 0039032, 2018 / 0028664, 2016 / 0317458, and 2013 / 0195920, the contents of all of which are incorporated herein by reference in their entireties.

[0261] In some embodiments, the ionizable lipid in the LNPs of the present disclosure is MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3), represented by the following structure: [ka]

[0262] The lipid DLin-MC3-DMA is described in Jayaraman et al., Angew. Chem. Int. Ed Engl. (2012), 51(34):8529-8533, the contents of which are incorporated herein by reference in their entirety.

[0263] In some embodiments, the ionizable lipid is N-[1-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC), 1, 2-Dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamidoethyl 1-3,4-di[oleyloxy]-benzamide (MVL5), dioctadecylamido-glycylspermine (DOGS), 3b-[N-(N',N'

[0039] The lipid may be selected from the group consisting of [(dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol), dioctadecyldimethylammonium bromide (DDAB), Saint lipids (e.g., SAINT-2, N-methyl-4-(dioleyl)methylpyridinium), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), 1,2-dioleoyl-3-dimethylhydroxyethylammonium bromide (DORIE), 1,2-dioleoyloxypropyl-3-dimethylhydroxyethylammonium chloride (DORI), dialkylated amino acids (DILA2) (e.g., C18:1-norArg-C16), dioleyldimethylammonium chloride (DODAC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC), and 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC).In some variations, the condensing agent, e.g., the cationic lipid, is, for example, dioctadecyldimethylammonium bromide (DDAB), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2-dioleo ...-MC3-DMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA)], 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-MC3-DMA)], 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA)], 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin- These lipids include diethylaminopropane (DODAP), 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA), morpholinocholesterol (Mo-CHOL), (R)-5-(dimethylamino)pentane-1,2-diyldioleate hydrochloride (DODAPen-C1), (R)-5-guanidinopentane-1,2-diyldioleate hydrochloride (DOPen-G), and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-aminium chloride (DOTAPen).

[0264] Formula (A) In some embodiments, the ionizable lipid in the LNPs of the present disclosure has formula (A): [ka] (A) (In the formula, R 1 and R 1’ are each independently, C 1-3 is alkylene, R 2 and R 2’ are each independently a linear or branched C 1-6 Alkylene, or C 3-6 is cycloalkylene, R 3 and R 3’ each independently represents an optionally substituted C 1-6 Alkyl or optionally substituted C 3-6 is cycloalkyl, or Alternatively, R 2 Branched C 1-6alkylene, and R 3 C 1-6 If it is alkyl, R 2 and R 3 together with the intervening N atom to form a 4- to 8-membered heterocyclyl, or Alternatively, R 2’ Branched C 1-6 alkylene, and R 3’ C 1-6 If it is alkyl, R 2’ and R 3’ together with the intervening N atom to form a 4- to 8-membered heterocyclyl; R 4 and R 4’ are each independently -CH, -CHCH, or -(CH)CH; R 5 and R 5’ are each independently hydrogen, C 1-20 Alkylene or C 2-20 is alkenylene, R 6 and R 6’ But for each occurrence, independently, C 1-20 Alkylene, C 3-20 Cycloalkylene or C 2-20 is alkenylene, m and n are each independently an integer selected from 1, 2, 3, 4, and 5, or a pharmaceutically acceptable salt thereof.

[0265] In some embodiments, R 2 and R 2’ are each independently, C 1-3 It is alkylene.

[0266] In some embodiments, R 1 or R 1’ Linear or branched C represented by 1-3 Alkylene, R 2 or R 2’ Linear or branched C represented by 1-6 Alkylene, and optionally substituted linear or branched C 1-6Each alkyl is optionally substituted with one or more halo and cyano groups.

[0267] In some embodiments, R 1 and R 2 Together, C 1-3 alkylene, and R 1’ and R 2’ Together, C 1-3 Alkylene, for example, ethylene.

[0268] In some embodiments, R 3 and R 3’ each independently represents an optionally substituted C 1-3 Alkyl, for example, methyl.

[0269] In some embodiments, R 4 and R 4’ are each -CH.

[0270] In some embodiments, R 2 is an optionally substituted branched C 1-6 alkylene, and R 2 and R 3 taken together with their intervening N atom form a 5- or 6-membered heterocyclyl. In some embodiments, R 2’ is an optionally substituted branched C 1-6 alkylene, and R 2’ and R 3’ taken together with their intervening N atom form a 5- or 6-membered heterocyclyl such as pyrrolidinyl or piperidinyl.

[0271] In some embodiments, R 4 is -C(R a )2CR a or -[C(R a )2]2CR a and R a is C 1-3 alkyl, and R 3 and R 4taken together with their intervening N atom form a 5- or 6-membered heterocyclyl. In some embodiments, R 4’ is -C(R a )2CR a or -[C(R a )2]2CR a and R a is C 1-3 alkyl, and R 3’ and R 4’ taken together with their intervening N atom form a 5- or 6-membered heterocyclyl such as pyrrolidinyl or piperidinyl.

[0272] In some embodiments, R 5 and R 5’ are each independently, C 1-10 Alkylene or C 2-10 In one embodiment, R is alkenylene. 5 and R 5’ are each independently, C 1-8 Alkylene or C 1-6 It is alkylene.

[0273] In some embodiments, R 6 and R 6’ is independently generated for each occurrence of C 1-10 Alkylene, C 3-10 Cycloalkylene, or C 2-10 In one embodiment, C is an alkenylene. 1-6 Alkylene, C 3-6 Cycloalkylene, or C 2-6 In one embodiment, C is an alkenylene. 3-10 Cycloalkylene or C 3-6 In some embodiments, m and n are each 3.

[0274] In some embodiments, the ionizable lipid in the LNPs of the present disclosure can be selected from any one of the lipids listed in Table 1 below, or a pharmaceutically acceptable salt thereof. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0275] Formula (B) In some embodiments, the ionizable lipid in the LNPs of the present disclosure has formula (B): [ka] (B) (In the formula, a is an integer ranging from 1 to 20 (e.g., a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); b is an integer ranging from 2 to 10 (e.g., b is 2, 3, 4, 5, 6, 7, 8, 9, or 10); R 1 is absent or (C2-C 20 ) alkenyl, -C(O)O(C2-C 20 ) alkyl, and (C2-C 20 ) cyclopropyl substituted with alkyl; R 2 However, (C2-C 20 ) alkyl) or a pharmaceutically acceptable salt thereof.

[0276] In a second embodiment of formula (B), the ionizable lipid of formula (B) has the formula (B-1): [ka] (B-1) wherein c and d are each independently an integer ranging from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and the remaining variables are as described in formula (B), or a pharmaceutically acceptable salt thereof.

[0277] In a third embodiment of Formula (B), c and d in Formula (B-1) are each independently an integer ranging from 2 to 8, from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5, from 4 to 8, from 4 to 7, from 4 to 6, from 5 to 8, from 5 to 7, or from 6 to 8, and the remaining variables are as described for Formula (B-1).

[0278] In a fourth embodiment of Formula (B), c in Formula (B-1) is 2, 3, 4, 5, 6, 7, or 8, and the remaining variables are as described for Formula (B), or the second or third embodiment of Formula (B). Alternatively, c and d in Formula (B-1) are each independently 1, 3, 5, or 7, and the remaining variables are as described for Formula (B), or the second or third embodiment of Formula (B).

[0279] In a fifth embodiment of Formula (B), d in the cationic lipid of Formula (B-1) is 2, 3, 4, 5, 6, 7, or 8, with the remaining variables being as described for Formula (B), or the second, third, or fourth embodiment of Formula (B). Alternatively, at least one of c and d in Formula (B-1) is 7, with the remaining variables being as described for Formula (B), or the second, third, or fourth embodiment of Formula (B).

[0280] In a sixth embodiment of formula (B), the ionizable lipid of formula (B) or formula (B-1) is of formula (B-2): [ka] (B-2) wherein the remaining variables are as described for Formula (B) or Formula (B-1), or a pharmaceutically acceptable salt thereof.

[0281] In a seventh embodiment of Formula (B), b in Formula (B), Formula (B-1), or Formula (B-2) is an integer ranging from 3 to 9, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, or sixth embodiment of Formula (B). Alternatively, b in Formula (B), Formula (B-1), or Formula (B-2) is an integer ranging from 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 5 to 9, 5 to 8, 5 to 7, 6 to 9, 6 to 8, or 7 to 9, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, or sixth embodiment of Formula (B). Alternatively, b in Formula (B), Formula (B-1), or Formula (B-2) is 3, 4, 5, 6, 7, 8, or 9, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, or sixth embodiment of Formula (B).

[0282] In an eighth embodiment of Formula (B), a in Formula (B), Formula (B-1), or Formula (B-2) is an integer ranging from 2 to 18, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, or seventh embodiment of Formula (B). Alternatively, a in formula (B), formula (B-1), or formula (B-2) is 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 18, 4 to 17, 4 to 16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 25-8, 5-7, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 7-18, 7-17, 7-16, 7 ~15, 7~14, 7~13, 7~12, 7~11, 7~10, 7~9, 8~18, 8~17, 8~16, 8~15, 8~14, 8~13, 8~12, 8~11, 8~10, 9~18, 9~17, 9~16, 9~15, 9~14, 9~13, 9~12, 9~11, 10~18, 10~17, 10~16, 10~15, 10~14, 10~13, 11~18, 11~17, 11~16, 11~1 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 124, 125, 136, 136, 137, 146, 147, 148, 1518, 1517, or 1618, and the remainder of the variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, or seventh embodiment of Formula (B). Alternatively, a in Formula (B), Formula (B-1), or Formula (B-2) is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, or seventh embodiment of Formula (B).

[0283] In a ninth embodiment of formula (B), R in formula (B), formula (B-1), or formula (B-2) 1 is absent or (C5-C 15 ) alkenyl, -C(O)O(C4-C 18 ) alkyl, and (C4-C 16 ) cyclopropyl substituted with alkyl, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B). Alternatively, R in Formula (B), Formula (B-1), or Formula (B-2) is selected from 1 is absent or (C5-C 15 ) alkenyl, -C(O)O(C4-C 16 ) alkyl, and (C4-C 16 ) cyclopropyl substituted with alkyl, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B). Alternatively, R in Formula (B), Formula (B-1), or Formula (B-2) is selected from 1 is absent or (C5-C 12 ) alkenyl, -C(O)O(C4-C 12 ) alkyl, and (C4-C 12 ) cyclopropyl substituted with alkyl, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B). In another alternative, R in the cationic lipid of Formula (B), Formula (B-1), or Formula (B-2) is selected from: 1 is absent or (C5-C 10 ) alkenyl, -C(O)O(C4-C 10 ) alkyl, and (C4-C 10 ) cyclopropyl substituted with alkyl, and the remainder of the variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B).

[0284] In a tenth embodiment of formula (B), R 1 is C 10alkenyl, and the remainder of the variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B).

[0285] In an eleventh embodiment of formula (B), R in formula (B), formula (B-1), or formula (B-2) 1 C(O)O(C2-C 20 ) alkyl, -C(O)O(C4-C 18 ) alkyl, -C(O)O(C4-C 12 ) alkyl, or -C(O)O(C4-C 10 ) alkyl is an unbranched alkyl, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh, eighth, or ninth embodiment of Formula (B). In one embodiment, R 1 is —C(O)O(C alkyl). Alternatively, R in formula (B), formula (B-1), or formula (B-2) 1 -C(O)O(C4-C 18 ) alkyl, -C(O)O(C4-C 12 ) alkyl, or -C(O)O(C4-C 10 The alkyl in alkyl is a branched alkyl, and the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, eighth, or ninth embodiment of Formula (B). In one embodiment, R 1 is -C(O)O(C 17 alkyl), and the remainder of the variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, eighth, or ninth embodiment of Formula (B).

[0286] In a twelfth embodiment of formula (B), R in formula (B), formula (B-1), or formula (B-2) 1is selected from any group listed in Table 2 below, where the wavy bond in each group indicates the point of attachment of the group to the remainder of the ionizable lipid molecule, and the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B). The present disclosure provides a method for treating R 1 and any one of the groups R in Table 3 of formula (B) 2 Further contemplated are combinations with any one of the groups, wherein the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B). [Table 2-1] [Table 2-2]

[0287] In a thirteenth embodiment, R in formula (B) 2 or a pharmaceutically acceptable salt thereof, selected from any of the groups listed in Table 3 below, where the wavy bond in each group indicates the point of attachment of the group to the remainder of the ionizable lipid molecule, and the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, or twelfth embodiment of Formula (B). [Table 3]

[0288] Table 4 below provides specific examples of ionizable lipids of Formula (B), including pharmaceutically acceptable salts, as well as ionized and neutral forms. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5]

[0289] Formula (C) In some embodiments, the ionizable lipid in the LNPs of the present disclosure has formula (C): [ka] (C) (In the formula, R 1 and R 1’ are each independently R a and (C1-C6) alkylene optionally substituted with one or more groups selected from R 2 and R 2’ are each independently (C1-C2) alkylene; R 3 and R 3’ are each independently R b or (C1-C6) alkyl optionally substituted with one or more groups selected from Alternatively, R 2 and R 3 and / or R 2’ and R 3’ together with the intervening N atom to form a 4- to 7-membered heterocyclyl; R 4 and R 4 each ' is (C2-C6)alkylene interrupted by -C(O)O-; R 5 and R 5 each independently being optionally interrupted by —C(O)O— or (C3-C6)cycloalkyl, (C2-C 30 ) alkyl or (C2-C 30 ) alkenyl, Ra and R b wherein each is halo or cyano, or a pharmaceutically acceptable salt thereof.

[0290] In a second embodiment of formula (C), R 1 and R 1 are each independently (C-C) alkylene, and the remaining variables are as described above for formula (C). Alternatively, R 1 and R 1’ is independently at each occurrence (C1-C3) alkylene, and the remainder of the variables are as described above for formula (C).

[0291] In a third embodiment of formula (C), the ionizable lipid of formula (C) has the formula (C-1): [ka] (C-1) (In the formula, R 2 and R 2’ , R 3 and R 3’ , R 4 and R 4 ', as well as R 5 and R 5 ' is represented by Formula (C), or as described above for the second embodiment of Formula (C), or a pharmaceutically acceptable salt thereof.

[0292] In a fourth embodiment, the ionizable lipid of formula (C) has formula (C-2) or formula (C-3): [ka] (C-2), or [ka] (C-3) (In the formula, R 4 and R 4 ' and R 5 and R 5' is as described above for formula (C)), or a pharmaceutically acceptable salt thereof.

[0293] In a fifth embodiment of formula (C), the ionizable lipid of formula (C) is of formula (C-4) or formula (C-5): [ka] (C-4), or [ka] (C-5) (In the formula, R 5 and R 5 ' is as described above for formula (C)), or a pharmaceutically acceptable salt thereof.

[0294] In a sixth embodiment of Formula (C), the ionizable lipid of Formula (C) is Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9): [ka] (C-6), [ka] (C-7), [ka] (C-8), or [ka] (C-9) (In the formula, R 5 and R 5 ' is as described above for formula (XV)), or a pharmaceutically acceptable salt thereof.

[0295] In a seventh embodiment of formula (C), R in formula (C), formula (C-1), formula (C-2), formula (C-3), formula (C-4), formula (C-5), formula (C-6), formula (C-7), formula (C-8), or formula (C-9) 5 and R 5’ is a branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 and R 5’ is a branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 is branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5’ is branched alkyl or branched alkenyl, and the remainder of the variables are as described above for Formula (C), or the second embodiment of Formula (C).

[0296] In an eighth embodiment of formula (C), R in formula (C), formula (C-1), formula (C-2), formula (C-3), formula (C-4), formula (C-5), formula (C-6), formula (C-7), formula (C-8), or formula (C-9) 5 are each optionally interrupted by —C(O)O— or (C-C)cycloalkyl, (C-C 26 ) alkyl or (C6-C 26) alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 are each optionally interrupted by —C(O)O— or (C3-C5)cycloalkyl, (C6-C 26 ) alkyl or (C6-C 26 ) alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 are each optionally interrupted by —C(O)O— or (C3-C5)cycloalkyl, (C7-C 26 ) alkyl or (C7-C 26 ) alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 are each optionally interrupted by —C(O)O— or (C3-C5)cycloalkyl, (C8-C 26 ) alkyl or (C8-C 26 ) alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 are each optionally interrupted by —C(O)O— or cyclopropyl, (C-C 24 ) alkyl or (C6-C 24) alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 is (C8-C 24 ) alkyl or (C8-C 24 ) alkenyl, and the (C8-C 24 )alkyl is optionally interrupted by -C(O)O- or cyclopropyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 is (C8-C 10 ) alkyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 is interrupted by cyclopropyl (C 14 -C 16 ) alkyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 is interrupted by -C(O)O- (C 10 -C 24 ) alkyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 is (C 16 -C 18) alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 -(CH2)3C(O)O(CH2)8CH3, -(CH2)5C(O)O(CH2)8CH3, -(CH2)7C(O)O(CH2)8CH3, - (CH2)7C(O)OCH[(CH2)7CH3]2, -(CH2)7-C3H6-(CH2)7CH3, -(CH2)7CH3, -(CH2)9CH 3、- (CH2) 16 CH3, -(CH2)7CH=CH(CH2)7CH3, or -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, and the remainder of the variables are as described above for Formula (C), or the second embodiment of Formula (C).

[0297] In the ninth embodiment, R in formula (C), formula (C-1), formula (C-2), formula (C-3), formula (C-4), formula (C-5), formula (C-6), formula (C-7), formula (C-8), or formula (C-9) 5’ is interrupted by -C(O)O- (C 15 -C 28 ) alkyl, and the remaining variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5’ is interrupted by -C(O)O- (C 17 -C 28 ) alkyl, and the remaining variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5’ is interrupted by -C(O)O- (C 19 -C 28) alkyl, and the remaining variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5’ is interrupted by -C(O)O- (C 17 -C 26 ) alkyl, and the remaining variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5’ is interrupted by -C(O)O- (C 19 -C 26 ) alkyl, and the remaining variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5’ is interrupted by -C(O)O- (C 20 -C 26 ) alkyl, and the remaining variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C). Alternatively, R 5’ is interrupted by -C(O)O- (C 22 -C 24 ) alkyl, and the remaining variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C). Alternatively, R 5’ is —(CH2)5C(O)OCH[(CH2)7CH3]2, —(CH2)7C(O)OCH[(CH2)7CH3]2, —(CH2)5C(O)OCH(CH2)2[(CH2)7CH3]2, or —(CH2)7C(O)OCH(CH2)2[(CH2)7CH3]2, and the remainder of the variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C).

[0298] In some embodiments, the ionizable lipid of Formula (C), Formula (C-1), Formula (C-3), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-7), Formula (C-8), or Formula (C-9) may be selected from any of the lipids listed in Table 5 below, or a pharmaceutically acceptable salt thereof. [Table 5-1] [Table 5-2]

[0299] Formula (D) In some embodiments, the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure has formula (D): [ka] (D) (In the formula, R' is absent, hydrogen, or C1-C6 alkyl, provided that when R' is hydrogen or C1-C6 alkyl, R', R 1 , and R 2 provided that all nitrogen atoms to which they are attached are positively charged, R 1 and R 2 are each independently hydrogen, C1-C6 alkyl, or C2-C6 alkenyl; R 3 But C1-C 12 Alkylene or C2-C 12 is alkenylene, R 4 But C1-C 18 Unbranched alkyl, C2-C 18 unbranched alkenyl, or [ka] where: R 4a and R 4b are each independently, C1-C 16 Unbranched alkyl or C2-C 16is an unbranched alkenyl, R 5 is absent, C1-C8 alkylene, or C2-C8 alkenylene; R 6a and R 6b are each independently, C7-C 16 Alkyl or C7-C 16 alkenyl, provided that R 6a and R 6b provided that the total number of carbon atoms in X 1 and X 2 each independently represents -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -SS-, -C(R a )=N-, -N=C(R a )-, -C(R a )=NO-, -ON=C(R a )-, -C(=O)NR a -, -NR a C(=O)-, -NR a C(=O)NR a -, -OC(=O)O-, -OSi(R a )2O-, -C(=O)(CR a 2) C(=O)O- or OC(=O)(CR a 2) C(=O)-, wherein: R a is independently at each occurrence hydrogen or C1-C6 alkyl; and n is an integer selected from 1, 2, 3, 4, 5, and 6, or a pharmaceutically acceptable salt thereof.

[0300] In a second embodiment of formula (D), X 1 and X 2 is the same, and all other remaining variables are as described for formula (C).

[0301] In a third embodiment of formula (D), X 1 and X 2are each independently -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, or -SS-; or X 1 and X 2 are each independently -C(=O)O-, -C(=O)S-, or -SS-, or X 1 and X 2 is each independently -C(=O)O- or -SS-, and all other remaining variables are as described for Formula (D), or the second embodiment of Formula (D).

[0302] In a fourth embodiment of Formula (D), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure has the formula (D-1): [ka] (D-1) wherein n is an integer selected from 1, 2, 3, and 4, and all other remaining variables are as described for Formula (D), or the second or third embodiment of Formula (D), or a pharmaceutically acceptable salt thereof.

[0303] In a fifth embodiment of Formula (D), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure is represented by Formula (D-2): [ka] (D-2) wherein n is an integer selected from 1, 2, and 3, and all other remaining variables are as described for Formula (D), or the second or third embodiment of Formula (D), or a pharmaceutically acceptable salt thereof.

[0304] In a sixth embodiment of Formula (D), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure is represented by Formula (D-3): [ka] (D-3) wherein all other remaining variables are as described for Formula (D), or the second or third embodiment of Formula (D), or a pharmaceutically acceptable salt thereof.

[0305] In a seventh embodiment of Formula (D), in an ionizable lipid, e.g., a cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), or the second or third embodiment of Formula (D), R 1 and R 2 are each independently hydrogen, C1-C6 alkyl or C2-C6 alkenyl, or C1-C5 alkyl or C2-C5 alkenyl, or C1-C4 alkyl or C2-C4 alkenyl, or C6 alkyl, or C5 alkyl, or C4 alkyl, or C3 alkyl, or C2 alkyl, or C1 alkyl, or C6 alkenyl, or C5 alkenyl, or C4 alkenyl, or C3 alkenyl, or C2 alkenyl, and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), or the second or third embodiment of Formula (D).

[0306] In an eighth embodiment of Formula (D), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure is represented by Formula (D-4): [ka] (D-4) wherein all other remaining variables are as described for the second, third, or seventh embodiment of Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), or Formula (D), or a pharmaceutically acceptable salt thereof.

[0307] In a ninth embodiment of Formula (D), in an ionizable lipid, e.g., a cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, or seventh embodiment of Formula (D), R 3is C1-C9 alkylene or C2-C9 alkenylene, C1-C7 alkylene or C2-C7 alkenylene, C1-C5 alkylene or C2-C5 alkenylene, or C2-C8 alkylene or C2-C8 alkenylene, or C3-C7 alkylene or C3-C7 alkenylene, or C5-C7 alkylene or C5-C7 alkenylene, or R 3 is C 12 Alkylene, C 11 Alkylene, C 10 Alkylene, C9 alkylene, or C8 alkylene, or C7 alkylene, or C6 alkylene, or C5 alkylene, or C4 alkylene, or C3 alkylene, or C2 alkylene, or C1 alkylene, or C 12 Alkenylene, C 11 Alkenylene, C 10 alkenylene, C9 alkenylene, or C8 alkenylene, or C7 alkenylene, or C6 alkenylene, or C5 alkenylene, or C4 alkenylene, or C3 alkenylene, or C2 alkenylene, and all other remaining variables are as described for the second, third, or seventh embodiment of Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or Formula (D).

[0308] In a tenth embodiment of Formula (D), in an ionizable lipid, e.g., a cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, or seventh embodiment of Formula (D), R 5 is absent, C-C alkylene, or C-C alkenylene, or R 5 is absent, C-C alkylene, or C-C alkenylene, or R 5 is absent or R 5is C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, or C alkenylene, and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, or ninth embodiment of Formula (D).

[0309] In an eleventh embodiment of Formula (D), in an ionizable lipid, e.g., a cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, or tenth embodiment of Formula (D), R 4 is C1-C 14 Unbranched alkyl, C2-C 14 unbranched alkenyl, or [ka] (In the formula, R 4a and R 4b are each independently, C1-C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl), or R 4 is C2-C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl, or R 4 is a C5-C7 unbranched alkyl or a C5-C7 unbranched alkenyl, or R 4 is C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C 13 Unbranched alkyl, C 12 Unbranched alkyl, C 11 Unbranched alkyl, C 10Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 unbranched alkyl, C1 unbranched alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C 14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl, or R 4 teeth, [ka] (In the formula, R 4a and R 4b are each independently, C2-C 10 Unbranched alkyl or C2-C 10 unbranched alkenyl), or R 4 teeth, [ka] (In the formula, R 4a and R 4b are each independently, C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C 13 Unbranched alkyl, C 12 Unbranched alkyl, C 11 Unbranched alkyl, C 10 Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 alkyl, C1 alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl), and all other remaining variables are as described for the second, third, seventh, ninth, or tenth embodiment of Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or Formula (D).

[0310] In a twelfth embodiment, in an ionizable lipid, e.g., a cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth, or eleventh embodiment of Formula (D), R 6a and R 6b are each independently C6-C 14 Alkyl or C6-C 14 alkenyl, or R 6a and R 6b are each independently C8-C 12 Alkyl or C8-C 12 alkenyl, or R 6a and R 6b are each independently, C 16 Alkyl, C 15 Alkyl, C 14 Alkyl, C 13 Alkyl, C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 16 Alkenyl, C 15 Alkenyl, C 14 Alkenyl, C 13 Alkenyl, C 12 Alkenyl, C 11 Alkenyl, C 10alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, provided that R 6a and R 6b is greater than 15, and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth, or eleventh embodiment of Formula (D).

[0311] In a thirteenth embodiment of Formula (D), in an ionizable lipid, e.g., a cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth, eleventh, or twelfth embodiment of Formula (D), or a pharmaceutically acceptable salt thereof, R 6a and R 6b contain an equal number of carbon atoms, or R 6a and R 6b are the same or R 6a and R 6b are both C 16 Alkyl, C 15 Alkyl, C 14 Alkyl, C 13 Alkyl, C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 16 Alkenyl, C 15 Alkenyl, C 14 Alkenyl, C 13 Alkenyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, provided that R 6a and R 6b is greater than 15, and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth, eleventh, or twelfth embodiment of Formula (D).

[0312] In a fourteenth embodiment of Formula (D), in an ionizable lipid, e.g., a cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth, eleventh, twelfth, or thirteenth embodiment of Formula (D), R as defined in any one of the preceding embodiments. 6a and R 6b each contain a different number of carbon atoms, or R 6a and R 6b The number of carbon atoms in R differs by one or two carbon atoms, or 6a and R 6b The number of carbon atoms in R differs by one carbon atom, or 6a is a C7 alkyl, and R 6a is C8 alkyl or R 6a is C8 alkyl, and R 6a is a C7 alkyl or R 6a is C8 alkyl, and R 6a is a C9 alkyl or R 6a is a C9 alkyl, and R 6a is C8 alkyl or R 6a is a C9 alkyl, and R 6a C 10 alkyl or R 6a C 10 alkyl, and R 6a is a C9 alkyl or R 6a C 10 alkyl, and R 6a C 11 alkyl or R 6a C 11 alkyl, and R 6a C 10 alkyl or R 6a C 11 alkyl, and R 6a C 12 alkyl or R 6a C 12 alkyl, and R 6a C 11 alkyl or R 6a is a C7 alkyl, and R6a is a C9 alkyl or R 6a is a C9 alkyl, and R 6a is a C7 alkyl or R 6a is C8 alkyl, and R 6a C 10 alkyl or R 6a C 10 alkyl, and R 6a is C8 alkyl or R 6a is a C9 alkyl, and R 6a C 11 alkyl or R 6a C 11 alkyl, and R 6a is a C9 alkyl or R 6a C 10 alkyl, and R 6a C 12 alkyl or R 6a C 12 alkyl, and R 6a C 10 alkyl or R 6a C 11 alkyl, and R 6a C 13 alkyl or R 6a C 13 alkyl, and R 6a C 11 is alkyl, and all other remaining variables are as described for the second, third, seventh, ninth, tenth, eleventh, twelfth, or thirteenth embodiment of Formula I, Formula II, Formula III, Formula IV, Formula V, or Formula (D).

[0313] In a fifteenth embodiment of formula (D), R 4 is C1-C 16 Unbranched alkyl, C2-C 16 unbranched alkenyl, or [ka] (In the formula, R 4a and R 4bis as described above for the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, or fourteenth embodiment of Formula (D).

[0314] In one embodiment, the ionizable lipid of the present disclosure, e.g., a cationic lipid, or an ionizable lipid of Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), or Formula (D-4), is any one lipid selected from the lipids listed in Table 6 below, or a pharmaceutically acceptable salt thereof. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

[0315] In one embodiment, the ionizable lipid in the LNP of the present disclosure is ionizable lipid 87: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0316] Formula (E) In some embodiments, the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure has the formula (E): [ka] (E) (In the formula, R' is absent, hydrogen, or C1-C3 alkyl, provided that when R' is hydrogen or C1-C3 alkyl, R', R 1 , and R 2provided that all nitrogen atoms to which they are attached are positively charged, R 1 and R 2 are each independently hydrogen or C1-C3 alkyl; R 3 But C3-C 10 Alkylene or C3-C 10 is alkenylene, R 4 But C1-C 16 Unbranched alkyl, C2-C 16 unbranched alkenyl, or [ka] where: R 4a and R 4b are each independently, C1-C 16 Unbranched alkyl or C2-C 16 is an unbranched alkenyl, R 5 is absent, C1-C6 alkylene, or C2-C6 alkenylene; R 6a and R 6b are each independently, C7-C 14 Alkyl or C7-C 14 is alkenyl, X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -SS-, -C(R a )=N-, -N=C(R a )-, -C(R a )=NO-, -ON=C(R a )-, -C(=O)NR a -, -NR a C(=O)-, -NR a C(=O)NR a -, -OC(=O)O-, -OSi(R a )2O-, -C(=O)(CR a 2) C(=O)O- or OC(=O)(CR a 2) C(=O)-, wherein: Ra is independently at each occurrence hydrogen or C1-C6 alkyl; and n is an integer selected from 1, 2, 3, 4, 5, and 6, or a pharmaceutically acceptable salt thereof.

[0317] In a second embodiment of Formula (E), in an ionizable lipid, e.g., a cationic lipid according to the first embodiment, or a pharmaceutically acceptable salt thereof, X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, or -SS-, and all other remaining variables are as described for Formula I or the first embodiment.

[0318] In a third embodiment of Formula (E), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure has the formula (E-1): [ka] (E-1) wherein n is an integer selected from 1, 2, 3, and 4, and all other remaining variables are as described for Formula (E), or the second embodiment of Formula (E), or a pharmaceutically acceptable salt thereof. Alternatively, n is an integer selected from 1, 2, and 3, and all other remaining variables are as described for Formula (E), or the second embodiment of Formula (E).

[0319] In a fourth embodiment of Formula (E), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure has the formula (E-2): [ka] (E-2) wherein all other remaining variables are as described for Formula (E), Formula (E-1), or the second embodiment of Formula (E), or a pharmaceutically acceptable salt thereof.

[0320] In a fifth embodiment of Formula (E), in an ionizable lipid, e.g., a cationic lipid, in an LNP of the present disclosure, R 1 and R 2 are each independently hydrogen or C1-C2 alkyl or C2-C3 alkenyl, or R', R 1 , and R 2 are each independently hydrogen, C1-C2 alkyl, and all other remaining variables are as described for Formula (E), Formula (E-1), or the second embodiment of Formula (E).

[0321] In a sixth embodiment of Formula (E), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure is represented by Formula (E-3): [ka] (E-3) wherein all other remaining variables are as described for the second or fifth embodiment of Formula (E), Formula (E-1), Formula (E-2), or Formula (E), or a pharmaceutically acceptable salt thereof.

[0322] In a seventh embodiment of Formula (E), in an ionizable lipid, e.g., a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), or the second or fifth embodiment of Formula (E), R 5 is absent or C1-C8 alkylene, or R 5 is absent, C-C alkylene, or C-C alkenylene, or R 5 is absent, C-C alkylene, or C-C alkenylene, or R 5 is absent or R 5is C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, or C alkenylene, and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), or the second or fifth embodiment of Formula (E).

[0323] In an eighth embodiment of Formula (E), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure has the formula (E-4): [ka] (E-4) wherein all other remaining variables are as described for the second, fifth, or seventh embodiment of Formula (E), Formula (E-1), Formula (E-2), (E-3), or Formula (E), or a pharmaceutically acceptable salt thereof.

[0324] In a ninth embodiment, in an ionizable lipid, e.g., a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, or seventh embodiment of Formula (E), or a pharmaceutically acceptable salt thereof, R 4 is C1-C 14 Unbranched alkyl, C2-C 14 unbranched alkenyl, or [ka] (In the formula, R 4a and R 4b are each independently, C1-C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl), or R 4 is C2-C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl, or R 4 is C5-C12 Unbranched alkyl or C5-C 12 unbranched alkenyl, or R 4 is C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C 13 Unbranched alkyl, C 12 Unbranched alkyl, C 11 Unbranched alkyl, C 10 Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 unbranched alkyl, C1 unbranched alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C 14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl, or R 4 teeth, [ka] (In the formula, R 4a and R 4b are each independently, C2-C 10 Unbranched alkyl or C2-C 10 unbranched alkenyl), or R 4 teeth, [ka] (In the formula, R 4a and R 4b are each independently, C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C 13Unbranched alkyl, C 12 Unbranched alkyl, C 11 Unbranched alkyl, C 10 Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 alkyl, C1 alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C 14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl), and all other remaining variables are as described for the second, fifth, or seventh embodiment of Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or Formula (E).

[0325] In a tenth embodiment, in an ionizable lipid, e.g., a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, seventh, or ninth embodiment of Formula (E), R 3 is C3-C8 alkylene or alkenylene, C3-C7 alkylene or alkenylene, or C3-C5 alkylene or alkenylene, or R 3is C alkylene, or C alkylene, or C alkylene, or C alkylene, or C alkylene, or C alkylene, or C alkylene, or C alkylene, or C alkenylene, or C alkenylene, or C alkenylene, or C alkenylene, or C alkenylene, and all other remaining variables are as described for the second, fifth, seventh, or ninth embodiment of Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or Formula (E).

[0326] In an eleventh embodiment, in an ionizable lipid, e.g., a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, seventh, ninth, or tenth embodiment of Formula (E), R 6a and R 6b are each independently C7-C 12 Alkyl or C7-C 12 alkenyl, or R 6a and R 6b are each independently C8-C 10 Alkyl or C8-C 10 alkenyl, or R 6a and R 6b are each independently, C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, and all other remaining variables are as described for the second, fifth, seventh, ninth, or tenth embodiment of Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or Formula (E).

[0327] In a twelfth embodiment, in an ionizable lipid, e.g., a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, seventh, ninth, tenth, or eleventh embodiment of Formula (E), R 6a and R 6b contain an equal number of carbon atoms, or R 6a and R 6b are the same or R 6a and R 6b are both C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, and all other remaining variables are as described for the second, fifth, seventh, ninth, tenth, or eleventh embodiment of Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or Formula (E).

[0328] In a thirteenth embodiment, in an ionizable lipid, e.g., a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), R as defined in any one of the preceding embodiments is 6a and R 6b each contain a different number of carbon atoms, or R 6a and R 6b The number of carbon atoms in R differs by one or two carbon atoms, or 6a and R 6b The number of carbon atoms in R differs by one carbon atom, or 6a is a C7 alkyl, and R 6a is C8 alkyl, R 6a is C8 alkyl, and R 6a is C7 alkyl, R 6a is C8 alkyl, and R 6a is C9 alkyl, R 6a is a C9 alkyl, and R 6ais C8 alkyl, R 6a is a C9 alkyl, and R 6a C 10 R is alkyl 6a C 10 alkyl, and R 6a is C9 alkyl, R 6a C 10 alkyl, and R 6a C 11 R is alkyl 6a C 11 alkyl, and R 6a C 10 R is alkyl 6a C 11 alkyl, and R 6a C 12 R is alkyl 6a C 12 alkyl, and R 6a C 11 R is alkyl 6a is a C7 alkyl, and R 6a is C9 alkyl, R 6a is a C9 alkyl, and R 6a is C7 alkyl, R 6a is C8 alkyl, and R 6a C 10 R is alkyl 6a C 10 alkyl, and R 6a is C8 alkyl, R 6a is a C9 alkyl, and R 6a C 11 R is alkyl 6a C 11 alkyl, and R 6a is C9 alkyl, R 6a C 10 alkyl, and R 6a C 12 R is alkyl 6a C 12 alkyl, and R 6a C 10is alkyl, and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, seventh, ninth, tenth, eleventh, or twelfth embodiment of Formula (E).

[0329] In a fourteenth embodiment, in an ionizable lipid, e.g., a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, seventh, ninth, tenth, eleventh, twelfth, or thirteenth embodiment of Formula (E), R' is absent, and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, seventh, ninth, tenth, eleventh, twelfth, or thirteenth embodiment of Formula (E).

[0330] In one embodiment, the ionizable lipid, e.g., cationic lipid, or cationic lipid of Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), in the LNP of the present disclosure is any one lipid selected from the lipids in Table 7, or a pharmaceutically acceptable salt thereof. [Table 7-1] [Table 7-2] [Table 7-3]

[0331] Specific examples are provided in the Exemplification section below and are included as part of the cationic or ionizable lipids described herein. Pharmaceutically acceptable salts as well as neutral forms are also included.

[0332] Cleavable lipids In some embodiments, the LNPs provided by the present disclosure include an ionizable lipid that is also a cleavable lipid. As used herein, the term "cleavable lipid," which may be used interchangeably with the term "SS-cleavable lipid," refers to an ionizable lipid that contains a disulfide bond (SS). The SS in the cleavable lipid is the cleavable unit. In one embodiment, the cleavable lipid includes an amine, e.g., a tertiary amine, and, e.g., a disulfide bond. In this cleavable lipid, the amine can become protonated in an acidic compartment (e.g., an endosome or lysosome), resulting in LNP destabilization, and the cleavable lipid can become cleaved in a reducing environment (e.g., the cytoplasm). Cleavable lipids also include pH-activated lipid-like materials, such as ss-OP lipids, ssPalm lipids, ss-M lipids, ss-E lipids, ss-EC lipids, ss-LC lipids, and ss-OC lipids.

[0333] According to some embodiments, the SS-cleavable lipids are described in International Patent Application Publication No. WO 2019188867, which is incorporated herein by reference in its entirety.

[0334] In one embodiment, the cleavable lipid may comprise three components: an amine head group, a linker group, and a hydrophobic tail. In one embodiment, the cleavable lipid comprises one or more phenyl ester bonds, one or more tertiary amino groups, and a disulfide bond. The tertiary amine groups provide pH responsiveness and induce endosomal escape, the phenyl ester bonds enhance the degradability (autolysis) of the structure, and the disulfide bonds become cleaved in a reducing environment.

[0335] In one embodiment, the cleavable lipid is a ss-OP lipid. In one embodiment, the ss-OP lipid comprises the structure of lipid A shown below: Lipid A [ka]

[0336] In one embodiment, the SS-cleavable lipid is SS-cleavable pH-activated lipid-like material (ssPalm). ssPalm lipid is well known in the art. For example, see Togashi et al., Journal of Controlled Release, 279 (2018) 262-270, the entire contents of which are incorporated herein by reference. In one embodiment, the ssPalm is ssPalmM lipid, comprising the structure of lipid B shown below. lipid B [ka]

[0337] In one embodiment, the ssPalmE lipid is a ssPalmE-P4-C2 lipid comprising the structure of lipid C below: lipid C [ka]

[0338] In one embodiment, the ssPalmE lipid is a ssPalmE-Paz4-C2 lipid comprising the structure of lipid D below: lipid D [ka]

[0339] In one embodiment, the cleavable lipid is a ss-M lipid. In one embodiment, the ss-M lipid comprises the structure shown in lipid E ​​below. Lipid E [ka]

[0340] In one embodiment, the cleavable lipid is an ss-E lipid. In one embodiment, the ss-E lipid comprises the structure shown in lipid F below. lipid F [ka]

[0341] In one embodiment, the cleavable lipid is a ss-EC lipid. In one embodiment, the ss-EC lipid comprises the structure shown for lipid G below. lipid G [ka]

[0342] In one embodiment, the cleavable lipid is a ss-LC lipid. In one embodiment, the ss-LC lipid comprises the structure shown for lipid H below. lipid H [ka]

[0343] In one embodiment, the cleavable lipid is a ss-OC lipid. In one embodiment, the ss-OC lipid comprises the structure shown for lipid J below. lipid J [ka]

[0344] Other lipids In some embodiments, the ionizable lipid in the LNPs of the present disclosure is N-[1-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DL EPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)aminolbutylcarboxamidoethyl 1-3,4-di[oleyloxy]-benzamide (MVL5), dioctadecylamido-glycylspermine (DOGS), 3b-[N-( N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol), dioctadecyldimethylammonium bromide (DDAB), Saint lipids (e.g., SAINT-2, N-methyl-4-(dioleyl)methylpyridinium), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), 1,2-dioleoyl-3-dimethylhydroxyethylammonium bromide (DORIE), 1,2-dioleoyloxypropyl-3-dimethylhydroxyethylammonium chloride (DORI), dialkylated amino acids (DILA2) (e.g., C18:1-norArg-C16), dioleyldimethylammonium chloride (DODAC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC), and 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC).In some variations, the condensing agent, e.g., the cationic lipid, is, for example, dioctadecyldimethylammonium bromide (DDAB), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2-dioleo ...-MC3-DMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA)], 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-MC3-DMA)], 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA)], 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin- These lipids include diethylaminopropane (DODAP), 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA), morpholinocholesterol (Mo-CHOL), (R)-5-(dimethylamino)pentane-1,2-diyldioleate hydrochloride (DODAPen-C1), (R)-5-guanidinopentane-1,2-diyldioleate hydrochloride (DOPen-G), and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-aminium chloride (DOTAPen).

[0345] In some embodiments, the ionizable lipid in the LNPs of the present disclosure has the following structure: [ka] , [ka] , [ka] (L369), [ka] , [ka] , [ka] (SM102), [ka] , [ka] , [ka] , [ka] , or [ka] (LP01) or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0346] B. Structured lipids In some embodiments, LNPs provided by the present disclosure comprise structured lipids. Without wishing to be bound by any particular theory, it is believed that structured lipids, when present in LNPs, contribute to the membrane integrity and stability of the LNPs.

[0347] In some embodiments, the structured lipid is a sterol, such as cholesterol, or a derivative thereof. In one embodiment, the structured lipid is cholesterol. In another embodiment, the structured lipid is a cholesterol derivative. Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholestanol, 5β-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol, non-polar analogs such as 5α-cholestan, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate, and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether. In some embodiments, the cholesterol derivative is cholestryl hemisuccinate (CHEMS).

[0348] Exemplary cholesterol derivatives are described in International Patent Application Publication No. 2009 / 127060 and US Patent Application Publication No. 2010 / 0130588, the contents of both of which are incorporated herein by reference in their entireties.

[0349] In some embodiments, the sterol in the LNPs of the present disclosure is selected from the group consisting of cholesterol, beta-sitosterol, stigmasterol, beta-sitostanol, campesterol, brassicasterol, and derivatives thereof, and any combination thereof. In one embodiment, the sterol is cholesterol. In another embodiment, the sterol is beta-sitosterol.

[0350] In some embodiments, the structured lipids, e.g., sterols, comprise about 20 mol% to about 50 mol% of the total lipids present in the LNP. In some embodiments, the structured lipids, e.g., sterols, comprise about 30 mol% to about 45 mol% of the total lipids present in the LNP. In some embodiments, the structured lipids, e.g., cholesterol, comprise about 30 mol% of the total lipids present in the LNP.

[0351] In some embodiments, the structured lipid is dexamethasone or dexamethasone palmitate.

[0352] C. Helper lipids Ceramide helper lipids In some embodiments, the LNPs provided by the present disclosure comprise a helper lipid. In some embodiments, the helper lipid is ceramide. The ceramides in the LNPs of the present disclosure are not conjugated to a polymer such as polyethylene glycol or PEG. Ceramides are sphingolipids, a class of cell membrane lipids. Ceramides comprise an N-acetylsphingosine (i.e., (E)-N-(1,3-dihydroxyoctadec-4-en-2-yl)acetamide) backbone and a fatty acid linked to an amide group. In some embodiments, the LNPs provided by the present disclosure comprise ceramide, wherein the fatty acid moiety of the ceramide is a fatty acid of a certain length or having a certain number of carbon atoms, as described below. As used herein, the term "helper lipid" refers to an amphipathic lipid comprising at least one nonpolar chain and at least one polar moiety. Without wishing to be bound by theory, it is believed that the helper lipid functions to avoid off-targeting of the LNPs to the blood compartment, increases the membrane fusogenicity of the lipid bilayer of the LNPs, and facilitates endosomal escape.

[0353] In some embodiments, the LNPs of the present disclosure comprise ceramide as a helper lipid. In some embodiments, the helper lipid has formula (II): [ka] Formula (II) (In the formula, [ka] is a single bond or a double bond, R 1 But C1-C 17 Alkyl or C2-C 17 is alkenyl, R 2 But C1-C 22 Alkyl or C2-C 22 is alkenyl, R 3 is hydrogen or C1-C2 alkyl, R4 is hydrogen or C1-C2 alkyl), or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0354] In some embodiments of Formula (II), [ka] is a single bond. In some embodiments of Formula (II), [ka] is a double bond.

[0355] In some embodiments of Formula (II), R 1 is C 10 -C 20 alkenyl, and R 2 is C 10 -C 20 alkyl, and R 3 is hydrogen.

[0356] In some embodiments of Formula (II), R 1 is C1-C 10 Alkyl or C2-C 10 It is alkenyl.

[0357] In some embodiments of Formula (II), R 1 is C1-C 10 Alkyl or C2-C 10 is alkenyl, R 2 is C1-C 22 Alkyl or C2-C 22 is alkenyl, R 3 is hydrogen or C1-C2 alkyl, R 4 is hydrogen or C1-C2 alkyl.

[0358] In some embodiments of Formula (II), R 3 and R 4are both hydrogen. In some embodiments of Formula (II), R 3 and R 4 are independently hydrogen or C1 alkyl.

[0359] In some embodiments of Formula (II), R 1 is C1-C7 alkyl or C2-C7 alkenyl. 1 is C1-C7 alkyl. In one embodiment, R 1 is a C1 alkyl.

[0360] The term "salt" when used to refer to a helper lipid represented by formula (II) means a pharmaceutically acceptable salt of the helper lipid represented by formula (II), including both acid and base addition salts. The salt of the helper lipid represented by formula (II) retains the biological effectiveness and properties of the free acid or free base form of the helper lipid represented by formula (II), which are not biologically or otherwise undesirable, and is formed with an inorganic or organic acid or base. Examples of inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Examples of organic acids include acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, and the like. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts.Salts derived from organic bases include, but are not limited to, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0361] The term "ester" when used to refer to a helper lipid represented by formula (II) refers to an ester of the helper lipid represented by formula (II). As a non-limiting example, the hydroxyl group of the helper lipid represented by formula (II) can be linked to an organic acid such as phosphoric acid or carboxylic acid through an esterification process to form an ester (e.g., a carboxylate or a phosphate) of the helper lipid represented by formula (II).

[0362] The term "deuterated analog," when used to refer to a helper lipid represented by formula (II), means an analog of a helper lipid represented by formula (II) in which any one or more hydrogen atoms of the helper lipid have been replaced with deuterium, an isotope of hydrogen.

[0363] In one embodiment of formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, [ka] is a double bond and R1 , R 2 , R 3 , and R 4 is as defined above. In an alternative embodiment of formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, [ka] is a single bond, and R 1 , R 2 , R 3 , and R 4 is as defined above.

[0364] In some embodiments of Formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is C1-C 15 Alkyl or C2-C 15 It is alkenyl.

[0365] In some embodiments of Formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is C1-C 15 Alkyl or C2-C 15 is alkenyl, R 2 is C1-C 22 Alkyl or C2-C 22 is alkenyl, R 3 is hydrogen or C1-C2 alkyl, R 4 is hydrogen or C1-C2 alkyl.

[0366] In some embodiments of Formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is C1-C 10 Alkyl or C2-C 10 It is alkenyl.

[0367] In some embodiments of Formula (II), or a salt or ester thereof, R 1 is C1-C 10 Alkyl or C2-C 10 is alkenyl, R 2 is C1-C 22 Alkyl or C2-C 22 is alkenyl, R 3 is hydrogen or C1-C2 alkyl, R 4 is hydrogen or C1-C2 alkyl.

[0368] In some embodiments of Formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is C1-C8 alkyl or C2-C8 alkenyl. 1 is C1-C8 alkyl. In some embodiments of Formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is C1-C7 alkyl or C2-C7 alkenyl. 1 is C1-C7 alkyl. In one embodiment of formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is C1-C7 alkyl, R 2 is C1-C 22 Alkyl or C2-C 22 is alkenyl, R 3 is hydrogen or C1-C2 alkyl, R 4 is hydrogen or C1-C2 alkyl.

[0369] In some embodiments of Formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1is a C1 alkyl, a C2 alkyl, a C3 alkyl, a C4 alkyl, a C5 alkyl, a C6 alkyl, or a C7 alkyl. In some embodiments of Formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is a C1 alkyl, a C2 alkyl, a C3 alkyl, a C4 alkyl, a C5 alkyl, a C6 alkyl, or a C7 alkyl. In some embodiments of Formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is a C1 alkyl, a C3 alkyl, a C5 alkyl, or a C7 alkyl. In one embodiment of Formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is C alkyl. In one embodiment of Formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is C alkyl. In one embodiment of Formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is C5 alkyl. In one embodiment of Formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is a C7 alkyl.

[0370] In some embodiments of Formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 2 is C3-C 15 Alkyl or C3-C 15 alkenyl, and R 1 , R 3 , and R 4 is as defined above. In some embodiments of formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 2 is C5-C 15 Alkyl or C3-C 15 alkenyl, and R 1 , R 3 , and R 4is as defined above. In some embodiments of formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 2 is C7-C 15 Alkyl or C3-C 15 alkenyl, and R 1 , R 3 , and R 4 is as defined above. In some embodiments of formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 2 is C9-C 15 Alkyl or C9-C 15 alkenyl, and R 1 , R 3 , and R 4 is as defined above. In some embodiments of formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 2 is C9 alkyl, C 10 Alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 14 Alkyl or C 15 alkyl, and R 1 , R 3 , and R 4 is as defined above. In some embodiments of formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 2 is a C9 alkyl, and R 1 , R 3 , and R 4 is as defined above. In some embodiments of formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 2 is C 11 alkyl, and R 1 , R 3 , and R 4 is as defined above. In some embodiments of formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 2 is C13 alkyl, and R 1 , R 3 , and R 4 is as defined above.

[0371] In some embodiments of Formula (II), or a salt or ester thereof, or a deuterated analog thereof, R 3 is hydrogen or C1 alkyl, and R 1 , R 2 , and R 4 is as defined above. In one embodiment of formula (II), or a salt or ester thereof, or a deuterated analog thereof, R 3 is hydrogen and R 1 , R 2 , and R 4 is as defined above. In one embodiment of formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 3 is a C1 alkyl, and R 1 , R 2 , and R 4 is as defined above.

[0372] In some embodiments of Formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 4 is hydrogen or C1 alkyl, and R 1 , R 2 , and R 3 is as defined above. In one embodiment of formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 4 is hydrogen and R 1 , R 2 , and R 3 is as defined above. In one embodiment of formula (II), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 4 is a C1 alkyl, and R 1 , R 2 , and R 3 is as defined above.

[0373] In some embodiments of Formula (II), R 1 is C1-C7 alkyl or C2-C7 alkenyl.

[0374] In some embodiments, R 1 is a C alkyl, a C alkyl, a C alkyl, or a C alkyl. In some embodiments, R 1 is a C1 alkyl.

[0375] In some embodiments of Formula (II), R 2 is C3-C 15 Alkyl or C3-C 15 In some embodiments, R is alkenyl. 2 is C 10 Alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 14 Alkyl or C 15 In some embodiments, R 2 is C 12 Alkyl, C 13 Alkyl or C 14 In some embodiments, R 2 is C 13 In some embodiments, R 2 is C 12 In some embodiments, R 2 is C 11 It is alkyl.

[0376] In some embodiments of Formula (II), R 1 and R 2 are both hydrogen, [ka] is a double bond.

[0377] In some embodiments of Formula (II), R 1 and R 2 are both hydrogen, [ka] is a double bond and R 1 is a C alkyl, a C alkyl, a C alkyl, or a C alkyl. In one embodiment, R 1 is C alkyl. In another embodiment, R 1 is C alkyl. In yet another embodiment, R 1 is C5 alkyl. In yet another embodiment, R 1 is a C7 alkyl.

[0378] In some embodiments of Formula (II), R 1 and R 2 are both hydrogen, [ka] is a double bond and R 1 is a C1 alkyl, a C3 alkyl, a C5 alkyl, or a C7 alkyl, and R 2 is C9 alkyl, C 11 , or C 13 In one embodiment, R 2 is C alkyl. In one embodiment, R 2 is C 11 In another embodiment, R 2 is C 13 It is alkyl.

[0379] In some embodiments of Formula (II), R 3 is hydrogen. In some embodiments of Formula (II), R 3 is a C1 alkyl.

[0380] In some embodiments of Formula (II), R 4 is hydrogen. In some embodiments of Formula (II), R 4 is a C1 alkyl.

[0381] Other helper lipids In some embodiments, the helper lipid included in the LNPs of the present disclosure is a phospholipid, a phosphatidylcholine, or a derivative thereof. In some embodiments, the helper lipid included in the LNPs of the present disclosure is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), hydrogenated soy PC (HSPC), phosphatidylserine (PS), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1-margalloyl-2-oleoyl-sn-glycero-3-phosphocholine (MOPC). ), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the helper lipid included in the LNPs of the present disclosure is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0382] In some embodiments, the helper lipid comprises about 1 mol% to about 40 mol%, or about 5 mol% to about 15 mol% of the total lipid present in the LNP. In some embodiments, the helper lipid comprises about 10 mol% to about 20 mol% of the total lipid present in the LNP, and such LNPs having about 10 mol% to about 20 mol% of the total lipid present in the LNP exhibit increased overall tolerability (e.g., as demonstrated by a reduced subject weight loss profile and cytokine response) compared to LNPs containing less than 10% of the same helper lipid.

[0383] D. Lipid-anchored polymers In some embodiments, LNPs provided by the present disclosure comprise at least one type of lipid-anchored polymer, e.g., a first lipid-anchored polymer and / or a second lipid-anchored polymer. As used herein, the term "lipid-anchored polymer" refers to a molecule comprising a lipid moiety covalently attached to a polymer, e.g., via a linker. Without wishing to be bound by theory, it is believed that lipid-anchored polymers can inhibit aggregation of LNPs and provide steric stabilization. In some embodiments, LNPs provided by the present disclosure comprise two lipid-anchored polymers, i.e., a first lipid-anchored polymer and a second lipid-anchored polymer.

[0384] In some embodiments, the first lipid-anchored polymer included in an LNP of the present disclosure is a polymer-conjugated lipid of the present disclosure, e.g., DODA-PG34, DODA-PG45, DODA-PG46, or DODA-PG58.

[0385] In some embodiments, LNPs of the present disclosure comprise two types of lipid-anchored polymers: a) a first lipid-anchored polymer, a polymer-conjugated lipid of the present disclosure, e.g., DODA-PG34, DODA-PG45, DODA-PG46, or DODA-PG58, and b) a second lipid-anchored polymer.

[0386] In some embodiments, the lipid-anchored polymer, e.g., the second lipid-anchored polymer according to the present disclosure, comprises: (i) a lipid moiety comprising at least one hydrophobic tail; (ii) a polymer conjugated to a lipid moiety, optionally via a linker.

[0387] In some embodiments, the lipid-anchored polymer, e.g., the second lipid-anchored polymer according to the present disclosure, comprises: (i) a lipid moiety comprising at least one hydrophobic tail; (ii) a polymer; and (iii) a linker, wherein the polymer is conjugated to the lipid moiety via the linker; and (iv) a targeting moiety conjugated to the polymer.

[0388] In some embodiments, the lipid-anchored polymer, e.g., the second lipid-anchored polymer according to the present disclosure, comprises: (i) a lipid moiety comprising at least one hydrophobic tail; (ii) a polymer; and (iii) a linker, wherein the polymer is conjugated to the lipid moiety via the linker; and (iv) a reactive species conjugated to a polymer, wherein the reactive species is functionalized to be conjugated to a targeting moiety.

[0389] In one embodiment, at least one (e.g., single or two) hydrophobic tail is a fatty acid. Non-limiting examples of at least one (e.g., single or two) hydrophobic tail containing 12 to 22 carbon atoms in a single aliphatic chain backbone include lauric acid, myristic acid, myristoleic acid, octadecylamine, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, and derivatives thereof.

[0390] The term "derivative," as used herein with respect to a hydrophobic tail in a lipid-anchored polymer, refers to a hydrophobic tail that has been modified compared to the original or native hydrophobic tail. In some embodiments, a derivative comprises one or more of the following modifications compared to the original or native hydrophobic tail: a) a carboxylate group has been replaced with an amine, amide, ether, or carbonate group; b) one or more saturation points, e.g., double bonds, have been introduced into the hydrophobic tail (e.g., via dehydrogenation); c) one or more saturation points, e.g., double bonds, have been removed from the hydrophobic tail (e.g., via hydrogenation); or d) if present, the configuration of one or more double bonds has been changed, e.g., from a cis configuration to a trans configuration or from a trans configuration to a cis configuration. A derivative comprises the same number of carbon atoms as its original or native hydrophobic tail.

[0391] As used herein, the term "single aliphatic chain backbone" refers to the main linear aliphatic chain or carbon chain, i.e., the longest continuous linear aliphatic chain or carbon chain, when referring to the hydrophobic tail in a lipid-anchored polymer. For example, the following alkyl chain with several branches contains 18 carbon atoms in the single aliphatic chain backbone, i.e., the longest continuous linear alkyl chain contains 18 carbon atoms. Note that one or two carbon atoms in the several branches (all indicated by *) are not included in the number of carbon atoms in the single aliphatic chain backbone. [ka]

[0392] In one embodiment, a second lipid-anchored polymer according to the present disclosure comprises a lipid moiety comprising at least one hydrophobic tail and a polymer conjugated to the lipid moiety, optionally via a linker, wherein the lipid moiety of the second lipid-anchored polymer is selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielaidoyl-sn-phosphatidyl Ethanolamine (DEPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 18-1-trans PE, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diphenyl ether (DPE), The lipid linker moiety of the second lipid-anchored polymer comprises a lipid linker moiety selected from the group consisting of dioctadecylamine (DODA), dihexadecylamine, distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and derivatives thereof. In some embodiments, the lipid moiety of the second lipid-anchored polymer comprises a lipid linker moiety selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, and derivatives thereof. In one particular embodiment, the lipid moiety of the second lipid-anchored polymer comprises DSPE. In one embodiment, the second lipid-anchored polymer further comprises a targeting moiety.

[0393] The lipid-anchored polymer of the present disclosure may also contain a reactive species. In some embodiments, the reactive species is conjugated to the polymer in the lipid-anchored polymer. The reactive species present in the lipid-anchored polymer of the present disclosure can be used, for example, for conjugation to a targeting moiety functionalized with a complementary reactive species, i.e., a reactive species capable of reacting with the reactive species contained in the lipid-anchored polymer of the present disclosure. In some embodiments, the reactive species conjugated to the lipid-anchored polymer of the present disclosure can be a reagent selected from the group consisting of a thiol reagent, a maleimide reagent, or a click chemistry reagent, for example, an alkyne reagent such as a dibenzocyclooctyne (DBCO) reagent, a transcyclooctene (TCO) reagent, a tetrazine (TZ) reagent, and an azide (AZ) reagent.

[0394] Linkers in lipid-anchored polymers In some embodiments, in the lipid-anchored polymers of the present disclosure, the lipid moiety is covalently attached to the polymer via a linker. In some embodiments, the linker in the lipid-anchored polymers of the present disclosure is an alkyl linker, a glycerol linker, a phosphate linker, a phosphate ester linker, an ether linker, an ester linker, a diester linker (e.g., a glutaryl linker, a succinyl linker, etc.), an amide linker, a diamide linker, an amine linker, a peptide linker, a phosphoethanolamine linker, a phosphocholine linker, a carbamate linker, a diamide alkyl linker, a cleavable linker, or any combination thereof. In some embodiments, the linker in the lipid-anchored polymer in the LNPs of the present disclosure is -(CH2) n -, -C(O)(CH2) n -, -C(O)O(CH2) n , -C(O)O(CH2) n -, -OC(O)(CH2) n C(O)O- and -NH(CH2) n C(O)O-, where n is a number integer ranging from 1 to 20. Thus, in some embodiments, the linker is selected from the group consisting of -C(O)(CH) n-, and in some embodiments, n is 2, 3, 4, 5, or 6.

[0395] In some embodiments, the linker of the second lipid-anchored polymer is a glycerol linker, a phosphate linker, an ether linker, an amide linker, an amine linker, a peptide linker, a phosphoethanolamine linker, a phosphocholine linker, a carbamate linker, or a -(CH) n -, -C(O)(CH2) n - or -C(O)O(CH2) n (wherein n is an integer ranging from 1 to 20), or any combination thereof.

[0396] As used herein, the term "linker lipid moiety" refers to a lipid moiety comprising at least one hydrophobic tail covalently attached to a linker. In some embodiments, the linker lipid moiety can be part of a lipid-anchored polymer.

[0397] As used herein, the term "derivative," when used in reference to a linker lipid moiety, means a linker-lipid moiety that contains one or more of the following modifications: a) if present, the phosphatidylethanolamine (PE) head group has been modified to convert the amino group to a methylamino or dimethylamino group; b) the modified linker lipid moiety contains one or more additional functional groups or moieties, e.g., -OH, -OCH3, -NH2, maleimide, azide, or cyclooctyne, such as dibenzocyclooctyne (DBCO).

[0398] Polymers in lipid-anchored polymers In some embodiments, the polymer included in the lipid-anchored polymer is selected from the group consisting of polyethylene (PE), polypropylene (PP), polyethylene glycol (PEG), polyglycerol (PG), polyvinyl alcohol (PVOH), polysarcosine (pSar), and combinations thereof. In one embodiment, the polymer is selected from the group consisting of polyethylene glycol (PEG), polyglycerol (PG), polysarcosine (pSar), poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), and combinations thereof. In one embodiment, the polymer is polyethylene glycol (PEG).

[0399] In some embodiments, the polymer included in the lipid-anchored polymer, e.g., the second lipid-anchored polymer, is selected from the group consisting of polyethylene (PE), polypropylene (PP), polyethylene glycol (PEG), polyvinyl alcohol (PVOH), polysarcosine (pSar), polyglycerol (PG), and derivatives of any of the foregoing.

[0400] In some embodiments, the polymer included in the lipid-anchored polymer of the present disclosure, e.g., the first lipid-anchored polymer and / or the second lipid-anchored polymer, is polyglycerol (PG) or a PG derivative. PG or a PG derivative can be linear or branched. PG derivatives can be carboxylated PG, e.g., glutarylated PG, such as 3-methylglutarylated PG, or 2-carboxycyclohexane-1-carboxylated PG. In some embodiments, PG or a PG derivative can contain an average of 5 to 100 monomer units. In some embodiments, PG or a PG derivative can contain an average of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 monomer units, or about 34, 45, 46, or 58. In one embodiment, the PG or a PG derivative contains an average of 34, 45, 46, or 58 monomer units.

[0401] In some embodiments, the polymer in the lipid-anchored polymer has a molecular weight of about 500 Da to about 5000 Da, e.g., about 1500 Da to about 5000 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 2000 Da.

[0402] targeting part In some embodiments, the LNPs of the present disclosure further comprise one or more targeting moieties. The targeting moiety targets the LNP for delivery to a specific site or tissue in a subject, for example, the liver. In some embodiments, the targeting moiety is capable of binding to specific liver cells, such as hepatocytes. The targeting moiety can be conjugated to a first lipid-anchored polymer, such as a polymer-conjugated lipid of the present disclosure, or a second lipid-anchored polymer, as described herein.

[0403] In one embodiment, the targeting moiety can bind to the asialoglycoprotein receptor (ASGPR), i.e., hepatocyte-specific ASGPR. In one embodiment, the targeting moiety comprises an N-acetylgalactosamine molecule (GalNAc) or a GalNAc derivative thereof. As used herein, "GalNAc derivative" refers to a modified GalNAc molecule or a conjugate of one or more GalNAc molecules (modified or unmodified) covalently linked to a lipid-anchored polymer, e.g., as defined herein. In one embodiment, the targeting moiety is a triantennary or trivalent GalNAc conjugate (i.e., GalNAc3), which is a ligand conjugate having three GalNAc molecules or three GalNAc derivatives. In one embodiment, the targeting moiety is a triantennary GalNAc represented by the following structural formula: [ka]

[0404] In one embodiment, the targeting moiety is a tetraantennary GalNAc conjugate. In one embodiment, the targeting moiety is a tetraantennary or tetravalent GalNAc conjugate (i.e., GalNAc4), which is a ligand having four GalNAc molecules or four GalNAc derivatives.

[0405] In one embodiment, the targeting moiety can bind to a low-density lipoprotein receptor (LDLR), e.g., a hepatocyte-specific LDLR. In one embodiment, the targeting moiety comprises an apolipoprotein E (ApoE) protein, an ApoE polypeptide (or peptide), an apolipoprotein B (ApoB) protein, an ApoB polypeptide (or peptide), a fragment of any of the foregoing, or a derivative of any of the foregoing. In one embodiment, the ApoE polypeptide, ApoB polypeptide, or fragment thereof is an ApoE polypeptide, ApoB polypeptide, or fragment thereof disclosed in International Patent Application Publication No. WO 2022 / 261101, the entire contents of which are incorporated herein by reference. In one embodiment, the ApoE protein is a modified ApoE protein, and the ApoB protein is a modified ApoB protein.

[0406] In one embodiment, the ApoE protein has the following amino acid sequence: MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVR (SEQ ID NO: 1). The ApoE protein has an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% sequence identity to SEQ ID NO:1. In one embodiment, the ApoE protein comprises the amino acid sequence set forth in SEQ ID NO:1. In one embodiment, the ApoE protein consists essentially of the amino acid sequence set forth in SEQ ID NO:1. In one embodiment, the ApoE protein consists of the amino acid sequence set forth in SEQ ID NO:1.

[0407] In one embodiment, the ApoE protein has the following amino acid sequence:MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRHHHHHH The ApoE protein has an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% sequence identity to SEQ ID NO:2 (SEQ ID NO:2). In one embodiment, the ApoE protein comprises the amino acid sequence set forth in SEQ ID NO:2. In one embodiment, the ApoE protein consists essentially of the amino acid sequence set forth in SEQ ID NO:2. In one embodiment, the ApoE protein consists of the amino acid sequence set forth in SEQ ID NO:2.

[0408] In one embodiment, the ApoE protein has the following amino acid sequence: It has an amino acid sequence that has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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, or at least about 99% sequence identity to MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVSGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVR (SEQ ID NO: 3). In one embodiment, the ApoE protein comprises the amino acid sequence set forth in SEQ ID NO: 3. In one embodiment, the ApoE protein consists essentially of the amino acid sequence set forth in SEQ ID NO: 3. In one embodiment, the ApoE protein consists of the amino acid sequence set forth in SEQ ID NO: 3.

[0409] In one embodiment, the ApoE protein has the following amino acid sequence: MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVSGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRHHHHHHGGSSGSGC (SEQ ID NO: 4) The ApoE protein has an amino acid sequence that shares about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% sequence identity with the ApoE protein. In one embodiment, the ApoE protein comprises the amino acid sequence set forth in SEQ ID NO:4. In one embodiment, the ApoE protein consists essentially of the amino acid sequence set forth in SEQ ID NO:4. In one embodiment, the ApoE protein consists of the amino acid sequence set forth in SEQ ID NO:4.

[0410] As used herein, the term "sequence identity," when used in reference to a polypeptide or protein, refers to the ratio of the number of identical amino acids between two aligned sequences over the aligned length, expressed as a percentage. In some embodiments, the two aligned sequences are identical in length, i.e., have the same number of amino acids.

[0411] In one embodiment, the targeting moiety in the LNPs of the present disclosure is an ApoE protein conjugate in an ApoB protein conjugate, e.g., a conjugate of one or more ApoE and / or ApoB protein molecules (native or modified) or fragments thereof covalently linked to a lipid-anchored polymer as defined herein. In one embodiment, the targeting moiety in the LNPs of the present disclosure is an ApoE polypeptide conjugate in an ApoB polypeptide conjugate, e.g., a conjugate of one or more ApoE and / or ApoB polypeptide molecules or fragments thereof covalently linked to a lipid-anchored polymer as defined herein.

[0412] In one embodiment, the targeting moiety is an antibody or antibody fragment, e.g., an antibody or antibody fragment capable of specifically binding to an antigen present on the surface of a cell. In one embodiment, the antibody or antibody fragment is a monoclonal antibody (mAb), a single-chain variable fragment (scFv), a heavy-chain antibody (hcAb), a nanobody (Nb), a heavy-chain-only immunoglobulin (HC1g), an immunoglobulin neo-antigen receptor (IgNAR), a variable domain of an immunoglobulin neo-antigen receptor (VNAR), a single-domain antibody, or a variable heavy-chain-only antibody (VHH).

[0413] In some embodiments, an LNP of the present disclosure comprises a polymer-conjugated lipid of the present disclosure, wherein a targeting moiety as defined herein (and including GalNAc, ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide, antibody, or antibody fragment) is conjugated to the polymer-conjugated lipid of the present disclosure.

[0414] In some embodiments, an LNP of the present disclosure can include a polymer-conjugated lipid of the present disclosure as a first lipid-anchoring polymer and a targeting moiety described herein conjugated to the polymer-conjugated lipid. In some embodiments, the polymer in the polymer-conjugated lipid, e.g., PG or a PG derivative, is conjugated to the targeting moiety.

[0415] In some embodiments, the targeting moiety can be conjugated to the polymer-conjugated lipid via a reactive species. In some embodiments, the reactive species can be a reagent selected from the group consisting of a thiol reagent, a maleimide reagent, or a click chemistry reagent, e.g., an alkyne reagent such as a dibenzocyclooctyne (DBCO) reagent, a transcyclooctene (TCO) reagent, a tetrazine (TZ) reagent, and an azide (AZ) reagent. Thus, in an exemplary embodiment, a polymer-conjugated lipid of the present disclosure comprising an azide reagent as a reactive species can be reacted with a targeting moiety functionalized with a DBCO reagent as a complementary reactive species to produce a polymer-conjugated lipid conjugated to the targeting moiety via a reactive species. In another exemplary embodiment, a polymer-conjugated lipid of the present disclosure comprising a thiol reagent can be reacted with a targeting moiety functionalized with a maleimide reagent to produce a polymer-conjugated lipid conjugated to the targeting moiety via a reactive species.

[0416] In some embodiments, an LNP of the present disclosure may comprise a polymer-conjugated lipid of the present disclosure as a first lipid-anchored polymer, a second lipid-anchored polymer, and a targeting moiety described herein conjugated to the second lipid-anchored polymer.

[0417] In some embodiments, the targeting moiety can be conjugated to the second lipid-anchored polymer via a reactive species. In some embodiments, the reactive species can be a reagent selected from the group consisting of a thiol reagent, a maleimide reagent, or a click chemistry reagent, e.g., an alkyne reagent such as a dibenzocyclooctyne (DBCO) reagent, a transcyclooctene (TCO) reagent, a tetrazine (TZ) reagent, and an azide (AZ) reagent. Thus, in an exemplary embodiment, a second lipid-anchored polymer of the present disclosure comprising an azide reagent as a reactive species can be reacted with a targeting moiety functionalized with a DBCO reagent as a complementary reactive species to produce a second lipid-anchored moiety conjugated to the targeting moiety via a reactive species. In another exemplary embodiment, a polymer-conjugated lipid of the present disclosure comprising a thiol reagent can be reacted with a targeting moiety functionalized with a maleimide reagent to produce a polymer-conjugated lipid comprising a targeting moiety.

[0418] Thus, in one embodiment of an LNP of the present disclosure, the LNP comprises a second lipid-anchored polymer, and a targeting moiety (including GalNAc, ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide, antibody, or antibody fragment) as defined herein is conjugated to the second lipid-anchored polymer, which comprises a lipid moiety conjugated to the polymer, optionally via a linker. In one embodiment, the second lipid-anchored polymer is selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielideyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl-2-oleoyl-sn-glycero- 3-phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 18-1-trans PE, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), and a moiety selected from the group consisting of dioctadecylamine (DODA), dihexadecylamine, distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and derivatives thereof. In one embodiment, the second lipid-anchored polymer comprises a linker lipid moiety selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, and derivatives thereof, hi another embodiment, the lipid moiety of the second lipid-anchored polymer comprises DSPE.

[0419] In one embodiment, an ApoE protein, an ApoB protein, an ApoE polypeptide, an ApoB polypeptide, an antibody, or a fragment thereof is covalently linked to a lipid-anchored polymer (e.g., a first lipid-anchored polymer or a second lipid-anchored polymer) or an LNP of the present disclosure via strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry, for example, via an azide-modified lipid-anchored polymer (e.g., DSG-PEG2000-azide, DSPE-PEG2000-azide, DSG-PEG3400-azide, DSPE-PEG3400-azide, DSG-PEG5000-azide, DSPE-PEG5000-azide), and a dibenzocyclooctyne (DBCO)-functionalized ApoE protein, ApoB protein, ApoE protein, ApoB protein, antibody, or a fragment thereof.

[0420] In an exemplary embodiment, the second lipid-anchored polymer conjugated to the targeting moiety is represented by the following structure: [ka]

[0421] In another exemplary embodiment, the second lipid-anchored polymer conjugated to the targeting moiety is represented by the following structure: [ka]

[0422] In some embodiments, LNPs of the present disclosure may comprise a first lipid-anchored polymer and a second lipid-anchored polymer. For example, LNPs of the present disclosure may comprise a first lipid-anchored polymer that does not include a targeting moiety and a second type of lipid-anchored polymer that includes a targeting moiety, such as GalNAc. For example, LNPs of the present disclosure may comprise DSG-PEG2000 modified to include an additional OCH3 group (DSG-PEG2000-OMe) as the first lipid-anchored polymer and DSPE-PEG2000-GalNAc3 as the second lipid-anchored polymer. In another example, LNPs of the present disclosure may comprise a polymer-conjugated lipid as the first lipid-anchored polymer of the present disclosure and a second anchor polymer, e.g., a second anchor polymer conjugated to a targeting moiety. For example, LNPs of the present disclosure may comprise DODA-PG45 as the first lipid-anchored polymer and DSPE-PEG2000-OH as the second lipid-anchored polymer.

[0423] In some embodiments, LNPs of the present disclosure can comprise a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the second lipid-anchored polymer comprises a targeting moiety. In some embodiments, the second lipid-anchored polymer comprises a lipid moiety selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, and derivatives thereof. In some embodiments, the first lipid-anchored polymer is any lipid-anchored polymer described above. In one specific embodiment, the first lipid-anchored polymer is a polymer-conjugated lipid of the present disclosure, such as DODA-PG34, DODA-PG45, DODA-PG46, or DODA-PG58. For example, LNPs of the present disclosure can comprise DODA-PG45 as the first lipid-anchored polymer and DSPE-PEG2000-GalNAc3 as the second lipid-anchored polymer.

[0424] In some embodiments, the targeting moiety is conjugated to a DSPE anchor polymer, hi some embodiments, the DSPE anchor polymer is DSPE-PEG or a derivative thereof.

[0425] In some embodiments, the targeting moiety is conjugated to a DSG anchor polymer, hi some embodiments, the DSG anchor polymer is DSG-PEG or a derivative thereof.

[0426] In some embodiments, an LNP provided by the present disclosure comprises a therapeutic nucleic acid (TNA), an ionizable lipid, a sterol, a helper lipid, and a first lipid-anchored polymer, wherein the first lipid-anchored polymer comprises a polymer-conjugated lipid.

[0427] In some embodiments, the ionizable lipids comprise between about 20 mol% and about 60 mol% of the total lipids present in the LNP. In some embodiments, the ionizable lipids comprise between about 35 mol% and about 50 mol% of the total lipids present in the LNP. In some embodiments, the sterols comprise between about 20 mol% and about 50 mol% of the total lipids present in the LNP. In some embodiments, the sterols comprise between about 30 mol% and about 45 mol% of the total lipids present in the LNP. In some embodiments, the helper lipids comprise between about 1 mol% and about 40 mol% of the total lipids present in the LNP. In some embodiments, the helper lipids comprise between about 5 mol% and about 15 mol% of the total lipids present in the LNP. In some embodiments, the first lipid-anchored polymer comprises between about 0.5 mol% and about 5 mol% of the total lipids present in the LNP. In some embodiments, the first lipid-anchored polymer comprises between about 1.5 mol% and about 3 mol% of the total lipids present in the LNP. In some embodiments, the second lipid-anchored polymer comprises about 0.05 mol % to about 5 mol % of the total lipid present in the LNP, hi some embodiments, the second lipid-anchored polymer comprises about 0.1 mol % to about 1 mol % of the total lipid present in the LNP.

[0428] Lipid nanoparticles (LNPs) containing ceDNA are disclosed in International Patent Application No. PCT / US2018 / 050042, filed September 7, 2018, which is incorporated herein in its entirety, and are contemplated for use in the methods and compositions disclosed therein.

[0429] The size of the LNPs can be determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK). In some embodiments, the LNPs of the present disclosure have an average diameter, as determined by light scattering, of less than about 90 nm, e.g., less than about 80 nm or less than about 75 nm. According to some embodiments, the LNPs of the present disclosure have an average diameter, as determined by light scattering, of between about 50 nm and about 75 nm, or between about 50 nm and about 70 nm.

[0430] The pKa of formulated cationic lipids can correlate with the effectiveness of LNPs for delivery of nucleic acids (see Jayaraman et al., Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al., Nature Biotechnology 28, 172-176 (2010), both of which are incorporated by reference in their entirety). In one embodiment, the pKa of each cationic lipid is determined in lipid nanoparticles using a fluorescence-based assay of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). LNPs in PBS at a concentration of 0.4 mM total lipid can be prepared using the in-line process described herein and elsewhere. TNS can be prepared as a 100 mM stock solution in distilled water. Vesicles can be diluted to 24 mM lipid in 2 mL of buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, with a pH ranging from 2.5 to 11. An aliquot of TNS solution can be added to obtain a final concentration of 1 mM. After vortex mixing, fluorescence intensity is measured at room temperature on an SLM Aminco Series 2 luminescence spectrophotometer using excitation and emission wavelengths of 321 nm and 445 nm. A sigmoidal best-fit analysis can be applied to the fluorescence data, and the pKa is determined when the pH yielded half-maximal fluorescence intensity.

[0431] In one embodiment, relative activity can be determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection. Activity is compared at 0.3 mg and 1.0 mg doses of ceDNA / kg and expressed as ng luciferase / g liver measured 4 hours after administration.

[0432] Without limitation, LNPs of the present disclosure include lipid formulations that can be used to deliver capsid-free, non-viral DNA vectors to a desired target site (e.g., a cell, tissue, organ, etc.). Generally, LNPs comprise a capsid-free, non-viral DNA vector and a cationic lipid or a salt thereof.

[0433] Further exemplary lipid-anchored polymers (e.g., second lipid-anchored polymers) include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, PG-lipid conjugates, polyamide lipid conjugates (such as ATTA-lipid conjugates), cationic polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the second lipid-anchored polymer is a PEGylated lipid, such as a (methoxypolyethylene glycol)-conjugated lipid, a PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEGylated phosphatidylethanolamine (PEG-PE), a PEG diacylglycerol succinate (PEGS-DAG) (such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), a PEG dialkoxypropylcarbamate, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or a mixture thereof.Additional exemplary PEG-lipid conjugates are described, for example, in International Patent Application Publication Nos. 1996 / 010392, 1998 / 051278, 2002 / 087541, 2005 / 026372, 2008 / 147438, 2009 / 086558, 2012 / 000104, 2013 / 000106, 2014 / 000108, 2015 / 000109, 2016 / 000109, 2017 / 000109, 2018 / 000109, 2019 / 000109, 2020 / 000109, 2021 / 000109, 2022 / 000109, 2023 / 000109, 2024 / 000109, 2025 / 000109, 2026 / 000109, 2027 / 000109, 2028 / 000109, 2029 / 000109, 2030 ... Nos. 017 / 117528, 2017 / 099823, 2015 / 199952, 2017 / 004143, 2015 / 095346, 2012 / 000104, 2012 / 000104, and 2010 / 006282, and U.S. Patent Application Publication No. 2003 / 007782 No. 9, No. 2005 / 0175682, No. 2008 / 0020058, No. 2011 / 0117125, No. 2013 / 0303587, No. 2 018 / 0028664, 2015 / 0376115, 2016 / 0376224, 2016 / 0317458, 2013 / 030 3587, 2013 / 0303587, and 20110123453, and U.S. Patent Nos. 5,885,613, 6,287,591, 6,320,017, and 6,586,559, the contents of all of which are incorporated herein by reference in their entireties.

[0434] Further examples of PEG-DAA PEGylated lipids include, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. PEG-lipids include PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol(1-[8'-(cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaotanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol) ), PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In one embodiment, the PEG-lipid is one or more of PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], [ka] , [ka] , [ka] , and [ka] It can be selected from the group consisting of:

[0435] Still further exemplary lipid-anchored polymers (e.g., second lipid-anchored polymers) include N-(carbonyl-methoxy PEG n )-1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEGn (wherein n is 350, 500, 750, 1000, or 2000)), N-(carbonyl-methoxyPEG n )-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG n (wherein n is 350, 500, 750, 1000, or 2000), DSPE-polyglycerin-cyclohexyl-carboxylic acid, DSPE-polyglycerin-2-methylglutaric-carboxylic acid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated polyethylene glycol (DSPE-PEG-OH), polyethylene glycol-dimyristylglycerol (PEG-DMG), or polyethylene glycol-distearoylglycerol (PEG-DSG). PEGn In some examples, where n is 350, 500, 750, 1000, or 2000, the PEG-lipid is N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG2,000). n In some examples, where n is 350, 500, 750, 1000, or 2000, the PEG-lipid is N-(carbonyl-methoxyPEG2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG2,000). In some embodiments, the PEG-lipid is DSPE-PEG-OH. In some embodiments, the PEG-lipid is a PEG-lipid having two C 14 PEG-DMG with a hydrophobic tail and PEG2000.

[0436] E. Therapeutic Nucleic Acids The LNPs provided by the present disclosure also comprise therapeutic nucleic acids (TNAs). According to embodiments, pharmaceutical compositions comprising the LNPs of the present disclosure are also provided.

[0437] Exemplary therapeutic nucleic acids in LNPs of the present disclosure include, but are not limited to, minigenes, plasmids, minicircles, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), ribozymes, deoxyribozymes, closed-ended double-stranded DNA (e.g., ceDNA, CELiD, covalently closed linear DNA ("ministring"), doggybone™, proteome closed-ended DNA, or dumbbell linear DNA), Dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), mRNA, tRNA, rRNA, gRNA, and DNA viral vectors, viral RNA vectors, non-viral vectors, and any combination thereof.

[0438] In any of the aspects and embodiments provided herein, the therapeutic nucleic acid can be therapeutic DNA, which can be ceDNA, ssDNA, CELiD, covalently closed linear DNA ("ministring" or otherwise), doggybone™, protelomeric closed-end DNA, dumbbell linear DNA, minigene, plasmid, or minicircle.

[0439] In one embodiment, the therapeutic nucleic acid can be a circular single-stranded polynucleotide consisting of at least three sections, two of which are sufficiently complementary to form a duplex, and an intervening sequence comprising the single-stranded nucleic acid to be delivered, as described in WO2021 / 058984, the contents of which are incorporated herein by reference in their entirety.

[0440] The present disclosure also contemplates that siRNA or miRNA, which can downregulate the intracellular levels of specific proteins through a process called RNA interference (RNAi), are nucleic acid therapeutics. After siRNA or miRNA is introduced into the cytoplasm of a host cell, these double-stranded RNA constructs can bind to a protein called RISC. The sense strand of siRNA or miRNA is removed by the RISC complex. When the RISC complex binds to complementary mRNA, it cleaves the mRNA and releases the cleaved strand. RNAi works by inducing specific destruction of mRNA, which leads to the downregulation of the corresponding protein.

[0441] Antisense oligonucleotides (ASOs) and ribozymes that inhibit mRNA translation into proteins can be used as nucleic acid therapeutics. In the case of antisense constructs, these single-stranded deoxyribonucleotides have sequences complementary to the target protein mRNA sequence and Watson bases that can bind to the mRNA by Crick base pairing. This binding blocks the translation of the target mRNA and / or induces RNase H degradation of the mRNA transcript. As a result, antisense oligonucleotides have increased specificity of action (i.e., downregulation of specific disease-related proteins).

[0442] In any of the aspects and embodiments provided herein, the therapeutic nucleic acid can be a therapeutic RNA. The therapeutic RNA can be a messenger RNA (mRNA) encoding a protein or peptide, an inhibitor of mRNA translation, an RNA interference agent (RNAi), a catalytically active RNA molecule (ribozyme), a transfer RNA (tRNA), an RNA that binds to an mRNA transcript (ASO), a protein or other molecular ligand (aptamer), or a guide RNA (gRNA). In any of the methods provided herein, the RNAi agent can be a double-stranded RNA, a single-stranded RNA, a microRNA, a short interfering RNA, a short hairpin RNA, or a triplex-forming oligonucleotide.

[0443] Closed-end DNA (ceDNA) vectors In some embodiments, the LNPs provided by the present disclosure comprise closed-ended DNA (ceDNA).

[0444] In some embodiments, the TNA comprises a closed-end linear double-stranded (ceDNA) vector capable of expressing a transgene (e.g., a therapeutic nucleic acid (TNA)). The ceDNA vectors disclosed herein do not have the packaging constraints imposed by the limited space within the viral capsid. In contrast to the enclosed AAV genome, ceDNA vectors represent a viable eukaryotic alternative to prokaryotically produced plasmid DNA vectors. This allows for the insertion of control elements, such as the regulatory switches disclosed herein, large transgenes, multiple transgenes, etc.

[0445] The ceDNA vector preferably has a linear, continuous structure rather than a discontinuous structure. A linear, continuous structure is believed to be more stable against attack by cellular endonucleases and less likely to undergo recombination, leading to mutagenesis. Therefore, a linear, continuous ceDNA vector is a preferred embodiment. A continuous, linear, single-stranded, intramolecular, double-stranded ceDNA vector can have covalently linked ends without the sequence encoding the AAV capsid protein. These ceDNA vectors are structurally different from plasmids (including the ceDNA plasmids described herein), which are circular, double-stranded nucleic acid molecules of bacterial origin. While the complementary strands of a plasmid can separate after denaturation to generate two nucleic acid molecules, in contrast, a ceDNA vector, while having complementary strands, is a single DNA molecule and therefore likely to remain a single molecule even when denatured. In some embodiments, ceDNA vectors, unlike plasmids, can be generated without prokaryotic-type DNA base methylation. Thus, ceDNA vectors and ceDNA-plasmids differ in terms of structure (specifically linear vs. circular), in terms of the methods used to generate and purify these different entities, and in terms of their DNA methylation, which is of prokaryotic type in the case of ceDNA-plasmids and of eukaryotic type in the case of ceDNA vectors.

[0446] Provided herein are capsid-free, non-viral ceDNA molecules (ceDNA) with covalently closed ends. These capsid-free, non-viral ceDNA molecules can be produced in permissive host cells from expression constructs (e.g., ceDNA plasmids, ceDNA-bacmids, ceDNA-baculoviruses, or integrative cell lines) containing a heterologous gene (e.g., a transgene, specifically a therapeutic transgene) positioned between two different inverted terminal repeat (ITR) sequences, where the ITRs differ from each other. In some embodiments, one of the ITRs is modified by deletion, insertion, and / or substitution compared to the wild-type ITR sequence (e.g., AAV ITR), and at least one of the ITRs contains a functional terminal separation site (trs) and a Rep binding site. The ceDNA vector is preferably double-stranded, e.g., self-complementary, over at least a portion of the molecule, such as an expression cassette (e.g., the ceDNA is not a double-stranded circular molecule). The ceDNA vectors have covalently closed ends and are therefore resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III), for example, at 37°C for 1 hour or more.

[0447] In one aspect, the ceDNA vector comprises, in the 5' to 3' direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette described herein), and a second AAV ITR. In one embodiment, the first ITR (5'ITR) and the second ITR (3'ITR) are asymmetric with respect to each other, i.e., they have different three-dimensional spatial arrangements. As an exemplary embodiment, the first ITR can be a wild-type ITR and the second ITR can be a mutated or modified ITR, or vice versa, in which case the first ITR can be a mutated or modified ITR and the second ITR can be a wild-type ITR. In one embodiment, the first ITR and the second ITR are both modified, but are of different sequences, or have different modifications, or are not the same modified ITR, but have different three-dimensional spatial arrangements. In other words, ceDNA vectors with asymmetric ITRs can have ITRs in which any changes in one ITR compared to the WT-ITR are not reflected in the other ITR, or, alternatively, the asymmetric ITRs can have different sequences and different three-dimensional shapes from each other if they have a modified asymmetric ITR pair.

[0448] In one embodiment, a ceDNA vector comprises, in the 5' to 3' direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette described herein), and a second AAV ITR, wherein the first ITR (5'ITR) and the second ITR (3'ITR) are symmetrical or substantially symmetrical to each other; i.e., the ceDNA vector can comprise ITR sequences with a symmetrical three-dimensional spatial arrangement, such that their structures have the same shape in geometric space or the same A, C-C', and B-B' loops in three-dimensional space. In such an embodiment, the symmetric or substantially symmetric ITR pair can be a modified ITR (e.g., a mod-ITR) that is not a wild-type ITR. A mod-ITR pair can have the same sequence with one or more modifications compared to the wild-type ITR and are reverse complements (inverted) of each other. In one embodiment, the modified ITR pair is substantially symmetrical as defined herein, i.e., the modified ITR pair can have different sequences but corresponding or identical symmetrical three-dimensional shapes. In some embodiments, the symmetrical or substantially symmetrical ITRs can be wild-type (WT-ITR) as described herein. That is, both ITRs have wild-type sequences derived from the same AAV serotype. In some other embodiments, the two wild-type ITRs can be derived from different AAV serotypes. For example, one WT-ITR can be derived from one AAV serotype, and the other WT-ITR can be derived from a different AAV serotype. In such embodiments, the WT-ITR pair is substantially symmetrical as defined herein, i.e., they can have one or more conservative nucleotide modifications while still retaining a symmetrical three-dimensional spatial configuration.

[0449] The wild-type, mutant, or otherwise modified ITR sequences provided herein represent DNA sequences contained in an expression construct (e.g., a ceDNA plasmid, a ceDNA bacmid, a ceDNA-baculovirus) for the generation of a ceDNA vector. Thus, the ITR sequences actually contained in a ceDNA vector generated from a ceDNA plasmid or other expression construct may or may not be identical to the ITR sequences provided herein as a result of naturally occurring variations (e.g., replication errors) that occur during the production process.

[0450] In one embodiment, the ceDNA vector in the LNP of the present disclosure, which contains an expression cassette with a transgene that is a therapeutic nucleic acid sequence, can be operably linked to one or more regulatory sequences that enable or control expression of the transgene. In one embodiment, the polynucleotide comprises a first ITR sequence and a second ITR sequence, and the nucleotide sequence of interest is flanked by the first ITR sequence and the second ITR sequence, and the first ITR sequence and the second ITR sequence are asymmetric or symmetric with respect to each other.

[0451] In one embodiment, the expression cassette is located between two ITRs and includes, in that order, one or more of a promoter operably linked to a transgene, a post-transcriptional regulatory element, and a polyadenylation and termination signal. In one embodiment, the promoter is regulatable, i.e., inducible or repressible. The promoter can be any sequence that facilitates transcription of the transgene. In one embodiment, the promoter is a CAG promoter or a variant thereof. The post-transcriptional regulatory element is a sequence that regulates the expression of the transgene, and, as a non-limiting example, is any sequence that creates a tertiary structure that enhances the expression of the transgene, which is a therapeutic nucleic acid sequence.

[0452] In one embodiment, the post-transcriptional regulatory element comprises a WPRE. In one embodiment, the polyadenylation and termination signal comprises a BGH polyA. Additionally, any cis-regulatory element known in the art, or combinations thereof, can be used, for example, the SV40 late polyA signal upstream enhancer sequence (USE) or other post-transcriptional processing elements (including, but not limited to, the thymidine kinase gene of herpes simplex virus or hepatitis B virus (HBV)). In one embodiment, the length of the expression cassette in the 5' to 3' direction exceeds the maximum length known to be encapsidated in AAV virions. In one embodiment, the length is greater than 4.6 kb, or greater than 5 kb, or greater than 6 kb, or greater than 7 kb. Various expression cassettes are exemplified herein.

[0453] In one embodiment, the expression cassette can comprise more than 4000 nucleotides, more than 5000 nucleotides, more than 10,000 nucleotides, or more than 20,000 nucleotides, or more than 30,000 nucleotides, or more than 40,000 nucleotides, or more than 50,000 nucleotides, or any range of about 4000-10,000 nucleotides, or 10,000-50,000 nucleotides, or more than 50,000 nucleotides. In some embodiments, the expression cassette can comprise a transgene that is a therapeutic nucleic acid sequence in the range of 500-50,000 nucleotides in length. In one embodiment, the expression cassette can comprise a transgene that is a therapeutic nucleic acid sequence in the range of 500-75,000 nucleotides in length. In one embodiment, the expression cassette can comprise a transgene that is a therapeutic nucleic acid sequence in the range of 500-10,000 nucleotides in length. In one embodiment, the expression cassette can comprise a transgene that is a therapeutic nucleic acid sequence in the range of 1000-10,000 nucleotides in length. In one embodiment, the expression cassette can contain a transgene, which is a therapeutic nucleic acid sequence ranging from 500 to 5,000 nucleotides in length. Because ceDNA vectors are not subject to the size limitations of encapsidated AAV vectors, they can deliver large expression cassettes into hosts. In one embodiment, the ceDNA vector lacks prokaryotic cell-specific methylation.

[0454] In one embodiment, the rigid therapeutic nucleic acid can be a plasmid.

[0455] In one embodiment, the ceDNA vectors disclosed herein are used for therapeutic purposes (e.g., medical, diagnostic, or veterinary uses) or immunogenic polypeptides.

[0456] The expression cassette can include any transgene that is a therapeutic nucleic acid sequence. In certain embodiments, the ceDNA vector includes any gene of interest in a subject, including a protein, an enzyme, one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, gRNAs, mRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen-binding fragments, or any combination thereof.

[0457] In one embodiment, the ceDNA expression cassette may include, for example, an expressible exogenous sequence (e.g., an open reading frame) encoding a protein that is either absent, inactive, or insufficiently active in the recipient subject, or a gene encoding a protein with a desired biological or therapeutic effect. In one embodiment, the exogenous sequence, such as the donor sequence, may encode a gene product that can function to correct the expression of a defective gene or transcript. In one embodiment, the expression cassette may also encode a corrective DNA strand, a polypeptide, a sense or antisense oligonucleotide, or a coding or non-coding RNA (e.g., siRNA, guide RNA, shRNA, microRNA, and their antisense counterparts (e.g., antagomir)). In one embodiment, the expression cassette may include an exogenous sequence encoding a reporter protein used for experimental or diagnostic purposes, such as β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others known in the art.

[0458] Thus, an expression cassette can include any gene that encodes a protein, polypeptide, or RNA that is reduced or absent due to a mutation, or that provides a therapeutic effect if overexpression is considered within the scope of this disclosure. A ceDNA vector can include a template or donor nucleotide sequence used as a corrective DNA strand to be inserted after a double-stranded break (or nick) provided by a nuclease. A ceDNA vector can include a template nucleotide sequence used as a corrective DNA strand to be inserted after a double-stranded break (or nick) provided by a guide RNA nuclease, meganuclease, or zinc finger nuclease.

[0459] single-stranded (ss) nucleic acid molecule In some embodiments, the TNAs included in the LNPs of the present disclosure can be single-stranded nucleic acids, such as single-stranded DNA or single-stranded RNA. In one embodiment, the TNAs can be single-stranded RNA, such as mRNA. In another embodiment, the TNAs can be single-stranded DNA (ssDNA) molecules, such as synthetic ssDNA molecules.

[0460] 3'-terminal stem-loop structure In some aspects, a TNA is a ssDNA molecule comprising at least one nucleic acid sequence of interest adjacent to at least one stem-loop structure at the 3' end. In some embodiments, the ssDNA molecule may further comprise at least one stem-loop structure at the 5' end. As described herein, the stem-loop structure at the 3' end may comprise a partial DNA duplex (e.g., with a free 3'-OH group) for priming replication or transcription. The partial DNA duplex functions, in part, to hold the stem-loop structure together.

[0461] According to some embodiments, the partial DNA duplex is 4 to 500 nucleotides, e.g., 4 to 10 nucleotides, 4 to 25 nucleotides, 4 to 50 nucleotides, 4 to 100 nucleotides, 4 to 200 nucleotides, 4 to 300 nucleotides, 4 to 400 nucleotides, 20 to 25 nucleotides, 20 to 50 nucleotides, 20 to 100 nucleotides, 20 to 200 nucleotides, 20 to 300 nucleotides, 20 to 400 nucleotides, 20 to 500 nucleotides, 50 to 100 nucleotides, 50 to 200 nucleotides, 50 to 300 nucleotides, 50 to 400 nucleotides. and 50 to 500 nucleotides, 150 to 200 nucleotides, 150 to 300 nucleotides, 150 to 400 nucleotides, 150 to 500 nucleotides, 200 to 300 nucleotides, 200 to 400 nucleotides, 200 to 500 nucleotides, 250 to 300 nucleotides, 250 to 400 nucleotides, 250 to 500 nucleotides, 300 to 400 nucleotides, 300 to 500 nucleotides, 350 to 400 nucleotides, 350 to 500 nucleotides, 400 to 500 nucleotides, or 450 to 500 nucleotides, and comprising at least one loop at the 3' end. According to some embodiments, the DNA duplex comprises at least 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nucleotides and comprises at least one loop at the 3' end.

[0462] According to some embodiments, the loop structure at the 3' end is at least 3 to 500 unlinked nucleotides, for example, 3 to 450 nucleotides, 3 to 400 nucleotides, 3 to 350 nucleotides, 3 to 300 nucleotides, 3 to 250 nucleotides, 3 to 200 nucleotides, 3 to 150 nucleotides, 3 to 100 nucleotides, 3 to 90 nucleotides, 3 to 80 nucleotides, 3 to 70 nucleotides, 3 to 60 nucleotides, 3 to 50 nucleotides, 3 to 40 nucleotides, 3 to 30 nucleotides, Nucleotide, 3-20 nucleotides, 3-10 nucleotides, 3-5 nucleotides, 10-450 nucleotides, 10-400 nucleotides, 10-350 nucleotides, 10-300 nucleotides, 10-250 nucleotides, 10-200 nucleotides, 10-150 nucleotides, 10-100 nucleotides, 10-90 nucleotides, 10-80 nucleotides, 10-70 nucleotides, 10-60 nucleotides, 10-50 nucleotides, 10-40 nucleotides , 10-30 nucleotides, 10-20 nucleotides, 50-450 nucleotides, 50-400 nucleotides, 50-350 nucleotides, 50-300 nucleotides, 50-250 nucleotides, 50-200 nucleotides, 50-150 nucleotides, 50-100 nucleotides, 50-90 nucleotides, 50-80 nucleotides, 50-70 nucleotides, 50-60 nucleotides, 100-450 nucleotides, 100-400 nucleotides, 100-350 Nucleotides, 100-300 nucleotides, 100-250 nucleotides, 100-200 nucleotides, 150-450 nucleotides, 150-400 nucleotides, 150-350 nucleotides, 150-300 nucleotides, 150-250 nucleotides, 150-200 nucleotides, 200-450 nucleotides, 200-400 nucleotides, 200-350 nucleotides, 200-300 nucleotides, 200-250 nucleotides, 250-450 nucleotides,It contains 250 to 400 nucleotides, 250 to 350 nucleotides, 250 to 300 nucleotides, 300 to 450 nucleotides, 300 to 400 nucleotides, 300 to 350 nucleotides, 350 to 450 nucleotides, 350 to 400 nucleotides, or 400 to 450 nucleotides.

[0463] In some embodiments, the stem portion of the stem-loop is 4 to 500 nucleotides in length, and the loop portion of the stem-loop is 3 to 500 nucleotides in length. In some embodiments, the stem portion of the stem-loop is 4 to 50 nucleotides in length, and the loop portion of the stem-loop is 3 to 50 nucleotides in length. In some embodiments, the stem portion of the stem-loop is 4 to 20 nucleotides in length, and the loop portion of the stem-loop is 3 to 20 nucleotides in length. In some embodiments, the stem portion of the stem-loop is 4 to 10 nucleotides in length, and the loop portion of the stem-loop is 3 to 10 nucleotides in length.

[0464] In some embodiments, the loop further comprises one or more nucleic acids or is used to stabilize the termini. In other embodiments, the loop further comprises one or more nucleic acids that may be used in therapeutic methods. In other embodiments, the loop further comprises one or more nucleic acids that may be used in diagnostic methods. In other embodiments, the loop further comprises one or more nucleic acids that may be used for research purposes.

[0465] According to some embodiments, the minimum nucleic acid structure required at the 3' end of the ssDNA is any structure that loops back on itself, i.e., a hairpin structure. However, it should be understood that various structures are contemplated at the 3' end, so long as there is at least one stem and one loop. For example, in some embodiments, the ssDNA described herein may contain at least one stem-loop structure at its 3' end. In some embodiments, the ssDNA may contain at least two stem-loop structures at its 3' end. In some embodiments, the ssDNA may contain at least three stem-loop structures at its 3' end. In some embodiments, the ssDNA may contain at least four stem-loop structures at its 3' end. In some embodiments, the ssDNA may contain at least five stem-loop structures at its 3' end.

[0466] In some embodiments, the 3'-terminal nucleotides form a cruciform DNA structure. A DNA cruciform structure can be formed when both strands form a stem-loop structure at the same position within the molecule and contain a four-way junction and two closed hairpin-shaped points.

[0467] In some embodiments, the 3'-terminal nucleotides form a hairpin DNA structure, where the hairpin loop structure in the nucleic acid consists of a base-paired stem structure and a loop sequence having unpaired or non-Watson-Crick paired nucleotides.

[0468] According to some embodiments, the 3' terminal nucleotides form a hammerhead DNA structure composed of three base-paired helices separated by short linkers of conserved sequence.

[0469] According to some embodiments, the 3'-terminal nucleotides form a quadruplex DNA structure. A G-quadruplex is a four-stranded DNA secondary structure (G4) formed from certain guanine-rich sequences.

[0470] According to some embodiments, the 3' terminal nucleotides form a bulge DNA structure.

[0471] According to some embodiments, the 3' terminal nucleotides form a multi-branched loop.

[0472] According to some embodiments, the 3' terminal nucleotides do not form two stem-loop structures.

[0473] According to some embodiments, the stem structure at the 3' end comprises one or more nucleotides modified to be exonuclease resistant. According to some embodiments, the stem structure at the 3' end comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or twenty or more nucleotides modified to be exonuclease resistant.

[0474] According to some embodiments, the stem structure at the 3'-end comprises one or more phosphorothioate-modified nucleotides. According to some embodiments, the stem structure at the 3'-end comprises about 2 to about 12 phosphorothioate-modified nucleotides. According to some embodiments, the stem structure at the 3'-end comprises about 4 to about 10, e.g., about 4 to about 5, about 4 to about 6, about 4 to about 7, about 4 to about 8, about 4 to about 9, about 4 to about 10, about 5 to about 6, about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 8 to about 9, about 8 to about 10, or about 9 to about 10 phosphorothioate-modified nucleotides. According to some embodiments, the stem structure comprises more than 10 phosphorothioate modified nucleotides.

[0475] According to some embodiments, the phosphorothioate modified nucleotides are located adjacent to one another.

[0476] According to some embodiments, one or more phosphorothioate-modified nucleotides at the 3'-end are resistant to exonuclease degradation. Boranophosphate-modified DNA is also resistant to nuclease degradation and can be considered an alternative to phosphorothioate modification.

[0477] According to a further embodiment, the stem structure may comprise at least one functional moiety. In one embodiment, the at least one functional moiety is an aptamer sequence. In a further embodiment, the aptamer sequence has a high binding affinity to a nuclear-localized protein.

[0478] According to some embodiments, the nucleotides in the loop are chemically modified with functional groups to alter their properties.

[0479] According to some embodiments, the loop further comprises one or more aptamers, according to some embodiments, the aptamers are identified from the publicly available Apta-index database of aptamers (aptagen.com / apta-index).

[0480] According to some embodiments, the loop further comprises one or more synthetic ribozymes.

[0481] According to some embodiments, the loop further comprises one or more antisense oligonucleotides (ASOs).

[0482] According to some embodiments, the loop further comprises one or more short interfering RNAs (siRNAs).

[0483] In ...

Claims

1. A polymer-conjugated lipid, (i) polyglycerol (PG) or a PG derivative; (ii) Formula (I) 【Chemical 119】 (I) (In the formula, R １ But absence, hydrogen, C １ -C ６ alkyl or a hydrophobic tail containing 10 to 30 carbon atoms; R ２ But absence, hydrogen, C １ -C ６ alkyl or a hydrophobic tail containing 10 to 30 carbon atoms; R １ and R ２ are hydrogen, C １ -C ６ N is positively charged when it is an alkyl or hydrophobic tail containing 10-30 carbon atoms; R ３ is a hydrophobic tail containing 10 to 30 carbon atoms, or a pharmaceutically acceptable salt thereof; (iii) a linker that conjugates the PG or the PG derivative to the lipid moiety, In formula (I), 【Chemical 120】 is a bond conjugating said lipid moiety and said linker.

2. The polymer-conjugated lipid of claim 1 , wherein the PG derivative is a carboxylated PG.

3. 3. The polymer-conjugated lipid of claim 2, wherein the carboxylated PG is glutarylated PG.

4. The polymer-conjugated lipid of claim 3, wherein the glutarylated PG is 3-methylglutarylated PG.

5. 3. The polymer-conjugated lipid of claim 2, wherein the carboxylated PG is 2-carboxycyclohexane-1-carboxylated PG.

6. The polymer-conjugated lipid according to any one of claims 1 to 5, wherein the PG or the PG derivative is linear or branched.

7. R １ is absent, and R ２ and R ３ is a hydrophobic tail containing 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon atoms.

8. R ２ and R ３ is a hydrophobic tail containing 16, 17, 18, 19, 20, 21, or 22 carbon atoms.

9. R ２ and R ３ is each independently a hydrophobic tail containing 18 carbon atoms, and the lipid moiety is dioctadecylamine (DODA).

10. The lipid moiety conjugated to a linker has the following structure: 【Chemistry 121】 10. The polymer-conjugated lipid of claim 9, represented by:

11. 11. The polymer-conjugated lipid of claim 1, wherein the PG or the PG derivative comprises about 5 to 100 monomer units.

12. 12. The polymer-conjugated lipid of claim 11, wherein the PG or PG derivative comprises about 5, 6, 7, 8, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 monomer units.

13. 13. The polymer-conjugated lipid of claim 12, wherein the PG or the PG derivative comprises about 8, 34, 45, 46, or 58 monomer units.

14. 14. The polymer-conjugated lipid of claim 13, wherein the PG or the PG derivative comprises about 8 monomer units.

15. 14. The polymer-conjugated lipid of claim 13, wherein the PG or the PG derivative comprises about 34 monomer units.

16. 14. The polymer-conjugated lipid of claim 13, wherein the PG or the PG derivative comprises about 45 monomer units.

17. 14. The polymer-conjugated lipid of claim 13, wherein the PG or the PG derivative comprises about 46 monomer units.

18. 14. The polymer-conjugated lipid of claim 13, wherein the PG or the PG derivative comprises about 58 monomer units.

19. 19. The polymer-conjugated lipid of any one of claims 1 to 18, wherein the linker is an alkyl linker, a glycerol linker, a phosphate linker, a phosphate ester linker, an ether linker, an ester linker, a diester linker, an amide linker, a diamide linker, an amine linker, a peptide linker, a phosphoethanolamine linker, a phosphocholine linker, a carbamate linker, a diamide alkyl linker, a cleavable linker, or any combination thereof.

20. The linker is -(CH ２ ) ｎ -, -C(O)(CH ２ ) ｎ -, -C(O)O(CH ２ ) ｎ , -OC(O)(CH ２ ) ｎ C(O)O- and -NH(CH ２ ) ｎ 20. The polymer-conjugated lipid of claim 19, wherein n is selected from the group consisting of: C(O)O—, wherein n is an integer ranging from 1 to 20.

21. 21. The polymer-conjugated lipid of claim 19 or 20, wherein the linker is a glutaryl linker or a succinyl linker.

22. The linker is —C(O)(CH ２ ) ｎ -, wherein n is 2, 3, 4, 5, or 6.

23. 23. The polymer-conjugated lipid of claim 22, wherein n is 4.

24. The following structure: 【Chemistry 122】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

25. The following structure: 【Chemical 123】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

26. The following structure: 【Chemistry 124】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

27. The following structure: 【Chemistry 125】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

28. The following structure: 【Chemistry 126】 (Wherein R is 【Chemistry 127-1】 or 【Chemistry 127-2】 a polymer-conjugated lipid represented by or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

29. 29. The polymer-conjugated lipid of any one of claims 1 to 28, further comprising a reactive species conjugated to said PG or said PG derivative, said reactive species being functionalized for conjugation to a targeting moiety.

30. 30. The polymer-conjugated lipid of claim 29, wherein the reactive species is a click chemistry reagent or a maleimide.

31. 31. The polymer-conjugated lipid of claim 30, wherein the click chemistry reagent is selected from the group consisting of a dibenzocyclooctyne (DBCO) reagent, a transcyclooctene (TCO) reagent, a tetrazine (Tz) reagent, an alkyne reagent, and an azide reagent.

32. 32. The polymer-conjugated lipid of any one of claims 29 to 31, further comprising a targeting moiety conjugated to said PG or said PG derivative via said reactive species.

33. 33. The polymer-conjugated lipid of claim 32, wherein the targeting moiety is conjugated to the PG or PG derivative via dibenzocyclooctyne (DBCO)-azide conjugation, azide-alkyne conjugation, TCO-Tz conjugation, or thiol-maleimide conjugation.

34. 34. The polymer-conjugated lipid of claim 32 or 33, wherein the targeting moiety is capable of binding to liver cells.

35. 35. The polymer-conjugated lipid of claim 34, wherein the liver cell is a hepatocyte.

36. 36. The polymer-conjugated lipid of claim 35, wherein the targeting moiety is N-acetylgalactosamine (GalNAc) or a GalNAc derivative.

37. 37. The polymer-conjugated lipid of claim 36, wherein the targeting moiety is a triantennary GalNAc conjugate or a tetraantennary GalNAc conjugate.

38. 34. The polymer-conjugated lipid of claim 32 or 33, wherein the targeting moiety is selected from the group consisting of an ApoE protein, an ApoE polypeptide, an ApoB protein, an ApoB polypeptide, and fragments or variants thereof.

39. 34. The polymer-conjugated lipid of claim 32 or 33, wherein the targeting moiety is an antibody or an antibody fragment, and the antibody or the antibody fragment is capable of specifically binding to an antigen present on the surface of a cell.

40. The antibody or antibody fragment may be a monoclonal antibody (mAb), a single chain variable fragment (scF ｖ ), heavy chain antibodies (hcAb), nanobodies (Nb), heavy chain-only immunoglobulins (HCIg), immunoglobulin neoantigen receptors (IgNAR), variable domains of immunoglobulin neoantigen receptors (V ＮＡＲ ), single domain antibodies, or antibodies with only variable heavy chains (V ＨＨ 40. The polymer-conjugated lipid of claim 39, wherein

41. A lipid nanoparticle (LNP), (i) a therapeutic nucleic acid (TNA); (ii) an ionizable lipid; and (iii) a sterol; and (iv) a first lipid-anchored polymer, wherein the first lipid-anchored polymer comprises the polymer-conjugated lipid of any one of claims 1 to 28.

42. 42. The LNP of claim 41, further comprising a helper lipid.

43. 43. The LNP of claim 42, wherein the helper lipid comprises a phospholipid or phosphatidylcholine (PC).

44. The helper lipid may be 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), hydrogenated soy PC (HSPC), phosphatidylserine (PS), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1-margalloyl-2-oleoyl-sn-glycero-3-phosphocholine (MOPC), 1-palmitoyl-2-linoleoyl- The LNP of claim 42 or 43, wherein the LNP is selected from the group consisting of sn-glycero-3-phosphocholine (PLPC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

45. The LNP of any one of claims 42 to 44, wherein the helper lipid is DSPC.

46. The helper lipid has the formula (II): 【Chemistry 128】 (II) (In the formula, 【Chemistry 129】 is a single bond or a double bond, R １ But C １ -C １７ Alkyl or C ２ -C １７ is alkenyl, R ２ But C １ -C ２２ Alkyl or C ２ -C ２２ is alkenyl, R ３ is hydrogen or C １ -C ２ is alkyl, R ４ is hydrogen or C １ -C ２ 43. The LNP of claim 42, wherein R is an integer from 1 to 10, and R is an integer from 1 to 10. 【Request Item 47】 【Chemistry 130】 is a double bond.

48. R １ is C １０ -C ２０ alkenyl, and R ２ is C １０ -C ２０ alkyl, and R ３ The LNP of claim 46 or 47, wherein is hydrogen.

49. The helper lipid represented by formula (II) 【Chemistry 131】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

50. The helper lipid represented by formula (II) 【Chemistry 132】 、 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

51. The helper lipid represented by formula (II) 【Chemistry 133】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

52. The helper lipid represented by formula (II) 【Chemistry 134】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

53. The helper lipid represented by formula (II) 【Chemistry 135】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

54. The helper lipid represented by formula (II) 【Transformation 136】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

55. 55. The LNP of any one of claims 41 to 54, wherein the sterol is selected from the group consisting of cholesterol, beta-sitosterol, stigmasterol, beta-sitostanol, campesterol, brassicasterol, and derivatives thereof.

56. 56. The LNP of claim 55, wherein the sterol is cholesterol.

57. The ionizable lipid is a) Formula (A): 【Chemistry 137】 Formula (A) (In the formula, R １ and R １’ each independently represents an optionally substituted linear or branched C １－３ is alkylene, R ２ and R ２’ each independently represents an optionally substituted linear or branched C １－６ is alkylene, R ３ and R ３’ each independently represents an optionally substituted linear or branched C １－６ Is it alkyl? Or, R ２ optionally substituted branched C １－６ When R is alkylene, ２ and R ３ together with the intervening N atom form a 4- to 8-membered heterocyclyl; or Or, R ２’ optionally substituted branched C １－６ When R is alkylene, ２’ and R ３’ together with the intervening N atom form a 4- to 8-membered heterocyclyl; R ４ and R ４’ each independently represents -CR ａ , -C(R ａ ) ２ CR ａ , or -[C(R ａ ) ２ ] ２ CR ａ and R ａ is, for each occurrence, independently H or C １－３ Is it alkyl? Or, R ４ -C(R ａ ) ２ CR ａ Or -[C(R ａ ) ２ ] ２ CR ａ and R ａ is C １－３ When R is alkyl, ３ and R ４ together with the intervening N atom form a 4- to 8-membered heterocyclyl; or Or, R ４’ -C(R ａ ) ２ CR ａ Or -[C(R ａ ) ２ ] ２ CR ａ and R ａ is C １－３ When R is alkyl, ３’ and R ４’ together with the intervening N atom form a 4- to 8-membered heterocyclyl; R ５ and R ５’ are each independently hydrogen, C １－２０ Alkylene or C ２－２０ is alkenylene, R ６ and R ６’ But for each occurrence, independently, C １－２０ Alkylene, C ３－２０ Cycloalkylene, or C ２－２０ is alkenylene, m and n are each independently an integer selected from 1, 2, 3, 4, and 5, or a pharmaceutically acceptable salt thereof; b) Formula (B): 【Chemistry 138】 Formula (B) (In the formula, a is an integer ranging from 1 to 20; b is an integer ranging from 2 to 10; R １ is absent, or (C ２ -C ２０ ) alkenyl, —C(O)O(C ２ -C ２０ ) alkyl, and (C ２ -C ２０ ) cyclopropyl substituted with alkyl; R ２ However, (C ２ -C ２０ ) alkyl) or a pharmaceutically acceptable salt thereof; or c) Formula (C): 【Chemistry 139】 Formula (C) (In the formula, R １ and R １’ are each independently R ａ (C １ -C ６ ) alkylene; R ２ and R ２’ are each independently １ -C ２ ) alkylene; R ３ and R ３’ are each independently R ｂ (C １ -C ６ ) alkyl or Or, R ２ and R ３ and / or R ２’ and R ３’ together with the intervening N atom form a 4- to 7-membered heterocyclyl; R ４ and R ４ ' are each interrupted by -C(O)O- (C ２ -C ６ ) alkylene; R ５ and R ５ Each ' is independently -C(O)O- or (C ３ -C ６ ) each optionally interrupted by cycloalkyl, (C ２ -C ３０ ) alkyl or (C ２ -C ３０ ) alkenyl, R ａ and R ｂ are each halo or cyano, or a pharmaceutically acceptable salt thereof; d) Formula (D): [Chemical 140] Formula (D) (In the formula, R' is absent, hydrogen, or C １ -C ６ alkyl, provided that R' is hydrogen or C １ -C ６ When R′, R １ , and R ２ are all positively charged nitrogen atoms, R １ and R ２ are each independently hydrogen, C １ -C ６ Alkyl, or C ２ -C ６ is alkenyl, R ３ But C １ -C １２ Alkylene or C ２ -C １２ is alkenylene, R ４ But C １ -C １８ Unbranched alkyl, C ２ -C １８ unbranched alkenyl, or 【Chemistry 141】 where: R ４ａ and R ４ｂ are each independently C １ -C １６ Unbranched alkyl or C ２ -C １６ is an unbranched alkenyl; R ５ But, absent, C １ -C ８ Alkylene or C ２ -C ８ is alkenylene, R ６ａ and R ６ｂ are each independently C ７ -C １６ Alkyl or C ７ -C １６ alkenyl, provided that R ６ａ and R ６ｂ provided that the total number of carbon atoms in X １ and X ２ each independently represents -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-、-S-S-、-C(R ａ )=N-、-N=C(R ａ )-、-C(R ａ )=NO-、-O-N=C(R ａ )-、-C(=O)NR ａ -、 -NR ａ C(=O)-, -NR ａ C(=O)NR ａ -, -OC(=O)O-, -OSi(R ａ ) ２ O-, -C(=O)(CR ａ ２ ) C(=O)O-, or OC(=O)(CR ａ ２ )C(=O)-, wherein R ａ is, at each occurrence, independently hydrogen or C １ -C ６ is alkyl, n is an integer selected from 1, 2, 3, 4, 5, and 6, or a pharmaceutically acceptable salt thereof; e) Formula (E): 【Chemistry 142】 Formula (E) (In the formula, R' is absent, hydrogen, or C １ -C ３ alkyl, provided that R' is hydrogen or C １ -C ３ When R′, R １ , and R ２ provided that all nitrogen atoms to which they are attached are positively charged, R １ and R ２ are each independently hydrogen or C １ -C ３ is alkyl, R ３ But C ３ -C １０ Alkylene or C ３ -C １０ is alkenylene, R ４ But C １ -C １６ Unbranched alkyl, C ２ -C １６ unbranched alkenyl, or 【Chemistry 143】 where: R ４ａ and R ４ｂ are each independently C １ -C １６ Unbranched alkyl or C ２ -C １６ is an unbranched alkenyl; R ５ But, absent, C １ -C ６ Alkylene or C ２ -C ６ is alkenylene, R ６ａ and R ６ｂ are each independently C ７ -C １４ Alkyl or C ７ -C １４ is alkenyl, Xが、-OC(=O)-、-SC(=O)-、-OC(=S)-、-C(=O)O-、-C(=O)S-、-S-S-、-C(R ａ )=N-、 -N=C(R ａ )-、-C(R ａ )=NO-、-O-N=C(R ａ )-、-C(=O)NR ａ -、-NR ａ C(=O)-、-NR ａ C(=O)NR ａ -、 -OC(=O)O-, -OSi(R ａ ) ２ O-, -C(=O)(CR ａ ) ２ C(=O)O- or OC(=O)(CR ａ ２ )C(=O)-, wherein R ａ is, at each occurrence, independently hydrogen or C １ -C ６ is alkyl, n is an integer selected from 1, 2, 3, 4, 5, and 6, or a pharmaceutically acceptable salt thereof; f) The LNP of any one of claims 41 to 56, wherein the lipid is an ionizable lipid selected from the group consisting of any of the ionizable lipids in Table 1, Table 4, Table 5, Table 6, or Table 7.

58. The ionizable lipid has the following structure: 【Chemistry 144】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

59. The ionizable lipid has the following structure: 【Chemistry 145】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

60. The ionizable lipid has the following structure: 【Chemistry 146】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

61. The ionizable lipid has the following structure: 【Chemistry 147】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

62. The ionizable lipid has the following structure: 【Chemistry 148】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

63. The ionizable lipid has the following structure: 【Chemistry 149】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

64. The ionizable lipid has the following structure: [Chemical 150] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

65. The ionizable lipid has the following structure: 【Chemistry 151】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

66. The ionizable lipid has the following structure: 【Chemistry 152】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

67. The ionizable lipid has the following structure: 【Chemistry 153】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

68. The ionizable lipid has the following structure: 【Chemistry 154】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

69. The ionizable lipid has the following structure: 【Chemistry 155】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

70. The ionizable lipid has the following structure: 【Chemistry 156】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

71. The ionizable lipid has the following structure: 【Chemistry 157】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

72. The ionizable lipid has the following structure: 【Chemistry 158】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

73. The ionizable lipid has the following structure: 【Chemistry 159】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

74. The LNP further comprises a second lipid-anchored polymer, the second lipid-anchored polymer comprising: (i) a lipid moiety comprising at least one hydrophobic tail; (ii) a polymer; and (iii) a linker, wherein the polymer is conjugated to the lipid moiety via the linker; and (iv) a reactive species conjugated to the polymer, wherein the reactive species is functionalized to be conjugated to a targeting moiety.

75. 75. The LNP of claim 74, wherein the polymer of the second lipid-anchored polymer is selected from the group consisting of polyethylene (PE), polypropylene (PP), polyethylene glycol (PEG), polyvinyl alcohol (PVOH), polysarcosine (pSar), poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), polyglycerol (PG), and derivatives of any of the foregoing.

76. 76. The LNP of claim 75, wherein the PG derivative is a carboxylated PG.

77. The LNP of claim 76, wherein the carboxylated PG is glutarylated PG or 2-carboxycyclohexane-1-carboxylated PG.

78. The LNP of claim 77, wherein the glutarylated PG is 3-methylglutarylated PG.

79. The LNP according to any one of claims 75 to 78, wherein the PG or the PG derivative is linear or branched.

80. The LNP of any one of claims 74 to 79, wherein the reactive species is a click chemistry reagent or a maleimide.

81. 81. The LNP of claim 80, wherein the click chemistry reagent is selected from the group consisting of a dibenzocyclooctyne (DBCO) reagent, a transcyclooctene (TCO) reagent, a tetrazine (Tz) reagent, an alkyne reagent, and an azide reagent.

82. The LNP of any one of claims 74-81, further comprising a targeting moiety conjugated to the polymer via the reactive species.

83. 83. The LNP of claim 82, wherein the targeting moiety is conjugated to the polymer via dibenzocyclooctyne (DBCO)-azide conjugation, azide-alkyne conjugation, TCO-Tz conjugation, or thiol-maleimide conjugation.

84. 84. The LNP of claim 82 or 83, wherein the targeting moiety is capable of binding to liver cells.

85. 85. The LNP of claim 84, wherein the liver cell is a hepatocyte.

86. The LNP of claim 84 or 85, wherein the targeting moiety is N-acetylgalactosamine (GalNAc) or a GalNAc derivative.

87. 87. The LNP of claim 86, wherein the targeting moiety is a triantennary GalNAc conjugate or a tetraantennary GalNAc conjugate.

88. 84. The LNP of claim 82 or 83, wherein the targeting moiety is selected from the group consisting of an ApoE protein, an ApoE polypeptide, an ApoB protein, an ApoB polypeptide, and fragments or variants thereof.

89. 84. The LNP of claim 82 or 83, wherein the targeting moiety is an antibody or an antibody fragment, and the antibody or antibody fragment is capable of specifically binding to an antigen present on the surface of a cell.

90. The antibody or antibody fragment may be a monoclonal antibody (mAb), a single chain variable fragment (scF ｖ ), heavy chain antibodies (hcAb), nanobodies (Nb), heavy chain-only immunoglobulins (HCIg), immunoglobulin neoantigen receptors (IgNAR), variable domains of immunoglobulin neoantigen receptors (V ＮＡＲ ), single domain antibodies, or antibodies with only variable heavy chains (V ＨＨ ) The LNP of claim 89.

91. The LNP of any one of claims 74 to 90, wherein the linker is selected from the group consisting of an alkyl linker, a glycerol linker, a phosphate linker, a phosphate ester linker, an ether linker, an ester linker, a diester linker, an amide linker, a diamide linker, an amine linker, a peptide linker, a phosphoethanolamine linker, a phosphocholine linker, a carbamate linker, a diamide alkyl linker, a cleavable linker, and any combination thereof.

92. The linker is -(CH ２ ) ｎ -, -C(O)(CH ２ ) ｎ -, -C(O)O(CH ２ ) ｎ -, -OC(O)(CH ２ ) ｎ C(O)O- and -NH(CH ２ ) ｎ 92. The LNP of claim 91, wherein n is selected from the group consisting of: C(O)O—, wherein n is an integer ranging from 1 to 20.

93. The LNP of claim 91 or 92, wherein the linker is a glutaryl linker or a succinyl linker.

94. The linker is —C(O)(CH ２ ) ｎ -, wherein n is 2, 3, 4, 5, or 6.

95. 95. The LNP of claim 94, wherein n is 4.

96. The lipid portion of the second lipid-anchored polymer has the formula (I): [Chemical 160] (I) (In the formula, R １ But absence, hydrogen, C １ -C ６ alkyl or a hydrophobic tail containing 10 to 30 carbon atoms; R ２ But absence, hydrogen, C １ -C ６ alkyl or a hydrophobic tail containing 10 to 30 carbon atoms; R １ and R ２ are hydrogen, C １ -C ６ N is positively charged when it is an alkyl or hydrophobic tail containing 10-30 carbon atoms; R ３ is a hydrophobic tail containing 10 to 30 carbon atoms), or a pharmaceutically acceptable salt thereof.

97. R １ is absent, and R ２ and R ３ is each independently a hydrophobic tail containing 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon atoms.

98. R ２ and R ３ is each independently a hydrophobic tail containing 16, 17, 18, 19, 20, 21, or 22 carbon atoms.

99. R ２ and R ３ is each independently a hydrophobic tail containing 18 carbon atoms, and the lipid moiety is dioctadecylamine (DODA).

100. The lipid moiety of the second lipid-anchored polymer may be selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielideyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl-2-oleo ...-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1-stearoyl-2-oleoyl-sn-glycero- 100. The LNP of any one of claims 74-99, comprising a moiety selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (SOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 18-1-trans PE, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), dioctadecylamine (DODA), dihexadecylamine, distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and derivatives thereof.

101. 101. The LNP of claim 100, wherein the lipid portion of the second lipid-anchored polymer comprises a moiety selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, and derivatives of any of the foregoing.

102. The LNP of claim 101, wherein the lipid portion of the second lipid-anchored polymer comprises DSPE.

103. The LNP of any one of claims 74 to 102, wherein the polymer of the second lipid-anchored polymer has an average molecular weight of about 500 Da to about 5000 Da.

104. The LNP of claim 103, wherein the polymer has an average molecular weight of about 1500 Da to about 5000 Da.

105. The LNP of claim 104, wherein the polymer has an average molecular weight of about 2000 Da.

106. The second lipid-anchored polymer has the following structure: 【Chemistry 161】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

107. The second lipid-anchored polymer has the following structure: 【Chemistry 162】 or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

108. The LNP of any one of claims 41 to 107, wherein the ionizable lipid is present in the LNP in an amount of about 20 mol% to about 60 mol% of the total lipid present in the LNP.

109. The LNP of claim 108, wherein the ionizable lipid is present in the LNP in an amount of about 35 mol% to about 50 mol% of the total lipid present in the LNP.

110. The LNP of any one of claims 41 to 109, wherein the sterol is present in the LNP in an amount of about 20 mol% to about 50 mol% of the total lipids present in the LNP.

111. The LNP of claim 110, wherein the sterol is present in the LNP in an amount of about 30 mol% to about 45 mol% of the total lipids present in the LNP.

112. The LNP of any one of claims 42 to 111, wherein the helper lipid is present in the LNP in an amount of about 1 mol% to about 40 mol% of the total lipid present in the LNP.

113. The LNP of claim 112, wherein the helper lipid is present in the LNP in an amount of about 5 mol% to about 15 mol% of the total lipid present in the LNP.

114. The LNP of any one of claims 41 to 113, wherein the first lipid-anchored polymer is present in the LNP in an amount of from about 0.5 mol% to about 5 mol% of the total lipid present in the LNP.

115. The LNP of claim 114, wherein the first lipid-anchored polymer is present in the LNP in an amount of about 1.5 mol% to about 3 mol% of the total lipid present in the LNP.

116. The LNP of any one of claims 41 to 115, wherein the second lipid-anchored polymer is present in the LNP in an amount of from about 0.05 mol% to about 5 mol% of the total lipid present in the LNP.

117. The LNP of claim 116, wherein the second lipid-anchored polymer is present in the LNP in an amount of about 0.1 mol% to about 1 mol% of the total lipid present in the LNP.

118. A lipid nanoparticle (LNP), (i) a therapeutic nucleic acid (TNA); (ii) an ionizable lipid, wherein the ionizable lipid has the following structure: 【Chemistry 163】 an ionizable lipid which is heptadecan-9-yl 9-((4-(dimethylamino)butanoyl)oxy)hexadecanoate having the formula: (iii) a sterol, wherein the sterol is cholesterol; (iv) a helper lipid, wherein the helper lipid is DSPC; and (v) a first lipid-anchored polymer, said first lipid-anchored polymer comprising DODA conjugated to a linear PG via a linker; (vi) a second lipid-anchored polymer, wherein the second lipid-anchored polymer comprises DSPE conjugated to PEG.

119. The LNP of claim 118, wherein the liner PG comprises about 30 to 60 monomer units.

120. 120. The LNP of claim 119, wherein the linear PG comprises about 34 monomer units or about 45 monomer units.

121. The LNP of any one of claims 118 to 120, wherein the PEG has an average molecular weight of about 1000 Da to about 5000 Da.

122. The LNP of claim 121, wherein the PEG has an average molecular weight of 2000 Da.

123. the ionizable lipid is present in the LNP in an amount of about 35 mol% to about 50 mol% of the total lipid present in the LNP; the sterol is present in the LNP in an amount of about 30 mol% to about 45 mol% of the total lipid present in the LNP; the helper lipid is present in the LNP in an amount of about 5 mol% to about 15 mol% of the total lipid present in the LNP; the first lipid-anchored polymer is present in the LNP in an amount of about 1.5 mol % to about 3 mol % of the total lipid present in the LNP; and The LNP of claim 118, wherein the second lipid-anchored polymer is present in the LNP in an amount of about 0.1 mol% to about 1 mol% of the total lipid present in the LNP.

124. The LNP of any one of claims 41 to 123, wherein the TNA is selected from the group consisting of a minigene, a plasmid, a minicircle, a small interfering RNA (siRNA), a microRNA (miRNA), a guide RNA (gRNA), an antisense oligonucleotide (ASO), a ribozyme, a deoxyribozyme, a closed-end DNA (ceDNA), a ssDNA, a ministring, doggybone™, a protelomeric closed-end DNA, a dumbbell-shaped linear DNA, a Dicer substrate dsRNA, a small hairpin RNA (shRNA), an asymmetric interfering RNA (aiRNA), an mRNA, a tRNA, a rRNA, a gRNA, a DNA viral vector, a viral RNA vector, a non-viral vector, and a combination thereof.

125. The LNP of claim 124, wherein the TNA is ceDNA.

126. The LNP of any one of claims 118 to 125, wherein the TNA is a single-stranded nucleic acid or a double-stranded nucleic acid.

127. The LNP of claim 126, wherein the single-stranded nucleic acid is mRNA.

128. The LNP of claim 126, wherein the single-stranded nucleic acid is a DNA molecule (ssDNA).

129. The LNP of claim 128, wherein the ssDNA is a linear ssDNA comprising at least one nucleic acid sequence of interest adjacent to at least one stem-loop structure at the 3' end.

130. 130. The LNP of claim 129, wherein said at least one stem-loop structure at the 3' end is sufficient to prime replication and / or transcription.

131. The LNP of claim 129 or 130, wherein the stem structure at the 3' end comprises a partial DNA duplex of 4 to 500 nucleotides.

132. The LNP of claim 131, wherein the stem structure at the 3' end comprises a partial DNA duplex of 4 to 50 nucleotides.

133. The LNP of any one of claims 129 to 132, wherein the loop structure at the 3' end comprises 3 to 500 unlinked nucleotides.

134. The LNP of any one of claims 129 to 132, wherein the loop structure at the 3' end comprises at least three unlinked nucleotides.

135. The LNP of any one of claims 128 to 134, wherein the ssDNA comprises at least two stem-loop structures at the 3' end.

136. The LNP of any one of claims 128 to 134, wherein the ssDNA comprises at least three stem-loop structures at the 3' end.

137. The LNP of any one of claims 128 to 134, wherein the ssDNA comprises at least four or more stem-loop structures at the 3' end.

138. The LNP of any one of claims 129 to 137, wherein the at least one stem-loop structure at the 3' end comprises a hairpin DNA structure.

139. The LNP of any one of claims 129 to 138, wherein the at least one stem-loop structure at the 3' end comprises a DNA structure selected from the group consisting of a cruciform DNA structure, a hammerhead DNA structure, a quadruplex DNA structure, a bulge DNA structure, and a multi-branched loop structure.

140. The LNP of any one of claims 129 to 139, wherein the at least one stem-loop structure at the 3' end does not include the A region, the A' region, the D region, and the D' region that would be present in a wild-type AAV ITR.

141. The LNP of any one of claims 129 to 139, wherein the at least one stem-loop structure at the 3' end does not include regions A, A', B, B', C, C', D, and D' that would be present in a wild-type AAV ITR.

142. The LNP of any one of claims 129 to 141, wherein the at least one stem-loop structure at the 3' end does not contain a rep binding element (RBE) that would be present in a wild-type ITR.

143. The LNP of any one of claims 129 to 142, wherein the at least one stem-loop structure at the 3' end does not contain a terminal resolution site (trs) that would be present in a wild-type ITR.

144. The LNP of any one of claims 129 to 142, wherein the stem structure at the 3' end comprises four or more nucleotides modified to be exonuclease resistant.

145. The LNP of claim 144, wherein the nucleotide is a phosphorothioate-modified nucleotide.

146. The LNP of any one of claims 129 to 145, wherein at least one stem-loop structure at the 3' end further comprises a functional moiety.

147. The LNP of any one of claims 129 to 146, wherein the ssDNA molecule further comprises a 5' end comprising at least one stem-loop structure.

148. The LNP of claim 147, wherein the ssDNA comprises at least two stem-loop structures at the 5' end.

149. The LNP of claim 147 or 148, wherein the ssDNA comprises at least three stem-loop structures at the 5' end.

150. The LNP of any one of claims 147 to 149, wherein the ssDNA comprises at least four or more stem-loop structures at the 5' end.

151. The LNP of any one of claims 147 to 150, wherein the at least one stem-loop structure at the 5' end comprises a hairpin DNA structure.

152. The LNP of any one of claims 147 to 151, wherein the at least one stem-loop structure at the 5' end comprises a DNA structure selected from the group consisting of a cruciform DNA structure, a hammerhead DNA structure, a quadruplex DNA structure, a bulge DNA structure, and a multi-branched loop structure.

153. The LNP of any one of claims 147 to 152, wherein the at least one stem-loop structure at the 5' end does not include the A region, the A' region, the D region, and the D' region that would be present in a wild-type AAV ITR.

154. The LNP of any one of claims 147 to 152, wherein the at least one stem-loop structure at the 5' end does not include regions A, A', B, B', C, C', D, and D' that would be present in a wild-type AAV ITR.

155. The LNP of any one of claims 147 to 154, wherein the at least one stem-loop structure at the 5' end does not contain a rep binding element (RBE) that would be present in a wild-type ITR.

156. The LNP of any one of claims 147 to 155, wherein the at least one stem-loop structure at the 5' end does not contain a terminal resolution site (trs) that would be present in a wild-type ITR.

157. The LNP of any one of claims 147 to 156, wherein the stem structure at the 5' end comprises four or more nucleotides modified to be exonuclease resistant.

158. The LNP of claim 157, wherein the nucleotide is a phosphorothioate-modified nucleotide.

159. The LNP of any one of claims 147 to 158, wherein the loop structure at the 5' end contains one or more nucleic acids to stabilize the end.

160. The LNP of any one of claims 147 to 159, wherein the loop structure at the 5' end further comprises one or more chemically modified nucleic acids.

161. The LNP of any one of claims 147 to 160, wherein the loop structure at the 5' end further comprises one or more aptamers.

162. The LNP of any one of claims 147 to 161, wherein the loop structure at the 5' end further comprises one or more synthetic ribozymes.

163. The LNP of any one of claims 147 to 162, wherein the loop structure at the 5' end further comprises one or more antisense oligonucleotides (ASO).

164. The LNP of any one of claims 147 to 163, wherein the loop structure at the 5' end further comprises one or more short interfering RNAs (siRNAs).

165. The LNP of any one of claims 147 to 164, wherein the loop structure at the 5' end further comprises one or more antiviral nucleoside analogs (ANA).

166. The LNP of any one of claims 147 to 165, wherein the loop structure at the 5' end further comprises one or more triplex-forming oligonucleotides.

167. The LNP of any one of claims 147 to 166, wherein the loop structure at the 5' end further comprises one or more gRNAs or gDNAs.

168. The LNP of any one of claims 147 to 167, wherein the loop structure at the 5' end further comprises one or more molecular probes.

169. The LNP of any one of claims 128-168, wherein the ssDNA molecule lacks any viral capsid protein coding sequence.

170. The LNP of any one of claims 128 to 169, wherein the ssDNA molecule is synthetically produced in vitro.

171. The LNP of claim 170, wherein the ssDNA molecule is synthetically produced in vitro in a cell-free environment.

172. The LNP of any one of claims 128 to 171, wherein the ssDNA molecule does not activate or only minimally activates immune pathways.

173. The LNP of claim 172, wherein the immune pathway is an innate immune pathway.

174. The LNP of claim 173, wherein the innate immune pathway is selected from the group consisting of the cGAS / STING pathway, the TLR9 pathway, the inflammasome-mediated pathway, and combinations thereof.

175. The LNP of any one of claims 128 to 174, wherein the ssDNA molecule is capable of expressing at least one therapeutic protein or therapeutic fragment thereof.

176. The LNP of claim 175, wherein the at least one therapeutic protein is selected from the group consisting of an antibody, an enzyme, a clotting factor, a transcription factor, a replication factor, a growth factor, a hormone, and a fusion protein.

177. The at least one therapeutic protein is effective in treating melanoma, hemophilia A (clotting factor VIII (FVIII) deficiency) and hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited liver metabolic disorders, Lesch-Nyhan syndrome, sickle cell disease, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharidosis (e.g., Hurler syndrome (MPS I), Scheie syndrome (MPS I S), Hurler-Scheie syndrome (MPS I H-S), Hunter syndrome (MPS II), Sanfilippo syndromes A, B, C, and D (MPS III MPS A, B, C, and D), Morquio syndrome types A and B (MPS IVA and MPS IVB), Maroteaux-Lamy syndrome (MPS VI), Sly syndrome (MPS VII), hyaluronidase deficiency (MPSIX), Niemann-Pick disease types A / B, C1, and C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandhoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis types I, II / III, and IV, sialidosis types I and II, glycogen storage disease types I and II (Pompe disease), Gaucher disease types I and II, and III, Fabry disease, cystinosis, Batten disease, aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidoses, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Haemophilus influenzae type 2 (HA), and leukemia. The LNP of claim 175 or 176 is useful for treating a genetic disorder selected from the group consisting of Chinton's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, infantile generalized arterial calcification (GACI), Leber's congenital amaurosis, Stargardt's macular dystrophy (ABCA4), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC) type I (ATP8B1 deficiency), type II (ABCB11), type III (ABCB4), or type IV (TJP2), and cathepsin A deficiency.

178. A pharmaceutical composition comprising the LNP of any one of claims 41 to 177 and a pharmaceutically acceptable carrier.

179. A method for treating a genetic disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the LNP of any one of claims 41 to 177 or the pharmaceutical composition of claim 178. The method.

180. 179. The method of claim 179, wherein the subject is a human.

181. The genetic disorder is sickle cell anemia, melanoma, hemophilia A (clotting factor VIII (FVIII) deficiency), hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited liver metabolic disorders, Lesch-Nyhan syndrome, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa , ataxia-telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharidosis, Niemann-Pick disease, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandhoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis (ML), sialidosis type II, glycogen storage disease (GSD), Gaucher disease, cystinosis, Batten disease, aspartylglucosaminuria, Salla disease, Danon disease (L AMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (NCL), sphingolipidosis, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy (SMA), Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa ( 181. The method of claim 179 or 180, wherein the inflammatory bowel disease is selected from the group consisting of: DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber's congenital amaurosis, Stargardt's disease, wet macular degeneration (wet AMD), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC), and cathepsin A deficiency.

182. A method for providing anti-tumor immunity to a subject in need thereof, the method comprising administering to the subject an effective amount of the LNP of any one of claims 41 to 177 or the pharmaceutical composition of claim 178.

183. A method of treating a subject having a disease, disorder, or condition associated with elevated expression of a tumor antigen, the method comprising administering to the subject an effective amount of the LNP of any one of claims 41 to 177 or the pharmaceutical composition of claim 178.

184. A method of treating a blood disease, disorder, or condition in a subject in need thereof, said method comprising administering to said subject an effective amount of the LNP of any one of claims 41 to 177 or the pharmaceutical composition of claim 178.

185. 29. A method for synthesizing a polymer-conjugated lipid according to any one of claims 1 to 28, comprising: a) reacting a lipid moiety to be conjugated to a linker with 2,3-epoxy-1-(1-ethoxyethoxy)propane (EEGE) in the presence of a base or an organic catalyst under an argon atmosphere to produce a lipid moiety conjugated to a linker and polymerized EEGE; b) subjecting the lipid moiety conjugated to a linker and the polymerized EEGE to acidic conditions to produce the polymer-conjugated lipid.

186. 186. The method of claim 185, wherein the base is a phosphazene base.

187. 187. The method of claim 186, wherein the phosphazene base is P4-t-Bu.

188. 186. The method of claim 185, wherein the organocatalyst is an N-heterocyclic carbene (NHC) or an N-heterocyclic olefin (NHO).

189. The acidic conditions include HCl, Br, HI, HClO ４ , HClO ３ , H ２ SO ４ , or HNO ３ 188. The method of any one of claims 185 to 187, comprising:

190. 190. The method of any one of claims 185-189, wherein the lipid moiety comprises DODA.

191. The lipid moiety conjugated to a linker has the following structure: 【Chemistry 164】 The method of any one of claims 185 to 190, represented by:

192. The polymer-conjugated lipid has the following structure: 【Chemistry 165】 191. The method of any one of claims 185 to 190, wherein n is a number in the range of 10 to 100.

193. 193. The method of claim 192, wherein n is about 34, 45, 46, or 58.

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