Novel polyglycerol conjugated lipids and lipid nanoparticle compositions comprising same

Abstract

The present disclosure provides novel polymer-conjugated lipids, for example, polymer-conjugated lipids comprising DODA conjugated to a polyglycerol or a polyglycerol derivative. The present disclosure also provides lipid nanoparticle (LNP) formulations using the polymer conjugated lipids and methods of treating disease by administering the LNP formulations.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 429,267, filed on December 1, 2022; U.S. Provisional Application No. 63 / 429,226, filed on December 1, 2022; U.S. Provisional Application No. 63 / 449,610, filed on March 3, 2023; U.S. Provisional Application No. 63 / 449,617, filed on March 3, 2023; U.S. Provisional Application No. 63 / 452,077, filed on March 14, 2023; U.S. Provisional Application No. 63 / 467,045, filed on May 17, 2023; U.S. Provisional Application No. 63 / 467,116, filed on May 17, 2023; and U.S. Provisional Application No. 63 / 592,852, filed on October 24, 2023. The entire contents of each of the aforementioned applications are expressly incorporated herein by reference in their entirety. Background Art

[0003] Lipid-based nanoparticles are used in COVID-19 vaccines and many other nanomedicines, such as and Nanoparticles have played a key role in the success of nanoparticles and are therefore considered a leader in nanoscale drug delivery systems. However, the effective targeted delivery of bioactive substances such as therapeutic nucleic acids represents an ongoing medical challenge. This severely limits the widespread application of nucleic acids such as mRNA and DNA in protein replacement therapy, gene therapy, gene editing, and vaccination.

[0004] The lack of effective methods and vehicles for intracellular delivery is a major obstacle to the widespread use of nucleic acid therapeutics. Intracellular delivery of mRNA or DNA is generally more challenging than that of small oligonucleotides, in part because mRNA and DNA molecules (which typically range from 300 kDa to 5,000 kDa, or about 1-15 kb) are significantly larger than other types of RNA, such as small interfering RNA (siRNA, which is typically about 14 kDa) or antisense oligonucleotides (ASOs, which typically range from 4 kDa to 10 kDa).

[0005] Furthermore, intracellular delivery of nucleic acid therapeutics to target cells is hampered by activation of innate and / or adaptive immune responses. While it is possible to circumvent RNA sensing by myeloid dendritic cells (MDCs) by chemically modifying RNA cargoes (e.g., with 1mΨ, 2′OMe, etc.), there are no known chemical modifications of DNA cargoes that can restrict pattern recognition receptor (PRR) sensing and still maintain transcriptional activity.

[0006] An alternative to gene therapy is the recombinant adeno-associated virus (rAAV) vector platform that packages heterologous DNA in a viral capsid. However, there are several major drawbacks to using rAAV vectors as gene delivery vectors. A major drawback associated with rAAV is its limited viral packaging capacity of approximately 4.5 kb of heterologous DNA. Another major drawback is capsid immunogenicity, which prevents re-administration to patients.

[0007] Therefore, there remains a need for effective delivery vehicles that can safely and efficiently deliver nucleic acid therapeutics to desired cell populations. Summary of the Invention

[0008] Thus, in some aspects, the present disclosure provides a polymer-conjugated lipid comprising:

[0009] (i) polyglycerol (PG) or PG derivatives;

[0010] (ii) lipid moiety represented by formula (I)

[0011]

[0012] or a pharmaceutically acceptable salt thereof, wherein:

[0013] R 1 Absent, hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10-30 carbon atoms;

[0014] R 2 Absent, hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10-30 carbon atoms;

[0015] When R 1 and R 2 When each is hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10-30 carbon atoms, N is positively charged; and

[0016] R 3 is a hydrophobic tail comprising 10-30 carbon atoms; and

[0017] (iii) a linker that conjugates the PG or the PG derivative to the lipid moiety,

[0018] wherein in formula (I) is the bond that conjugates the lipid moiety and the linker.

[0019] 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.

[0020] In some embodiments, the PG or the PG derivative is linear or branched. In some embodiments, in the lipid portion represented by formula (I), R 1 does not exist, and where R 2 and R 3 Each independently comprises a hydrophobic tail of 14, 15, 16, 17, 18, 19, 20, 21 or 22 carbon atoms. 2 and R 3 Each independently comprises a hydrophobic tail of 16, 17, 18, 19, 20, 21 or 22 carbon atoms. 2 and R 3 Each independently comprises a hydrophobic tail comprising 18 carbon atoms, and wherein the lipid moiety is dioctadecylamine (DODA).

[0021] In some embodiments, the lipid moiety conjugated to the linker is represented by the structure:

[0022]

[0023] In some embodiments, the PG or the PG derivative comprises about 5 to 100 monomer units, or an average of 5 to 100 monomer units.

[0024] In some embodiments, the PG or the 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 monomeric 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 monomeric units.

[0025] In some embodiments, the PG or the PG derivative comprises about 8, 34, 45, 46 or 58 monomeric units, or an average of 8, 34, 45, 46 or 58 monomeric units. In some embodiments, the PG or the PG derivative comprises about 8 monomeric units or an average of 8 monomeric units. In some embodiments, the PG or the PG derivative comprises about 34 monomeric units or an average of 34 monomeric units. In some embodiments, the PG or the PG derivative comprises about 45 monomeric units or an average of 45 monomeric units. In some embodiments, the PG or the PG derivative comprises about 46 monomeric units or an average of 46 monomeric units. In some embodiments, the PG or the PG derivative comprises about 58 monomeric units or an average of 58 monomeric units.

[0026] 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 phosphorylcholine linker, a carbamate linker, a diamide alkyl linker, a cleavable linker, or 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 C(O)O-, wherein 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)(CH2) n -, and wherein n is 2, 3, 4, 5, or 6. In some embodiments, n is 4.

[0027] In some aspects, the present disclosure provides a polymer-conjugated lipid represented by the following structure:

[0028]

[0029] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0030] In some aspects, the present disclosure provides a polymer-conjugated lipid represented by the following structure:

[0031]

[0032] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0033] In some aspects, the present disclosure provides a polymer-conjugated lipid represented by the following structure:

[0034]

[0035] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0036] In some aspects, the present disclosure provides a polymer-conjugated lipid represented by the following structure:

[0037]

[0038] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0039] In some aspects, the present disclosure provides a polymer-conjugated lipid represented by the following structure:

[0040]

[0041] Where R is

[0042] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0043] In some embodiments, the polymer-conjugated lipid of the present disclosure further comprises an active species conjugated to the PG or the PG derivative, wherein the active species is functionalized to conjugate with a targeting moiety. In some embodiments, the active species is a click chemistry reagent or a maleimide. In some embodiments, the click chemistry reagent is selected from the group consisting of: dibenzocyclooctyne (DBCO) reagents, trans-cyclooctene (TCO) reagents, tetrazine (Tz) reagents, alkyne reagents, and azide reagents.

[0044] In some embodiments, the polymer-conjugated lipid of the present disclosure further comprises a targeting moiety conjugated to the PG or the PG derivative via the active species. In some embodiments, the targeting moiety is conjugated to the PG or the 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 hepatocytes. In some embodiments, the liver cell is a hepatocyte. 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.

[0045] In some embodiments, the targeting moiety is selected from the group consisting of: ApoE protein, ApoE polypeptide, ApoB protein, 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 (scFv), or a combination thereof. v ), heavy chain antibodies (hcAb), nanobodies (Nb), heavy chain immunoglobulins (HC1g), immunoglobulin new antigen receptors (IgNARs), variable domains of immunoglobulin new antigen receptors (V NAR ), single domain antibodies or variable heavy chain antibodies only (V HH ).

[0046] In some aspects, the present disclosure also provides a lipid nanoparticle (LNP) comprising:

[0047] (i) Therapeutic nucleic acids (TNAs);

[0048] (ii) ionizable lipids;

[0049] (iii) sterols; and

[0050] (iv) a first lipid-anchored polymer, wherein the first lipid-anchored polymer comprises a polymer-conjugated lipid of the present disclosure.

[0051] In some embodiments, the LNP further comprises a helper lipid. In some embodiments, the helper lipid comprises a phospholipid or a phosphatidylcholine (PC). In some embodiments, the helper lipid is selected from the group consisting of: 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-heptadecanoyl-2-oleoyl-sn-glycero-3-phosphocholine In one embodiment, the helper lipid is DSPC.

[0052] In some embodiments, the helper lipid is represented by formula (II):

[0053]

[0054] or a pharmaceutically acceptable salt or ester thereof, wherein:

[0055] Is a single bond or a double bond;

[0056] R 1 C1-C 17 Alkyl or C2-C 17 alkenyl;

[0057] R 2 C1-C 22 Alkyl or C2-C 22 alkenyl;

[0058] R 3 is hydrogen or C1-C2 alkyl; and

[0059] R 4 is hydrogen or C1-C2 alkyl.

[0060] In some embodiments, It's a double bond.

[0061] In some embodiments, R 1 C 10 -C 20 Alkenyl, R 2 C 10 -C 20 Alkyl, and R 3 For hydrogen.

[0062] In some embodiments, the helper lipid represented by formula (II) is:

[0063]

[0064] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0065] In some embodiments, the helper lipid represented by formula (II) is:

[0066]

[0067] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0068] In some embodiments, the helper lipid represented by formula (II) is:

[0069]

[0070] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0071] In some embodiments, the helper lipid represented by formula (II) is:

[0072]

[0073] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0074] In some embodiments, the helper lipid represented by formula (II) is:

[0075]

[0076] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0077] In some embodiments, the helper lipid represented by formula (II) is:

[0078]

[0079] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0080] In some embodiments, the sterol is selected from the group consisting of cholesterol, β-sitosterol, stigmasterol, β-sitostanol, campesterol, brassicasterol, and derivatives thereof. In one embodiment, the sterol is cholesterol.

[0081] In some embodiments, the ionizable lipid is represented by:

[0082] a) Formula (A):

[0083]

[0084] or a pharmaceutically acceptable salt thereof, wherein:

[0085] R 1 and R 1′ Each is independently an optionally substituted linear or branched C 1-3 alkylene;

[0086] R 2 and R 2′ Each is independently an optionally substituted linear or branched C 1-6 alkylene;

[0087] R 3 and R 3′ Each is independently an optionally substituted linear or branched C 1-6 alkyl;

[0088] Or alternatively, when R 2 is an optionally substituted branched C 1-6 When alkylene, R 2 and R 3 Together with its central nitrogen atom, it forms a 4- to 8-membered heterocyclic group;

[0089] Or alternatively, when R 2′ is an optionally substituted branched C 1-6 When alkylene, R 2' and R 3′ Together with its central nitrogen atom, it forms a 4- to 8-membered heterocyclic group;

[0090] R 4 and R 4′ Each independently is -CR a 、-C(R a )2CR a or -[C(R a )2]2CR a ;

[0091] R a H or C independently at each occurrence 1-3 alkyl;

[0092] Or alternatively, when R4 -C(R a )2CR a or -[C(R a )2]2CR a When R a C 1-3 When alkyl, R 3 and R 4 Together with its central nitrogen atom, it forms a 4- to 8-membered heterocyclic group;

[0093] Or alternatively, when R 4′ -C(R a )2CR a or -[C(R a )2]2CR a When R a C 1-3 When alkyl, R 3′ and R 4′ Together with its central nitrogen atom, it forms a 4- to 8-membered heterocyclic group;

[0094] R 5 and R 5′ are independently hydrogen, C 1-20 Alkylene or C 2-20 alkenylene;

[0095] R 6 and R 6′ C independently at each occurrence 1-20 Alkylene, C 3-20 Cycloalkylene or C 2-20 alkenylene; and

[0096] m and n are each independently an integer selected from 1, 2, 3, 4 and 5; or

[0097] b) Formula (B):

[0098]

[0099] or a pharmaceutically acceptable salt thereof, wherein:

[0100] a is an integer ranging from 1 to 20;

[0101] B is an integer ranging from 2 to 10;

[0102] R 1 Not present or selected from (C2-C 20 )alkenyl, -C(O)O(C2-C 20 ) alkyl and (C2-C 20 ) alkyl-substituted cyclopropyl; and

[0103] R2 (C2-C 20 )alkyl; or

[0104] c) Formula (C):

[0105]

[0106] or a pharmaceutically acceptable salt thereof, wherein:

[0107] R 1 and R 1′ Each independently is optionally selected from one or more of R a (C1-C6)alkylene substituted with a group;

[0108] R 2 and R 2′ are each independently (C1-C2)alkylene;

[0109] R 3 and R 3′ Each independently is optionally selected from one or more of R b (C1-C6) alkyl substituted with a group;

[0110] Or alternatively, R 2 and R 3 and / or R 2′ and R 3′ Together with its central nitrogen atom, it forms a 4- to 7-membered heterocyclic group;

[0111] R 4 and R 4 ' are each (C2-C6)alkylene interrupted by -C(O)O-;

[0112] R 5 and R 5 ' are each independently (C2-C 30 )alkyl or (C2-C 30 )alkenyl, each of which is optionally interrupted by -C(O)O- or (C3-C6)cycloalkyl; and

[0113] R a and R b each is halo or cyano; or

[0114] d) Formula (D):

[0115]

[0116] or a pharmaceutically acceptable salt thereof, wherein:

[0117] R' is absent, hydrogen or C1-C6 alkyl; provided that when R' is hydrogen or C1-C6 alkyl, R', R1 and R 2 All nitrogen atoms are positively charged;

[0118] R 1 and R 2 Each is independently hydrogen, C1-C6 alkyl or C2-C6 alkenyl;

[0119] R 3 C1-C 12 Alkylene or C2-C 12 alkenylene;

[0120] R 4 C1-C 18 Unbranched alkyl, C2-C 18 Unbranched alkenyl or in:

[0121] R 4a and R 4b Each independently is C1-C 16 Unbranched alkyl or C2-C 16 unbranched alkenyl;

[0122] R 5 Not present, C1-C8 alkylene or C2-C8 alkenylene;

[0123] R 6a and R 6b Each independently is C7-C 16 Alkyl or C7-C 16 alkenyl; provided that the combined R 6a and R 6b The total number of carbon atoms in is greater than 15;

[0124] 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 a2) C(=O)-; wherein:

[0125] R a is independently at each occurrence hydrogen or C1-C6 alkyl; and

[0126] n is an integer selected from 1, 2, 3, 4, 5 and 6;

[0127] Wherein, in some embodiments, R 4 C1-C 16 Unbranched alkyl, C2-C 16 Unbranched alkenyl or where R 4a and R 1b as defined above; or

[0128] e) Formula (E):

[0129]

[0130] or a pharmaceutically acceptable salt thereof, wherein:

[0131] R' is absent, hydrogen or C1-C3 alkyl; provided that when R' is hydrogen or C1-C3 alkyl, R', R 1 and R 2 The nitrogen atoms they are connected to have a positive charge;

[0132] R 1 and R 2 Each is independently hydrogen or C1-C3 alkyl;

[0133] R 3 C3-C 10 Alkylene or C3-C 10 alkenylene;

[0134] R 4 C1-C 16 Unbranched alkyl, C2-C 16 Unbranched alkenyl or in:

[0135] R 4a and R 4b Each independently is C1-C 16 Unbranched alkyl or C2-C 16 unbranched alkenyl;

[0136] R 5 Not present, C1-C6 alkylene or C2-C6 alkenylene;

[0137] R 6a and R 6b Each independently is C7-C 14 Alkyl or C7-C14 alkenyl;

[0138] 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:

[0139] R a is independently at each occurrence hydrogen or C1-C6 alkyl; and

[0140] n is an integer selected from 1, 2, 3, 4, 5 and 6; or

[0141] f) an ionizable lipid selected from the group consisting of any one of the ionizable lipids in Tables 1, 4, 5, 6 or 7.

[0142] In some embodiments, the ionizable lipid is lipid 87, which is represented by the following structure:

[0143]

[0144] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0145] In some embodiments, the ionizable lipid is represented by the structure:

[0146]

[0147] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0148] In some embodiments, the ionizable lipid is represented by the structure:

[0149]

[0150] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0151] In some embodiments, the ionizable lipid is represented by the structure:

[0152]

[0153] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0154] In some embodiments, the ionizable lipid is represented by the structure:

[0155]

[0156] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0157] In some embodiments, the ionizable lipid is represented by the structure:

[0158]

[0159] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0160] In some embodiments, the ionizable lipid is represented by the structure:

[0161]

[0162] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0163] In some embodiments, the ionizable lipid is represented by the structure:

[0164]

[0165] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0166] In some embodiments, the ionizable lipid is represented by the structure:

[0167]

[0168] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0169] In some embodiments, the ionizable lipid is represented by the structure:

[0170]

[0171] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0172] In some embodiments, the ionizable lipid is represented by the structure:

[0173]

[0174] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0175] In some embodiments, the ionizable lipid is represented by the structure:

[0176]

[0177] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0178] In some embodiments, the ionizable lipid is lipid Z, which is represented by the structure:

[0179]

[0180] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0181] In some embodiments, the ionizable lipid is represented by the structure:

[0182]

[0183] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0184] In some embodiments, the ionizable lipid is lipid A, which is represented by the following structure:

[0185]

[0186] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0187] In some embodiments, the ionizable lipid is represented by the structure:

[0188]

[0189] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0190] In some embodiments, the LNPs of the present disclosure further comprise a second lipid-anchored polymer, wherein the second lipid-anchored polymer comprises:

[0191] (i) a lipid portion comprising at least one hydrophobic tail;

[0192] (ii) polymers;

[0193] (iii) a linker, wherein the polymer is conjugated to the lipid moiety via the linker; and

[0194] (iv) an active species conjugated to the polymer, wherein the active species is functionalized to be conjugated to a targeting moiety.

[0195] In some embodiments, the polymer of the second lipid anchoring 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.

[0196] In some embodiments, the PG or the PG derivative is linear or branched.

[0197] In some embodiments, the active 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 trans-cyclooctene (TCO) reagent, a tetrazine (Tz) reagent, an alkyne reagent, and an azide reagent.

[0198] In certain embodiments, the LNP of the present disclosure further comprises a targeting moiety conjugated via the active species and the polymer. In certain embodiments, the targeting moiety is conjugated via dibenzocyclooctyne (DBCO)-azide, azide-alkyne, TCO-Tz or thiol-maleimide and the polymer. In certain embodiments, the targeting moiety can be combined with hepatocytes. In certain embodiments, the hepatocyte is a hepatocyte. In certain embodiments, the targeting moiety is N-acetylgalactosamine (GalNAc) or a GalNAc derivative. In certain embodiments, the targeting moiety is a triantennary GalNAc conjugate or a tetraantennary GalNAc conjugate.

[0199] 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.

[0200] 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 (scFv), or a v), heavy chain antibodies (hcAb), nanobodies (Nb), heavy chain immunoglobulins (HC1g), immunoglobulin new antigen receptors (IgNARs), variable domains of immunoglobulin new antigen receptors (V NAR ), single domain antibodies or variable heavy chain antibodies only (V HH ).

[0201] 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 phosphorylcholine 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 C(O)O-, wherein 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)(CH2) n -, and wherein n is 2, 3, 4, 5 or 6. In one embodiment, n is 4.

[0202] In some embodiments, in the LNPs of the present disclosure, the lipid portion of the second lipid-anchored polymer is represented by formula (I)

[0203]

[0204] or a pharmaceutically acceptable salt thereof, wherein:

[0205] R 1 Absent, hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10-30 carbon atoms;

[0206] R 2 Absent, hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10-30 carbon atoms;

[0207] When R 1 and R 2 When each is hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10-30 carbon atoms, N is positively charged; and

[0208] R 3 It is a hydrophobic tail containing 10-30 carbon atoms.

[0209] In some embodiments, R 1 does not exist, and where R 2 and R 3 Each independently comprises a hydrophobic tail of 14, 15, 16, 17, 18, 19, 20, 21 or 22 carbon atoms. 2 and R 3 Each independently comprises a hydrophobic tail of 16, 17, 18, 19, 20, 21 or 22 carbon atoms. 2 and R 3 Each independently comprises a hydrophobic tail comprising 18 carbon atoms, and wherein the lipid moiety is dioctadecylamine (DODA).

[0210] In some embodiments, the lipid portion of the second lipid-anchoring polymer comprises a moiety 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-dioleoyl-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), 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-anchoring polymer comprises a portion selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, and derivatives of any one of the foregoing. In some embodiments, the lipid portion of the second lipid-anchoring polymer comprises DSPE.

[0211] In some embodiments, the average molecular weight of the polymer of the second lipid-anchored polymer is about 500 Da to about 5000 Da. In some embodiments, the average molecular weight of the polymer is about 1500 Da to about 5000 Da. In some embodiments, the average molecular weight of the polymer is about 2000 Da.

[0212] In some embodiments, the second lipid-anchored polymer is represented by the structure:

[0213]

[0214] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0215] In some embodiments, the second lipid-anchored polymer is represented by the structure:

[0216]

[0217] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0218] In certain embodiments, the ionizable lipid is present in the LNP in an amount of from about 20mol% to about 60mol% of the total lipid present in the LNP. In certain embodiments, the ionizable lipid is present in the LNP in an amount of from about 35mol% to about 50mol% of the total lipid present in the LNP.

[0219] 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. In 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.

[0220] In some embodiments, the helper lipid is present in the LNP in an amount of from about 1 mol % to about 40 mol % of the total lipids present in the LNP. In some embodiments, the helper lipid is present in the LNP in an amount of from about 5 mol % to about 15 mol % of the total lipids present in the LNP.

[0221] 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 lipids present in the LNP. In 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 lipids present in the LNP.

[0222] 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 lipids present in the LNP. In 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 lipids present in the LNP.

[0223] In some aspects, the present disclosure also provides a lipid nanoparticle (LNP) comprising:

[0224] (i) Therapeutic nucleic acids (TNAs);

[0225] (ii) an ionizable lipid, wherein the ionizable lipid is heptadecan-9-yl 9-((4-(dimethylamino)butanoyl)oxy)hexadecanoate, and the ionizable lipid has the following structure:

[0226]

[0227] (iii) a sterol, wherein the sterol is cholesterol;

[0228] (iv) a helper lipid, wherein the helper lipid is DSPC;

[0229] (v) a first lipid-anchored polymer, wherein the first lipid-anchored polymer comprises DODA conjugated to a linear PG via a linker; and

[0230] (vi) a second lipid-anchored polymer, wherein the second lipid-anchored polymer comprises DSPE conjugated to PEG.

[0231] In some embodiments, the linear PG comprises about 30-60 monomer units or an average of 30-60 monomer units.

[0232] In some embodiments, the linear PG comprises about 34 monomeric units or about 45 monomeric units, or an average of 34 monomeric units or an average of 45 monomeric units.

[0233] In some embodiments, the average molecular weight of the PEG is about 1000 Da to about 5000 Da. In some embodiments, the average molecular weight of the PEG is 2000 Da.

[0234] In some embodiments, in the LNPs of the present disclosure:

[0235] The ionizable lipid is present in the LNP in an amount from about 35 mol % to about 50 mol % of the total lipid present in the LNP;

[0236] 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;

[0237] The helper lipid is present in the LNP in an amount from about 5 mol % to about 15 mol % of the total lipids present in the LNP;

[0238] The first lipid-anchoring polymer is present in the LNP in an amount from about 1.5 mol % to about 3 mol % of the total lipid present in the LNP; and

[0239] The second lipid-anchoring polymer is present in the LNP in an amount from about 0.1 mol % to about 1 mol % of the total lipid present in the LNP.

[0240] In some embodiments, the TNA contained in the LNP of the present disclosure is selected from the group consisting of: minigene, plasmid, minicircle, small interfering RNA (siRNA), microRNA (miRNA), guide RNA (gRNA), antisense oligonucleotide (ASO), ribozyme, deoxyribozyme, end-blocked DNA (ceDNA), ssDNA, ministring, doggybone TM , prototelomere end-blocked DNA, dumbbell-shaped linear DNA, Dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), mRNA, tRNA, rRNA, gRNA, DNA viral vectors, viral RNA vectors, non-viral vectors and combinations thereof.

[0241] In some embodiments, the TNA is ceDNA.

[0242] 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).

[0243] In some embodiments, the ssDNA is a linear ssDNA comprising at least one nucleic acid sequence of interest flanked by 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 initiate replication and / or transcription. In some embodiments, the stem structure at the 3' end comprises a partial DNA duplex of 4-500 nucleotides. In some embodiments, the stem structure at the 3' end comprises a partial DNA duplex of 4-50 nucleotides.

[0244] In some embodiments, the loop structure at the 3' end comprises 3-500 unbound nucleotides. In some embodiments, the loop structure at the 3' end comprises a minimum of 3 unbound nucleotides.

[0245] 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, the at least one stem-loop structure at the 3′ end comprises a hairpin DNA structure. In some embodiments, 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 protruding DNA structure, and a multi-branched loop structure. In some embodiments, the at least one stem-loop structure at the 3′ end does not comprise the A, A′, D, and D′ regions present in wild-type AAV ITRs. In some embodiments, the at least one stem-loop structure at the 3′ end does not comprise the A, A′, B, B′, C, C′, D, and D′ regions present in wild-type AAV ITRs. In some embodiments, the at least one stem-loop structure at the 3′ end does not comprise the rep binding element (RBE) present in wild-type ITRs. In some embodiments, the at least one stem-loop structure at the 3' end does not comprise a terminal melting site (trs) present in wild-type ITRs.

[0246] In some embodiments, the stem structure at the 3' end comprises four or more nucleotides modified to confer exonuclease resistance. In some embodiments, the nucleotides are phosphorothioate-modified nucleotides.

[0247] In some embodiments, at least one stem-loop structure at the 3' end further comprises a functional moiety.

[0248] In some embodiments, the ssDNA molecule further comprises a 5' end, the 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, the at least one stem-loop structure at the 5' end comprises a hairpin DNA structure. In some embodiments,

[0249] 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.

[0250] In some embodiments, the at least one stem-loop structure at the 5' end does not comprise the A, A', D, and D' regions present in wild-type AAV ITRs. In some embodiments, the at least one stem-loop structure at the 5' end does not comprise the A, A', B, B', C, C', D, and D' regions present in wild-type AAV ITRs. In some embodiments, the at least one stem-loop structure at the 5' end does not comprise the rep binding element (RBE) present in wild-type ITRs. In some embodiments, the at least one stem-loop structure at the 5' end does not comprise the terminal melting site (trs) present in wild-type ITRs.

[0251] In some embodiments, the stem structure at the 5' end comprises four or more nucleotides modified to confer exonuclease resistance. In some embodiments, the nucleotides are phosphorothioate-modified nucleotides.

[0252] In some embodiments, the loop structure at the 5' end further comprises one or more nucleic acids to stabilize the end. In some embodiments, the loop structure at the 5' end further comprises one or more chemically modified nucleic acids. In some embodiments, the loop structure at the 5' end further comprises one or more aptamers. In some embodiments, the loop structure at the 5' end further comprises one or more synthetic ribozymes.

[0253] In some embodiments, the loop structure at the 5' end further comprises one or more antisense oligonucleotides (ASOs). In some embodiments, the loop structure at the 5' end further comprises one or more short interfering RNAs (siRNAs). In some embodiments, the loop structure at the 5' end further comprises one or more antiviral nucleoside analogs (ANAs).

[0254] In some embodiments, the loop structure at the 5' end further comprises one or more triplex-forming oligonucleotides. In some embodiments, the loop structure at the 5' end further comprises one or more gRNAs or gDNAs. In some embodiments, the loop structure at the 5' end further comprises one or more molecular probes.

[0255] 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.

[0256] In some embodiments, the ssDNA molecule does not activate or 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: a cGAS / STING pathway, a TLR9 pathway, an inflammasome-mediated pathway, and combinations thereof.

[0257] In some embodiments, the ssDNA molecule is capable of expressing at least one therapeutic protein or a therapeutic fragment thereof. In some embodiments, the at least one therapeutic protein is selected from the group consisting of an antibody, an enzyme, a coagulation 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 can be used to treat a genetic disease selected from the group consisting of melanoma, hemophilia A (factor VIII (FVIII) deficiency) and hemophilia B (factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited liver metabolic disorders, Lesch Nyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi's anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharidosis (e.g.,Hurler syndrome (MPS type I), Scheie syndrome (MPS type I S), Hurler-Scheie syndrome (MPS type I HS), Hunter syndrome (MPS type II), Sanfilippo types A, B, C, and D (MPS types III A, B, C, and D), Morquio types A and B (MPS IVA and MPS IVB), Maroteaux-Lamy syndrome (MPS type VI), Sly syndrome (MPS type VII), hyaluronidase deficiency (MPS type IX), Niemann-Pick disease types A / B, C1, and C2, Fabry disease, Schindlerdisease, GM2-gangliosidosis type II (Sandhoff disease), 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, Battendisease, aspartate glucosamineuria, sialidosis (Salla disease), Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidosis, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease 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 congenital amaurosis, Stargardt 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.

[0258] In some aspects, the present disclosure also provides a pharmaceutical composition comprising the LNP of the present disclosure and a pharmaceutically acceptable carrier.

[0259] In some aspects, the present disclosure also provides a method of treating a genetic disease in a subject in need thereof, the method comprising administering to the subject an effective amount of the LNP of the present disclosure or the pharmaceutical composition of the present disclosure.

[0260] In some embodiments, the subject is a human.

[0261] In some embodiments, the genetic disease is selected from the group consisting of: sickle cell anemia; melanoma; hemophilia A (coagulation factor VIII (FVIII) deficiency); hemophilia B (coagulation 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's anemia; Retinitis pigmentosa; Ataxia-telangiectasia; Bloom's syndrome; Retinoblastoma; Mucopolysaccharidoses; 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; Cyst Aminopathy; Batten disease; Aspartate glucosamineuria; Sialidosis; Dannon 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 trophoblastic syndrome dystrophy (DMD); Becker muscular dystrophy (BMD); dystrophic epidermolysis bullosa (DEB); ectonucleotide pyrophosphatase 1 deficiency; systemic arterial calcification of infancy (GACI); Leber 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.

[0262] In some aspects, the present disclosure also provides a method of providing anti-tumor immunity to a subject in need thereof, the method comprising administering to the subject an effective amount of the LNP of the present disclosure or the pharmaceutical composition of the present disclosure.

[0263] In some aspects, the present disclosure also provides a method of treating a subject suffering from 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 the present disclosure or the pharmaceutical composition of the present disclosure.

[0264] In some aspects, the present disclosure also provides a method of treating a blood disease, disorder, or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of the LNP of the present disclosure or the pharmaceutical composition of the present disclosure.

[0265] In some aspects, the present disclosure also provides a method for synthesizing the polymer-conjugated lipid of the present disclosure, the method comprising:

[0266] a) reacting the lipid moiety conjugated to a linker with 2,3-epoxy-1-(1-ethoxyethoxy)propane (EEGE) under an argon atmosphere in the presence of a base or in the presence of an organic catalyst to produce the lipid moiety conjugated to a linker and polymeric EEGE; and

[0267] b) subjecting the lipid moiety conjugated to a linker and the polymeric EEGE to acidic conditions to produce the polymer-conjugated lipid.

[0268] In some embodiments, the base is a phosphazene base. In some embodiments, the phosphazene base is P4-t-Bu.

[0269] In some embodiments, the organic catalyst is an N-heterocyclic carbene (NHC) or an N-heterocyclic olefin (NHO). In some embodiments, the acidic conditions comprise HCl, Br, HI, HClO4, HClO3, H2SO4, or HNO3.

[0270] In some embodiments, the lipid moiety comprises DODA.

[0271] In some embodiments, the lipid moiety conjugated to the linker is represented by the structure:

[0272]

[0273] In some embodiments, the polymer-conjugated lipid is represented by the structure:

[0274]

[0275] Where n is a number ranging from 10 to 100.

[0276] In some embodiments, n is about 34, 45, 46, or 58. BRIEF DESCRIPTION OF THE DRAWINGS

[0277] Figure 1A The MALDI-TOF spectrum of DODA-PG34 is depicted.

[0278] Figure 1B The MALDI-TOF spectrum of DODA-PG45 is depicted.

[0279] Figure 1C The MALDI-TOF spectrum of DODA-PG58 is depicted.

[0280] Figure 1D"Scheme 1" illustrating the synthesis of DODA-PG41 and DODA-PG46 is shown.

[0281] Figure 1E "Scheme 2" showing the synthesis of DODA-PG45 and DODA-PG58 is shown.

[0282] Figure 2A Shown are the total flux measured by total photon counts per region of interest (ie, liver) measured by an in vivo imaging system (IVIS) in mice on day 4 after administration of the LNP formulations of the present disclosure and a negative control (PBS).

[0283] Figure 2B Shown are the total flux measured by total photon counts through each region of interest (ie, liver) measured by IVIS in mice on day 7 after administration of the LNP formulations of the present disclosure and a negative control (PBS).

[0284] Figure 2C Shown are the total flux measured by total photon counts through each region of interest (ie, liver) measured by IVIS in mice over two collection days (days 4 and 7) following administration of the disclosed LNP formulations and negative control (PBS).

[0285] Figure 2D Shown are the percent changes in body weight (BW) of mice on day 1 after dosing with the LNP formulations of the present disclosure.

[0286] Figure 3 Shown are the luciferase activities of LNP formulations of the present disclosure containing different lipid-anchored polymers.

[0287] Figure 4A is a schematic diagram depicting the proposed mechanism of opsonization-driven cellular uptake of LNPs.

[0288] Figure 4B is a schematic diagram depicting the assay used to evaluate opsonization-driven cellular uptake of LNPs.

[0289] Figure 4C Shown are the DiD fluorescence areas normalized to the area of ​​the viable nuclei measured for LNP formulations of the present disclosure containing different lipid-anchored polymers.

[0290] Figure 5 Shown are the DiD fluorescence areas normalized to the area of ​​viable nuclei for LNP formulations of the present disclosure containing varying amounts of polyglycerol-conjugated lipids and controls.

[0291] Figure 6Shown is 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 varying amounts of polyglycerol-conjugated lipids and controls.

[0292] Figure 7 Shown are the whole blood clearance rates of control LNPs and different lipid Z-carrying LNPs of the present disclosure.

[0293] Figure 8A Shown are the total flux quantified by total photon counts through each region of interest (ie, liver) measured by IVIS in mice on day 7 after dosing with the disclosed LNP formulations and negative control (DPBS).

[0294] Figure 8B Shown are the percent changes in body weight on day 1 of mice injected with the LNP formulations of the present disclosure.

[0295] FIG9 shows various cytokines involved in the regulation of innate immune responses, namely IFN-α ( Figure 9A ), IFN-γ( Figure 9B ), IL-6( Figure 9C ) and IL-18( Figure 9D ) level.

[0296] Figure 10 Shown are DiD fluorescence areas normalized to the area of ​​viable nuclei for LNP formulations of the present disclosure containing varying amounts of polyglycerol-conjugated lipids and controls and formulated with DSPE-PEG5K-N3 using a molar percentage of 0.5%. DETAILED DESCRIPTION

[0297] The present disclosure provides polymer-conjugated lipids comprising, for example, polyglycerol (PG) conjugated to dioctadecylamine (DODA), such as DODA-PG34, DODA-PG45, and DODA-PG58, and methods for synthesizing the same. The present disclosure also provides lipid nanoparticles (LNPs), comprising, inter alia, the polymer-conjugated lipids of the present disclosure, and methods for treating various conditions comprising administering the LNPs of the present disclosure to a subject in need thereof. It has been surprisingly discovered that LNPs comprising the polymer-conjugated lipids of the present disclosure are characterized by low levels of undesirable opsonization-driven LNP uptake into non-target cells, balanced with desired levels of endosomal escape, thereby achieving a favorable stealth / endosomal escape trade-off, as described herein.

[0298] I. Definition

[0299] Unless otherwise defined herein, scientific and technical terms used in conjunction with this application shall have the meanings commonly understood by those of ordinary skill in the art to which this disclosure pertains. It should be understood that this disclosure is not limited to the specific methods, protocols, and reagents described herein and may vary accordingly. The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of this disclosure, which is defined solely by the claims. Definitions of commonly used terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th ed., Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), Fields Virology, 6th ed., Lippincott Williams & Wilkins, Philadelphia, PA, USA (2013); Knipe, DM and Howley, PM (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc.), 1995 (ISBN 1-56081-569-8); Werner Luttmann, Immunology, Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, Jones & Bartlett Publishers, 2014 (ISBN 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual =The following are examples of the following: Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.), Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, Inc. Sons), 2014 (ISBN 047150338X, 9780471503385); Current Protocols in Protein Science (CPPS), John E.Coligan (ed.), John Wiley & Sons, 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.), John Wiley & Sons, 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated herein by reference in their entirety.

[0300] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0301] As used herein, when referring to a measurable value such as an amount, duration, etc., the term "about" is meant to encompass deviations of ±20% or ±10% (more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%) from the specified value, as such deviations are suitable for performing the disclosed methods.

[0302] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the range, and where appropriate, fractions thereof (such as tenths and hundredths of integers).

[0303] As used herein, "comprise," "comprising," and "comprises" are meant to be synonymous with "include," "including," "includes," or "contain," "containing," "contains," and are inclusive or open-ended terms that specify the presence of, for example, components below, and do not exclude or preclude the presence of additional, unrecited components, features, elements, components, steps known in the art or disclosed herein.

[0304] The term "consisting of" refers to compositions, methods, processes, and corresponding components thereof as described herein, and the term excludes any elements not recited in the described description of the embodiments.

[0305] As used herein, the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of the described embodiments of the disclosure.

[0306] 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 and include the simultaneous and sequential introduction of one or more compositions or agents. "Administration" may refer to, for example, treatment, pharmacokinetics, diagnosis, research, placebo, and experimental methods. "Administration" also encompasses in vitro and ex vivo treatments. The 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 local. Administration includes self-administration and administration by another person. Administration may be performed by any suitable route. A suitable route of administration allows 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 vein of the subject.

[0307] 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 undesirable immune response in a subject after the LNP of the present disclosure or the composition comprising the LNP of the present disclosure is administered to the subject. In some embodiments, the immune response before and after administration of a composition comprising an LNP of the present disclosure can be measured by measuring the levels of one or more proinflammatory cytokines. Exemplary proinflammatory cytokines that can be used to determine the immunogenicity of the LNPs of the present disclosure or compositions comprising the LNPs of the present disclosure include, but are not limited to, granulocyte colony-stimulating factor (G-CSF), interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), 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-alpha), interferon beta (IFN-beta), interferon gamma (IFN-gamma), CXC 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-alpha), and combinations thereof.

[0308] As used herein, the term "off-target delivery" refers to the delivery of LNP of the present disclosure to non-target cells. For example, LNP targeting LNP of the present disclosure comprising GalNAc is delivered to hepatocytes, and the off-target delivery of LNP refers to the delivery of LNP to random non-target cells that are not, for example, hepatocytes. In certain embodiments, the non-target cells can be blood cells, such as leukocytes, neutrophils, eosinophils, basophils, macrophages, or monocytes. In certain embodiments, the non-target cells can be immune cells, such as T cells, B cells, or macrophages. In certain embodiments, the non-target cells can be liver sinusoidal endothelial cells (LSEC cells), splenocytes, or Kupfer cells.

[0309] After administration to a subject, the LNP can be delivered to non-target cells, such as one or more of the blood cells listed above, and can result in expression of the therapeutic nucleic acid (TNA) in the non-target cells, or can be degraded once engulfed by, for example, macrophages. In some embodiments, a reference LNP that does not contain a polyglycerol-conjugated lipid can be characterized by a higher rate of delivery to non-target cells, such as one or more of the blood cells listed above, compared to the LNPs of the present disclosure. In some embodiments, the LNPs of the present disclosure result in a lower level of uptake of TNAs (e.g., ceDNA or mRNA) in non-target cells, such as blood cells, than the level of uptake of the reference LNP. In some embodiments, the reference LNP is an LNP that does not contain a polymer-conjugated lipid. 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.

[0310] As used herein, the term "aqueous solution" refers to a composition that comprises in whole or in part water.

[0311] As used herein, the term "base" includes purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine and natural analogs, 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.

[0312] As used herein, the terms "carrier" and "excipient" are meant 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 may also be incorporated into the compositions. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce toxic, allergic, or similar adverse reactions when administered to a host.

[0313] As used herein, the term "ceDNA" refers to a capsid-free, end-closed, linear, double-stranded (ds) duplex DNA for non-viral gene transfer, synthetic or otherwise. According to some embodiments, the ceDNA is an end-closed linear duplex (CELiD) CELiD DNA. According to some embodiments, the ceDNA is a DNA-based minicircle. According to some embodiments, the ceDNA is a minimalistic, immunologically defined gene expression (MIDGE) vector. According to some embodiments, the ceDNA is a ministring DNA. According to some embodiments, the ceDNA is a dumbbell-shaped, linear duplex end-closed DNA comprising two hairpin structures of ITRs in the 5' and 3' ends of the expression cassette. According to some embodiments, the ceDNA is a doggybone TM DNA. A detailed description of ceDNA is described 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 generating ceDNA comprising 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 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 comprising 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.

[0314] As used herein, the term "end-blocked DNA vector" refers to a capsid-free DNA vector having at least one covalently blocked end, and wherein at least a portion of the vector has an intramolecular duplex structure.

[0315] As used herein, the term "ceDNA vector" is used interchangeably with "ceDNA" and refers to an end-closed DNA vector comprising at least one terminal palindrome. In some embodiments, the ceDNA comprises two covalently closed ends.

[0316] As used herein, the term "ceDNA-bacmid" refers to an infectious baculovirus genome comprising the ceDNA genome as an intermolecular duplex that is capable of propagation as a plasmid in Escherichia coli (E. coli) and can therefore be manipulated as a baculovirus shuttle vector.

[0317] As used herein, the term "ceDNA-baculovirus" refers to a baculovirus that comprises a ceDNA genome as an intermolecular duplex within the baculoviral genome.

[0318] 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 ceDNA-baculovirus.

[0319] As used herein, the term "ceDNA genome" refers to an expression cassette further incorporated with 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 duplex polynucleotide of DNA.

[0320] 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, protein degradation signals, and the like, 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 the encoded polypeptide.

[0321] "ITR" can be artificially synthesized using a set of oligonucleotides comprising one or more desired functional sequences (e.g., palindromic sequences, RBS). The ITR sequence can be an AAV ITR, an artificial non-AAV ITR, or an ITR physically derived from a viral AAV ITR (e.g., an ITR fragment removed from the viral genome). For example, ITR can be derived from the Parvoviridae family, which encompasses parvoviruses and dependent viruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19), or the SV40 hairpin serving as the SV40 origin of 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: the Parvovirinae family, which infects vertebrates, and the Densovirinae family, which infects invertebrates. The genus Dependovirus includes a family of viruses called adeno-associated viruses (AAVs) that are capable of replicating in vertebrate hosts, including but not limited to humans, primates, cattle, dogs, horses, and sheep species. Generally speaking, ITR sequences can be derived not only from AAV, but also from parvoviruses, lentiviruses, goose viruses, and B19 in configurations of wild-type, "doggy bone," and "dumbbell-shaped," symmetrical, or even asymmetrical ITR orientations. Although ITRs are typically present in both the 5' and 3' ends of an AAV vector, ITRs can be present only at one end of the end of a linear vector. For example, an ITR can be present only at the 5' end. In some other cases, an ITR can be present only at the 3' end in a synthetic AAV vector. For convenience herein, the ITR located at the 5' ("upstream") end of the expression cassette in the synthetic AAV vector is referred to as the "5' ITR" or "left ITR," and the ITR located at the 3' ("downstream") end of the expression cassette in the vector or synthetic AAV is referred to as the "3' ITR" or "right ITR."

[0322] As used herein, "wild-type ITR" or "WT-ITR" refers to a sequence of an ITR sequence naturally occurring in an AAV genome or other dependent virus that retains, for example, Rep binding activity and Rep nicking ability. Due to the degeneracy or drift of the genetic code, the nucleotide sequence of the WT-ITR from any AAV serotype may differ slightly from the typical naturally occurring sequence, and therefore, the WT-ITR sequences contemplated for use herein include those resulting from naturally occurring variations (e.g., replication errors).

[0323] As used herein, the term "substantially symmetrical WT-ITR" or "substantially symmetrical WT-ITR pair" refers to a pair of WT-ITRs within a synthetic AAV vector, both of which are wild-type ITRs, having reverse complementary sequences across their entire length. For example, even if the ITR has one or more nucleotides that deviate from a typical naturally occurring canonical sequence, the sequence can be considered a wild-type sequence as long as these changes do not affect the physical and functional properties and overall three-dimensional structure (secondary and tertiary structure) of the sequence. In some aspects, the deviated nucleotides represent conservative sequence changes. As a non-limiting example, a sequence has at least 95%, 96%, 97%, 98% or 99% sequence identity to the canonical sequence (as measured, for example, using BLAST under default settings) and also has a symmetrical three-dimensional spatial organization with another WT-ITR such that its 3D structure has the same shape in geometric space. Substantially symmetrical WT-ITRs have identical A, C-C' and BB' loops in 3D space. It can be functionally confirmed as WT by confirming that the substantially symmetrical WT-ITR has an operable Rep binding site (RBE or RBE') and a terminal melting site (trs) that is paired with the appropriate Rep protein. Other functions can optionally be tested, including transgene expression under permissive conditions.

[0324] As used herein, the phrases "modified ITR" or "mod-ITR" or "mutant ITR" are used interchangeably and refer to an ITR that has a mutation in at least one or more nucleotides compared to a WT-ITR from the same serotype. The mutation may result in a change in one or more of the A, C, C', B, or B' regions of the ITR and may result in a change in the three-dimensional organization (i.e., its 3D structure in geometric space) compared to the 3D organization of a WT-ITR from the same serotype.

[0325] As used herein, the term "asymmetric ITR", also referred to as an "asymmetric ITR pair", refers to a pair of ITRs within a single synthetic AAV genome, the sequences of which are not reverse complementary sequences over their entire length. As a non-limiting example, an asymmetric ITR pair does not have a symmetrical three-dimensional spatial organization with its cognate ITR, such that its 3D structure has a different shape in geometric space. In other words, the asymmetric ITR pair has a different overall geometric structure, i.e., the pair has a different organization of its A, CC' and BB' loops in 3D space (e.g., one ITR may have a short CC' arm and / or a short BB' 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 in the asymmetric ITR pair may be a wild-type AAV ITR sequence, and the other ITR is a modified ITR as defined herein (e.g., a non-wild-type or synthetic ITR sequence). In another embodiment, neither ITR in the asymmetric ITR pair is a wild-type AAV sequence, and both ITRs are modified ITRs having different shapes in geometric space (i.e., different overall geometric structures). In some embodiments, one mod-ITR in the asymmetric ITR pair can have a short CC' arm, and the other ITR can have a different modification (e.g., a single arm or a short BB' arm, etc.) such that it has a different three-dimensional spatial organization compared to the homologous asymmetric mod-ITR.

[0326] As used herein, the term "symmetrical ITR" refers to a pair of ITRs within a single-stranded AAV genome, the sequences of which are wild-type or mutated (e.g., modified relative to wild-type) dependent viral ITR sequences and are reverse complementary sequences over their entire length. In one non-limiting example, both ITRs are wild-type ITR sequences from AAV2. In another example, neither of the ITRs is a wild-type ITR AAV2 sequence (i.e., the sequences are modified ITRs, also referred to as mutant ITRs), and may differ in sequence from the wild-type ITR due to nucleotide additions, deletions, substitutions, truncations, or point mutations. For convenience herein, the ITR located 5' (upstream) of the expression cassette in the synthetic AAV vector is referred to as the "5' ITR" or "left ITR," and the ITR located 3' (downstream) of the expression cassette in the synthetic AAV vector is referred to as the "3' ITR" or "right ITR."

[0327] As used herein, the term "substantially symmetrical modified ITR" or "substantially symmetrical mod-ITR pair" refers to a pair of modified ITRs within a synthetic AAV that have reverse complementary sequences across their entire length. For example, even if the modified ITR has some nucleotide sequence that deviates from the reverse complementary sequence, the sequence can be considered to be substantially symmetrical as long as these changes do not affect the properties and overall shape. As a non-limiting example, a sequence has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a typical sequence (as measured using BLAST at default settings) and also has a symmetrical three-dimensional spatial organization with its homologous modified ITR such that its 3D structure has the same shape in geometric space. In other words, a substantially symmetrical modified ITR pair has the same A, CC' and BB' loops organized in 3D 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 produce the same overall 3D shape. For example, one ITR in a mod-ITR pair (e.g., the 5' ITR) 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 (e.g., if the 5' ITR has a deletion in the C region, then the homologous modified 3' ITR from the different serotype also has a deletion at the corresponding position in the C' region), such that the modified ITR pair has the same symmetrical three-dimensional spatial organization. In such embodiments, each ITR in the modified ITR pair can be from a different serotype (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), such as a combination of AAV2 and AAV6, wherein the modification in one ITR is reflected in the corresponding position in the homologous ITR from the different serotype. In one embodiment, a substantially symmetrical modified ITR pair refers to a pair of modified ITRs (mod-ITRs) as long as the differences in nucleotide sequence between the ITRs do not affect the properties or overall shape and they have substantially the same shape in 3D space. As non-limiting examples, the mod-ITRs have at least 95%, 96%, 97%, 98% or 99% sequence identity to a typical mod-ITR as determined by standard methods well known in the art, such as BLAST (Basic Local Alignment Search Tool) or BLASTN under default settings, and also have a symmetrical three-dimensional spatial organization such that their 3D structures have the same shape in geometric space.A substantially symmetric mod-ITR pair has identical A, C-C', and BB' loops in 3D space. For example, if a modified ITR in a substantially symmetric mod-ITR pair lacks the CC' arm, then the corresponding homologous mod-ITR lacks the CC' loop and also has a similar 3D structure with the remaining A and BB' loops being identically shaped in the geometric space of its cognate mod-ITR.

[0328] As used herein, the phrase "effective amount" or "therapeutically effective amount" of an active agent or therapeutic agent, such as a therapeutic nucleic acid, is an amount sufficient to produce the desired effect, e.g., 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 expression of a target gene or target sequence include, for example, examining protein or RNA levels using techniques known to those skilled in the art (e.g., dot blotting, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function), as well as phenotypic assays known to those skilled in the art.

[0329] As used herein, the term "expression" refers to the cellular processes involved in the production of RNA and protein, and secretion of protein when appropriate, including, 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), as well as polypeptides obtained by translating mRNA transcribed from a gene.

[0330] As used herein, the term "expression vector" refers to a vector that directs the expression of an RNA or polypeptide from a sequence linked to a transcriptional regulatory sequence on the vector. The expressed sequence is usually, but not necessarily, heterologous to the host cell. The expression vector may contain additional elements, for example, the expression vector may have two replication systems, thereby allowing it to be maintained in two organisms, for example, for expression in human cells and for cloning and amplification in a prokaryotic host. The expression vector may be a recombinant vector.

[0331] As used herein, the term "flanking" refers to the relative position of one nucleic acid sequence relative to another nucleic acid sequence. Typically, in the sequence ABC, B is flanked by A and C. The same is true for the arrangement AxBxC. Thus, a flanking sequence precedes or follows the flanking sequence, but need not be adjacent to or immediately adjacent to the flanking sequence.

[0332] As used herein, the term "spacer region" means the intermediate sequence of the functional elements in a separation vector or genome. In certain embodiments, the spacer region is maintained at a desired distance for optimal functionality for two functional elements. In certain embodiments, the spacer region provides or increases the genetic stability of a vector or genome. In certain embodiments, the spacer region promotes the ready genetic manipulation of the genome by providing a convenient location for a cloning site and the gap of a designed number of nucleotides.

[0333] 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 of a DNA vector, such as a synthetic AAV vector. Suitable promoters include, for example, tissue-specific promoters. The promoter may also be of AAV origin.

[0334] As used herein, a "vector" or "expression vector" is a replicon that can be a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, the vector in its final form can be of viral or non-viral origin. For the purposes of this disclosure, a "vector" generally refers to a synthetic, capsid-free AAV, such as a single-stranded (ss) synthetic vector or a nicked ceDNA vector. Thus, the term "vector" encompasses any genetic element that is capable of replication or expression and can transfer a gene sequence to a cell when associated with appropriate control elements. In some embodiments, a vector can be a recombinant vector or an expression vector. It should be understood that, as used herein, the term "single-stranded (ss) synthetic vector" includes single-stranded AAV-like vectors that may not have any viral sequences.

[0335] As used herein, the term "genetic disease" refers to a disease that is caused, in part or in whole, directly or indirectly, by one or more abnormalities in the genome, particularly conditions that are present from birth and that can be treated by single-stranded (ssDNA) molecules as described herein. The abnormality can be a mutation, insertion, or deletion. The abnormality can affect the coding sequence of a gene or its regulatory sequences. The genetic disease may be, but is not limited to, phenylketonuria (PKU), melanoma, hemophilia A (coagulation factor VIII (FVIII) deficiency) and hemophilia B (coagulation 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, Leschnihan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom's syndrome, retinoblastoma, and mucopolysaccharidoses (e.g., Hurler syndrome (MPS type I), Shay syndrome (MPS type I S), Hurler-Shay syndrome (MPS type I HS), Hunter syndrome (MPS type II), Sanfilippo A, B, C, and D (MPS type III A, B, C, and D), Morque A and B (MPS IVA and MPS IVB), Mallo-Lami syndrome (MPS type VI), Sly syndrome (MPS type VII), hyaluronidase deficiency (MPS type 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, aspartate glucosamineuria, sialic acid storage disease (Salladisease), Dannon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CL N1-8, INCL, and LINCL), sphingolipid storage diseases, and galactosialidosis. Genetic diseases 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), epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, systemic arterial calcification of infancy (GACI), Leber congenital amaurosis (LCA, e.g., LCA10 [CEP290]), Stargardt macular dystrophy (ABCA4), or cathepsin A deficiency.

[0336] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length, which may include chemically or biochemically modified or derivatized amino acids and polypeptides having modified peptide backbones.

[0337] According to some embodiments, the polypeptides of the present disclosure are ApoE or ApoB polypeptides. According to some embodiments, the ApoE polypeptide is a functional fragment (or functional portion) of a full-length ApoE polypeptide. According to some embodiments, the ApoE polypeptide is a functional fragment (or functional portion) of a full-length ApoB polypeptide. According to some embodiments, the ApoE polypeptide is 30 amino acids or less in length. According to some embodiments, the ApoB polypeptide is 30 amino acids or less in length.

[0338] As used herein, the term "lipid" refers to a group of organic compounds including, but not limited to, esters of fatty acids and characterized by being insoluble in water but soluble in many organic solvents. The lipids are generally divided into at least three categories: (1) "simple lipids," which include fats and oils as well as waxes; (2) "compound lipids," which include phospholipids and glycolipids; and (3) "derivative lipids," such as steroids.

[0339] The 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 lacking in phosphorus, such as sphingolipids, sphingolipid family, diacylglycerol and β-acyloxy acid, are also designated as the group of amphipathic lipids. In addition, above-mentioned amphipathic lipids can be mixed with other lipids (comprising triglyceride and sterol).

[0340] As used herein, the term "polyglycerol" refers to an organic compound that is a polymerized condensation product of glycerol. The polyglycerol obtained from the dehydration of glycerol can have a linear, branched, or cyclic structure. In some embodiments, the polyglycerol of the present disclosure is linear or branched. In one embodiment, the polyglycerol is linear. In one embodiment, the polyglycerol is branched. In some embodiments, the term "polyglycerol" encompasses a population of polyglycerol molecules. A population of polyglycerol molecules can comprise a distribution of polyglycerol molecules of different lengths, i.e., a distribution of polyglycerol molecules comprising different numbers of monomeric units. Therefore, when referring to polyglycerol herein, 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. When used herein with reference to the number of monomeric units present in a polyglycerol, the term "average" refers to the average number of monomeric units per polyglycerol molecule in the population of polyglycerol molecules. Therefore, when used herein with reference to the number of monomeric units present in a polyglycerol, language such as "an average of 45 monomeric units" means an average of 45 monomeric units per polyglycerol molecule in the 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.

[0341] In some embodiments, the polyglycerol can comprise an average of 8 to 100 monomer units, such as 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 polyglycerol of the present disclosure comprises 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.

[0342] As used herein, the term "straight chain" when referring to a polyglycerol or aliphatic hydrocarbon chain means that the chain is unbranched.

[0343] 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.

[0344] In some embodiments, the term "polyglycerol derivative" encompasses a population of polyglycerol derivative molecules. A population of polyglycerol derivative molecules may comprise a distribution of polyglycerol derivative molecules of different lengths, i.e., a distribution of polyglycerol derivative molecules comprising different numbers of monomeric units. Therefore, when referring to a polyglycerol derivative in this article, 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 reference to the number of monomeric units present in a polyglycerol derivative, the term "average" refers to the average number of monomeric units of each polyglycerol derivative molecule in the population of polyglycerol derivative molecules. Therefore, when used herein with reference to the number of monomeric units present in a polyglycerol derivative, language such as "an average of 8 monomeric units" refers to an average of 8 monomeric units per polyglycerol derivative molecule in the population of polyglycerol derivative molecules. The average number of monomeric units per polyglycerol derivative molecule can be calculated based on the average molecular weight of the polyglycerol derivative.

[0345] In some embodiments, the polyglycerol derivatives of the present disclosure may comprise an average of 8 to 100 monomer units, such as 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 polyglycerol derivatives of the present disclosure comprise 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.

[0346] In some embodiments, the polyglycerol derivative can be a carboxylated polyglycerol, i.e., a polyglycerol in which the free alcohol groups have been modified by converting them into a moiety comprising one or more carboxylate groups, such as 2-carboxycyclohexane-1-carboxylated polyglycerol. In some embodiments, the polyglycerol derivative can be a glutarylated polyglycerol, i.e., a polyglycerol in which the free alcohol groups have been modified by converting them into 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) as described herein. In some embodiments, the polyglycerol derivative conjugated to a lipid moiety is represented by the following structural formula:

[0347]

[0348] in:

[0349] n is an integer ranging from 8 to 100; and

[0350] R chooses freedom The group composed of.

[0351] As used herein, the term "hydrophobic tail" refers to a hydrocarbon chain, i.e., a chain containing carbon and hydrogen atoms, which may be saturated or unsaturated. In one embodiment, the hydrophobic tail may comprise 10-30 carbon atoms, such as 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon atoms. In certain embodiments, the hydrocarbon chain of the hydrophobic tail is unsaturated, i.e., does not comprise a double bond or a triple bond. In other embodiments, the hydrocarbon chain of the hydrophobic tail is unsaturated, comprising one or more double bonds and / or one or more triple bonds. In certain embodiments, the hydrocarbon chain may be a straight chain. In other embodiments, the hydrocarbon chain may be a branched chain. 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, palmitic acid, homosapiens acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, trans-linoleic acid, α-trans-linoleic acid, arachidonic acid, eicosapentaenoic acid, and derivatives thereof.

[0352] As used herein, the term "click chemistry reaction product" refers to the moiety formed by the two click chemistry reagents in 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 trans-cyclooctene reagent (i.e., a reagent comprising a trans-cyclooctene 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.

[0353] As used herein, the term "lipid-anchored polymer" can be used interchangeably with the term "lipid conjugate" in this article and refers to a molecule comprising a lipid portion covalently attached to a polymer via a linker. Without wishing to be bound by a particular theory, it is believed that lipid-anchored polymers can inhibit the aggregation of LNPs and provide steric stabilization. Exemplary lipid-anchored polymers include, but are not limited to, polymer-conjugated lipids as described herein, PEGylated lipids such as PEG coupled to a dialkyloxypropyl group (e.g., PEG-DAA conjugate), PEG coupled to diacylglycerol (e.g., PEG-DAG conjugate), PEG coupled to dimyristyl glycerol (e.g., PEG-DMG), PEG coupled to distearoylglycerol (e.g., PEG-DSG), PEG coupled to poly (2-methacryloyloxyethylphosphorylcholine) (e.g., PEG-PMPC), PEG coupled to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (e.g., PEG-DS

[0013] Examples of the present invention include PEG (e.g., PEG-PEG), polyglycerol coupled to dioctadecylamine (e.g., DODA-PG), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine, and PEG conjugated to ceramide (see, e.g., U.S. Patent No. 5,885,613), cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates (e.g., POZ-DAA conjugates; see, e.g., U.S. Provisional Application No. 61 / 294,828, filed January 13, 2010, and U.S. Provisional Application No. 61 / 295,140, ​​filed January 14, 2010), polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof. Additional examples of POZ-lipid conjugates are described in International Patent Application Publication No. WO 2010 / 006282. PEG, PG, or POZ can be conjugated directly to the lipid or can be linked to the lipid via a linker moiety. Can use be suitable for any linker moiety with PEG, PG or POZ and lipid coupling, comprise for example the linker moiety that does not contain ester and the linker moiety that contains ester.In some preferred embodiment, use the linker moiety that does not contain ester, as amides or carbamate.The disclosure of each in the above-mentioned patent document is incorporated to this paper with its entirety by reference for all purposes.

[0354] Exemplary linkers that can be used to conjugate the lipid moiety to the polymer in the lipid-anchored polymer of the present disclosure can be 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 (e.g., a glutaryl linker, a succinyl linker), an amide linker, a diamide linker, an amine linker, a peptide linker, a phosphoethanolamine linker, a phosphorylcholine linker, a carbamate linker, a diamide alkyl linker, a cleavable linker, a click reaction product, and any combination thereof. In some embodiments, the linker can be 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 C(O)O-, wherein n is an integer ranging from 1 to 20. In some embodiments, the linker is -C(O)(CH2) n -, and wherein n is 2, 3, 4, 5 or 6. In a specific embodiment, n is 4.

[0355] As used herein, the term "encapsulated lipid" refers to a lipid particle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g., ceDNA), that is fully encapsulated, partially encapsulated, or both. In a preferred embodiment, the nucleic acid is fully encapsulated in the lipid particle (e.g., to form a nucleic acid containing lipid particle).

[0356] As used herein, the term "lipid granule" or "lipid nanoparticle" or "LNP" refers to a lipid formulation that can be used to deliver therapeutic agents such as nucleic acid therapeutics to a target site of interest (e.g., cells, tissues, organs, etc.). In one embodiment, lipid granules of the present disclosure are lipid granules containing nucleic acids, which are typically formed by cationic lipids, non-cationic lipids, and optionally conjugated lipids that prevent particle aggregation. In other preferred embodiments, therapeutic agents such as therapeutic nucleic acids can be encapsulated in the lipid portion of the granule, thereby protecting it from enzymatic degradation. In one embodiment, lipid granules comprise nucleic acids (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.

[0357] According to some embodiments, the average diameter of the lipid particles of the present disclosure is generally 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 75 nm, about 50 nm to about 70 nm, about 60 nm to about 75 nm, about 60 nm to about 75 nm, about 65 nm to about 70 nm. 70 nm, about 71 nm, about 72 nm, about 73 nm, about 74 nm, or about 75 nm (± 3 nm).

[0358] In general, the LNPs of the present disclosure have an average diameter selected to provide the desired therapeutic effect. For example, the LNPs of the present disclosure have an average diameter that is compatible with the target organ, such that the LNPs of the present disclosure can diffuse through fenestrations of the target organ (e.g., liver) or target cell subpopulations (e.g., hepatocytes).

[0359] According to some embodiments, the lipid particles of the present disclosure generally have a mean 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, less than about 20 nm.

[0360] As used herein, term " cationic lipid " refers to any lipid with positive charge at physiological pH.The cationic lipid in lipid granule can include for example one or more cationic lipids, such as 1,2-bis-linoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-bis-linoleoyloxy-N, N-dimethylaminopropane (DLenDMA), 1,2-bis-γ-linoleoyloxy-N, N-dimethylaminopropane (γ-DLenDMA), 2,2-bis-linoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-bis-linoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), " SS can cut lipid " or its mixture.In certain embodiments, cationic lipid can also be ionizable lipid, i.e. ionizable cationic lipid. That is, the term "cationic lipid" also encompasses positively charged lipids at any pH, for example, lipids comprising quaternary amine groups, i.e., quaternary lipids. Any cationic lipid comprising primary, secondary or tertiary amine groups as described herein can be, for example, by being converted into corresponding quaternary lipids by processing with acetonitrile (CH3CN) and chloroform (CHCl3) in methyl chloride (CH3Cl).

[0361] As used herein, the term "ionizable lipid" refers to a lipid having at least one protonatable or deprotonatable group, such as a cationic lipid, such that the lipid is positively charged at or below physiological pH (e.g., pH 7.4) and is neutral at a second pH, preferably at or above physiological pH. It will be understood by those of ordinary skill in the art that the addition or removal of protons as a function of pH is an equilibrium process, and reference to charged or neutral lipids refers to the properties of the predominant species, without requiring that all lipids exist in a charged or neutral form. Generally speaking, the pKa of the protonatable group of an ionizable lipid ranges from about 4 to about 7. In some embodiments, the ionizable lipid may include a "cleavable lipid" or a "SS cleavable lipid."

[0362] As used herein, the term "neutral lipid" refers to any of a plurality of lipid species that exist as uncharged or neutral zwitterionic forms at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, cephalins, cholesterol, cerebrosides, and diacylglycerols.

[0363] As used herein, the term "non-cationic lipid" refers to any amphiphilic helper lipid as well as any other neutral lipid or anionic lipid.

[0364] As used herein, the term "cleavable lipid" or "SS cleavable lipid" refers to an ionizable lipid comprising a disulfide cleavable unit. The cleavable lipid may include a cleavable disulfide bond ("ss") containing lipid-like material comprising a pH-sensitive amine, such as a tertiary amine and a self-degradable phenyl ester. For example, the SS cleavable lipid may be a ss-OP lipid ( SS-OP), ss-M lipid ( SS-M), ss-E lipids ( SS-E), ss-EC lipids ( SS-EC), ss-LC lipids ( SS-LC), ss-OC lipids ( SS-OC) and ss-PalmE lipids (see, e.g., Formulas I-IV) or lipids described by Togashi et al., (2018) Journal of Controlled Release "A hepatic pDNA delivery system based on anintracellular environment sensitive vitamin E-scaffold lipid-like material with the aid of an anti-inflammatory drug" 279:262-270. Additional examples of cleavable lipids are described in U.S. Pat. No. 9,708,628 and U.S. Pat. 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 acidic compartments, e.g., endosomes or lysosomes for membrane destabilization and a disulfide bond that can be cleaved in a reducing environment (e.g., 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.

[0365] As used herein, the term "organic lipid solution" refers to a composition comprising all or part of an organic solvent with lipids.

[0366] As used herein, the term "liposome" refers to lipid molecules assembled in a spherical configuration that encapsulates an internal aqueous volume separated from an aqueous exterior. Liposomes are vesicles with at least one lipid bilayer. In the context of pharmaceutical research and development, liposomes are typically used as carriers for drug / therapeutic agent delivery. Liposomes work by fusing with cell membranes and repositioning their lipid structure to deliver drugs or active pharmaceutical ingredients. The liposome compositions for this type of delivery are typically composed of phospholipids (particularly compounds with phosphatidylcholine groups), but these compositions can also include other lipids.

[0367] As used herein, the term "local delivery" refers to the direct delivery of an active agent, such as an interfering RNA (e.g., siRNA), to a target site within an organism. For example, an agent can be delivered locally by direct injection into a disease site, such as a tumor or other target site, such as an inflammatory site or a target organ, such as the liver, heart, pancreas, kidney, etc.

[0368] As used herein, the term "nucleic acid" refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in single-stranded or double-stranded form and includes DNA, RNA, and hybrids thereof. The 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, a chromosomal DNA, or derivatives and combinations of these groups. The DNA can be in the form of a minicircle, a plasmid, a bacmid, a minigene, a ministring DNA (linear covalently closed DNA vector), an end-blocked linear duplex DNA (CELiD or ceDNA), a doggybone TM The nucleic acid may be in the form of a DNA, dumbbell-shaped DNA, minimalistic immunologically defined gene expression (MIDGE) vector, viral vector or non-viral vector. RNA may be in the form of small interfering RNA (siRNA), Dicer substrate dsRNA, small 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 bonds, which are synthetic, naturally occurring and non-naturally occurring, and which have binding properties similar to reference nucleic acids. Examples of such analogs and / or modified residues include, but are not limited to, phosphorothioates, diamidophosphorothioate morpholino oligomers (morpholinos), phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2′-O-methyl ribonucleotides, locked nucleic acids (LNAs). TM) and peptide nucleic acids (PNA). Unless otherwise 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 sequences explicitly indicated.

[0369] As used herein, the phrases "nucleic acid therapeutic," "therapeutic nucleic acid," and "TNA" are used interchangeably and refer to any modality of therapeutic agent that uses nucleic acid as the active ingredient of a therapeutic agent for treating a disease or condition. As used herein, these phrases refer to RNA-based therapeutics and DNA-based therapeutics. Non-limiting examples of RNA-based therapeutics include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), Dicer substrate dsRNA, small 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., lentiviral or AAV genomes) or non-viral synthetic DNA vectors, end-blocked linear duplex DNA (ceDNA / CELiD), single-stranded DNA (ssDNA) molecules, plasmids, bacmids, DOGGYBONE TM DNA vectors, minimalist immunologically defined gene expression (MIDGE) vectors, non-viral ministring DNA vectors (linear covalently enclosed DNA vectors), or dumbbell-shaped minimal DNA vectors ("dumbbell DNA").

[0370] As used herein, a "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. The nucleotides are linked together by the phosphate groups.

[0371] 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 flanked by 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), such as a stem-loop structure, such as an inverted terminal repeat or portion thereof, at one 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.

[0372] As used herein, the terms "single-stranded (ss) synthetic DNA molecules," "single-stranded (ss) synthetic vectors," "synthetic production of ss DNA molecules," and "synthetic production of ss vectors" refer to single-stranded (ss) synthetic DNA molecules (ssDNA), single-stranded vectors, and methods for synthetic production thereof in a completely cell-free environment. The production can involve one or more molecules without the need for replication or other multiplication of the molecules by or within cells or using cell extracts. Synthetic production avoids contamination of the produced molecules with cellular contaminants, such as cellular proteins or nucleic acids, viral proteins or DNA, insect proteins or DNA, and further minimizes unwanted cell-specific modifications of the molecules, such as methylation or glycosylation or other post-translational modifications, during the production process.

[0373] As used herein, the term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, emulsions such as oil / water or water / oil, and various types of wetting agents. The term also encompasses any agent approved by U.S. federal regulatory agencies 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 abrogate the biological activity and properties of the administered compound.

[0374] As used herein, the term "gap" refers to an intercalated portion of a synthetic DNA vector of the present disclosure that creates a single-stranded DNA portion within an otherwise double-stranded ceDNA. In one strand of the duplex DNA, the length of the gap can be 1 nucleotide to 100 nucleotides. 55, 56, 57, 58, 59, or 60 nt in length. Exemplary gaps designed and produced by the methods herein and synthetic vectors produced by the methods 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.

[0375] As used herein, the term "nick" refers to a discontinuity in a double-stranded DNA molecule in which the phosphodiester bonds between adjacent nucleotides of one strand are no longer present, typically by damage or enzymatic action. It will be understood that one or more nicks allow for the release of torsion in the strand during DNA replication, and nicks are also believed to play a role in facilitating the binding of the transcription machinery.

[0376] As used herein, the term "ceDNA" refers to a capsid-free, end-closed, linear, double-stranded (ds) duplex DNA for non-viral gene transfer, synthesis, or other forms. A detailed description of ceDNA is described 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 generating ceDNA comprising various inverted terminal repeat (ITR) sequences and configurations using cell-based methods are described in Example 1 of International Application Nos. PCT / US18 / 49996, filed September 7, 2018, and 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 comprising various ITR sequences and configurations are described, for example, in international application PCT / US2019 / 14122 filed on 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 minimalistic immunologically defined gene expression (MIDGE) vector. According to some embodiments, the ceDNA is a ministring DNA. According to some embodiments, the ceDNA is a doggybone TM 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.

[0377] As used herein, the term "neDNA" or "nicked ceDNA" refers to end-closed DNA having a nick or gap of 1-100 nucleotides in the stem or spacer region upstream of the open reading frame (e.g., the promoter and transgene to be expressed).

[0378] 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 comprising a partial duplex and at least one loop.

[0379] 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 "stem") and at least one single-stranded region (referred to herein as "loop"). In some embodiments, the stem-loop structure is a hairpin structure. In some embodiments, the stem-loop structure comprises more than one stem and more than one loop. In some embodiments, the loop is located at the end of the stem (so that a single loop connects the two chains of the duplex stem, for example, as in a hairpin structure). In some embodiments, the loop can be located between the two stems (which can be referred to herein as a "bulge" or "bubble") so that the loop connects the two chains of different stems. In some embodiments, as described in more detail herein, the stem-loop structure can comprise a more complex secondary structure comprising multiple stems and multiple loops.

[0380] According to some embodiments, the 5' and / or 3' ends of certain ssDNA molecules contain inverted terminal repeats (ITRs) of approximately 145 nucleotides at either end or at fragments thereof. The terminal 125 nucleotides in each ITR form a palindromic double-stranded T-shaped hairpin structure, in which the A-A' palindrome forms the stem and two smaller palindromes B-B' and C-C' form the cross arms of the T. The other 20 nucleotides in the ITR remain single-stranded and are referred to as D sequences. A D(-) sequence (also referred to herein as a "ssD(-) sequence") is at the 3' end, and a complementary D(+) sequence (also referred to herein as a "ssD(+) sequence") is at the 5' end. Second-strand DNA synthesis converts both the ssD(-) and ssD(+) sequences into double-stranded (ds)D(±) sequences, each of which comprises a D region and a D' region. Ling et al. J Virol. 2015 Jan 15;89(2):952-61, WO2016081927A2, herein incorporated by reference in their entirety, describe ssAAV genomes substituted with ssD(+)-sequences. ssD(-) and ssD(+) have been reported to 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, herein incorporated by reference in their entirety).

[0381] According to some embodiments, the ITR can be a viral ITR (e.g., AAV or other dependent virus), a sequence derived or modified (e.g., truncated, deleted, substituted, inserted and / or added) from a viral ITR, or a completely artificial sequence (e.g., the ITR does not contain a sequence derived from a virus). The ITR can further comprise a stem-loop structure (e.g., a "hairpin") or more than one stem-loop structure. For example, the ITR can comprise two stem-loop structures (e.g., a "hammerhead," "doggy-bone," or "dumbbell"), three stem-loop structures (e.g., a "cross"), or a more complex structure (e.g., a quadruplex stem-loop structure). The ITR can comprise an aptamer sequence or one or more chemical modifications. The ITR can be made entirely of an aptamer sequence having at least one stem region and at least one loop region.

[0382] As used herein, the term "substantially symmetrical WT-ITR" or "substantially symmetrical WT-ITR pair" refers to a pair of WT-ITRs within a single-stranded DNA (ssDNA) molecule, both of which are wild-type ITRs, having reverse complementary sequences across their entire length. For example, even if an ITR has one or more nucleotides that deviate from a typical naturally occurring canonical sequence, the sequence can be considered a wild-type sequence as long as these changes do not affect the physical and functional properties and overall three-dimensional structure (secondary and tertiary structure) of the sequence. In some aspects, the deviated nucleotides represent conservative sequence changes. As a non-limiting example, a sequence has at least 95%, 96%, 97%, 98% or 99% sequence identity to the canonical sequence (as measured, for example, using BLAST at default settings) and also has a symmetrical three-dimensional spatial organization with another WT-ITR, such that their 3D structures have the same shape in geometric space. Substantially symmetrical WT-ITRs have identical ssD(-) / ssD(+), A-A', C-C' and B-B' loops in 3D space. By confirming that the substantially symmetrical WT-ITR has an operable Rep binding site (RBE or RBE') and a terminal melting site (TRS) that are paired with the appropriate Rep protein, it can be functionally confirmed as WT. Other functions can optionally be tested, including transgene expression under permissive conditions.

[0383] As used herein, the phrases "modified ITR" or "mod-ITR" or "mutant ITR" are used interchangeably and refer to an ITR that has a mutation in at least one or more nucleotides compared to a WT-ITR from the same serotype. The mutation may result in a change in one or more of the ssD(-) or ssD(+), A, A', C, C', B, B' regions of the ITR, and may result in a change in the three-dimensional organization (i.e., its 3D structure in geometric space) compared to the 3D organization of a WT-ITR from the same serotype.

[0384] The terms "polynucleotide" and "nucleic acid" used interchangeably herein refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derived nucleotide bases. An "oligonucleotide" generally refers to a polynucleotide of about 5 to about 100 nucleotides of single-stranded or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also referred to as "oligomers" or "oligos" and can be isolated from genes or chemically synthesized by methods known in the art. It should be understood that the terms "polynucleotide" and "nucleic acid" include single-stranded (e.g., sense or antisense) and double-stranded polynucleotides (if applicable in the described embodiment). According to some embodiments, a nucleic acid is a single-stranded DNA (ssDNA) molecule described by the present disclosure. The DNA can be in the form of, for example, an antisense molecule, plasmid DNA, DNA-DNA duplex, precondensed DNA, PCR product, vector (P1, PAC, BAC, YAC, artificial chromosome), expression cassette, chimeric sequence, chromosomal DNA, or derivatives and combinations of these groups. The DNA can be in the form of a minicircle, plasmid, bacmid, minigene, ministring DNA (linear covalently closed DNA vector), end-blocked linear duplex DNA (CELiD or ceDNA), doggybone (dbDNA), or a combination of these groups. TM ) DNA, dumbbell-shaped DNA, minimalist immunologically defined gene expression (MIDGE) vectors, viral vectors or non-viral vectors. RNA can be in the form of small interfering RNA (siRNA), Dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, rRNA, tRNA, viral RNA (vRNA) and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring and non-naturally occurring, and have binding properties similar to reference nucleic acids. Examples of such analogs and / or modified residues include, but are not limited to, phosphorothioates, phosphorodiamidate morpholino oligomers (morpholinos), phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methyl ribonucleotides, locked nucleic acids (LNAs) TM) and peptide nucleic acids (PNA). Unless otherwise 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 sequences explicitly indicated.

[0385] As used herein, an "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 and RNA molecules, for example, RNAi molecules that encode the actual inhibitory species, such as DNA molecules that encode ribozymes.

[0386] A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. The nucleotides are linked together by the phosphate groups.

[0387] "Bases" include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine and natural analogs, 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.

[0388] "Receptor" means 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 particularly include those regions sufficient for specific binding of the ligand.

[0389] As used herein, the term "subject" refers to a human or animal to which treatment with a therapeutic nucleic acid according to the present disclosure is provided, including preventive and therapeutic treatment. Generally speaking, an animal is a vertebrate, such as, but not limited to, a primate, a rodent, a domestic animal, or a wild animal. Primates include, but are not limited to, chimpanzees, crab-eating macaques, spider monkeys, and macaques, such as rhesus macaques. Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Domestic animals and wild animals include, but are not limited to, cattle, horses, pigs, deer, bison, buffaloes, feline species (e.g., house 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. In addition, the subject can be an infant or a child. In certain embodiments, the subject can be a newborn or unborn subject, e.g., the subject is in the womb. Preferably, the subject is a mammal. Mammals can be people, non-human primates, mice, rats, dogs, cats, horses or cattle, but are not limited to these examples. Mammals other than people can advantageously be used as the subject of the animal model representing disease and illness. In addition, methods and compositions described herein can be used for domestic animals and / or pets. Human subjects can have any age, sex, race or ethnic group, for example, Caucasians (white people), Asians, Africans, blacks, African Americans, African Europeans, Spaniards, Middle Easterners etc. In certain embodiments, the subject can be a patient or other subject in a clinical setting. In certain embodiments, the subject has been treated. In certain embodiments, the subject is an embryo, fetus, newborn, baby, child, teenager or adult. In certain embodiments, the subject is a human fetus, human newborn, human baby, human child, human teenager or human adult. In certain embodiments, the subject is an animal embryo or a non-human embryo or a non-human primate embryo. In certain embodiments, the subject is a human embryo.

[0390] As used herein, unless the context and usage of the phrase indicate otherwise, the phrase "a subject in need thereof" refers to a subject to whom (i) a ceDNA lipid particle according to the disclosure (or a pharmaceutical composition comprising ceDNA lipid particles) as described will be administered, (ii) is currently receiving a ceDNA lipid particle according to the disclosure (or a pharmaceutical composition comprising ceDNA lipid particles); or (iii) has received a ceDNA lipid particle according to the disclosure (or a pharmaceutical composition comprising ceDNA lipid particles).

[0391] As used herein, the terms "suppress," "reduce," "interfere with," "inhibit," and / or "reduce" (and similar terms) generally refer to the act of directly or indirectly decreasing a concentration, level, function, activity, or behavior relative to natural, expected, or average conditions, or relative to a control condition.

[0392] As used herein, the term "systemic delivery" refers to the delivery of lipid particles that produce a wide range of biodistributions of active agents such as interfering RNA (e.g., siRNA) in vivo. Some administration techniques can produce systemic delivery of certain medicaments rather than other medicaments. Systemic delivery means exposing a useful amount (preferably, a therapeutic amount) of the medicament to most parts of the body. In order to obtain a wide range of biodistribution, a blood life span is usually required so that the medicament will not be rapidly degraded or cleared (e.g., by first-pass organs (liver, lungs, etc.) or by fast, non-specific cell binding) before arriving at the disease site at the distal end of the administration site. The systemic delivery of LNP can be carried out by any means known in the art (including, for example, intravenous, subcutaneous, and intraperitoneal). In a preferred embodiment, the systemic delivery of LNP is by intravenous delivery.

[0393] As used herein, the term "effective amount" can 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 as described herein), which refers to an amount sufficient to provide the intended benefit of a treatment or effect, e.g., expression of a target sequence or inhibition of expression 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, examining protein or RNA levels using techniques known to those skilled in the art (e.g., dot blots, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function), as well as phenotypic assays known to those skilled in the art. Dosage levels are based on various factors, including the type of injury, the patient's age, weight, sex, medical condition, severity of the condition, route of administration, and the specific active agent employed. 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 the described prophylactic or preventative amounts of the compositions of the present invention. In the described preventive or prophylactic application of the present invention, to the patient who is susceptible to disease, illness or condition or otherwise is in the risk of the disease, illness or condition with enough to eliminate or reduce the risk of the disease, illness or condition, alleviate its severity or delay the amount of its outbreak, administer pharmaceutical composition or medicine, the disease, illness or condition include the biochemistry of the disease, illness or condition, histology and / or behavioral symptoms, its complication and the intermediate pathological phenotype presented during the development of the disease, illness or condition. It is usually preferred to use the maximum dose, that is, the highest safe dose according to some medical judgments. The terms "dose (dose)" and "dosage (dosage)" are used interchangeably in this article. In one aspect of any aspect or embodiment of this article, "therapeutic amount", "therapeutically effective dose" and "pharmaceutically effective dose" refer to non-preventive or non-preventive applications.

[0394] As used herein, the term "therapeutic effect" refers to the result of a treatment that is determined to be desirable and beneficial. A therapeutic effect can directly or indirectly include the containment, reduction, or elimination of disease manifestations. A therapeutic effect can also directly or indirectly include the containment, reduction, or elimination of the progression of disease manifestations.

[0395] For any therapeutic agent described herein, a therapeutically effective amount can be initially determined based on preliminary in vitro studies and / or animal models. A therapeutically effective dose can also be determined based on human data. The administered dose can be adjusted based on the relative bioavailability and the efficacy of the administered compound. It is within the capabilities of ordinary technicians to adjust the dose to achieve maximum efficacy based on the above methods and other well-known methods. The general principles for determining therapeutic effectiveness are summarized below, which can be found in Chapter 1 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th edition, McGraw-Hill (New York) (2001), which is incorporated herein by reference.

[0396] Pharmacokinetic principles provide the basis for modifying the dosage regimen to obtain the desired degree of therapeutic efficacy with minimal unacceptable side effects. Additional guidance for dosage modifications can be obtained where the plasma concentration of the drug can be measured and related to the therapeutic window.

[0397] As used herein, the terms "treat," "treating," and / or "treatment" include eliminating, inhibiting, slowing, or reversing the progression of a condition, improving the clinical symptoms of a condition, or preventing the appearance of clinical symptoms of a condition, obtaining a beneficial or desired clinical outcome. Treatment further refers to accomplishing one or more of: (a) reducing the severity of the condition; (b) limiting the development of symptoms specific to the condition being treated; (c) limiting the worsening of symptoms specific to the condition being treated; (d) limiting the recurrence of the condition in patients who previously had the condition; and (e) limiting the recurrence of symptoms in patients who were previously asymptomatic for the condition. In one aspect of any aspect or embodiment herein, the terms "treat," "treating," and / or "treatment" include eliminating, inhibiting, slowing, or reversing the progression of a condition, or improving the clinical symptoms of a condition.

[0398] Beneficial or desired clinical results, such as pharmacological and / or physiological effects, include, but are not limited to: preventing the development of a disease, disorder or condition in a subject who may be susceptible to the disease, disorder or condition but who does not yet experience or exhibit symptoms of the disease (prophylactic or preventive treatment); alleviating the symptoms of the disease, disorder or condition; reducing the extent 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 palliating the disease, disorder or condition; and combinations thereof, and prolonging survival as compared to expected survival if not receiving treatment.

[0399] As used herein, the term "combination therapy" refers to a treatment regimen comprising two or more therapeutic agents for clinical indications. Therefore, the term refers to such a treatment regimen, wherein the first therapy comprising the first composition (such as active ingredient) is combined with the second therapy comprising the second composition (active ingredient) and is administered to the patient, for the purpose of treating identical or overlapping diseases or clinical symptoms. The first composition and the second composition can both act on the same cell target or discrete cell target. In the context of combination therapy, phrase "combination" means that in the subject receiving combination therapy, the therapeutic effect of the first therapy is temporarily and / or spatially overlapped with the therapeutic effect of the second therapy. Therefore, combination therapy can be formulated into a single preparation for simultaneous administration, or formulated into a separate preparation for the sequential administration of therapy.

[0400] As used herein, the term "alkyl" refers to a saturated monovalent hydrocarbon group having 1 to 20 carbon atoms (i.e., C 1-20 Alkyl). "Monovalent" means that the alkyl group has one point of attachment to the rest of the molecule. In one embodiment, the alkyl group 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, the alkyl group 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. Straight or branched chain alkyl groups, such as “straight or branched chain C 1-6 Alkyl", "straight or branched chain C 1-4 Alkyl" or "straight or branched C 1-3 "Alkyl" means a saturated monovalent hydrocarbon group that is straight or branched. As used herein, the term "straight chain" as used in reference to an aliphatic hydrocarbon chain means that the chain is unbranched.

[0401] 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), examples of which include, but are not limited to, alkylene groups having the same core structure as the alkyl groups exemplified above. "Divalent" means that the alkylene group has two points of attachment to the rest of the molecule. In one embodiment, the alkylene group has 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 group 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 1-3 Alkylene), ethylene or methylene. Straight chain or branched chain alkylene, such as "straight chain or branched chain C 1-6 Alkylene", "straight or branched C 1-4 "Alkylene" or "straight or branched C 1-3 "Alkylene" means a saturated divalent hydrocarbon group which is straight or branched.

[0402] The term "alkenyl" refers to a straight or branched aliphatic hydrocarbon group having one or more (e.g., one or two) carbon-carbon double bonds, wherein the alkenyl group includes groups having "cis" and "trans" orientations, or, by alternative nomenclature, "E" and "Z" orientations.

[0403] The term "alkenylene" refers to an aliphatic divalent hydrocarbon radical of 2 to 20 carbon atoms (i.e., C2-20 "Alkenylene"), wherein the alkenylene includes groups having "cis" and "trans" orientations, or by alternative nomenclature, "E" and "Z" orientations. "Divalent" means that the alkenylene has two points of attachment to the rest of the molecule. In one embodiment, the alkenylene has 2 to 12 carbon atoms (i.e., C 2-12 alkenyl), 2 to 10 carbon atoms (i.e., C 2-10 In one embodiment, the alkenylene group has 2 to 4 carbon atoms (C 2-4 Examples include, but are not limited to, ethylenylene or vinylene (-CH=CH-), allyl (-CH2CH=CH-), etc. Straight-chain or branched alkenylene, such as "straight-chain or branched C 2-6 Alkenylene", "straight or branched chain C 2-4 Alkenylene" or "straight or branched C 2-3 "Alkenylene" means an unsaturated divalent hydrocarbon group which is straight or branched.

[0404] As used herein, term " cycloalkylidene " refers to the divalent saturated carbocyclic radical with 3 to 12 carbon atoms as monocycle or 7 to 12 carbon atoms as bicyclic. " divalent " means that cycloalkylidene has two connection points with the remainder of molecule. In one embodiment, cycloalkylidene is 3 to 7 yuan monocycle or 3 to 6 yuan monocycle. The example of monocyclic cycloalkyl includes but is not limited to cyclopropene, cyclobutylidene, cyclopentylidene, cyclohexylidene, cycloheptylidene, cyclooctylidene, cyclononylidene, cyclodecylidene, cycloundecylidene, cyclododecylidene etc. In one embodiment, cycloalkylidene is cyclopropene.

[0405] The terms "heterocycle", "heterocyclyl", heterocyclic and "heterocyclic ring" are used interchangeably herein and refer to a cyclic group containing at least one N atom, with a heteroatom and optionally 1 to 3 other heteroatoms selected from N and S, and are non-aromatic (i.e., partially or completely saturated). The heterocycle can be monocyclic or bicyclic (bridged or fused). The example of heterocycle includes but is not limited to aziridine, diaziridine, thiaaziridine, azetidinyl, diazetidinyl, triazetidinyl, thiadiazetidinyl, thiazetidinyl, pyrrolidinyl, pyrazolidinyl, imidazolinyl, isothiazolidinyl, thiazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl, azepanyl, azooctanyl etc. Heterocycle contains 1 to 4 heteroatoms selected from N and S that may be the same or different. In one embodiment, 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" means a group 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" means a group 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), particularly Chapters 1, 3, 4, 6, 7, and 9; The Chemistry of Heterocyclic Compounds, A Series of Monographs (John Wiley & Sons, New York, 1950-present), particularly Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82: 5566. Where possible, the heterocyclic group can be carbon (carbon-bonded) or nitrogen (nitrogen-bonded) attached to the rest of the molecule.

[0406] If a group is described as "optionally substituted," the group may be (1) unsubstituted or (2) substituted. If 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, if any, may be replaced individually and / or collectively with independently selected optional substituents.

[0407] Suitable substituents for alkyl, alkylene, alkenylene, cycloalkylene and heterocyclyl groups are substituents that do not significantly adversely affect the biological activity of the molecule. Unless otherwise specified, exemplary substituents for these groups include straight chain, branched chain or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, aryl, heteroaryl, heterocyclyl, halogen, guanidinium salts [-NH(C=NH)NH2], -OR 100 NR 101 R 102 、-NO2、-NR 101 COR 102 、-SR 100 , by-SOR 101 Sulfoxide represented by -SO2R 101 Sulfone, sulfonate -SO3M, sulfate -OSO3M, -SO2NR 101 R 102 Sulfonamide, cyano, azide, -COR 101 、-OCOR 101 、-OCONR 101 R 102 and polyethylene glycol units (-OCH2CH2) n R 101 , where M is H or a cation (such as Na + or K + );R 101 、R 102 and R 103 Each independently selected from H, a linear, branched or cyclic alkyl group, an alkenyl or alkynyl group 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 group having 6 to 10 carbon atoms, a heterocyclic ring having 3 to 10 carbon atoms, and a heteroaryl group having 5 to 10 carbon atoms; and R 104 is H or a straight or branched chain alkyl group having 1 to 4 carbon atoms, wherein R 100 、R 101 、R 102 、R 103 and R 104The alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclic groups in the group represented by are optionally substituted by one or more (e.g., 2, 3, 4, 5, 6 or more) substituents independently selected from halogen, -OH, -CN, -NO2 and unsubstituted straight or branched alkyl groups having 1 to 4 carbon atoms. Preferably, the substituents of the above-mentioned optionally substituted alkyl, alkylene, alkenylene, cycloalkylene and heterocyclic groups are selected from the group consisting of halogen, -CN, -NR 101 R 102 、-CF3、-OR 100 , aryl, heteroaryl, heterocyclic, -SR 101 、-SOR 101 、-SO2R 101 Alternatively, suitable substituents are selected from the group consisting of halogen, -OH, -NO2, -CN, C 1-4 Alkyl, -OR 100 NR 101 R 102 、-NR 101 COR 102 、-SR 100 、-SO2R 101 、-SO2NR 101 R 102 、-COR 101 、-OCOR 101 and-OCONR 101 R 102 , where R 100 、R 101 and R 102 Each independently is -H or C 1-4 alkyl.

[0408] As used herein, the term "halogen" refers to F, Cl, Br, or I. "Cyano" is -CN.

[0409] The terms "amine" or "amino" are used interchangeably herein and refer to a functional group containing a single basic nitrogen atom.

[0410] 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 ester, isonicotinic acid, lactic acid, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, sugar ester, formate, benzoate, glutamate, methanesulfonate "mesylate", ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-hydroxy-1,3-diol)-1,3-diol) and 1,1'-methylene-bis-(2-hydroxy-3-hydroxy-1,3-diol)-1,3-diol). -naphthoate) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. Pharmaceutically acceptable salts may involve the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of a pharmaceutically acceptable salt may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0411] The grouping of the alternative elements of the present disclosure disclosed herein or embodiments should not be construed as limiting.Each group member can be mentioned and claimed individually or with any combination of other elements found by other members of the group or this paper.For the purpose of convenience and / or patentability, one or more members in a group can be included in the group or deleted from the group.When any such inclusion or lack occurs, description is considered herein as comprising the group after amendment, thereby meeting the written description of all Markush groups (Markush group) used in the appended claims.

[0412] 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 may cause suffering to the humans or animals without any substantial medical benefit to the humans or animals, and animals resulting from such processes.

[0413] Additional terms are defined herein within the description of various aspects of the disclosure.

[0414] All patents and other publications cited in this application in full, including references, granted patents, published patent applications, and co-pending patent applications, are expressly incorporated herein by reference for the purpose of describing and disclosing the methods that can be used in combination with the technology described herein, such as those described in such publications. Only the disclosures of these publications prior to the filing date of this application are provided. In this regard, nothing should be construed as admitting that the inventor has no right to disclose in advance by means of prior disclosure or for any other reason. All statements about the dates or contents of these documents are based on information available to the applicant and do not constitute any admission to the correctness of the dates or contents of these documents.

[0415] 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. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications can be made within the scope of the present disclosure as will be appreciated by those skilled in the relevant art. For example, although method steps or functions are presented in a given order, alternative embodiments can perform functions in a different order or can perform functions substantially in parallel. The teachings of the present disclosure provided herein can be appropriately applied to other programs or methods. The various embodiments described herein can be combined to provide additional embodiments. If necessary, various aspects of the present disclosure can be modified to provide further embodiments of the present disclosure using the compositions, functions, and concepts in the above-mentioned references and applications. In addition, due to biological functional equivalence considerations, some changes can be made in the protein structure without affecting the type or amount of biological or chemical effects. These and other changes can be made to the present disclosure based on the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0416] The specific elements of any of the foregoing embodiments may be combined with or replace the elements in other embodiments. In addition, although the advantages associated with certain embodiments of the present disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the present disclosure.

[0417] The technology described herein is further illustrated by the following examples, but these examples should not be construed as further limiting. It should be understood that the present disclosure is not limited in any way to the specific methods, protocols, reagents, etc. described herein and can vary accordingly. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present disclosure, which is defined solely by the claims.

[0418] II. Polymer-conjugated lipids

[0419] In some embodiments, the present disclosure provides a polymer-conjugated lipid comprising: (i) polyglycerol (PG) or a PG derivative; (ii) a lipid portion; and (iii) a linker that conjugates the PG or the PG derivative to the lipid portion.

[0420] Polyglycerol and polyglycerol derivatives

[0421] The PG or PG derivative contained in the polymer-conjugated lipid of the present disclosure can be linear or branched. In a specific embodiment, the PG or the PG derivative is linear. In another embodiment, the PG or the PG derivative is branched.

[0422] The polymer-conjugated lipids of the present disclosure may comprise PG or a PG derivative comprising an average of 5 to 100 monomer units, such as an average of 10 to 100 monomer units, such as an average of 10 to 40 monomer units, an average of 15-75 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 comprise 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.

[0423] In one embodiment, the PG or PG derivative of the present disclosure comprises an average of 8 monomer units.

[0424] In one embodiment, the PG or PG derivative of the present disclosure comprises an average of 34 monomer units.

[0425] In one embodiment, the PG or PG derivative of the present disclosure comprises an average of 45 monomer units.

[0426] In one embodiment, the PG or PG derivative of the present disclosure comprises an average of 46 monomer units.

[0427] In one embodiment, the PG or PG derivative of the present disclosure comprises an average of 58 monomer units.

[0428] In one embodiment, the polymer-conjugated lipid of the present disclosure comprises PG.

[0429] In one embodiment, the polymer-conjugated lipids of the present disclosure comprise a PG derivative.

[0430] The PG derivative contained in the polymer-conjugated lipid of the present disclosure may be a carboxylated PG, for example, 2-carboxycyclohexane-1-carboxylated polyglycerol.

[0431] The PG derivative contained in the polymer-conjugated lipid of the present disclosure may also be glutarylated PG, for example, 3-methylglutarylated PG.

[0432] In some embodiments, the PG derivative contained in the polymer-conjugated lipid of the present disclosure is represented by the following structural formula:

[0433]

[0434] in:

[0435] n is an integer ranging from 8 to 100; and

[0436] R chooses freedom The group composed of.

[0437] lipid part

[0438] In some embodiments, the lipid moiety contained in the polymer-conjugated lipid of the present disclosure is represented by formula (I)

[0439]

[0440] or a pharmaceutically acceptable salt thereof, wherein:

[0441] R 1 Absent, hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10-30 carbon atoms;

[0442] R 2 Absent, hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10-30 carbon atoms;

[0443] When R 1 and R 2 When each is hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10-30 carbon atoms, N is positively charged; and

[0444] R 3 A hydrophobic tail comprising 10-30 carbon atoms;

[0445] wherein in formula (I) is the bond that conjugates the lipid moiety and the linker.

[0446] In some embodiments, R 1 does not exist, and R 2 and R 3 Each independently comprises a hydrophobic tail of 14, 15, 16, 17, 18, 19, 20, 21 or 22 carbon atoms. 2 and R 3 Each independently comprises a hydrophobic tail of 16, 17, 18, 19, 20, 21 or 22 carbon atoms. 2 and R 3 Each is independently a hydrophobic tail comprising 18 carbon atoms, and the lipid moiety is dioctadecylamine (DODA).

[0447] connector

[0448] The linker conjugating the polyglycerol or polyglycerol derivative to the lipid portion of 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, etc.), an amide linker, a diamide linker, an amine linker, a peptide linker, a phosphoethanolamine linker, a phosphorylcholine linker, a carbamate linker, a diamide alkyl linker, a cleavable linker, or any combination thereof.

[0449] In some embodiments, the linker can be 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 C(O)O-, wherein n is an integer ranging from 1 to 20. In some embodiments, the linker is -C(O)(CH2) n -, and n is 2, 3, 4, 5 or 6. In one embodiment, n is 4.

[0450] In some embodiments, the polymer-conjugated lipid of the present disclosure is DODA-PG34 represented by the following structure:

[0451]

[0452] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0453] In some embodiments, the polymer-conjugated lipid of the present disclosure is DODA-PG45 represented by the following structure:

[0454]

[0455] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0456] In some embodiments, the polymer-conjugated lipid of the present disclosure is DODA-PG46 represented by the following structure:

[0457]

[0458] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0459] In some embodiments, the polymer-conjugated lipid of the present disclosure is DODA-PG58 represented by the following structure:

[0460]

[0461] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0462] In some embodiments, the polymer-conjugated lipids of the present disclosure can be represented by the following structure

[0463]

[0464] Where R is

[0465] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0466] The polymer-conjugated lipids of the present disclosure may also comprise a targeting moiety. Various targeting moieties that may be included in the polymer-conjugated lipids of the present disclosure are described herein in the "Targeting Moiety" section.

[0467] The polymer-conjugated lipids of the present disclosure may also include an active species conjugated to the PG or the PG derivative. The active species present in the polymer-conjugated lipids of the present disclosure can be used for conjugation, for example, with a targeting moiety that has been functionalized with a complementary active species, i.e., an active species capable of reacting with the active species contained in the polymer-conjugated lipids of the present disclosure. In some embodiments, the active species conjugated to the polymer-conjugated lipids of the present disclosure can be a thiol reagent, a maleimide reagent, or a click chemistry reagent, for example, a reagent selected from the group consisting of: an alkyne reagent, such as a dibenzocyclooctyne (DBCO) reagent, a trans-cyclooctene (TCO) reagent, a tetrazine (TZ) reagent, and an azide (AZ) reagent.

[0468] In some embodiments, the polymer-conjugated lipids of the present disclosure may comprise a targeting moiety that has been conjugated to the polymer-conjugated lipid by an active species. For example, a polymer-conjugated lipid comprising an azide reagent as an active species of the present disclosure can be reacted with a targeting moiety functionalized with a DBCO reagent as a complementary active species to produce a polymer-conjugated lipid comprising a targeting moiety. In another example, a polymer-conjugated lipid comprising a thiol reagent of the present disclosure can be reacted 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 a polymer-conjugated lipid of the present disclosure.

[0469] Synthesis of polymer-conjugated lipids

[0470] The polymer-conjugated lipids of the present disclosure wherein the polymer is PG can be synthesized by a method comprising:

[0471] (a) reacting the lipid portion conjugated to a linker with 2,3-epoxy-1-(1-ethoxyethoxy)propane (EEGE) in the presence of a base or in the presence of an organic catalyst under an argon atmosphere to produce the lipid portion conjugated to a linker and polymeric EEGE; and

[0472] (b) subjecting the lipid moiety conjugated to a linker and the polymeric EEGE to acidic conditions to produce the polymer-conjugated lipid.

[0473] In some embodiments, the molar ratio of the lipid moiety conjugated to the linker to the EEGE can vary from about 1:20 to about 1:100. For example, in step (a), the molar ratio of the lipid moiety conjugated to the linker to the 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, such as 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 the lipid moiety conjugated to the linker to the EEGE can be about 1:50. In another embodiment, the ratio of lipid moiety conjugated to the linker to EEGE may be about 1:60.

[0474] In some embodiments, the ratio of the linker-conjugated lipid moiety to the EEGE in step (a) determines the average number of monomer units present in the PG moiety of the polymer-conjugated lipid in the final product.

[0475] The base that can be used to carry out step (a) of the process may be a phosphazene base, such as P4-t-Bu.

[0476] In some embodiments, the organic catalyst useful in performing step (a) of the method may be an N-heterocyclic carbene (NHC) or an N-heterocyclic olefin (NHO).

[0477] In some embodiments, step (a) can be performed overnight.

[0478] Acidic conditions that can be used to perform step (b) of the 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 (HClO4), chloric acid (HClO3), sulfuric acid (H2SO4) and nitric acid (HNO3).

[0479] In some embodiments, the lipid moiety is DODA, and the lipid moiety conjugated to the linker is represented by the following structure:

[0480]

[0481] In some embodiments, the polymer-conjugated lipid is DODA-PG, wherein the PG comprises an average of 5-100 monomer units. In some embodiments, the polymer-conjugated lipid is DODA-PG34, DODA-PG45, DODA-PG46, or DODA-PG58.

[0482] III. Lipid Nanoparticles (LNPs)

[0483] The present disclosure also provides lipid nanoparticles (LNPs) comprising: (i) a therapeutic nucleic acid (TNA); (ii) an ionizable lipid; (iii) a sterol; and (iv) a first lipid-anchored polymer, and optionally further comprising a helper lipid, wherein the first lipid-anchored polymer comprises a polymer-conjugated lipid of the present disclosure. Also provided herein are LNPs consisting essentially of: (i) a therapeutic nucleic acid (TNA); (ii) an ionizable lipid; (iii) a sterol; and (iv) a first lipid-anchored polymer, and 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 are LNPs consisting of: (i) a therapeutic nucleic acid (TNA); (ii) an ionizable lipid; (iii) a sterol; and (iv) a first lipid-anchored polymer, and optionally further consisting of a helper lipid, wherein the first lipid-anchored polymer comprises a polymer-conjugated lipid of the present disclosure.

[0484] A. Ionizable lipids

[0485] In some embodiments, the ionizable lipid is present in the LNPs provided herein in an amount of about 20 mol% to about 60 mol%, about 35 mol% to about 50 mol% of the total lipids present in the LNP.

[0486] In some embodiments, the LNPs provided herein comprise ionizable lipids. Exemplary ionizable lipids in the LNPs disclosed herein are described in International Patent Application Publication Nos. WO2015 / 095340, WO2015 / 199952, WO2018 / 011633, WO2017 / 049245, WO2015 / 061467, WO2012 / 040184, WO2012 / 000104, WO2015 / 074085, WO2016 / 081029, WO2017 / 004143, WO2017 / 075531, WO2017 / 117528, WO2011 / 022460, and WO2012 / 030966. , WO2013 / 148541, WO2013 / 116126, WO2011 / 153120, WO2012 / 044638, WO2012 / 054365, WO2011 / 090965, WO2013 / 016058, WO2012 / 162210, WO2008 / 042973, WO2010 / 129709, WO2010 / 144740, WO2012 / 099755, WO2013 / 049328, WO2013 / 086322, WO2013 / 08 6373, WO2011 / 071860, WO2009 / 132131, WO2010 / 048536, WO2010 / 088537, WO2010 / 054401, WO2010 / 054406, WO2010 / 054405, WO2010 / 054384, WO2012 / 016184, WO2009 / 086558, WO2010 / 042877, WO2011 / 000106, WO2011 / 000107, WO2005 / 120152, WO2012 / 016184, WO2012 / 086558, WO2010 / 042877, WO2011 / 000106, WO2011 / 000107, WO2005 / 120152, WO2012 11 / 141705, WO2013 / 126803, WO2006 / 007712, WO2011 / 038160, WO2005 / 121348, WO2011 / 066651, WO2009 / 127060, WO2011 / 141704, WO2006 / 069782, WO2012 / 031043, WO2013 / 006825, WO2013 / 033563, WO2013 / 089151, WO2017 / 099823, WO2015 / 095346,Nos. WO2013 / 086354 and WO2021 / 102411 and U.S. Patent Application Publication Nos. US2016 / 0311759, US2015 / 0376115, US2016 / 0151284, US2017 / 0210697, US2015 / 0140070, US2013 / 0178541, US2013 / 0303587, US2015 / 0141678, US2015 / 0239926, US2016 / 0376224, US2017 / 0119904, U.S. Patent Application Publication Nos. S2012 / 0149894, US2015 / 0057373, US2013 / 0090372, US2013 / 0274523, US2013 / 0274504, US2013 / 0274504, US2009 / 0023673 No., US2012 / 0128760, US2010 / 0324120, US2014 / 0200257, US2015 / 0203446, US2018 / 0005363, US2014 / 0308304, US2013 / 033 No. 8210, No. US2012 / 0101148, No. US2012 / 0027796, No. US2012 / 0058144, No. US2013 / 0323269, No. US2011 / 0117125, No. US2011 / 0256175, No. US2012 / 0202871, No. US2011 / 0076335, No. US2006 / 0083780, No. US2013 / 0123338, No. US2015 / 0064242, No. US2006 / 0051405, No. US2013 / 0065939, No. US Nos. 2006 / 0008910, 2003 / 0022649, 2010 / 0130588, 2013 / 0116307, 2010 / 0062967, 2013 / 0202684, 2014 / 0141070, 2014 / 0255472, 2014 / 0039032, 2018 / 0028664, 2016 / 0317458, and 2013 / 0195920, the contents of which are hereby incorporated by reference in their entirety.

[0487] In some embodiments, the ionizable lipid in the LNPs of the present disclosure is MC3 (6Z, 9Z, 28Z, 31Z)-heptatriacontano-6, 9, 28, 31-tetraen-19-yl-4-(dimethylamino) butyrate (DLin-MC3-DMA or MC3), which is represented by the following structural formula:

[0488]

[0489] 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.

[0490] In some embodiments, the ionizable lipid is selected from the group consisting of: 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-[bis(3-amino-propyl)amino]butylcarboxamidoethyl]-3,4-bis[oleyloxy]-benzamide (MVL5); dioctadecylamino-glycylamine amine (DOGS); 3b-[N-(N′,N′-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol); dioctadecyldimethylammonium bromide (DDAB); Saint lipids (e.g., SAINT-2,N-methyl-4-(dioleyl)methylpyridinium ion); 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE); 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium 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., a cationic lipid, is a lipid such as dioctadecyldimethylammonium bromide (DDAB), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,31-dioxolane] (DLin-KC2-DMA), heptathriacontane-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA), 1,2-dioleoyl oxy-3-dimethylaminopropane (DODAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), morpholinocholesterol (Mo-CHOL), (R)-5-(dimethylamino)pentane-1,2-dioleic acid diester hydrochloride (DODAPen-C1), (R)-5-guanidinopentane-1,2-dioleic acid diester hydrochloride (DOPen-G), and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-ammonium chloride (DOTAPen).

[0491] Formula (A)

[0492] In some embodiments, the ionizable lipid in the LNPs of the present disclosure is represented by formula (A):

[0493]

[0494] or a pharmaceutically acceptable salt thereof, wherein:

[0495] R 1 and R 1′ Each independently is C 1-3 alkylene;

[0496] R 2 and R 2′ Each independently is a straight chain or branched C 1-6 Alkylene or C 3-6 cycloalkylene;

[0497] R 3 and R 3′ are each independently an optionally substituted C 1-6 Alkyl or optionally substituted C 3-6 Cycloalkyl;

[0498] Or alternatively, when R 2 For branched chain C 1-6 When alkylene, and when R 3 C 1-6 When alkyl, R 2 and R 3 Together with its central nitrogen atom, it forms a 4- to 8-membered heterocyclic group;

[0499] Or alternatively, when R 2′ For branched chain C 1-6 When alkylene, and when R 3′ C 1-6 When alkyl, R 2′ and R 3' Together with its central nitrogen atom, it forms a 4- to 8-membered heterocyclic group;

[0500] R 4 and R 4' are each independently -CH, -CH2CH or -(CH2)2CH;

[0501] R 5 and R 5' are independently hydrogen, C 1-20 Alkylene or C 2-20 alkenylene;

[0502] R 6 and R 6' C independently at each occurrence 1-20 Alkylene, C 3-20 Cycloalkylene or C 2-20 alkenylene; and

[0503] m and n are each independently an integer selected from 1, 2, 3, 4 and 5.

[0504] In some embodiments, R 2 and R 2' Each independently is C 1-3 Alkylene.

[0505] In some embodiments, R 1 or R 1' The straight or branched C 1-3 Alkylene, R 2 or R 2' The straight or branched C 1-6 Alkylene and the optionally substituted straight or branched C 1-6 The alkyl groups are each optionally substituted with one or more halo and cyano groups.

[0506] In some embodiments, R 1 and R 2 Together for C 1-3 Alkylene, and R 1′ and R 2′ Together for C 1-3 Alkylene groups, such as ethylene.

[0507] In some embodiments, R 3 and R 3' are each independently an optionally substituted C 1-3Alkyl groups, such as methyl.

[0508] In some embodiments, R 4 and R 4' Each is -CH.

[0509] In some embodiments, R 2 is an optionally substituted branched C 1-6 alkylene; and R 2 and R 3 Together with its central nitrogen atom, it forms a 5-membered or 6-membered heterocyclic group. 2' is an optionally substituted branched C 1-6 alkylene; and R 2' and R 3' Together with its central nitrogen atom, it forms a 5-membered or 6-membered heterocyclic group, such as pyrrolidinyl or piperidinyl.

[0510] In some embodiments, R 4 -C(R a )2CR a or -[C(R a )2]2CR a , and R a C 1-3 alkyl; and R 3 and R 4 Together with its central nitrogen atom, it forms a 5-membered or 6-membered heterocyclic group. 4' -C(R a )2CR a or -[C(R a )2]2CR a , and R a C 1-3 alkyl; and R 3' and R 4' Together with its central nitrogen atom, it forms a 5-membered or 6-membered heterocyclic group, such as pyrrolidinyl or piperidinyl.

[0511] In some embodiments, R 5 and R 5' Each independently is C 1-10 Alkylene or C 2-10 In one embodiment, R 5 and R 5' Each independently is C 1-8 Alkylene or C 1-6 Alkylene.

[0512] In some embodiments, R 6 and R 6' C independently at each occurrence 1-10 Alkylene, C 3-10Cycloalkylene or C 2-10 In one embodiment, C 1-6 Alkylene, C 3-6 Cycloalkylene or C 2-6 In one embodiment, the C 3-10 Cycloalkylene or C 3-6 In some embodiments, m and n are each 3.

[0513] In some embodiments, the ionizable lipid in the LNP of the present disclosure can be selected from any one of the lipids listed in Table 1 below, or a pharmaceutically acceptable salt thereof.

[0514] Table 1

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524] Formula (B)

[0525] In some embodiments, the ionizable lipid in the LNPs of the present disclosure is represented by formula (B):

[0526]

[0527] or a pharmaceutically acceptable salt thereof, wherein:

[0528] 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);

[0529] b is an integer ranging from 2 to 10 (e.g., b is 2, 3, 4, 5, 6, 7, 8, 9, or 10);

[0530] R 1 Not present or selected from (C2-C20 )alkenyl, -C(O)O(C2-C 20 ) alkyl and (C2-C 20 ) alkyl-substituted cyclopropyl; and

[0531] R 2 (C2-C 20 )alkyl.

[0532] In a second embodiment of formula (B), the ionizable lipid of formula (B) is represented by formula (B-1):

[0533]

[0534] or a pharmaceutically acceptable salt thereof, 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 wherein the remaining variables are as described for Formula (B).

[0535] In a third embodiment of Formula (B), c and d in Formula (B-1) are each independently an integer ranging from 2 to 8, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 8, 4 to 7, 4 to 6, 5 to 8, 5 to 7 or 6 to 8, with the remaining variables being as described for Formula (B-1).

[0536] In a fourth embodiment of Formula (B), c in Formula (B-1) is 2, 3, 4, 5, 6, 7, or 8, with the remaining variables 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, with the remaining variables as described for Formula (B) or the second or third embodiment of Formula (B).

[0537] 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, wherein the remaining variables are 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, wherein the remaining variables are as described for Formula (B) or the second, third or fourth embodiment of Formula (B).

[0538] In a sixth embodiment of formula (B), the ionizable lipid of formula (B) or formula (B-1) is represented by formula (B-2):

[0539]

[0540] or a pharmaceutically acceptable salt thereof, wherein the remaining variables are as described for Formula (B) or Formula (B-1).

[0541] In a seventh embodiment of Formula (B), b in Formula (B), (B-1) or (B-2) is an integer ranging from 3 to 9, with the remaining variables as described for Formula (B) or the second, third, fourth, fifth or sixth embodiment of Formula (B). Alternatively, b in Formula (B), (B-1) or (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, with the remaining variables as described for Formula (B) or the second, third, fourth, fifth or sixth embodiment of Formula (B). Alternatively, b in Formula (B), (B-1) or (B-2) is 3, 4, 5, 6, 7, 8 or 9, with the remaining variables as described for Formula (B) or the second, third, fourth, fifth or sixth embodiment of Formula (B).

[0542] In an eighth embodiment of formula (B), a in formula (B), (B-1) or (B-2) is an integer ranging from 2 to 18, with the remaining variables being as described for formula (B) or the second, third, fourth, fifth, sixth or seventh embodiment of formula (B). Alternatively, a in formula (B), (B-1) or (B-2) is an integer ranging from 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 20, 4 to 30, 4 to 50, 4 to 60, 4 to 70, 4 to 80 to 17, 4 to 16, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 25 to 8, 5 to 7, 6 to 18, 6 to 17, 6 to 16, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 6 to 9, 6 to 8, 7 to 1 8, 7 to 17, 7 to 16, 7 to 15, 7 to 14, 7 to 13, 7 to 12, 7 to 11, 7 to 10, 7 to 9, 8 to 18, 8 to 17, 8 to 16, 8 to 15, 8 to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 9 to 18, 9 to 17, 9 to 16, 9 to 15, 9 to 14, 9 to 13, 9 to 12, 9 to 11, 10 to 18, 10 to 17, 10 to 16, 10 to 15, 10 to 14, 10 to 13, 11 17, 14 to 16, 15 to 18, 15 to 17, 14 to 16, 15 to 18, 15 to 17, or 16 to 18, wherein 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), (B-1) or (B-2) is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18, with the remaining variables being as described for Formula (B) or the second, third, fourth, fifth, sixth or seventh embodiment of Formula (B).

[0543] In the ninth embodiment of formula (B), R in formula (B), formula (B-1) or formula (B-2) 1 Not present or selected from (C5-C 15 )alkenyl, -C(O)O(C4-C 18) alkyl and (C4-C 16 )alkyl-substituted cyclopropyl, wherein 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) 1 Not present or selected from (C5-C 15 )alkenyl, -C(O)O(C4-C 16 ) alkyl and (C4-C 16 )alkyl-substituted cyclopropyl, wherein 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) 1 Not present or selected from (C5-C 12 )alkenyl, -C(O)O(C4-C 12 ) alkyl and (C4-C 12 )alkyl-substituted cyclopropyl, wherein 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) 1 Not present or selected from (C5-C 10 )alkenyl, -C(O)O(C4-C 10 ) alkyl and (C4-C 10 )alkyl-substituted cyclopropyl, wherein the remaining variables are as described for Formula (B) or the second, third, fourth, fifth, sixth, seventh or eighth embodiment of Formula (B).

[0544] In the tenth embodiment of formula (B), R 1 C 10 alkenyl, wherein the remaining variables are as described for Formula (B) or the second, third, fourth, fifth, sixth, seventh or eighth embodiment of Formula (B).

[0545] In the 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, wherein 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 1is -C(O)O(C9 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 )alkyl is a branched alkyl, 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, eighth or ninth embodiment of Formula (B). In one embodiment, R 1 -C(O)O(C 17 alkyl), 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, eighth, or ninth embodiment of Formula (B).

[0546] In the twelfth embodiment of formula (B), R in formula (B), formula (B-1) or formula (B-2) 1 is selected from any of the groups listed in Table 2 below, wherein the wavy bond in each group indicates the point of attachment of the group to the rest of the ionizable lipid molecule, and 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). The present disclosure also contemplates R in Table 2 in Formula (B) 1 Any of the groups and R in Table 3 2 A combination of 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).

[0547] Table 2

[0548]

[0549] In the thirteenth embodiment, R in formula (B) 2 or a pharmaceutically acceptable salt thereof is selected from any group listed in Table 3 below, wherein the wavy bond in each of the groups indicates the point of attachment of the group to the rest of the ionizable lipid molecule, and 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, ninth, tenth, eleventh or twelfth embodiment of Formula (B).

[0550] Table 3

[0551]

[0552] Specific examples of ionizable lipids of formula (B) are provided in Table 4 below. Pharmaceutically acceptable salts as well as ionized and neutral forms are also included.

[0553] Table 4

[0554]

[0555]

[0556]

[0557]

[0558] Formula (C)

[0559] In some embodiments, the ionizable lipid in the LNPs of the present disclosure is represented by formula (C):

[0560]

[0561] or a pharmaceutically acceptable salt thereof, wherein:

[0562] R 1 and R 1' Each independently is optionally selected from one or more of R a (C1-C6)alkylene substituted with a group;

[0563] R 2 and R 2' are each independently (C1-C2)alkylene;

[0564] R 3 and R 3' Each independently is optionally selected from one or more of R b (C1-C6) alkyl substituted with a group;

[0565] Or alternatively, R 2 and R 3 and / or R 2' and R 3′ Together with its central nitrogen atom, it forms a 4- to 7-membered heterocyclic group;

[0566] R 4 and R 4 'Each is a (C2-C6) alkylene group interrupted by -C(O)O-;

[0567] R 5 and R 5 ' are each independently (C2-C 30 )alkyl or (C2-C 30)alkenyl, each of which is optionally interrupted by -C(O)O- or (C3-C6)cycloalkyl; and

[0568] R a and R b Each is halo or cyano.

[0569] In the second embodiment of formula (C), R 1 and R 1 are each independently (C1-C6)alkylene, wherein the remaining variables are as described above for formula (C). Alternatively, R 1 and R 1' is each independently (C1-C3)alkylene, wherein the remaining variables are as described above for Formula (C).

[0570] In a third embodiment of formula (C), the ionizable lipid of formula (C) is represented by formula (C-1):

[0571]

[0572] or a pharmaceutically acceptable salt thereof, wherein R 2 and R 2' 、R 3 and R 3' 、R 4 and R 4 ' and R 5 and R 5 'As described above for Formula (C) or the second embodiment of Formula (C).

[0573] In a fourth embodiment, the ionizable lipid of formula (C) is represented by formula (C-2) or formula (C-3):

[0574]

[0575] or a pharmaceutically acceptable salt thereof, wherein R 4 and R 4 ' and R 5 and R 5 'As described above for formula (C).

[0576] In a fifth embodiment of formula (C), the ionizable lipid of formula (C) is represented by formula (C-4) or (C-5):

[0577]

[0578] or a pharmaceutically acceptable salt thereof, wherein R 5 and R 5 'As described above for formula (C).

[0579] In a sixth embodiment of formula (C), the ionizable lipid of formula (C) is represented by formula (C-6), (C-7), (C-8), or (C-9):

[0580]

[0581] or a pharmaceutically acceptable salt thereof, wherein R 5 and R 5 'As described above for formula (XV).

[0582] In the seventh embodiment of formula (C), R in formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8) or (C-9) 5 and R 5' At least one of R is a branched alkyl or alkenyl group, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is 5 and R 5′ One of R is a branched alkyl or alkenyl group, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is 5 is a branched alkyl or alkenyl group, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is 5' is a branched alkyl or alkenyl group, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C).

[0583] In an eighth embodiment of formula (C), R in formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) 5 (C6-C 26 )alkyl or (C6-C 26)alkenyl, each of which is optionally interrupted by -C(O)O- or (C3-C6)cycloalkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9 5 (C6-C 26 )alkyl or (C6-C 26 )alkenyl, each of which is optionally interrupted by -C(O)O- or (C3-C5)cycloalkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9 5 (C7-C 26 )alkyl or (C7-C 26 )alkenyl, each of which is optionally interrupted by -C(O)O- or (C3-C5)cycloalkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9 5 (C8-C 26 )alkyl or (C8-C 26 )alkenyl, each of which is optionally interrupted by -C(O)O- or (C3-C5)cycloalkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9 5 (C6-C 24 )alkyl or (C6-C 24 )alkenyl, each of which 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), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9 5 (C8-C 24 )alkyl or (C8-C 24 ) alkenyl, wherein 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), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9 5 (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), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (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), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) 5 (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), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) 5 For (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), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is 5 is -(CH2)3C(O)O(CH2)8CH3, -(CH2)5C(O)O(CH2)8CH3, -(CH2)7C(O)O(CH2)8CH3,

[0584] -(CH2)7C(O)OCH[(CH2)7CH3]2, -(CH2)7-C3H6-(CH2)7CH3, -(CH2)7CH3, -(CH2)9CH3, -(CH2) 16 CH3, -(CH2)7CH=CH(CH2)7CH3 or

[0585] -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C).

[0586] In a ninth embodiment, R in formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) 5' (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), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) 5′ (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), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) 5' (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), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) 5' (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), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) 5' (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), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) 5' (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' (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 remaining variables are as described above for Formula (C) or the second or eighth embodiment of Formula (C).

[0587] In some embodiments, the ionizable lipid of Formula (C), (C-1), (C-3), (C-4), (C-5), (C-7), (C-8), or (C-9) can be selected from any of the lipids listed in Table 5 below, or a pharmaceutically acceptable salt thereof.

[0588] Table 5

[0589]

[0590]

[0591] Formula (D)

[0592] In some embodiments, the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by formula (D):

[0593]

[0594] or a pharmaceutically acceptable salt thereof, wherein:

[0595] R' is absent, hydrogen or C1-C6 alkyl; provided that when R' is hydrogen or C1-C6 alkyl, R', R 1 and R 2 The nitrogen atoms they are connected to have a positive charge;

[0596] R 1 and R 2 Each is independently hydrogen, C1-C6 alkyl or C2-C6 alkenyl;

[0597] R 3 C1-C 12 Alkylene or C2-C 12 alkenylene;

[0598] R 4 C1-C 18 Unbranched alkyl, C2-C 18 Unbranched alkenyl or in:

[0599] R 4a and R 4b Each independently is C1-C 16 Unbranched alkyl or C2-C 16 unbranched alkenyl;

[0600] R 5 Not present, C1-C8 alkylene or C2-C8 alkenylene;

[0601] R 6a and R 6b Each independently is C7-C 16 Alkyl or C7-C 16 alkenyl; provided that the combined R 6a and R 6b The total number of carbon atoms in is greater than 15;

[0602] 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:

[0603] R a is independently at each occurrence hydrogen or C1-C6 alkyl; and

[0604] n is an integer selected from 1, 2, 3, 4, 5 and 6.

[0605] In the second embodiment of formula (D), X 1 and X 2 and all other remaining variables are as described for Formula (C).

[0606] In the third embodiment of formula (D), X 1 and X 2 are 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 each is independently -C(=O)O- or -SS-; and all other remaining variables are as described for Formula (D) or the second embodiment of Formula (D).

[0607] In a fourth embodiment of formula (D), the ionizable lipid, such as a cationic lipid, in the LNP of the present disclosure is represented by formula (D-1):

[0608]

[0609] or a pharmaceutically acceptable salt thereof, 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).

[0610] 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):

[0611]

[0612] or a pharmaceutically acceptable salt thereof, 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).

[0613] 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):

[0614]

[0615] or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (D) or the second or third embodiment of Formula (D).

[0616] 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 2each is 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).

[0617] In an eighth embodiment of Formula (D), the ionizable lipid, such as a cationic lipid, in the LNP of the present disclosure is represented by Formula (D-4):

[0618]

[0619] or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3) or the second, third or seventh embodiment of Formula (D).

[0620] In a ninth embodiment of Formula (D), in an ionizable lipid, such as 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 3 is 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 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 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, or seventh embodiment of Formula (D).

[0621] In a tenth embodiment of Formula (D), in an ionizable lipid, such as 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 does not exist, is C1-C6 alkylene or C2-C6 alkenylene; or R 5 does not exist, is C1-C4 alkylene or C2-C4 alkenylene; or R 5 does not exist; or R 5 is C8 alkylene, C7 alkylene, C6 alkylene, C5 alkylene, C4 alkylene, C3 alkylene, C2 alkylene, C1 alkylene, C8 alkenylene, C7 alkenylene, C6 alkenylene, C5 alkenylene, C4 alkenylene, C3 alkenylene or C2 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).

[0622] In an eleventh embodiment of Formula (D), in an ionizable lipid, such as 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 C1-C 14 Unbranched alkyl, C2-C 14 Unbranched alkenyl or where R 4a and R 4b Each independently is C1-C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl; or R 4 C2-C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl; or R 4 is a C5-C7 unbranched alkyl group or a C5-C7 unbranched alkenyl group; or R 4 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, 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; or R 4 for where R 4a and R 4b Each independently is C2-C 10 Unbranched alkyl or C2-C 10 unbranched alkenyl; or R 4 for where R 4a and R 4b Each independently 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 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 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).

[0623] In a twelfth embodiment, in the ionizable lipid, such as 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 Each independently is C6-C 14 Alkyl or C6-C 14alkenyl; or R 6a and R 6b Each independently is C8-C 12 Alkyl or C8-C 12 alkenyl; or R 6a and R 6b Each independently is 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 the combined R 6a and R 6b the total number of carbon atoms in 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).

[0624] In the thirteenth embodiment of Formula (D), in the ionizable lipid, such as a cationic lipid, or a pharmaceutically acceptable salt thereof 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), R 6a and R 6b contain an equal number of carbon atoms; or R 6a and R 6b Same; or R 6a and R 6b All 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 the combined R 6a and R 6b the total number of carbon atoms in 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).

[0625] In the fourteenth embodiment of Formula (D), in the ionizable lipid, such as 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 of the preceding embodiments 6a and R 6b Each contains a different number of carbon atoms from the other; or R 6a and R 6b The number of carbon atoms in the 6a and R 6b The number of carbon atoms in the 6a is a C7 alkyl group and R 6a is C8 alkyl, R 6a is a C8 alkyl group and R 6a is a C7 alkyl group, R 6a is a C8 alkyl group and R 6a is C9 alkyl, R 6a is a C9 alkyl group and R 6a is C8 alkyl, R 6a is a C9 alkyl group and R 6a C 10 Alkyl, R 6a C 10 Alkyl and R 6a is C9 alkyl, R 6a C 10 Alkyl and R 6a C 11 Alkyl, R 6a C 11 Alkyl and R 6a C 10 Alkyl, R 6a C 11 Alkyl, and R 6a C 12 Alkyl, R 6a C 12 Alkyl and R 6a C 11 Alkyl, R 6a is a C7 alkyl group and R 6a is C9 alkyl, R6a is a C9 alkyl group, and R 6a is a C7 alkyl group, R 6a is a C8 alkyl group and R 6a C 10 Alkyl, R 6a C 10 Alkyl and R 6a is C8 alkyl, R 6a is a C9 alkyl group and R 6a C 11 Alkyl, R 6a C 11 Alkyl and R 6a is C9 alkyl, R 6a C 10 Alkyl and R 6a C 12 Alkyl, R 6a C 12 Alkyl and R 6a C 10 Alkyl, R 6a C 11 Alkyl and R 6a C 13 Alkyl, or R 6a C 13 Alkyl and R 6a C 11 alkyl, etc.; 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).

[0626] In the fifteenth embodiment of formula (D), R 4 C1-C 16 Unbranched alkyl, C2-C 16 Unbranched alkenyl or where R 4a and R 4b As described above for the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, eleventh, twelfth, thirteenth or fourteenth embodiment of Formula (D).

[0627] In one embodiment, the ionizable lipid of the present disclosure, such as 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:

[0628] Table 6

[0629]

[0630]

[0631]

[0632]

[0633] In one embodiment, the ionizable lipid in the LNP of the present disclosure is ionizable lipid 87:

[0634]

[0635] Heptadec-9-yl 9-((4-dimethylamino)butanoyl)oxy)hexadecanoate or a pharmaceutically acceptable salt or ester or a deuterated analog thereof.

[0636] Formula (E)

[0637] In some embodiments, the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure is represented by formula (E):

[0638]

[0639] or a pharmaceutically acceptable salt thereof, wherein:

[0640] R' is absent, hydrogen or C1-C3 alkyl; provided that when R' is hydrogen or C1-C3 alkyl, R', R 1 and R 2 The nitrogen atoms they are connected to have a positive charge;

[0641] R 1 and R 2 Each is independently hydrogen or C1-C3 alkyl;

[0642] R 3 C3-C 10 Alkylene or C3-C 10 alkenylene;

[0643] R 4 C1-C 16 Unbranched alkyl, C2-C 16 Unbranched alkenyl or in:

[0644] R 4a and R 4b Each independently is C1-C 16 Unbranched alkyl or C2-C 16 unbranched alkenyl;

[0645] R 5 Not present, C1-C6 alkylene or C2-C6 alkenylene;

[0646] R 6a and R 6b Each independently is C7-C 14 Alkyl or C7-C 14 alkenyl;

[0647] 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:

[0648] R a is independently at each occurrence hydrogen or C1-C6 alkyl; and

[0649] n is an integer selected from 1, 2, 3, 4, 5 and 6.

[0650] In a second embodiment of Formula (E), in an ionizable lipid according to the first embodiment, e.g., a cationic lipid 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.

[0651] In a third embodiment of formula (E), the ionizable lipid, such as a cationic lipid, in the LNP of the present disclosure is represented by formula (E-1):

[0652]

[0653] or a pharmaceutically acceptable salt thereof, 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). 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).

[0654] In a fourth embodiment of formula (E), the ionizable lipid, such as a cationic lipid, in the LNP of the present disclosure is represented by formula (E-2):

[0655]

[0656] or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (E), Formula (E-1) or the second embodiment of Formula (E).

[0657] In a fifth embodiment of formula (E), in the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure, R 1 and R 2 are independently hydrogen or C1-C2 alkyl or C2-C3 alkenyl; or R', R 1 and R 2 each is 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).

[0658] 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):

[0659]

[0660] or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2) or the second or fifth embodiment of Formula (E).

[0661] 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 does not exist or is C1-C8 alkylene; or R 5 does not exist, is C1-C6 alkylene or C2-C6 alkenylene; or R 5 does not exist, is C1-C4 alkylene or C2-C4 alkenylene; or R 5 does not exist; or R 5 is C8 alkylene, C7 alkylene, C6 alkylene, C5 alkylene, C4 alkylene, C3 alkylene, C2 alkylene, C1 alkylene, C8 alkenylene, C7 alkenylene, C6 alkenylene, C5 alkenylene, C4 alkenylene, C3 alkenylene or C2 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).

[0662] In an eighth embodiment of Formula (E), the ionizable lipid, such as a cationic lipid, in the LNP of the present disclosure is represented by Formula (E-4):

[0663]

[0664] or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3) or the second, fifth or seventh embodiment of Formula (E).

[0665] In a ninth embodiment, in the ionizable lipid, such as 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), R 4 C1-C 14 Unbranched alkyl, C2-C 14 Unbranched alkenyl, or where R 4a and R 4b Each independently is C1-C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl; or R 4 C2-C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl; or R 4 C5-C 12 Unbranched alkyl or C5-C 12 unbranched alkenyl; or R 4 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 10unbranched 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 yes where R 4a and R 4b Each independently is C2-C 10 Unbranched alkyl or C2-C 10 unbranched alkenyl; or R 4 yes where R 4a and R 4b Each independently 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 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 Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth or seventh embodiment of Formula (E).

[0666] In a tenth embodiment, in the ionizable lipid, e.g., 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 C3-C8 alkenylene, C3-C7 alkylene or C3-C7 alkenylene or C3-C5 alkylene or C3-C5 alkenylene; or R 3is C8 alkylene, or C7 alkylene, or C6 alkylene, or C5 alkylene, or C4 alkylene, or C3 alkylene, or C1 alkylene, or C8 alkenylene, or C7 alkenylene, or C6 alkenylene, or C5 alkenylene, or C4 alkenylene or C3 alkenylene; 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 or ninth embodiment of Formula (E).

[0667] In an eleventh embodiment, in the ionizable lipid, e.g., 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 Each independently is C7-C 12 Alkyl or C7-C 12 alkenyl; or R 6a and R 6b Each independently is C8-C 10 Alkyl or C8-C 10 alkenyl; or R 6a and R 6b Each independently is C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 Alkenyl, C 11 Alkenyl, C 10 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, or tenth embodiment of Formula (E).

[0668] In a twelfth embodiment, in the ionizable lipid, e.g., 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 Same; or R 6a and R 6b All C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 Alkenyl, C 11 Alkenyl, C 10and 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 or eleventh embodiment of Formula (E).

[0669] In a thirteenth embodiment, in an ionizable lipid, such as a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), or Formula (E-4), R as defined in any one of the preceding embodiments 6a and R 6b Each contains a different number of carbon atoms from the other; or R 6a and R 6b The number of carbon atoms in the 6a and R 6b The number of carbon atoms in the 6a is a C7 alkyl group and R 6a is C8 alkyl, R 6a is a C8 alkyl group and R 6a is a C7 alkyl group, R 6a is a C8 alkyl group and R 6a is C9 alkyl, R 6a is a C9 alkyl group and R 6a is C8 alkyl, R 6a is a C9 alkyl group and R 6a C 10 Alkyl, R 6a C 10 Alkyl and R 6a is C9 alkyl, R 6a C 10 Alkyl and R 6a C 11 Alkyl, R 6a C 11 Alkyl and R 6a C 10 Alkyl, R 6a C 11 Alkyl and R 6a C 12 Alkyl, R 6a C 12 Alkyl and R 6a C 11 Alkyl, R 6a is a C7 alkyl group and R 6a is C9 alkyl, R 6a is a C9 alkyl group and R 6a is a C7 alkyl group, R 6a is a C8 alkyl group and R 6a C 10 Alkyl, R6a C 10 Alkyl and R 6a is C8 alkyl, R 6a is a C9 alkyl group and R 6a C 11 Alkyl, R 6a C 11 Alkyl and R 6a is C9 alkyl, R 6a C 10 Alkyl and R 6a C 12 Alkyl, R 6a C 12 Alkyl and R 6a C 10 alkyl, etc.; 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).

[0670] In a fourteenth embodiment, in an ionizable lipid, e.g., a cationic lipid, according to the second, fifth, seventh, ninth, tenth, eleventh, twelfth or thirteenth embodiment of Formula (E), (E-1), (E-2), (E-3), (E-4) or (E), R′ is absent; and all other remaining variables are as described for the second, fifth, seventh, ninth, tenth, eleventh, twelfth or thirteenth embodiment of Formula (E), (E-1), (E-2), (E-3), (E-4) or (E).

[0671] 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), or 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:

[0672] Table 7

[0673]

[0674]

[0675]

[0676] Specific examples are provided in the Exemplary section below and are included as part of the cationic lipids or ionizable lipids described herein. Pharmaceutically acceptable salts and neutral forms are also included.

[0677] Cleavable lipids

[0678] In certain embodiments, the LNP provided by the present disclosure comprises an ionizable lipid that is also a cleavable lipid. As used herein, the term "cleavable lipid" that can be used interchangeably with the term "SS cleavable lipid" refers to an ionizable lipid comprising a disulfide bond ("SS"). The SS in the cleavable lipid is a cleavable unit. In one embodiment, the cleavable lipid comprises an amine, such as a tertiary amine, such as and a disulfide bond. In this cleavable lipid, amine can be protonated in an acidic compartment (for example, in endosome or lysosome), thereby making the LNP unstable, and the cleavable lipid can be cut in a reducing environment (for example, in cytoplasm). Cleavable lipid also includes 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 etc.

[0679] According to some embodiments, the SS-cleavable lipids are described in International Patent Application Publication No. WO2019188867, which is herein incorporated by reference in its entirety.

[0680] 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 of the tertiary amino groups, and a disulfide bond. The tertiary amino group provides pH responsiveness and induces endosomal escape, the phenyl ester bond enhances the degradability (self-degradability) of the structure, and the disulfide bond is cleaved in a reducing environment.

[0681] In one embodiment, the cleavable lipid is a ss-OP lipid. In one embodiment, the ss-OP lipid comprises the structure of lipid A as shown below:

[0682] Lipid A

[0683]

[0684] In one embodiment, the SS cleavable lipid is an SS cleavable and pH activated lipid-like material (ssPalm). ssPalm lipids are well known in the art. See, for example, 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 an ssPalmM lipid comprising the structure of lipid B shown below:

[0685] Lipid B

[0686]

[0687] In one embodiment, the ssPalmE lipid is a ssPalmE-P4-C2 lipid comprising the following lipid C structure:

[0688] Lipid C

[0689]

[0690] In one embodiment, the ssPalmE lipid is a ssPalmE-Paz4-C2 lipid comprising the following lipid D structure:

[0691] Lipid D

[0692]

[0693] In one embodiment, the cleavable lipid is an ss-M lipid. In one embodiment, the ss-M lipid comprises the structure shown in the following lipid E:

[0694] Lipid E

[0695]

[0696] In one embodiment, the cleavable lipid is an ss-E lipid. In one embodiment, the ss-E lipid comprises the structure shown in the following lipid F:

[0697] Lipid F

[0698]

[0699] In one embodiment, the cleavable lipid is a ss-EC lipid. In one embodiment, the ss-EC lipid comprises the structure shown for the following lipid G:

[0700] Lipid G

[0701]

[0702] 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:

[0703] Lipid H

[0704]

[0705] In one embodiment, the cleavable lipid is a ss-OC lipid. In one embodiment, the ss-OC lipid comprises the structure shown for the following lipid J:

[0706] Lipid J

[0707]

[0708] Other lipids

[0709] In some embodiments, the ionizable lipids in the LNPs of the present disclosure are selected from the group consisting of: 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 (DOEPC); 1,2-Dimyristoyl-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-[bis(3-amino-propyl)amino]butylcarboxamidoethyl]-3,4-bis[oleyloxy]-benzamide (MVL5); Dioctadecylamino- Glycyl spermine (DOGS); 3b-[N-(N′,N′-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol); dioctadecyldimethylammonium bromide (DDAB); Saint lipids (e.g., SAINT-2,N-methyl-4-(dioleyl)methylpyridinium ion); 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE); 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide ammonium (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., a cationic lipid, is a lipid such as dioctadecyldimethylammonium bromide (DDAB), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,31-dioxolane] (DLin-KC2-DMA), heptathriacontane-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA), 1,2-dioleoyl oxy-3-dimethylaminopropane (DODAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), morpholinocholesterol (Mo-CHOL), (R)-5-(dimethylamino)pentane-1,2-dioleic acid diester hydrochloride (DODAPen-C1), (R)-5-guanidinopentane-1,2-dioleic acid diester hydrochloride (DOPen-G), and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-ammonium chloride (DOTAPen).

[0710] In some embodiments, the ionizable lipid in the LNP of the present disclosure is represented by the following structure:

[0711]

[0712]

[0713]

[0714] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0715] B. Structural lipids

[0716] In some embodiments, the LNPs provided by the present disclosure comprise structural lipids. Without wishing to be bound by a particular theory, it is believed that structural lipids, when present in LNPs, contribute to the membrane integrity and stability of the LNPs.

[0717] In some embodiments, the structured lipid is a sterol, for example, cholesterol or a derivative thereof. In one embodiment, the structured lipid is cholesterol. In another embodiment, the structured lipid is a derivative of cholesterol. Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholestanol, 5β-coprosterol, cholesteryl-(2′-hydroxy)-ethyl ether, cholesteryl-(4′-hydroxy)-butyl ether and 6-ketocholestanol; non-polar analogs such as 5α-cholestane, cholesterenone, 5α-cholestanone, 5β-cholestanone and cholesteryl; 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 cholesterol hemisuccinate (CHEMS).

[0718] Exemplary cholesterol derivatives are described in International Patent Application Publication No. WO 2009 / 127060 and US Patent Application Publication No. US 2010 / 0130588, the contents of both of which are herein incorporated by reference in their entireties.

[0719] In some embodiments, the sterol in the LNP of the present disclosure is selected from the group consisting of: cholesterol, β-sitosterol, stigmasterol, β-sitostanol, campesterol, brassicasterol and derivatives thereof, and any combination thereof. In one embodiment, the sterol is cholesterol. In another embodiment, the sterol is β-sitosterol.

[0720] In some embodiments, the structural lipid, e.g., sterol, constitutes about 20 mol% to about 50 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid, e.g., sterol, constitutes about 30 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid, e.g., cholesterol, constitutes about 30 mol% of the total lipid present in the LNP.

[0721] In some embodiments, the structured lipid is dexamethasone or dexamethasone-palmitate.

[0722] C. Helper lipids

[0723] Ceramide Helper Lipid

[0724] In some embodiments, the LNP provided by the present disclosure includes a helper lipid. In some embodiments, the helper lipid is a ceramide. The ceramide in the LNP of the present disclosure is not conjugated to a polymer such as polyethylene glycol or PEG. Ceramide is a sphingolipid, which is a class of cell membrane lipids. Ceramide contains N-acetyl sphingosine (i.e., (E)-N-(1,3-dihydroxyoctadec-4-ene-2-yl) acetamide) backbone and a fatty acid connected to an amide group. In some embodiments, the LNP provided by the present disclosure includes ceramide, whereby the fatty acid portion of the ceramide has a certain length or is a fatty acid with 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 non-polar chain and at least one polar part. Without wishing to be bound by a particular theory, it is believed that helper lipids function to escape the off-target effect of LNP on the blood compartment, to increase the pathogenicity of the lipid bilayer of LNP and to promote endosomal escape.

[0725] In some embodiments, the LNPs of the present disclosure comprise ceramide as a helper lipid. In some embodiments, the helper lipid is represented by formula (II):

[0726]

[0727] or a salt or ester thereof or a deuterated analog of any of the foregoing, wherein:

[0728] Is a single bond or a double bond;

[0729] R 1 C1-C 17 Alkyl or C2-C 17 alkenyl;

[0730] R 2 C1-C 22 Alkyl or C2-C 22 alkenyl;

[0731] R 3 is hydrogen or C1-C2 alkyl; and

[0732] R 4 is hydrogen or C1-C2 alkyl.

[0733] In some embodiments of Formula (II), Is a single bond. In some embodiments of formula (II), It's a double bond.

[0734] In some embodiments of Formula (II), R 1 C 10 -C 20 Alkenyl, R 2 C 10 -C 20 Alkyl, and R 3 For hydrogen.

[0735] In some embodiments of Formula (II), R 1 C1-C 10 Alkyl or C2-C 10 Alkenyl.

[0736] In some embodiments of Formula (II),

[0737] R 1 C1-C 10 Alkyl or C2-C 10 alkenyl;

[0738] R 2 C1-C 22 Alkyl or C2-C 22 alkenyl;

[0739] R 3 is hydrogen or C1-C2 alkyl; and

[0740] R4 is hydrogen or C1-C2 alkyl.

[0741] In some embodiments of Formula (II), R 3 and R 4 In some embodiments of formula (II), R 3 and R 4 are independently hydrogen or C1 alkyl.

[0742] In some embodiments of Formula (II), R 1 is C1-C7 alkyl or C2-C7 alkenyl. In one embodiment, R 1 is a C1-C7 alkyl group. In one embodiment, R 1 It is a C1 alkyl group.

[0743] When used to refer to a helper lipid represented by formula (II), the term "salt" means a pharmaceutically acceptable salt of the helper lipid represented by formula (II), including both acid addition salts and base addition salts. The salt of the helper lipid represented by formula (II) retains the biological effectiveness and properties of the free acid form or free base form of the helper lipid represented by formula (II), which is not biologically or otherwise undesirable and is formed from an inorganic acid or organic acid or an inorganic base or an organic base. Examples of inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; and examples of organic acids include, but are not limited to, 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, hexanoic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexanoic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, gluconic acid, heptanoic acid, gluconic acid. , glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Salts derived from organic bases include, but are not limited to, salts of 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, dimethylaminoethanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0744] When used to refer to a helper lipid represented by formula (II), the term "ester" means 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 a carboxylic acid, through an esterification process to form an ester (e.g., a formate or phosphate ester) of the helper lipid represented by formula (II).

[0745] The term "deuterated analog," when used in reference to a helper lipid represented by formula (II), means an analog of the helper lipid represented by formula (II) whereby any one or more hydrogen atoms of the helper lipid are replaced by deuterium, an isotope of hydrogen.

[0746] In one embodiment of Formula (II) or a salt or ester thereof or a deuterated analog of any of the foregoing, is a double bond; R 1 、R 2 、R 3 and R 4 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, is a single bond; R 1 、R 2 、R 3 and R 4 As defined above.

[0747] In some embodiments of Formula (II) or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 C1-C 15 Alkyl or C2-C 15 Alkenyl.

[0748] In some embodiments of Formula (II) or a salt or ester thereof, or a deuterated analog of any of the foregoing:

[0749] R 1 C1-C 15 Alkyl or C2-C 15 alkenyl;

[0750] R 2 C1-C 22 Alkyl or C2-C 22 alkenyl;

[0751] R 3 is hydrogen or C1-C2 alkyl; and

[0752] R 4 is hydrogen or C1-C2 alkyl.

[0753] In some embodiments of Formula (II) or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 C1-C10 Alkyl or C2-C 10 Alkenyl.

[0754] In some embodiments of Formula (II) or a salt or ester thereof:

[0755] R 1 C1-C 10 Alkyl or C2-C 10 alkenyl;

[0756] R 2 C1-C 22 Alkyl or C2-C 22 alkenyl;

[0757] R 3 is hydrogen or C1-C2 alkyl; and

[0758] R 4 is hydrogen or C1-C2 alkyl.

[0759] 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. In one embodiment, R 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. In one embodiment, R 1 Is C1-C7 alkyl. In one embodiment of formula (II) or a salt or ester thereof or a deuterated analogue of any of the foregoing:

[0760] R 1 is a C1-C7 alkyl group;

[0761] R 2 C1-C 22 Alkyl or C2-C 22 alkenyl;

[0762] R 3 is hydrogen or C1-C2 alkyl; and

[0763] R 4 is hydrogen or C1-C2 alkyl.

[0764] 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 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl or C7 alkyl. In some embodiments of Formula (II) or a salt or ester thereof or a deuterated analogue of any of the foregoing, R 1is C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl or C7 alkyl. In some embodiments of Formula (II) or a salt or ester thereof or a deuterated analogue of any of the foregoing, R 1 is C1 alkyl, C3 alkyl, C5 alkyl or C7 alkyl. In one embodiment of Formula (II) or a salt or ester thereof or a deuterated analogue of any of the foregoing, R 1 In one embodiment of Formula (II) or a salt or ester thereof or a deuterated analog of any of the foregoing, R 1 In one embodiment of Formula (II) or a salt or ester thereof or a deuterated analogue of any of the foregoing, R 1 In one embodiment of Formula (II) or a salt or ester thereof or a deuterated analog of any of the foregoing, R 1 It is a C7 alkyl group.

[0765] In some embodiments of Formula (II) or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 2 C3-C 15 Alkyl or C3-C 15 alkenyl; and R 1 、R 3 and R 4 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 C5-C 15 Alkyl or C3-C 15 alkenyl; and R 1 、R 3 and R 4 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 C7-C 15 Alkyl or C3-C 15 alkenyl; and R 1 、R 3 and R 4 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 C9-C 15 Alkyl or C9-C 15 alkenyl; and R 1 、R 3 and R 4 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 C9 alkyl, C 10 Alkyl, C 11 Alkyl, C 12 Alkyl, C13 Alkyl, C 14 Alkyl or C 15 alkyl; and R 1 、R 3 and R 4 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 group; and R 1 、R 3 and R 4 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 C 11 alkyl; and R 1 、R 3 and R 4 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 C 13 alkyl; and R 1 、R 3 and R 4 As defined above.

[0766] 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 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 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 group; and R 1 、R 2 and R 4 As defined above.

[0767] 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 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 3As 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 group; and R 1 、R 2 and R 3 As defined above.

[0768] In some embodiments of Formula (II), R 1 It is a C1-C7 alkyl group or a C2-C7 alkenyl group.

[0769] In some embodiments, R 1 is C1 alkyl, C3 alkyl, C5 alkyl or C7 alkyl. In some embodiments, R 1 It is a C1 alkyl group.

[0770] In some embodiments of Formula (II), R 2 C3-C 15 Alkyl or C3-C 15 In some embodiments, R 2 C 10 Alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 14 Alkyl or C 15 In some embodiments, R 2 C 12 Alkyl, C 13 Alkyl or C 14 In some embodiments, R 2 C 13 In some embodiments, R 2 C 12 In some embodiments, R 2 C 11 alkyl.

[0771] In some embodiments of Formula (II), R 1 and R 2 are hydrogen; and It's a double bond.

[0772] In some embodiments of Formula (II), R 1 and R 2 are hydrogen and is a double bond; and R 1 is C1 alkyl, C3 alkyl, C5 alkyl or C7 alkyl. In one embodiment, R 1 In another embodiment, R 1 In another embodiment, R 1In another embodiment, R 1 It is a C7 alkyl group.

[0773] In some embodiments of Formula (II), R 1 and R 2 are hydrogen and is a double bond; R 1 is C1 alkyl, C3 alkyl, C5 alkyl or C7 alkyl, and R 2 C9 alkyl, C 11 or C 13 In one embodiment, R 2 In one embodiment, R 2 C 11 In another embodiment, R 2 C 13 alkyl.

[0774] In some embodiments of Formula (II), R 3 In some embodiments of formula (II), R 3 It is a C1 alkyl group.

[0775] In some embodiments of Formula (II), R 4 In some embodiments of formula (II), R 4 It is a C1 alkyl group.

[0776] Other helper lipids

[0777] In some embodiments, the helper lipid contained in the LNP of the present disclosure is a phospholipid, a phosphatidylcholine, or a derivative thereof. In some embodiments, the helper lipid contained in the LNP of the present disclosure is selected from the group consisting of: 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-heptadecanoyl-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 LNP of the present disclosure is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0778] 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 lipids present in the LNP. In some embodiments, the helper lipid comprises about 10% mol% to about 20 mol% of the total lipids present in the LNP, and such LNPs having about 10% mol% to about 20 mol% of the total lipids present in the LNP exhibit increased overall tolerability (e.g., as shown by a weight loss curve and reduced cytokine response in a subject) compared to LNPs comprising less than 10% of the same helper lipid.

[0779] D. Lipid-anchored polymers

[0780] In some embodiments, the LNP provided by the present disclosure comprises 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 portion covalently attached to a polymer, e.g., via a linker. Without wishing to be bound by a particular theory, it is believed that lipid-anchored polymers can inhibit the aggregation of LNPs and provide steric stabilization. In some embodiments, the LNP provided by the present disclosure comprises two lipid-anchored polymers, i.e., a first lipid-anchored polymer and a second lipid-anchored polymer.

[0781] In some embodiments, the first lipid-anchoring polymer included in the LNPs of the present disclosure is a polymer-conjugated lipid of the present disclosure, such as DODA-PG34, DODA-PG45, DODA-PG46, or DODA-PG58.

[0782] In some embodiments, the LNPs of the present disclosure comprise two types of lipid-anchoring polymers: a) a polymer-conjugated lipid of the present disclosure, such as DODA-PG34, DODA-PG45, DODA-PG46, or DODA-PG58 as a first lipid-anchoring polymer, and b) a second lipid-anchoring polymer.

[0783] In some embodiments, a lipid-anchored polymer, e.g., a second lipid-anchored polymer according to the present disclosure, comprises:

[0784] (i) a lipid portion comprising at least one hydrophobic tail; and

[0785] (ii) a polymer conjugated to the lipid moiety, optionally via a linker.

[0786] In some embodiments, a lipid-anchored polymer, e.g., a second lipid-anchored polymer according to the present disclosure, comprises:

[0787] (i) a lipid portion comprising at least one hydrophobic tail;

[0788] (ii) polymers;

[0789] (iii) a linker, wherein the polymer is conjugated to the lipid moiety via the linker; and

[0790] (iv) a targeting moiety conjugated to the polymer.

[0791] In some embodiments, a lipid-anchored polymer, e.g., a second lipid-anchored polymer according to the present disclosure, comprises:

[0792] (i) a lipid portion comprising at least one hydrophobic tail;

[0793] (ii) polymers;

[0794] (iii) a linker, wherein the polymer is conjugated to the lipid moiety via the linker; and

[0795] (iv) an active species conjugated to the polymer, wherein the active species is functionalized to be conjugated to a targeting moiety.

[0796] 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 comprising 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, palmitic acid, homosapiens acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, trans-linoleic acid, α-trans-linoleic acid, arachidonic acid, eicosapentaenoic acid, and derivatives thereof.

[0797] When used herein with reference to a hydrophobic tail in a lipid-anchored polymer, the term "derivative" refers to a hydrophobic tail that has been modified compared to the original or native hydrophobic tail. In some embodiments, the derivative contains 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); and d) the configuration of one or more double bonds (if any) has been changed, e.g., from a cis configuration to a trans configuration or vice versa. A derivative contains the same number of carbon atoms as its original or native hydrophobic tail.

[0798] As used herein, when referring to the hydrophobic tail in a lipid-anchored polymer, the term "single aliphatic chain backbone" refers to the main straight aliphatic chain or carbon chain, i.e., the longest continuous straight aliphatic chain or carbon chain. For example, the following alkyl chain with several branches contains 18 carbon atoms in the single aliphatic chain backbone, i.e., the longest continuous straight 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.

[0799]

[0800] In one embodiment, the second lipid-anchored polymer according to the present disclosure comprises a lipid portion comprising at least one hydrophobic tail; and a polymer optionally conjugated to the lipid portion via a linker, wherein the lipid portion of the second lipid-anchored polymer comprises a lipid-linker portion 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-dioleoyl-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 dioctadecylamine (DODA), dihexadecylamine, distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and their derivatives. In some embodiments, the lipid portion of the second lipid-anchoring polymer comprises a lipid-linker moiety selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, and derivatives thereof. In a specific embodiment, the lipid portion of the second lipid-anchoring polymer comprises DSPE. In one embodiment, the second lipid-anchoring polymer further comprises a targeting moiety.

[0801] The lipid-anchored polymers of the present disclosure may also contain active species. In some embodiments, the active species is conjugated to the polymer in the lipid-anchored polymer. The active species present in the lipid-anchored polymers of the present disclosure can be used for conjugation, for example, with a targeting moiety that has been functionalized with a complementary active species, i.e., an active species capable of reacting with the active species contained in the lipid-anchored polymers of the present disclosure. In some embodiments, the active species conjugated to the lipid-anchored polymers of the present disclosure can be a thiol reagent, a maleimide reagent, or a click chemistry reagent, for example, a reagent selected from the group consisting of: an alkyne reagent, such as a dibenzocyclooctyne (DBCO) reagent, a trans-cyclooctene (TCO) reagent, a tetrazine (TZ) reagent, and an azide (AZ) reagent.

[0802] Linkers in lipid-anchored polymers

[0803] 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 phosphorylcholine 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 LNP of the present disclosure is selected from the group consisting of: -(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-, wherein n is a numeric integer ranging from 1 to 20. Thus, in some embodiments, the linker is -C(O)(CH2) n -, and in some embodiments, n is 2, 3, 4, 5 or 6.

[0804] 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 phosphorylcholine linker, a carbamate linker, or a -(CH2) 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.

[0805] 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.

[0806] As used herein, the term "derivative" when used with reference to a linker-lipid moiety means a linker-lipid moiety containing one or more of the following modifications: a) the phosphatidylethanolamine (PE) head group (if any) is modified to convert the amino group to a methylamino group or a dimethylamino group; b) the modified linker-lipid moiety contains one or more additional functional groups or moieties, such as -OH, -OCH3, -NH2, maleimide, azide or cyclooctyne, such as dibenzocyclooctyne (DBCO).

[0807] Polymers in lipid-anchored polymers

[0808] 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).

[0809] In some embodiments, the lipid-anchoring polymer, e.g., the polymer comprised in the second lipid-anchoring 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.

[0810] In some embodiments, the polymer contained in the lipid-anchored polymer of the present disclosure, for example, the first lipid-anchored polymer and / or the second lipid-anchored polymer, is polyglycerol (PG) or a PG derivative. The PG or the PG derivative can be linear or branched. The PG derivative can be a carboxylated PG, for example, a glutarylated PG, such as 3-methylglutarylated PG or 2-carboxycyclohexane-1-carboxylated PG. In some embodiments, the PG or the PG derivative can contain an average of 5-100 monomer units. In some embodiments, the PG or the PG derivative may comprise 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, or about 34, 45, 46 or 58, monomeric units. In one embodiment, the PG or the PG derivative comprises an average of 34, 45, 46 or 58, monomeric units.

[0811] In some embodiments, the molecular weight of the polymer in the lipid-anchored polymer is about 500 Da to about 5000 Da, such as about 1500 Da to about 5000 Da. In some embodiments, the average molecular weight of the polymer in the lipid-anchored polymer is about 2000 Da.

[0812] Targeting moiety

[0813] In certain embodiments, the LNP of the present disclosure further comprises one or more targeting moieties. The targeting moiety targets the LNP to be delivered to a specific site or tissue in a subject, such as the liver. In certain embodiments, the targeting moiety is capable of binding to a specific liver cell, such as a hepatocyte. 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.

[0814] In one embodiment, the targeting moiety is capable of binding to an sialoglycoprotein receptor (ASGPR), i.e., a 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 attached to, for example, a lipid-anchored polymer as defined herein. In one embodiment, the targeting moiety is a triantennary or trivalent GalNAc conjugate (i.e., GalNAc3), which is a ligand conjugate with three GalNAc molecules or three GalNAc derivatives. In one embodiment, the targeting moiety is a triantennary GalNAc represented by the following structural formula:

[0815]

[0816] 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 with four GalNAc molecules or four GalNAc derivatives.

[0817] In one embodiment, the targeting moiety is capable of binding to a low-density lipoprotein receptor (LDLR), such as 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, the ApoB polypeptide, or a fragment thereof is an ApoE polypeptide, an ApoB polypeptide, or a fragment thereof as disclosed in International Patent Application Publication No. WO2022 / 261101, which is incorporated herein by reference in its entirety. In one embodiment, the ApoE protein is a modified ApoE protein, and the ApoB protein is a modified ApoB protein.

[0818] In one embodiment, the ApoE protein 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 the following amino acid sequence: MKVEQAVET EPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVR (SEQ ID NO: 1). In one embodiment, the ApoE protein comprises the amino acid sequence shown in SEQ ID NO: 1. In one embodiment, the ApoE protein consists essentially of the amino acid sequence shown in SEQ ID NO: 1. In one embodiment, the ApoE protein consists of the amino acid sequence shown in SEQ ID NO: 1.

[0819] In one embodiment, the ApoE protein 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 the following amino acid sequence: MKVEQAVETEPE PELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRHHHHHH (SEQ ID NO: 2). In one embodiment, the ApoE protein comprises the amino acid sequence shown in SEQ ID NO: 2. In one embodiment, the ApoE protein consists essentially of the amino acid sequence shown in SEQ ID NO: 2. In one embodiment, the ApoE protein consists of the amino acid sequence shown in SEQ ID NO: 2.

[0820] In one embodiment, the ApoE protein 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 the following amino acid sequence:

[0821] MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVSGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVR (SEQ ID NO: 3). In one embodiment, the ApoE protein comprises the amino acid sequence shown in SEQ ID NO: 3. In one embodiment, the ApoE protein consists essentially of the amino acid sequence shown in SEQ ID NO: 3. In one embodiment, the ApoE protein consists of the amino acid sequence shown in SEQ ID NO: 3.

[0822] In one embodiment, the ApoE protein 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 the following amino acid sequence:

[0823] MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVSGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRHHHHHHGGSSGSGC (SEQ ID NO: 4). In one embodiment, the ApoE protein comprises the amino acid sequence shown in SEQ ID NO: 4. In one embodiment, the ApoE protein consists essentially of the amino acid sequence shown in SEQ ID NO: 4. In one embodiment, the ApoE protein consists of the amino acid sequence shown in SEQ ID NO: 4.

[0824] 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 to the length of the alignment, expressed as a percentage. In some embodiments, the two aligned sequences are the same length, i.e., have the same number of amino acids.

[0825] In one embodiment, the targeting moiety in the LNP of the present disclosure is an ApoE protein conjugate in an ApoB protein conjugate, which is 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, e.g., as defined herein. In one embodiment, the targeting moiety in the LNP of the present disclosure is an ApoE polypeptide conjugate in an ApoB polypeptide conjugate, which is a conjugate of one or more ApoE and / or ApoB polypeptide molecules or fragments thereof covalently linked to a lipid-anchored polymer, e.g., as defined herein.

[0826] In one embodiment, the targeting moiety is an antibody or antibody fragment, such as an antibody or antibody fragment that can specifically bind 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 immunoglobulin only (HClg), an immunoglobulin new antigen receptor (IgNAR), a variable domain of an immunoglobulin new antigen receptor (VNAR), a single domain antibody, or a variable heavy chain antibody only (VHH).

[0827] In some embodiments, the LNPs of the present disclosure comprise a polymer-conjugated lipid of the present disclosure, and 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.

[0828] In some embodiments, the LNPs of the present disclosure may comprise a polymer-conjugated lipid of the present disclosure as the first lipid anchoring polymer, and a targeting moiety as 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 a targeting moiety.

[0829] In some embodiments, the targeting moiety can be conjugated to a polymer-conjugated lipid via an active species. In some embodiments, the active species can be a thiol reagent, a maleimide reagent, or a click chemistry reagent, for example, a reagent selected from the group consisting of: an alkyne reagent, such as a dibenzocyclooctyne (DBCO) reagent, a trans-cyclooctene (TCO) reagent, a tetrazine (TZ) reagent, and an azide (AZ) reagent. Therefore, in an exemplary embodiment, the polymer-conjugated lipid comprising an azide reagent as an active species of the present disclosure can react with a targeting moiety functionalized with a DBCO reagent as a complementary active species to produce a polymer-conjugated lipid conjugated to a targeting moiety via an active species. In another exemplary embodiment, the polymer-conjugated lipid comprising a thiol reagent of the present disclosure can react with a targeting moiety functionalized with a maleimide reagent to produce a polymer-conjugated lipid conjugated to a targeting moiety via an active species.

[0830] In some embodiments, the LNPs of the present disclosure may comprise a polymer-conjugated lipid of the present disclosure as a first lipid-anchoring polymer, a second lipid-anchoring polymer, and a targeting moiety as described herein conjugated to the second lipid-anchoring polymer.

[0831] In some embodiments, the targeting moiety can be conjugated to a second lipid-anchored polymer via an active species. In some embodiments, the active species can be a thiol reagent, a maleimide reagent, or a click chemistry reagent, for example, a reagent selected from the group consisting of: an alkyne reagent, such as a dibenzocyclooctyne (DBCO) reagent, a trans-cyclooctene (TCO) reagent, a tetrazine (TZ) reagent, and an azide (AZ) reagent. Therefore, in one exemplary embodiment, the second lipid-anchored polymer of the present disclosure comprising an azide reagent as an active species can react with a targeting moiety functionalized with a DBCO reagent as a complementary active species to produce a second lipid-anchored moiety conjugated to a targeting moiety via an active species. In another exemplary embodiment, the polymer-conjugated lipid comprising a thiol reagent of the present disclosure can react with a targeting moiety functionalized with a maleimide reagent to produce a polymer-conjugated lipid comprising a targeting moiety.

[0832] Thus, in one embodiment of the LNPs of the present disclosure, the LNP comprises a second lipid-anchored polymer, and 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 second lipid-anchored polymer. The second lipid-anchored polymer contains a lipid portion optionally conjugated to the polymer via a linker. In one embodiment, the second lipid-anchoring polymer comprises a moiety 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-dioleoyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG). Acyl-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 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 portion selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG and derivatives thereof. In another embodiment, said lipid portion of said second lipid-anchoring polymer comprises DSPE.

[0833] In one embodiment, the ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide, antibody, or fragment thereof is covalently linked to a lipid-anchoring polymer (e.g., a first lipid-anchoring polymer or a second lipid-anchoring polymer) or is covalently linked to the LNP of the present disclosure via strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry, such as via an azide-modified lipid-anchoring 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 polypeptide, ApoB polypeptide, antibody, or fragment thereof.

[0834] In an exemplary embodiment, the second lipid-anchored polymer conjugated to the targeting moiety is represented by the following structure:

[0835]

[0836] In another exemplary embodiment, the second lipid-anchored polymer conjugated to the targeting moiety is represented by the following structure:

[0837]

[0838] In some embodiments, the LNP of the present disclosure may comprise a first lipid-anchored polymer and a second lipid-anchored polymer. For example, the LNP of the present disclosure may comprise a first lipid-anchored polymer that does not comprise a targeting moiety and a second type of lipid-anchored polymer comprising a targeting moiety, such as GalNAc. For example, the LNP of the present disclosure may comprise DSG-PEG2000 modified to comprise 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, the LNP of the present disclosure may comprise a polymer-conjugated lipid as the first lipid-anchored polymer of the present disclosure and a second anchored polymer, such as a second anchored polymer conjugated to a targeting moiety. For example, the LNP of the present disclosure may comprise DODA-PG45 as the first lipid-anchored polymer and DSPE-PEG2000-OH as the second lipid-anchored polymer.

[0839] In some embodiments, the LNPs of the present disclosure may 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 as described above. In a 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, the LNPs of the present disclosure may comprise DODA-PG45 as the first lipid-anchored polymer and DSPE-PEG2000-GalNAc3 as the second lipid-anchored polymer.

[0840] In some embodiments, the targeting moiety is conjugated to a DSPE anchoring polymer. In some embodiments, the DSPE anchoring polymer is DSPE-PEG or a derivative thereof.

[0841] In some embodiments, the targeting moiety is conjugated to a DSG anchoring polymer. In some embodiments, the DSG anchoring polymer is DSG-PEG or a derivative thereof.

[0842] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA); an ionizable lipid; a sterol; a helper lipid, and a first lipid-anchoring polymer, wherein the first lipid-anchoring polymer comprises a polymer-conjugated lipid.

[0843] In some embodiments, the ionizable lipid constitutes about 20 mol% to about 60 mol% of the total lipid present in the LNP. In some embodiments, the ionizable lipid constitutes about 35 mol% to about 50 mol% of the total lipid present in the LNP. In some embodiments, the sterol constitutes about 20 mol% to about 50 mol% of the total lipid present in the LNP. In some embodiments, the sterol constitutes about 30 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the helper lipid constitutes about 1 mol% to about 40 mol% of the total lipid present in the LNP. In some embodiments, the helper lipid constitutes about 5 mol% to about 15 mol% of the total lipid present in the LNP. In some embodiments, the first lipid-anchored polymer constitutes about 0.5 mol% to about 5 mol% of the total lipid present in the LNP. In some embodiments, the first lipid-anchored polymer constitutes about 1.5 mol% to about 3 mol% of the total lipid present in the LNP. In some embodiments, the second lipid-anchored polymer constitutes about 0.05 mol% to about 5 mol% of the total lipid present in the LNP. In some embodiments, the second lipid-anchoring polymer comprises about 0.1 mol% to about 1 mol% of the total lipid present in the LNP.

[0844] 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 contemplated for use in the methods and compositions disclosed herein.

[0845] The size of the LNPs can be determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, United Kingdom (UK)). In some embodiments, the average diameter of the LNPs of the present disclosure as determined by light scattering is less than about 90 nm, e.g., less than about 80 nm or less than about 75 nm. According to some embodiments, the average diameter of the LNPs of the present disclosure as determined by light scattering is from about 50 nm to about 75 nm or from about 50 nm to about 70 nm.

[0846] The pKa of the formulated cationic lipid can be correlated with the effectiveness of the LNP for delivering 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 entireties). In one embodiment, the pKa of each cationic lipid is determined in lipid nanoparticles using a fluorescence-based assay of 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS). LNPs with a concentration of 0.4 mM total lipid in PBS can be prepared using the online method described herein, among others. 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 a buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, with a pH range of 2.5 to 11. An aliquot of the TNS solution can be added to a final concentration of 1 mM, and after vortex mixing, fluorescence intensity can be measured at room temperature in an SLM Aminco Series 2 luminescence spectrophotometer using excitation and emission wavelengths of 321 nm and 445 nm. Sigma-Aldrich best-fit analysis can be applied to the fluorescence data, and the pKa can be measured as the pH that produces half-maximal fluorescence intensity.

[0847] 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 doses of 0.3 and 1.0 mg ceDNA / kg and expressed as ng luciferase / g liver measured 4 hours after administration.

[0848] Without limitation, the LNPs of the present disclosure include lipid formulations that can be used to deliver uncoated non-viral DNA vectors to target sites of interest (e.g., cells, tissues, organs, etc.). Typically, the LNPs comprise uncoated non-viral DNA vectors and cationic lipids or salts thereof.

[0849] Additional exemplary lipid anchoring polymers (e.g., second lipid anchoring polymers) include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, PG-lipid conjugates, polyamide-lipid conjugates (e.g., ATTA-lipid conjugates), cationic polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the second lipid anchoring polymer is a PEGylated lipid, e.g., a (methoxypolyethylene glycol)-conjugated lipid. PEG-diacylglycerol (DAG) (e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkoxypropyl (DAA), PEG-phospholipid, PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (e.g., 4-O-(2′,3′-di-tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), PEG dialkoxypropyl carbamate, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or mixtures thereof. Additional exemplary PEG-lipid conjugates are described, for example, in International Patent Application Publication No. WO 20 ... 1996 / 010392, WO1998 / 051278, WO2002 / 087541, WO2005 / 026372, WO2008 / 147438, WO2009 / 086558, WO2012 / 000104, WO2017 / 117528, WO2017 / 0 No. 99823, No. WO2015 / 199952, No. WO2017 / 004143, No. WO2015 / 095346, No. WO2012 / 000104, No. WO2012 / 000104 and No. WO2010 / 006282, U.S. Patent Application Publication No. US2003 / 0077829, No. US200 No. 5 / 0175682, US2008 / 0020058, US2011 / 0117125, US2013 / 0303587, US2018 / 0028664, US2015 / 0376115, US2016 / 0376224, US2016 / 0317458, US2013 / 0303587, US2013 / 0303587 and US20110123453, 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 entirety.

[0850] Additional examples of PEG-DAAPEGylated lipids include, for example, PEG-dilauroyloxypropyl, PEG-dimyristoyloxypropyl, PEG-dipalmitoyloxypropyl, or PEG-distearoyloxypropyl. The PEG-lipid can be one or more of the following: PEG-DMG, PEG-dilauroylglycerol, PEG-dipalmitoylglycerol, PEG-distearoylglycerol, PEG-dilauroylglycinamide, PEG-dimyristoylglycinamide, PEG-dipalmitoylglycinamide, PEG-distearoylglycinamide, PEG-cholesterol (1-[8′-(cholest-5-en-3[β]-oxy)carboxamide-3′,6′-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol)), PEG-DMB (3,4-ditetradecylbenzyl-[ω]-methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In one embodiment, the PEG-lipid can be selected from the group consisting of PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000],

[0851]

[0852] as well as

[0853]

[0854] Yet further exemplary lipid-anchoring polymers (e.g., second lipid-anchoring polymers) include N-(carbonyl-methoxy PEG n )-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG n , wherein n is 350, 500, 750, 1000 or 2000), N-(carbonyl-methoxy PEG n )-1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG n , wherein n is 350, 500, 750, 1000 or 2000), DSPE-polyglycerol-cyclohexyl-carboxylic acid, DSPE-polyglycerol-2-methylglutaraldehyde-carboxylic acid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated with polyethylene glycol (DSPE-PEG-OH), polyethylene glycol-dimyristoylglycerol (PEG-DMG) or polyethylene glycol-distearoylglycerol (PEG-DSG). DMPE-PEG wherein n is 350, 500, 750, 1000 or 2000 nIn some examples, the PEG-lipid is N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG 2,000). In some examples, the DSPE-PEG wherein n is 350, 500, 750, 1000, or 2000 n In some embodiments, the PEG-lipid is N-(carbonyl-methoxy PEG 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG 2,000). 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 hydrophobic tail and PEG2000.

[0855] E. Therapeutic Nucleic Acids

[0856] The LNPs provided by the present disclosure further comprise a therapeutic nucleic acid (TNA).According to an embodiment, a pharmaceutical composition comprising the LNPs of the present disclosure is also provided.

[0857] Illustrative therapeutic nucleic acids in the LNPs of the present disclosure can include, but are not limited to, minigenes, plasmids, minicircles, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASOs), ribozymes, deoxyribozymes, end-blocked double-stranded DNA (e.g., ceDNA, CELiD, linear covalently blocked DNA ("ministring"), doggybone TM , protelomeric end-blocked DNA or dumbbell-shaped 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.

[0858] In any of the aspects and embodiments provided herein, the therapeutic nucleic acid can be a therapeutic DNA. The therapeutic DNA can be ceDNA, ssDNA, CELiD, linear covalently enclosed DNA ("mini-string" or other), doggybone TM , prototelomere end-blocked DNA, dumbbell-shaped linear DNA, minigene, plasmid or minicircle.

[0859] In one embodiment, the therapeutic nucleic acid can be a circular single-stranded polynucleotide comprising at least three segments, two of which have sufficient complementarity to form a duplex, and an intermediate sequence containing 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.

[0860] The siRNA or miRNA that can reduce the intracellular level of a specific protein by a process called RNA interference (RNAi) is also considered by the present disclosure as a nucleic acid therapeutic agent. After siRNA or miRNA are introduced into the cytoplasm of the host cell, these double-stranded RNA constructs can be combined with a protein called RISC. The sense strand of the siRNA or miRNA is removed by the RISC complex. When the RISC complex is combined with the complementary mRNA, the mRNA is cut and the cut chain is released. RNAi is the reduction of the corresponding protein by inducing the specific destruction of mRNA.

[0861] Antisense oligonucleotides (ASOs) and ribozymes that suppress mRNA translation into protein can be used as nucleic acid therapeutic agents. For antisense constructs, these single-stranded deoxynucleic acids have sequences complementary to the sequence of target protein mRNA, and can be combined with mRNA by Watson-Crick base pairing. This combination prevents the translation of target mRNA, and / or triggers the RNase H degradation of mRNA transcripts. Therefore, antisense oligonucleotides have increased action specificity (that is, the downward regulation of specific disease-related proteins).

[0862] In any aspect and embodiment provided herein, therapeutic nucleic acid can be therapeutic RNA. The therapeutic RNA can be messenger RNA (mRNA) encoding a protein or peptide, an inhibitor of mRNA translation, an agent (RNAi) of RNA interference, a catalytically active RNA molecule (ribozyme), a transfer RNA (tRNA), an RNA (ASO) bound to an mRNA transcript, a protein or other molecular ligand (aptamer) or a guide RNA (gRNA). In any method provided herein, the agent of RNAi can be double-stranded RNA, single-stranded RNA, microRNA, short interfering RNA, short hairpin RNA or a triplex forming oligonucleotide.

[0863] End-closed DNA (ceDNA) vectors

[0864] In some embodiments, LNPs provided by the present disclosure comprise end-capped DNA (ceDNA).

[0865] In some embodiments, the TNA comprises a closed-end linear duplex (ceDNA) vector that can express a transgene (e.g., a therapeutic nucleic acid (TNA)). The ceDNA vectors described herein do not have the packaging limitations imposed by the limited space within the viral capsid. In contrast to encapsulated AAV genomes, ceDNA vectors represent a viable eukaryotic production alternative to prokaryotic production of plasmid DNA vectors. This allows for the insertion of control elements, e.g., regulatory switches, large transgenes, multiple transgenes, and the like, as disclosed herein.

[0866] ceDNA vectors preferably have linear and continuous structures rather than discontinuous structures. It is believed that linear and continuous structures are more stable when attacked by cellular endonucleases, and are less likely to reorganize and cause mutagenesis. Therefore, ceDNA vectors with linear and continuous structures are preferred embodiments. Continuous linear single-stranded intramolecular duplex ceDNA vectors can have covalently bound ends without a sequence encoding AAV capsid protein. These ceDNA vectors are structurally different from plasmids (including ceDNA plasmids described herein), which are circular duplex nucleic acid molecules of bacterial origin. The complementary chain of the plasmid can be separated to produce two nucleic acid molecules after denaturation, while in contrast, although the ceDNA vector has a complementary chain, it is a single DNA molecule, and therefore even if denatured, it may still remain a single molecule. In some embodiments, unlike plasmids, ceDNA vectors can be produced in the absence of prokaryotic type DNA base methylation. Thus, ceDNA vectors and ceDNA plasmids differ in terms of structure (specifically linear vs. circular) and also in terms of the methods used to produce and purify these different objects and in terms of their DNA methylation, i.e. ceDNA plasmids are of the prokaryotic type and ceDNA vectors are of the eukaryotic type.

[0867] Provided herein are non-viral, capsid-free ceDNA molecules (ceDNA) with covalently closed ends. These non-viral, capsid-free ceDNA molecules can be produced in permissive host cells by expression constructs (e.g., ceDNA-plasmids, ceDNA-bacmids, ceDNA-baculoviruses, or integrating cell lines) containing a heterologous gene (e.g., a transgene, specifically a therapeutic transgene) positioned between two different inverted terminal repeat (ITR) sequences, wherein the ITRs are different from each other. In some embodiments, one of the ITRs is modified by deletion, insertion, and / or substitution compared to a wild-type ITR sequence (e.g., AAV ITR); and at least one of the ITRs comprises a functional terminal melting site (trs) and a Rep binding site. The ceDNA vector is preferably duplexed on at least a portion of the molecule (e.g., an expression cassette), e.g., self-complementary (e.g., the ceDNA is not a double-stranded circular molecule). The ceDNA vector has covalently closed ends and is therefore resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III), e.g., for greater than one hour at 37°C.

[0868] 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 as described herein), and a second AAV ITR. In one embodiment, the first ITR (5' ITR) and the second ITR (3' ITR) are asymmetric relative to each other, i.e., they have different 3D spatial configurations from each other. 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, wherein the first ITR can be a mutated or modified ITR and the second ITR is a wild-type ITR. In one embodiment, both the first ITR and the second ITR are modified but have different sequences, or have different modifications, or are ITRs that are not identically modified and have different 3D spatial configurations. In other words, ceDNA vectors with asymmetric ITRs have ITRs in which any changes in one ITR relative to the WT-ITR are not reflected in the other ITR; or alternatively, in which the asymmetric ITRs have been modified. Asymmetric ITR pairs can have different sequences and different three-dimensional shapes relative to each other.

[0869] In one embodiment, 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 as described herein), and a second AAV ITR, wherein the first ITR (5' ITR) and the second ITR (3' ITR) are symmetrical or substantially symmetrical with respect to each other, that is, the ceDNA vector can comprise ITR sequences having a symmetrical three-dimensional spatial organization, such that their structures have the same shape in geometric space, or have the same A, CC', and BB' loops in 3D space. In such embodiments, the symmetrical ITR pair or substantially symmetrical ITR pair can be a modified ITR (e.g., mod-ITR) that is not a wild-type ITR. The mod-ITR pair can have the same sequence with one or more modifications relative to the wild-type ITR and are the reverse complement (inverse) of each other. In one embodiment, the modified ITR pair, as defined herein, is substantially symmetrical, that is, the modified ITR pair can have different sequences but have corresponding or identical symmetrical three-dimensional shapes. In some embodiments, the symmetrical or substantially symmetrical ITRs can be wild-type ITRs (WT-ITRs) as described herein. That is, both ITRs have wild-type sequences from the same AAV serotype. In some other embodiments, the two wild-type ITRs can be from different AAV serotypes. For example, one WT-ITR can be from one AAV serotype, while the other WT-ITR can be from a different AAV serotype. In such embodiments, the WT-ITR pair is substantially symmetrical, as defined herein, that is, it can have one or more conservative nucleotide modifications while still retaining a symmetrical three-dimensional spatial organization.

[0870] The wild-type or mutated or otherwise modified ITR sequences provided herein represent DNA sequences included in expression constructs (e.g., ceDNA-plasmids, ceDNA bacmids, ceDNA-baculoviruses) for use in generating ceDNA vectors. Thus, the ITR sequences actually contained in ceDNA vectors generated from ceDNA-plasmids or other expression constructs may or may not be identical to the ITR sequences provided herein due to naturally occurring variations during the production process (e.g., replication errors).

[0871] In one embodiment, the ceDNA vector in the LNP of the present disclosure comprising an expression cassette with a transgene as a therapeutic nucleic acid sequence can be operably linked to one or more regulatory sequences that allow or control the expression of the transgene. In one embodiment, the polynucleotide comprises a first ITR sequence and a second ITR sequence, wherein the nucleotide sequence of interest is flanked by the first and second ITR sequences, and the first and second ITR sequences are asymmetric to each other, or symmetric to each other.

[0872] In one embodiment, the expression cassette is positioned between two ITRs that have one or more of the following in the following order: a promoter operably linked to the 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 promotes transcription of the transgene. In one embodiment, the promoter is a CAG promoter or a variant thereof. A 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 expression of a transgene that is a therapeutic nucleic acid sequence.

[0873] In one embodiment, the post-transcriptional regulatory element comprises a WPRE. In one embodiment, the polyadenylation and termination signal comprises a BGHpolyA. Any cis-regulatory element known in the art or a combination thereof can be used in addition, such as 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 is greater than the maximum length known to be encapsulated in the AAV virion. 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 illustrated herein.

[0874] In one embodiment, the expression cassette can comprise greater than 4000 nucleotides, 5000 nucleotides, 10,000 nucleotides or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides or 50,000 nucleotides, or any range between about 4000-10,000 nucleotides or 10,000-50,000 nucleotides, or greater than 50,000 nucleotides. In some embodiments, the expression cassette can comprise a transgene, which is a therapeutic nucleic acid sequence with a length range of 500 to 50,000 nucleotides. In one embodiment, the expression cassette can comprise a transgene, which is a therapeutic nucleic acid sequence with a length range of 500 to 75,000 nucleotides. In one embodiment, the expression cassette can comprise a transgene, which is a therapeutic nucleic acid sequence with a length range of 500 to 10,000 nucleotides. In one embodiment, the expression cassette can comprise a transgene that is a therapeutic nucleic acid sequence ranging in length from 1000 to 10,000 nucleotides. In one embodiment, the expression cassette can comprise a transgene that is a therapeutic nucleic acid sequence ranging in length from 500 to 5,000 nucleotides. ceDNA vectors do not have the size limitations of encapsulated AAV vectors and are therefore capable of delivering large expression cassettes to the host. In one embodiment, ceDNA vectors lack prokaryotic-specific methylation.

[0875] In one embodiment, the rigid therapeutic nucleic acid can be a plasmid.

[0876] In one embodiment, the ceDNA vectors disclosed herein are used for therapeutic purposes (e.g., for medical, diagnostic, or veterinary use) or immunogenic polypeptides.

[0877] The expression cassette can contain any transgene that is a therapeutic nucleic acid sequence. In certain embodiments, the ceDNA vector comprises any gene of interest in a subject, including a protein, an enzyme, one or more polypeptides, a peptide, a ribozyme, a peptide nucleic acid, an siRNA, a gRNA, an mRNA, an RNAi, an antisense oligonucleotide, an antisense polynucleotide, an antibody, an antigen-binding fragment, or any combination thereof.

[0878] In one embodiment, the ceDNA expression cassette may include, for example, an expressible exogenous sequence (e.g., an open reading frame), the exogenous sequence encoding a protein that does not exist in the recipient subject, is inactive or lacks activity, or encodes a gene for a protein with a desired biological or therapeutic effect. In one embodiment, exogenous sequences such as donor sequences may encode a gene product that can play a role in correcting the expression of defective genes or transcripts. In one embodiment, the expression cassette may also encode a corrective DNA chain, an encoded polypeptide, a sense or antisense oligonucleotide, or encoding RNA or non-coding RNA (e.g., siRNA, guide RNA, shRNA, microRNA, and its antisense counterpart (e.g., antagoMiR)). In one embodiment, the expression cassette may include an exogenous sequence encoding a reporter protein 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 other reporter proteins well known in the art.

[0879] Thus, the expression cassette may include any gene encoding a protein, polypeptide, or RNA that is reduced or deleted due to mutation or that conveys a therapeutic benefit when overexpressed that is considered within the scope of the present disclosure. The ceDNA vector may comprise a template or donor nucleotide sequence that serves as a corrective DNA strand inserted after a double-strand break (or nick) provided by a nuclease. The ceDNA vector may comprise a template nucleotide sequence that serves as a corrective DNA strand inserted after a double-strand break (or nick) provided by a guide RNA nuclease, a meganuclease, or a zinc finger nuclease.

[0880] single-stranded (ss) nucleic acid molecules

[0881] In some embodiments, the TNA present in the LNP of the present disclosure can be a single-stranded nucleic acid, such as single-stranded DNA or single-stranded RNA. In one embodiment, the TNA can be a single-stranded RNA, such as mRNA. In another embodiment, the TNA can be a single-stranded DNA (ssDNA) molecule, such as a synthetic ssDNA molecule.

[0882] 3′ stem-loop structure

[0883] In some aspects, a TNA is an ssDNA molecule comprising at least one nucleic acid sequence of interest flanked by at least one stem-loop structure at its 3' end. In some embodiments, the ssDNA molecule may further comprise at least one stem-loop structure at its 5' end. As described herein, the stem-loop structure at the 3' end may comprise a partial DNA duplex (e.g., having a free 3'-OH group) to initiate replication or transcription. The partial DNA duplex serves, in part, to hold the stem-loop structure together.

[0884] According to some embodiments, the partial DNA duplex comprises 4-500 nucleotides, such as 4-10 nucleotides, 4-25 nucleotides, 4-50 nucleotides, 4-100 nucleotides, 4-200 nucleotides, 4-300 nucleotides, 4-400 nucleotides, 20-25 nucleotides, 20-50 nucleotides, 20-100 nucleotides, 20-200 nucleotides, 20-300 nucleotides, 20-400 nucleotides, 20-500 nucleotides, 50-100 nucleotides, 50-200 nucleotides, 50-300 nucleotides, 50-400 nucleotides. , 50-500 nucleotides, 150-200 nucleotides, 150-300 nucleotides, 150-400 nucleotides, 150-500 nucleotides, 200-300 nucleotides, 200-400 nucleotides, 200-500 nucleotides, 250-300 nucleotides, 250-400 nucleotides, 250-500 nucleotides, 300-400 nucleotides, 300-500 nucleotides, 350-400 nucleotides, 350-500 nucleotides, 400-500 nucleotides or 450-500 nucleotides, and at least one loop on 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 at least one loop at the 3′ end.

[0885] According to some embodiments, the loop structure at the 3′ end comprises a minimum of 3-500 unbound nucleotides, such as 3-450 nucleotides, 3-400 nucleotides, 3-350 nucleotides, 3-300 nucleotides, 3-250 nucleotides, 3-200 nucleotides, 3-150 nucleotides, 3-100 nucleotides, 3-90 nucleotides, 3-80 nucleotides, 3-70 nucleotides, 3-60 nucleotides, 3-50 nucleotides, 3-40 nucleotides, 3-30 nucleotides, 3-20 nucleotides, 3-10 nucleotides, 3-50 nucleotides, 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, 50-3 00 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, 250-400 nucleotides, 250-350 nucleotides, 250-300 nucleotides, 300-450 nucleotides, 300-400 nucleotides, 300-350 nucleotides, 350-450 nucleotides, 350-400 nucleotides, or 400-450 nucleotides.

[0886] According to some embodiments, the stem portion of the stem-loop is 4-500 nucleotides in length, and the loop portion of the stem-loop is 3-500 nucleotides in length. According to some embodiments, the stem portion of the stem-loop is 4-50 nucleotides in length, and the loop portion of the stem-loop is 3-50 nucleotides in length. According to some embodiments, the stem portion of the stem-loop is 4-20 nucleotides in length, and the loop portion of the stem-loop is 3-20 nucleotides in length. According to some embodiments, the stem portion of the stem-loop is 4-10 nucleotides in length, and the loop portion of the stem-loop is 3-10 nucleotides in length.

[0887] According to some embodiments, the loop further comprises one or more nucleic acids or nucleic acids used to stabilize the ends. According to other embodiments, the loop further comprises one or more nucleic acids useful in therapeutic methods. According to other embodiments, the loop further comprises one or more nucleic acids useful in diagnostic methods. According to other embodiments, the loop further comprises one or more nucleic acids useful for research purposes.

[0888] According to some embodiments, the minimum nucleic acid structure required at the 3' end of the ssDNA is any self-looping structure, i.e., a hairpin structure. However, it should be understood that as long as there is at least one stem and one loop, various structures can be envisioned at the 3' end. For example, in some embodiments, the ssDNA described herein can include at least one stem-loop structure at the 3' end. In some embodiments, the ssDNA can include at least two stem-loop structures at the 3' end. In some embodiments, the ssDNA can include at least three stem-loop structures at the 3' end. In some embodiments, the ssDNA can include at least four stem-loop structures at the 3' end. In some embodiments, the ssDNA can include at least five stem-loop structures at the 3' end.

[0889] According to some embodiments, the nucleotides at the 3' end form a cross-shaped DNA structure. When the two chains form a stem-loop structure at the same position in the molecule, a DNA cross-shaped structure can be formed, and includes a four-way junction and two closed hairpin-shaped points.

[0890] According to some embodiments, the nucleotides at the 3' end form a hairpin DNA structure.A hairpin loop structure in a nucleic acid consists of a base-paired stem structure and a loop sequence with unpaired or non-Watson-Crick paired nucleotides.

[0891] According to some embodiments, the nucleotides at the 3' end form a hammerhead DNA structure composed of three base-paired helices separated by short linkers of conserved sequence.

[0892] According to some embodiments, the nucleotides at the 3' end form a quadruplex DNA structure. G-quadruplex is a quadruplex DNA secondary structure (G4s) formed by certain guanine-rich sequences.

[0893] According to some embodiments, the nucleotides at the 3' end form a bulged DNA structure.

[0894] According to some embodiments, the nucleotides at the 3' end form a multi-branched loop.

[0895] According to some embodiments, the nucleotides at the 3' end do not form a 2-stem-loop structure.

[0896] According to some embodiments, the stem structure at the 3 ' end comprises one or more nucleotides modified to have exonuclease resistance. According to some embodiments, the stem structure at the 3 ' end comprises two or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more or 20 or more nucleotides modified to have exonuclease resistance.

[0897] According to some embodiments, the stem structure at the 3' end comprises one or more thiophosphate-modified nucleotides. According to some embodiments, the stem structure at the 3' end comprises about 2 to about 12 thiophosphate-modified nucleotides. According to some embodiments, the stem structure at the 3' end comprises about 4 to about 10 thiophosphate-modified nucleotides, for example, 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. According to some embodiments, the stem structure comprises greater than 10 thiophosphate-modified nucleotides.

[0898] According to some embodiments, the phosphorothioate-modified nucleotides are positioned adjacent to each other.

[0899] According to some embodiments, the one or more phosphorothioate-modified nucleotides at the 3' end are resistant to exonuclease degradation.Borophosphate-modified DNA is also resistant to nuclease degradation and can be considered an alternative to phosphorothioate modification.

[0900] According to further embodiments, the stem structure may comprise at least one functional moiety. In one embodiment, the at least one functional moiety is an aptamer sequence. In further embodiments, the aptamer sequence has a high binding affinity for a nuclear localization protein.

[0901] According to some embodiments, the nucleotides in the loop are chemically modified with functional groups in order to alter their properties.

[0902] 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).

[0903] According to some embodiments, the loop further comprises one or more synthetic ribozymes.

[0904] According to some embodiments, the loop further comprises one or more antisense oligonucleotides (ASOs).

[0905] According to some embodiments, the loop further comprises one or more short interfering RNAs (siRNAs).

[0906] According to some embodiments, the loop further comprises one or more antiviral nucleoside analogs (ANA).

[0907] According to some embodiments, the loop further comprises one or more triplex-forming oligonucleotides.

[0908] According to some embodiments, the loop further comprises one or more gRNAs or gDNAs.

[0909] According to some embodiments, the ring further comprises one or more molecular probes, such as nucleic acid-based fluorescent probes.

[0910] According to some embodiments, "click" azide-alkyne cycloaddition (Kolb et al., Applied Chemistry International English Edition 2001, 40, 2004-2021) is used to modify nucleotides in the ring. Click chemistry has been developed to join organic molecules together under mild conditions in the presence of various functional groups. By introducing novel base analogs, connecting fluorophores or isotopic elements for molecular imaging, forming interchain bonds between oligonucleotides, and for biological conjugation of molecules, most of the modifications mediated by click are performed on nitrogenous bases. The best example of click chemistry is Huisgen's [3+2] azide-alkyne cycloaddition reaction of Cu ICatalytic version (Angewandte Chemie International Edition 1963, 2, 633-645), independently discovered by Sharpless and Meldal (CuAAC reaction) (Angewandte Chemie International Edition 2002, 41, 2596-2599).

[0911] According to some embodiments, the introduction of reactive amino or thiol groups into synthetic oligonucleotides provides acceptors for, for example, subsequent chemifluorescent labeling.

[0912] According to some embodiments, the stem-loop structure may comprise alternative or modified nucleotides, including but not limited to ribonucleic acid (RNA), peptide-nucleic acid (PNA), locked nucleic acid (LNA). According to some embodiments, the ring portion of the stem-loop structure may comprise a chemical structure that does not comprise a nucleic acid.

[0913] IV. Preparation of Lipid Nanoparticles (LNPs)

[0914] Lipid nanoparticles (LNPs) can form spontaneously upon mixing a therapeutic nucleic acid (e.g., ceDNA, ssDNA, synthetic AAV, etc., as described herein) and a pharmaceutically acceptable excipient comprising a lipid.

[0915] Generally, LNP can be formed by any method known in the art.For example, LNP can be prepared by the method described in for example US2013 / 0037977, US2010 / 0015218, US2013 / 0156845, US2013 / 0164400, US2012 / 0225129 and US2010 / 0130588, and the content of each in said patent is incorporated herein by reference in its entirety. In certain embodiments, LNP can use continuous mixing method, direct d...

Claims

1. A polymer-conjugated lipid comprising: (i) polyglycerol (PG) or PG derivatives; (ii) lipid moiety represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein: R 1 Absent, hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10-30 carbon atoms; R 2 Absent, hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10-30 carbon atoms; When R 1 and R 2 When each is hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10-30 carbon atoms, N is positively charged; and R 3 is a hydrophobic tail comprising 10-30 carbon atoms; and (iii) a linker that conjugates the PG or the PG derivative to the lipid moiety, wherein in formula (I) is the bond that conjugates the lipid moiety and the linker.

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

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

4. The polymer-conjugated lipid according to claim 3, wherein the glutarylated PG is 3-methylglutarylated PG. The polymer-conjugated lipid according to 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. The polymer-conjugated lipid according to any one of claims 1 to 6, wherein R 1 does not exist, and where R 2 and R 3 Each independently comprises a hydrophobic tail of 14, 15, 16, 17, 18, 19, 20, 21 or 22 carbon atoms.

8. The polymer-conjugated lipid according to claim 7, wherein R 2 and R 3 Each independently comprises a hydrophobic tail of 16, 17, 18, 19, 20, 21 or 22 carbon atoms.

9. The polymer-conjugated lipid according to claim 8, wherein R 2 and R 3 Each independently comprises a hydrophobic tail comprising 18 carbon atoms, and wherein the lipid moiety is dioctadecylamine (DODA).

10. The polymer-conjugated lipid according to claim 9, wherein the lipid portion conjugated to the linker is represented by the following structure:

11. The polymer-conjugated lipid according to any one of claims 1 to 10, wherein the PG or the PG derivative comprises about 5 to 100 monomer units.

12. The polymer-conjugated lipid of claim 11, wherein the PG or the 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. 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. The polymer-conjugated lipid according to claim 13, wherein the PG or the PG derivative comprises about 8 monomer units.

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

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

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

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

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 phosphorylcholine linker, a carbamate linker, a diamide alkyl linker, a cleavable linker, or any combination thereof.

20. The polymer-conjugated lipid of claim 19, wherein 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 C(O)O—, wherein n is an integer ranging from 1 to 20.

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

22. The polymer-conjugated lipid of claim 20, wherein the linker is -C(O)(CH2) n -, and wherein n is 2, 3, 4, 5 or 6.

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

24. A polymer-conjugated lipid represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

25. A polymer-conjugated lipid represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

26. A polymer-conjugated lipid represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

27. A polymer-conjugated lipid represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

28. A polymer-conjugated lipid represented by the following structure: Where R is or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

29. The polymer-conjugated lipid according to any one of claims 1 to 28, further comprising an active species conjugated to the PG or the PG derivative, wherein the active species is functionalized to be conjugated to a targeting moiety.

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

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 trans-cyclooctene (TCO) reagent, a tetrazine (Tz) reagent, an alkyne reagent, and an azide reagent.

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

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

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

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

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

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

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

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

40. The polymer-conjugated lipid of claim 39, wherein the antibody or antibody fragment is a monoclonal antibody (mAb), a single chain variable fragment (scF v ), heavy chain antibodies (hcAb), nanobodies (Nb), heavy chain immunoglobulins (HC1g), immunoglobulin new antigen receptors (IgNARs), variable domains of immunoglobulin new antigen receptors (V NAR ), single domain antibodies or variable heavy chain antibodies only (V HH ).

41. A lipid nanoparticle (LNP), comprising: (i) Therapeutic nucleic acids (TNAs); (ii) ionizable lipids; (iii) sterols; and (iv) a first lipid-anchored polymer, wherein the first lipid-anchored polymer comprises the polymer-conjugated lipid according to any one of claims 1 to 28.

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

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

44. The LNP of claim 42 or 43, wherein the helper lipid is selected from the group consisting of: 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-heptadecanoyl-2-oleoyl-sn- Glycerol-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).

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

46. ​​The LNP of claim 42, wherein the helper lipid is represented by formula (II): or a pharmaceutically acceptable salt or ester thereof, wherein: Is a single bond or a double bond; R 1 C1-C 17 Alkyl or C2-C 17 alkenyl; R 2 C1-C 22 Alkyl or C2-C 22 alkenyl; R 3 is hydrogen or C1-C2 alkyl; and R 4 is hydrogen or C1-C2 alkyl.

47. The LNP of claim 46, wherein It's a double bond.

48. The LNP of claim 46 or 47, wherein R 1 C 10 -C 20 Alkenyl, R 2 C 10 -C 20 Alkyl, and R 3 For hydrogen.

49. The LNP of any one of claims 46 to 48, wherein the helper lipid represented by formula (II) is: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

50. The LNP of any one of claims 46 to 48, wherein the helper lipid represented by formula (II) is: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

51. The LNP of any one of claims 46 to 48, wherein the helper lipid represented by formula (II) is: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

52. The LNP of any one of claims 46 to 48, wherein the helper lipid represented by formula (II) is: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

53. The LNP of any one of claims 46 to 48, wherein the helper lipid represented by formula (II) is: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

54. The LNP of any one of claims 46 to 48, wherein the helper lipid represented by formula (II) is: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

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

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

57. The LNP of any one of claims 41 to 56, wherein the ionizable lipid is represented by: a) Formula (A): or a pharmaceutically acceptable salt thereof, wherein: R 1 and R 1′ Each is independently an optionally substituted linear or branched C 1-3 alkylene; R 2 and R 2′ Each is independently an optionally substituted linear or branched C 1-6 alkylene; R 3 and R 3′ Each is independently an optionally substituted linear or branched C 1-6 alkyl; Or alternatively, when R 2 is an optionally substituted branched C 1-6 When alkylene, R 2 and R 3 Together with its central nitrogen atom, it forms a 4- to 8-membered heterocyclic group; Or alternatively, when R 2′ is an optionally substituted branched C 1-6 When alkylene, R 2′ and R 3' Together with its central nitrogen atom, it forms a 4- to 8-membered heterocyclic group; R 4 and R 4' Each independently is -CR a 、-C(R a )2CR a or -[C(R a )2]2CR a ; R a H or C independently at each occurrence 1-3 alkyl; Or alternatively, when R 4 -C(R a )2CR a or -[C(R a )2]2CR a When R a C 1-3 When alkyl, R 3 and R 4 Together with its central nitrogen atom, it forms a 4- to 8-membered heterocyclic group; Or alternatively, when R 4' -C(R a )2CR a or -[C(R a )2]2CR a When R a C 1-3 When alkyl, R 3′ and R 4' Together with its central nitrogen atom, it forms a 4- to 8-membered heterocyclic group; R 5 and R 5′ are independently hydrogen, C 1-20 Alkylene or C 2-20 alkenylene; R 6 and R 6' C independently at each occurrence 1-20 Alkylene, C 3-20 Cycloalkylene or C 2-20 alkenylene; and m and n are each independently an integer selected from 1, 2, 3, 4 and 5; or b) formula (B): or a pharmaceutically acceptable salt thereof, wherein: a is an integer ranging from 1 to 20; b is an integer ranging from 2 to 10; R 1 Not present or selected from (C2-C 20 )alkenyl, -C(O)O(C2-C 20 ) alkyl and (C2-C 20 ) alkyl-substituted cyclopropyl; and R 2 (C2-C 20 )alkyl; or c) Formula (C): or a pharmaceutically acceptable salt thereof, wherein: R 1 and R 1′ Each independently is optionally selected from one or more of R a (C1-C6)alkylene substituted with a group; R 2 and R 2′ are each independently (C1-C2)alkylene; R 3 and R 3′ Each independently is optionally selected from one or more of R b (C1-C6) alkyl substituted with a group; Or alternatively, R 2 and R 3 and / or R 2′ and R 3′ Together with its central nitrogen atom, it forms a 4- to 7-membered heterocyclic group; R 4 and R 4′ Each is a (C2-C6)alkylene group interrupted by -C(O)O-; R 5 and R 5′ Each independently is (C2-C 30 )alkyl or (C2-C 30 )alkenyl, each of which is optionally interrupted by -C(O)O- or (C3-C6)cycloalkyl; and R a and R b each is halo or cyano; or d) Formula (D): or a pharmaceutically acceptable salt thereof, wherein: R' is absent, hydrogen or C1-C6 alkyl; provided that when R' is hydrogen or C1-C6 alkyl, R', R 1 and R 2 All nitrogen atoms are positively charged; R 1 and R 2 Each is independently hydrogen, C1-C6 alkyl or C2-C6 alkenyl; R 3 C1-C 12 Alkylene or C2-C 12 alkenylene; R 4 C1-C 18 Unbranched alkyl, C2-C 18 Unbranched alkenyl or Where: R 4a and R 4b Each independently is C1-C 16 Unbranched alkyl or C2-C 16 Unbranched alkenyl; R 5 Not present, C1-C8 alkylene or C2-C8 alkenylene; R 6a and R 6b Each independently is C7-C 16 Alkyl or C7-C 16 alkenyl; provided that the combined R 6a and R 6b The total number of carbon atoms in is greater than 15; 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 e) Formula (E): or a pharmaceutically acceptable salt thereof, wherein: R' is absent, hydrogen or C1-C3 alkyl; provided that when R' is hydrogen or C1-C3 alkyl, R', R 1 and R 2 The nitrogen atoms they are connected to have a positive charge; R 1 and R 2 Each is independently hydrogen or C1-C3 alkyl; R 3 C3-C 10 Alkylene or C3-C 10 alkenylene; R 4 C1-C 16 Unbranched alkyl, C2-C 16 Unbranched alkenyl or in: R 4a and R 4b Each independently is C1-C 16 Unbranched alkyl or C2-C 16 unbranched alkenyl; R 5 Not present, C1-C6 alkylene or C2-C6 alkenylene; R 6a and R 6b Each independently is C7-C 14 Alkyl or C7-C 14 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; and n is an integer selected from 1, 2, 3, 4, 5 and 6; or f) an ionizable lipid selected from the group consisting of any one of the ionizable lipids in Tables 1, 4, 5, 6 or 7.

58. The LNP of any one of claims 41 to 56, wherein the ionizable lipid is lipid 87, which is represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

59. The LNP of any one of claims 41 to 56, wherein the ionizable lipid is represented by the structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

60. The LNP of any one of claims 41 to 56, wherein the ionizable lipid is represented by the structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

61. The LNP of any one of claims 41 to 56, wherein the ionizable lipid is represented by the structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

62. The LNP of any one of claims 41 to 56, wherein the ionizable lipid is represented by the structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

63. The LNP of any one of claims 41 to 56, wherein the ionizable lipid is represented by the structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

64. The LNP of any one of claims 41 to 56, wherein the ionizable lipid is represented by the structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

65. The LNP of any one of claims 41 to 56, wherein the ionizable lipid is represented by the structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

66. The LNP of any one of claims 41 to 56, wherein the ionizable lipid is represented by the structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

67. The LNP of any one of claims 41 to 56, wherein the ionizable lipid is represented by the structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

68. The LNP of any one of claims 41 to 56, wherein the ionizable lipid is represented by the structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

69. The LNP of any one of claims 41 to 56, wherein the ionizable lipid is represented by the structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

70. The LNP of any one of claims 41 to 56, wherein the ionizable lipid is represented by the structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

71. The LNP of any one of claims 41 to 56, wherein the ionizable lipid is represented by the structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

72. The LNP of any one of claims 41 to 56, wherein the ionizable lipid is lipid A, represented by the following structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

73. The LNP of any one of claims 41 to 56, wherein the ionizable lipid is represented by the structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

74. The LNP of any one of claims 41 to 73, wherein the LNP further comprises a second lipid-anchored polymer, wherein the second lipid-anchored polymer comprises: (i) a lipid portion comprising at least one hydrophobic tail; (ii) polymers; (iii) a linker, wherein the polymer is conjugated to the lipid moiety via the linker; and (iv) an active species conjugated to the polymer, wherein the active species is functionalized to be conjugated to a targeting moiety.

75. The LNP of claim 74, wherein the polymer of the second lipid-anchoring 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.

76. The LNP of claim 75, wherein the PG derivative is 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 of 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 active species is a click chemistry reagent or a maleimide.

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

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

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

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

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

90. The LNP of claim 89, wherein the antibody or antibody fragment is a monoclonal antibody (mAb), a single chain variable fragment (scF v ), heavy chain antibodies (hcAb), nanobodies (Nb), heavy chain immunoglobulins (HC1g), immunoglobulin new antigen receptors (IgNARs), variable domains of immunoglobulin new antigen receptors (V NAR ), single domain antibodies or variable heavy chain antibodies only (V HH ).

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 phosphorylcholine linker, a carbamate linker, a diamide alkyl linker, a cleavable linker, and any combination thereof.

92. The LNP of claim 91, wherein 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 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 LNP of claim 92, wherein the linker is -C(O)(CH2) n -, and wherein n is 2, 3, 4, 5 or 6.

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

96. The LNP of any one of claims 74 to 95, wherein the lipid portion of the second lipid-anchored polymer is represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein: R 1 Absent, hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10-30 carbon atoms; R 2 Absent, hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10-30 carbon atoms; When R 1 and R 2 When each is hydrogen, C1-C6 alkyl, or a hydrophobic tail containing 10-30 carbon atoms, N is positively charged; and R 3 It is a hydrophobic tail containing 10-30 carbon atoms.

97. The LNP of claim 96, wherein R 1 does not exist, and where R 2 and R 3 Each independently comprises a hydrophobic tail of 14, 15, 16, 17, 18, 19, 20, 21 or 22 carbon atoms.

98. The LNP of claim 97, wherein R 2 and R 3 Each independently comprises a hydrophobic tail of 16, 17, 18, 19, 20, 21 or 22 carbon atoms.

99. The LNP of claim 98, wherein R 2 and R 3 Each independently comprises a hydrophobic tail comprising 18 carbon atoms, and wherein the lipid moiety is dioctadecylamine (DODA).

100. The LNP of any one of claims 74 to 99, wherein the lipid portion of the second lipid-anchoring polymer comprises a moiety 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-dioleoyl-sn-phospholipids acylethanolamine (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), dioctadecylamine (DODA), dihexadecylamine, distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and their derivatives.

101. The LNP of claim 100, wherein the lipid portion of the second lipid-anchoring 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-anchoring polymer comprises DSPE.

103. The LNP of any one of claims 74 to 102, wherein the average molecular weight of the polymer of the second lipid-anchored polymer is from 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 LNP of claim 74, wherein the second lipid-anchored polymer is represented by the structure: or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

107. The LNP of claim 74, wherein the second lipid-anchored polymer is represented by the structure: 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 lipids 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 lipids present in the LNP.

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

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

118. A lipid nanoparticle (LNP), comprising: (i) Therapeutic nucleic acids (TNAs); (ii) an ionizable lipid, wherein the ionizable lipid is heptadecan-9-yl 9-((4-(dimethylamino)butanoyl)oxy)hexadecanoate, and the ionizable lipid has the following structure: (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.

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

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

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

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

123. The LNP of claim 118, wherein: The ionizable lipid is present in the LNP in an amount from 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 lipids present in the LNP; The helper lipid is present in the LNP in an amount from about 5 mol % to about 15 mol % of the total lipids present in the LNP; The first lipid-anchoring polymer is present in the LNP in an amount from about 1.5 mol % to about 3 mol % of the total lipid present in the LNP; and The second lipid-anchoring polymer is present in the LNP in an amount from 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, end-blocked DNA (ceDNA), ssDNA, a ministring, a doggybone TM , prototelomere end-blocked DNA, dumbbell-shaped linear DNA, Dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), mRNA, tRNA, rRNA, gRNA, DNA viral vectors, viral RNA vectors, non-viral vectors and combinations 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 flanked at the 3' end by at least one stem-loop structure.

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

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

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

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

134. The LNP of any one of claims 129 to 132, wherein the loop structure at the 3' end comprises a minimum of 3 unbound 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 comprise the A, A', D, and D' regions present in wild-type AAV ITRs.

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 comprise the A, A', B, B', C, C', D, and D' regions present in wild-type AAV ITRs.

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 comprise a rep binding element (RBE) present in wild-type ITRs.

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 comprise a terminal melting site (trs) present in wild-type ITRs.

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 confer exonuclease resistance.

145. The LNP of claim 144, wherein the nucleotides are phosphorothioate-modified nucleotides.

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 any one of claims 147 to 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 comprise the A, A', D, and D' regions present in wild-type AAV ITRs.

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 comprise the A, A', B, B', C, C', D, and D' regions present in wild-type AAV ITRs.

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 comprise a rep binding element (RBE) present in wild-type ITRs.

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 comprise a terminal melting site (trs) present in wild-type ITRs.

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 confer exonuclease resistance.

158. The LNP of claim 157, wherein the nucleotides are phosphorothioate-modified nucleotides.

159. The LNP of any one of claims 147 to 158, wherein the loop structure at the 5' end further comprises 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 (ASOs).

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 ring 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 to 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 minimally activates an immune pathway.

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 a cGAS / STING pathway, a TLR9 pathway, an 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 coagulation factor, a transcription factor, a replication factor, a growth factor, a hormone, and a fusion protein.

177. The LNP of claim 175 or 176, wherein the at least one therapeutic protein is useful for treating a genetic disease selected from the group consisting of melanoma, hemophilia A (factor VIII (FVIII) deficiency) and hemophilia B (factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited liver metabolic disorders, Lesch Nyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi's anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharidoses (e.g.,Hurler syndrome (MPS type I), Scheie syndrome (MPS type I S), Hurler-Scheie syndrome (MPS type I HS), Hunter syndrome (MPS type II), Sanfilippo types A, B, C, and D (MPS types III A, B, C, and D), Morquio types A and B (MPS IVA and MPS IVB), Maroteaux-Lamy syndrome (MPS type VI), Sly syndrome (MPS type VII), hyaluronidase deficiency (MPS type IX), Niemann-Pick disease types A / B, C1, and C2, Fabry disease, Schindlerdisease, GM2-gangliosidosis type II (Sandhoff disease), 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, Battendisease, aspartate glucosamineuria, sialidosis (Salla disease), Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidosis, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease 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 congenital amaurosis, Stargardt 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 according to any one of claims 41 to 177 and a pharmaceutically acceptable carrier.

179. A method of treating a genetic disease 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.

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

181. The method of claim 179 or 180, wherein the genetic disease is selected from the group consisting of: sickle cell anemia; melanoma; hemophilia A (coagulation factor VIII (FVIII) deficiency); hemophilia B (coagulation 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 syndrome; retinoblastoma; mucopolysaccharidoses; 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; aspartate glucosamiduria; sialic acid storage diseases; Dannon disease (LAMP-2 deficiency); lysosomal acid lipase (LAL) deficiency; neuronal ceroid lipofuscinosis (NCL); sphingolipid storage diseases, 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; systemic arterial calcification of infancy (GACI); Leber 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.

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

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

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

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

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

189. The method of any one of claims 185 to 187, wherein the acidic conditions comprise HCl, Br, HI, HClO4, HClO3, H2SO4, or HNO3.

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

191. The method of any one of claims 185 to 190, wherein the lipid moiety conjugated to a linker is represented by the structure:

192. The method of any one of claims 185 to 190, wherein the polymer-conjugated lipid is represented by the structure: Where n is a number ranging from 10 to 100.

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

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