Lipid nanoparticles comprising nucleic acids, ionizable lipids, sterols, lipid-anchored polymers, and helper lipids, and uses thereof

Abstract

Lipid-based nanoparticles have played a key role in the success of many other nanopharmaceuticals and are therefore considered a leading candidate among nanoscale drug delivery systems. However, effective targeted delivery of biologically active substances, such as therapeutic nucleic acids, remains a continuing medical challenge. Provided herein are lipid nanoparticle (LNP) compositions (e.g., pharmaceutical compositions) containing therapeutic nucleic acids (TNAs), where the LNPs comprise an ionizable lipid, a "helper" lipid, e.g., ceramide or distearoylphosphatidylcholine (DSPC), a structural lipid, e.g., a sterol, and one or more types of lipid-anchored polymers, as well as uses thereof.

Description

[Technical Field]

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

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

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

[0004] Furthermore, viral delivery of nucleic acid therapeutics to target cells is largely hindered by the activation of innate and / or adaptive immune responses. While it is possible to avoid RNA sensing by myeloid dendritic cells (MDCs) by chemically modifying RNA cargo (e.g., with 1mΨ, 2'OMe, etc.), no known chemical modifications to DNA cargo can limit pattern recognition receptor (PRR) sensing and still maintain transcriptional activity. An alternative approach to gene therapy is the recombinant adeno-associated virus (rAAV) vector platform, which packages heterologous DNA into the viral capsid. However, there are several major disadvantages to using rAAV vectors as gene delivery vectors. One major drawback associated with rAAV is the limited viral packaging capacity of approximately 4.5 kb of heterologous DNA. Another major drawback is capsid immunogenicity, which prevents re-administration to patients.

[0005] Thus, there remains a need for effective non-viral delivery vehicles that allow for the safe and effective delivery of nucleic acid therapeutics to desired cell populations. Summary of the Invention

[0006] The present disclosure provides lipid nanoparticles (LNPs) and LNP compositions (e.g., pharmaceutical compositions) containing therapeutic nucleic acids (TNAs), e.g., gene expression vectors such as closed-end DNA (ceDNA), single-stranded DNA (ssDNA) vectors, or messenger RNA (mRNA). The LNPs of the present disclosure comprise structural LNP components including an ionizable lipid, a "helper" lipid such as ceramide or distearoylphosphatidylcholine (DSPC), a structural lipid such as a sterol, and one or more types of lipid-anchored polymers. The LNPs disclosed herein offer surprising and unexpected properties compared to known LNPs. For example, helper lipids in LNPs function to increase the membrane fusogenicity of the lipid bilayer of LNPs and facilitate endosomal escape; structural lipids in LNPs contribute to the membrane integrity and stability of LNPs; and lipid-anchored polymers in LNPs can inhibit LNP aggregation and provide steric stabilization (e.g., enhancing the stealth properties of the overall LNP characteristics in the circulation (e.g., blood compartment) by minimizing interactions between opsonins present in the blood and the surface of the LNP). Furthermore, the disclosed LNP compositions are characterized by reduced LNP-associated toxicity, as evidenced by serum levels of immune response markers (see the Examples herein). Furthermore, the disclosed LNPs with a certain molecular proportion of sterols (e.g., 30%-45% molecular proportion of total lipids) are characterized by a diameter of approximately 80 nm or less, making them particularly useful for therapeutic administration specifically targeted to certain tissues / organs that have size limitations for effective delivery.

[0007] According to one aspect, the present disclosure provides a lipid nanoparticle (LNP), comprising: a therapeutic nucleic acid (TNA); an ionizable lipid; Sterols and A first lipid-anchored polymer, the lipid-anchored polymer comprising: i) a polymer; ii) a lipid moiety comprising at least one hydrophobic tail; iii) optionally a linker that attaches the polymer to the lipid moiety; a first lipid-anchored polymer, wherein at least one hydrophobic tail comprises 12 to 22 carbon atoms in a single aliphatic chain backbone; Formula (I): [ka] Formula (I) (In the formula, [ka] is a single bond or a double bond, A is hydrogen, [ka] , or [ka] and R 1 But C1-C 17 Alkyl or C2-C 17 is alkenyl, R 2 But C1-C 22 Alkyl or C2-C 22 is alkenyl, R 3 is hydrogen or C1-C2 alkyl, R 4 is hydrogen or C1-C2 alkyl), or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0008] According to another aspect, the present disclosure provides a lipid nanoparticle (LNP), comprising: a therapeutic nucleic acid (TNA); an ionizable lipid; Sterols and A first lipid-anchored polymer, the lipid-anchored polymer comprising: i) a polymer; ii) a lipid moiety comprising at least two hydrophobic tails; iii) a linker that connects the polymer to the lipid moiety; a first lipid-anchored polymer, wherein at least two hydrophobic tails each contain 16 to 22 carbon atoms in a single aliphatic chain backbone; Formula (I): [ka] Formula (I) (In the formula, [ka] is a single bond or a double bond, A is hydrogen, [ka] , or [ka] and R 1 But C1-C 17 Alkyl or C2-C 17 is alkenyl, R 2 But C1-C 22 Alkyl or C2-C 22 is alkenyl, R 3 is hydrogen or C1-C2 alkyl, R 4 is hydrogen or C1-C2 alkyl), or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0009] According to yet another aspect, the present disclosure provides a lipid nanoparticle (LNP), comprising: a therapeutic nucleic acid (TNA); an ionizable lipid; Sterols and A first lipid-anchored polymer, the lipid-anchored polymer comprising: i) a polymer; ii) a lipid moiety comprising at least two hydrophobic tails; iii) a linker that connects the polymer to the lipid moiety; a first lipid-anchored polymer, wherein at least two hydrophobic tails each contain 12 to 15 carbon atoms in a single aliphatic chain backbone; Formula (I): [ka] Formula (I) (In the formula, [ka] is a single bond or a double bond, A is hydrogen, [ka] , or [ka] and R 1 But C1-C 17 Alkyl or C2-C 17 is alkenyl, R 2 But C1-C 22 Alkyl or C2-C 22 is alkenyl, R 3 is hydrogen or C1-C2 alkyl, R 4 is hydrogen or C1-C2 alkyl), or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0010] According to a further aspect, the present disclosure provides a lipid nanoparticle (LNP), comprising: a therapeutic nucleic acid (TNA); an ionizable lipid; Sterols and A first lipid-anchored polymer, the lipid-anchored polymer comprising: i) a polymer; ii) a lipid moiety comprising a single hydrophobic tail; iii) a linker that connects the polymer to the lipid moiety; a first lipid-anchored polymer, wherein the single hydrophobic tail comprises 18 to 22 carbon atoms in a single aliphatic chain backbone; Formula (I): [ka] Formula (I) (In the formula, [ka] is a single bond or a double bond, A is hydrogen, [ka] , or [ka] and R 1 But C1-C 17 Alkyl or C2-C 17 is alkenyl, R 2 But C1-C 22 Alkyl or C2-C 22 is alkenyl, R 3 is hydrogen or C1-C2 alkyl, R 4 is hydrogen or C1-C2 alkyl), or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0011] In some embodiments, the helper lipid in the LNPs provided herein is represented by the structure of formula (II): [ka] Formula (II) or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0012] In some embodiments, the helper lipid is represented by the structure of formula (III): [ka] Formula (III) or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0013] In some embodiments, the helper lipid is represented by the structure of formula (IV): [ka] Formula (IV) or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0014] In some embodiments, the LNPs of the present disclosure do not comprise distearoylphosphatidylcholine (DSPC), provided that a helper lipid represented by (I), (II), (III), or (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present. In some embodiments, the LNPs of the present disclosure do not comprise 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), provided that a helper lipid represented by (I), (II), (III), or (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present. In some embodiments, the LNPs of the present disclosure do not comprise 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), provided that a helper lipid represented by (I), (II), (III), or (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present.

[0015] In some embodiments, in Formula (I), Formula (II), Formula (III), or Formula (IV), R 1 is C1-C 10 Alkyl or C2-C 10 alkenyl, and R 2 , R 3 , and R 4 is as defined above.

[0016] In some embodiments, in Formula (I), Formula (II), Formula (III), or Formula (IV): [ka] is a double bond and R 1 , R 2 , R 3 , and R 4 is as defined above.

[0017] In some embodiments, in Formula (I), Formula (II), Formula (III), or Formula (IV), R 1 is C1-C8 alkyl or C2-C8 alkenyl, and R 2 , R 3 , and R 4 is as defined above. In some embodiments, in Formula (I), Formula (II), Formula (III), or Formula (IV), R 1 is C1-C7 alkyl or C2-C7 alkenyl, and R 2 , R 3 , and R 4 is as defined above. In some embodiments, in Formula (I), Formula (II), Formula (III), or Formula (IV), R 1 is a C1 alkyl, a C3 alkyl, a C5 alkyl, or a C7 alkyl, and R 2 , R 3 , and R 4 is as defined above. In some embodiments, in Formula (I), Formula (II), Formula (III), or Formula (IV), R 1 is a C1 alkyl, and R 2 , R 3 , and R4 is as defined above.

[0018] In some embodiments, in Formula (I), Formula (II), Formula (III), or Formula (IV), R 2 is C3-C 15 Alkyl or C3-C 15 alkenyl, and R 1 , R 3 , and R 4 is as defined above. In some embodiments, in Formula (I), Formula (II), Formula (III), or Formula (IV), R 2 is C9 alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl or C 15 alkyl, and R 1 , R 3 , and R 4 is as defined above. In some embodiments, R 2 is C 12 Alkyl, C 13 Alkyl or C 14 alkyl, and R 1 , R 3 , and R 4 is as defined above. In some embodiments, in Formula (I), Formula (II), Formula (III), or Formula (IV), R 2 is C 13 alkyl, and R 1 , R 3 , and R 4 is as defined above.

[0019] In some embodiments, in Formula (I), Formula (II), Formula (III), or Formula (IV), R 3 is hydrogen and R 1 , R 2 , and R 4 is as defined above. In some embodiments, in Formula (I), Formula (II), Formula (III), or Formula (IV), R 3 is a C1 alkyl, and R 1 , R 2 , and R 4is as defined above.

[0020] In some embodiments, in Formula (I), Formula (II), Formula (III), or Formula (IV), R 4 is hydrogen and R 1 , R 2 , and R 3 is as defined above. In some embodiments, R 4 is a C1 alkyl, and R 1 , R 2 , and R 3 is as defined above.

[0021] In some embodiments, the helper lipid represented by formula (I) is selected from any of the helper lipids listed in Table 8, or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0022] In some embodiments, the helper lipid represented by formula (I) is [ka] ; [ka] ; [ka] ; [ka] ; [ka] , and Selected from the structure or [ka] ; or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0023] In some embodiments, the helper lipid represented by Formula (I) or Formula (II) has the structure: [ka] ; or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0024] In some embodiments, the lipid represented by Formula (I), Formula (III), or Formula (IV) has the structure: [ka] ; or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0025] According to another aspect, the present disclosure provides a lipid nanoparticle (LNP), comprising: a therapeutic nucleic acid (TNA); an ionizable lipid; Sterols and A first lipid-anchored polymer, the first lipid-anchored polymer comprising: i) a polymer; ii) a lipid moiety comprising at least two hydrophobic tails; iii) a linker that connects the polymer to the lipid moiety; a first lipid-anchored polymer, wherein at least two hydrophobic tails each contain 16 to 22 carbon atoms in a single aliphatic chain backbone; a helper lipid selected from distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), The LNPs have a whole blood half-life (t 1 / 2 The present invention provides lipid nanoparticles (LNPs) having a

[0026] In some embodiments, the at least two hydrophobic tails of the first lipid-anchored polymer each have 18 to 22 carbon atoms in the single aliphatic chain backbone. In some embodiments, the at least two hydrophobic tails of the first lipid-anchored polymer each have 18 to 20 carbon atoms in the single aliphatic chain backbone. In some embodiments, the at least two hydrophobic tails of the first lipid-anchored polymer each have 18 carbon atoms in the single aliphatic chain backbone.

[0027] In some embodiments, a helper lipid selected from the group consisting of distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) is present in the LNP in an amount of about 2 mol% to about 40 mol%, or about 5 mol% to about 35 mol%, or about 5 mol% to about 30 mol%, or about 5 mol% to about 25 mol%, or about 5 mol% to about 20 mol%, or about 5 mol% to about 15 mol%, or about 5 mol% to about 10 mol%, or about 10 mol% to about 15 mol% of the total lipid present in the LNP. In some embodiments, a helper lipid selected from the group consisting of distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) is present in the LNP in an amount of about 10 mol%. In one embodiment, the helper lipid is DSPC. In one embodiment, the DSPC helper lipid is present in an amount of about 10 mol%.

[0028] In some embodiments, LNPs comprising a helper lipid selected from the group consisting of DSPC, DOPC, and DOPE have a whole blood half-life (t) of about 3 hours to about 24 hours, or about 3 hours to about 18 hours, or about 3 hours to about 15 hours, or about 3 hours to about 12 hours, or about 3 hours to about 10 hours, or about 3 hours to about 9 hours, or about 3 hours to about 8 hours, or about 3 hours to about 7.5 hours, or about 3 hours to about 6.5 hours, or about 3 hours to about 6 hours. 1 / 2In some embodiments, the LNP has a whole blood half-life (t) of about 3 hours to about 3.5 hours, or about 3 hours to about 4 hours, or about 3 hours to about 4.5 hours, or about 3 hours to about 5 hours, or about 3 hours to about 5.5 hours, or about 3.5 hours to about 4 hours, or about 3.5 hours to about 4.5 hours, or about 3.5 hours to about 5 hours, or about 3.5 hours to about 5.5 hours, or about 4 hours to about 4.5 hours, or about 4 hours to about 5 hours, or about 4 hours to about 5.5 hours, or about 4.5 hours to about 5 hours, or about 4.5 hours to about 5.5 hours, or about 5 hours to about 5.5 hours. 1 / 2 In some embodiments, in comparison, the reference LNP has a whole blood half-life (t) of about 3 hours or less, e.g., about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 1 hour, about 2 hours, or about 2.5 hours. 1 / 2 ). The reference LNP does not include a first lipid-anchored polymer having at least two hydrophobic tails with 16-22 carbon atoms in a single aliphatic chain backbone. In one embodiment, the reference LNP includes a first lipid-anchored polymer having at least two hydrophobic tails with 12-15 carbon atoms each in a single aliphatic chain backbone. In one embodiment, the first lipid-anchored polymer is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG, also referred to as PEG-DMG). In one embodiment, the reference LNP includes DMG-PEG and a helper lipid selected from the group consisting of DSPC, DOPC, and DOPE.

[0029] In some embodiments, LNPs comprising a helper lipid selected from the group consisting of DSPC, DOPC, and DOPE have a whole blood clearance rate (Cl) of about 10 mL / min / kg to about 50 mL / min / kg, or about 10 mL / min / kg to about 45 mL / min / kg, or about 10 mL / min / kg to about 40 mL / min / kg. In some embodiments, LNPs have a whole blood clearance rate (Cl) of about 30 mL / min / kg to about 40 mL / min / kg, or about 35 mL / min / kg to about 40 mL / min / kg, or about 10 mL / min / kg to about 20 mL / min / kg, or about 10 mL / min / kg to about 18 mL / min / kg, or about 10 mL / min / kg to about 15 mL / min / kg. In some embodiments, in comparison, the reference LNP has a whole blood clearance rate (Cl) that is at least twice the value of the whole blood clearance rate (Cl) of the LNP comprising a helper lipid selected from the group consisting of DSPC, DOPC, and DOPE described above, e.g., greater than about 50 mL / min / kg, or about 50-100 mL / min / kg, or about 50-150 mL / min / kg, or about 50-200 mL / min / kg, or about 50-250 mL / min / kg, or about 50-300 mL / min / kg, or about 50-350 mL / min / kg, or about 100-150 mL / min / kg, or about 100-200 mL / min / kg, or about 100-250 mL / min / kg, or about 100-300 mL / min / kg, or about 100-350 mL / min / kg. In one embodiment, the reference LNP does not include a first lipid-anchored polymer having at least two hydrophobic tails with 16-22 carbon atoms in a single aliphatic chain backbone. In one embodiment, the reference LNP includes a lipid-anchored polymer including at least two hydrophobic tails, each containing 12-15 carbon atoms in a single aliphatic chain backbone. In one embodiment, the reference lipid-anchored polymer is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG, also referred to as PEG-DMG). In one embodiment, the reference includes DMG-PEG and a helper lipid selected from the group consisting of DSPC, DOPC, and DOPE.

[0030] In some embodiments, LNPs comprising a helper lipid selected from the group consisting of DSPC, DOPC, and DOPE have a whole blood end-point exposure (AUC last ) in whole blood. In one embodiment, the final time point is 24 hours. In other embodiments, the final time point is about 18 hours, about 20 hours, about 22 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 38 hours, or about 40 hours. In some embodiments, the LNPs have a whole blood final time point exposure (AUC) of about 50 h*ng / mL to about h*ng / mL, or about 100 h*ng / mL to about 750 h*ng / mL, or about 150 h*ng / mL to about 750 h*ng / mL, or about 200 h*ng / mL to about 700 h*ng / mL. last )

[0031] In some embodiments, LNPs comprising a helper lipid selected from the group consisting of DSPC, DOPC, and DOPE have a whole blood end-point exposure (AUC) of about 200 h*ng / mL to about 250 h*ng / mL, or about 200 h*ng / mL to about 300 h*ng / mL, or about 500 h*ng / mL to about 700 h*ng / mL, or about 500 h*ng / mL to about 550 h*ng / mL, or about 500 h*ng / mL to about 600 h*ng / mL, or about 550 h*ng / mL to about 600 h*ng / mL, or about 600 h*ng / mL to about 700 h*ng / mL, or about 600 h*ng / mL to about 650 h*ng / mL, or about 650 h*ng / mL to about 700 h*ng / mL. last In some embodiments, in comparison, the reference LNP has a whole blood end-point exposure (AUC last) or 50 h*ng / mL or less, e.g., about 40-45 h*ng / mL, or about 35-40 h*ng / mL, or about 30-35 h*ng / mL, or about 25-30 h*ng / mL, or about 20-25 h*ng / mL, or about 15-20 h*ng / mL, or about 10-15 h*ng / mL, or about 5-10 h*ng / mL. In one embodiment, the reference LNP does not include a first lipid-anchored polymer having at least two hydrophobic tails with 16-22 carbon atoms in a single aliphatic chain backbone. In one embodiment, the reference LNP includes a reference lipid-anchored polymer having at least two hydrophobic tails each with 12-15 carbon atoms in a single aliphatic chain backbone. In one embodiment, the reference lipid-anchored polymer is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG). In one embodiment, the reference comprises DMG-PEG and a helper lipid selected from the group consisting of DSPC, DOPC, and DOPE.

[0032] In some embodiments, the first lipid-anchored polymer in an LNP of the present disclosure comprises a lipid moiety comprising one hydrophobic tail or two hydrophobic tails. In one embodiment, the first lipid-anchored polymer in an LNP of the present disclosure comprises a lipid moiety comprising two hydrophobic tails. In one embodiment, each of the two hydrophobic tails is a fatty acid. In some embodiments, the two hydrophobic tails each independently comprise 16, 17, 18, 19, 20, 21, or 22 carbon atoms. In some embodiments, the two hydrophobic tails each independently comprise 16, 17, 18, 19, 20, or 21 carbon atoms. In some embodiments, the two hydrophobic tails each independently comprise 16, 17, 18, 19, or 20 carbon atoms. In some embodiments, the two hydrophobic tails each independently comprise 16, 17, 18, 19, or 20 carbon atoms. In some embodiments, the two hydrophobic tails each independently comprise 16, 17, 18, or 19 carbon atoms. In some embodiments, the two hydrophobic tails each independently comprise 16, 17, 18, or 19 carbon atoms. In some embodiments, the two hydrophobic tails each independently comprise 16, 17, or 18 carbon atoms. In one embodiment, the two hydrophobic tails each comprise 16 carbon atoms. In one embodiment, the two hydrophobic tails each comprise 18 carbon atoms. In one embodiment, the two hydrophobic tails each comprise 20 carbon atoms. In some embodiments, the two hydrophobic tails are each independently selected from the group consisting of octadecylamine, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, and derivatives thereof.

[0033] In some embodiments, the two hydrophobic tails each independently comprise 12, 13, 14, or 15 carbon atoms. In some embodiments, the two hydrophobic tails each independently comprise 12, 13, or 14 carbon atoms. In one embodiment, the two hydrophobic tails each comprise 12 carbon atoms. In one embodiment, the two hydrophobic tails each comprise 14 carbon atoms. In some embodiments, the two hydrophobic tails are each independently selected from the group consisting of lauric acid, myristic acid, myristoleic acid, and derivatives thereof.

[0034] In one embodiment, the first lipid-anchored polymer in the LNP of the present disclosure comprises a lipid moiety comprising a single hydrophobic tail. In one embodiment, the single hydrophobic tail is a fatty acid. In some embodiments, the single hydrophobic tail comprises 12, 14, 16, 18, 20, or 22 carbon atoms. In some embodiments, the single hydrophobic tail comprises 12, 14, 16, or 18 carbon atoms. In one embodiment, the single hydrophobic tail comprises 14 carbon atoms. In one embodiment, the single hydrophobic tail comprises 16 carbon atoms. In one embodiment, the single hydrophobic tail comprises 18 carbon atoms. In some embodiments, the single hydrophobic tail is selected from the group consisting of lauric acid, myristic acid, myristoleic acid, octadecylamine, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, and derivatives thereof.

[0035] In some embodiments, the first lipid-anchored polymer is a glycerolipid. In some embodiments, the first lipid-anchored polymer is a phospholipid. In some embodiments, the first lipid-anchored polymer does not comprise distearoylphosphatidylcholine (DSPC), provided that a helper lipid represented by (I), (II), (III), or (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present.

[0036] In some embodiments, the first lipid-anchored polymer is selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielideyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl- The lipid moiety comprises a linker lipid moiety selected from the group consisting of 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), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), derivatives thereof, and combinations of any of the foregoing. In some embodiments, the linker lipid moiety in the first lipid-anchored polymer is selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, derivatives thereof, and combinations of any of the foregoing.

[0037] In some embodiments, the first lipid-anchored polymer is selected from the group consisting of 1,2-dimyristoyl-rac-glycero-3-methoxy (DMG), R-3-[(ω-methoxycarbamoyl)]-1,2-dimyristyloxyl-propyl-3-amine, derivatives thereof, and combinations of any of the foregoing. In some embodiments, the first lipid-anchored polymer comprises DMG.

[0038] In some embodiments, the 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 some embodiments, the polymer is selected from the group consisting of polyethylene glycol (PEG), polyglycerol (PG), polysarcosine (pSar), or combinations thereof.

[0039] In some embodiments, the polymer has a molecular weight of about 1000 Da to about 5000 Da. In some embodiments, the polymer has a molecular weight of about 2000 Da to about 5000 Da. In some embodiments, the polymer has a molecular weight of about 2000 Da. In some embodiments, the polymer has a molecular weight of about 3200 Da to about 3500 Da.

[0040] In some embodiments, the polymer is polyethylene glycol (PEG).

[0041] In some embodiments, the sterol is selected from the group consisting of cholesterol, beta-sitosterol, stigmasterol, beta-sitostanol, campesterol, brassicasterol, and derivatives thereof, and combinations thereof. In some embodiments, the sterol is cholesterol. In some embodiments, the sterol is beta-sitosterol.

[0042] In some embodiments, the ionizable lipid is a) Formula (A): [ka] Formula (A) (In the formula, R 1 and R 1’ each independently represents an optionally substituted linear or branched C 1-3 is alkylene, R2 and R 2’ each independently represents an optionally substituted linear or branched C 1-6 is alkylene, R 3 and R 3’ each independently represents an optionally substituted linear or branched C 1-6 Is it alkyl? Alternatively, R 2 optionally substituted branched C 1-6 When R is alkylene, 2 and R 3 together with the intervening N atom to form a 4- to 8-membered heterocyclyl, or Alternatively, R 2’ optionally substituted branched C 1-6 When R is alkylene, 2’ and R 3’ together with the intervening N atom to form a 4- to 8-membered heterocyclyl; R 4 and R 4’ are each independently -CR a , -C(R a )2CR a , or -[C(R a )2]2CR a and R a each occurrence independently represents H or C 1-3 Is it alkyl? Alternatively, R 4 -C(R a )2CR a or -[C(R a )2]2CR a and R a C 1-3 If it is alkyl, R 3 and R 4 together with the intervening N atom to form a 4- to 8-membered heterocyclyl, or Alternatively, R 4’ -C(R a )2CR a or -[C(R a )2]2CR a and Ra C 1-3 If it is alkyl, R 3’ and R 4’ together with the intervening N atom to form a 4- to 8-membered heterocyclyl; R 5 and R 5’ are each independently hydrogen, C 1-20 Alkylene or C 2-20 is alkenylene, R 6 and R 6’ But for each occurrence, independently, C 1-20 Alkylene, C 3-20 Cycloalkylene or C 2-20 is alkenylene, wherein m and n are each independently an integer selected from 1, 2, 3, 4, and 5, or a pharmaceutically acceptable salt thereof; b) Formula (B): [ka] Formula (B) (In the formula, a is an integer ranging from 1 to 20; b is an integer ranging from 2 to 10; R 1 is absent or (C2-C 20 ) alkenyl, -C(O)O(C2-C 20 ) alkyl, and (C2-C 20 ) cyclopropyl substituted with alkyl; R 2 However, (C2-C 20 ) alkyl), or a pharmaceutically acceptable salt thereof; or c) Formula (C): [ka] Formula (C) (In the formula, R 1 and R 1’ are each independently R aand (C1-C6) alkylene optionally substituted with one or more groups selected from R 2 and R 2’ are each independently (C1-C2) alkylene; R 3 and R 3’ are each independently R b or (C1-C6) alkyl optionally substituted with one or more groups selected from Alternatively, R 2 and R 3 and / or R 2’ and R 3’ together with the intervening N atom to form a 4- to 7-membered heterocyclyl; R 4 and R 4 each ' is (C2-C6)alkylene interrupted by -C(O)O-; R 5 and R 5 each independently being optionally interrupted by —C(O)O— or (C3-C6)cycloalkyl, (C2-C 30 ) alkyl or (C2-C 30 ) alkenyl, R a and R b wherein each is halo or cyano, or a pharmaceutically acceptable salt thereof; d) Formula (D): [ka] Formula (D) (In the formula, R' is absent, hydrogen, or C1-C6 alkyl, provided that when R' is hydrogen or C1-C6 alkyl, R', R 1 , and R 2 are all positively charged nitrogen atoms, R 1 and R 2 are each independently hydrogen, C1-C6 alkyl, or C2-C6 alkenyl; R3 But C1-C 12 Alkylene or C2-C 12 is alkenylene, R 4 But C1-C 18 Unbranched alkyl, C2-C 18 unbranched alkenyl, or [ka] wherein: R 4a and R 4b are each independently, C1-C 16 Unbranched alkyl or C2-C 16 is an unbranched alkenyl, R 5 is absent, C1-C8 alkylene, or C2-C8 alkenylene; R 6a and R 6b are each independently, C7-C 16 Alkyl or C7-C 16 alkenyl, provided that R 6a and R 6b the total number of carbon atoms in the 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 ais independently at each occurrence hydrogen or C1-C6 alkyl; n is an integer selected from 1, 2, 3, 4, 5, and 6; In some embodiments, R 4 But C1-C 16 Unbranched alkyl, C2-C 16 unbranched alkenyl, or [ka] where R 4a and R 4b is as defined above), or a pharmaceutically acceptable salt thereof; or e) Formula (E): [ka] Formula (E) (In the formula, R' is absent, hydrogen, or C1-C3 alkyl, provided that when R' is hydrogen or C1-C3 alkyl, R', R 1 , and R 2 provided that all nitrogen atoms to which they are attached are positively charged, R 1 and R 2 are each independently hydrogen or C1-C3 alkyl; R 3 But C3-C 10 Alkylene or C3-C 10 is alkenylene, R 4 But C1-C 16 Unbranched alkyl, C2-C 16 unbranched alkenyl, or [ka] wherein: R 4a and R 4b are each independently, C1-C 16 Unbranched alkyl or C2-C 16 is an unbranched alkenyl, R 5is absent, C1-C6 alkylene, or C2-C6 alkenylene; R 6a and R 6b are each independently, C7-C 14 Alkyl or C7-C 14 is alkenyl, X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -SS-, -C(R a )=N-, -N=C(R a )-, -C(R a )=NO-, -ON=C(R a )-, -C(=O)NR a -, -NR a C(=O)-, -NR a C(=O)NR a -, -OC(=O)O-, -OSi(R a )2O-, -C(=O)(CR a 2) C(=O)O- or OC(=O)(CR a 2) C(=O)-, wherein: R a is independently at each occurrence hydrogen or C1-C6 alkyl; n is an integer selected from 1, 2, 3, 4, 5, and 6, or a pharmaceutically acceptable salt thereof; f) An ionizable lipid selected from any of the ionizable lipids in Table 1, Table 4, Table 5, Table 6, or Table 7.

[0043] In some embodiments, the LNPs of the present disclosure further comprise a targeting moiety.

[0044] In some embodiments, the LNP comprises a second lipid-anchored polymer, and a targeting moiety is conjugated to the second lipid-anchored polymer. In some embodiments, the second lipid-anchored polymer is selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielaidoyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl- The lipid moiety comprises a linker lipid moiety selected from the group consisting of 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), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), derivatives thereof, and combinations of any of the foregoing. In some embodiments, the linker lipid moiety in the first lipid-anchored polymer is selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, derivatives thereof, and combinations of any of the foregoing.

[0045] In some embodiments, the first lipid anchor polymer and the second lipid anchor polymer are different lipid anchor polymers, and the first lipid anchor polymer and the second lipid anchor polymer are any of the following combinations: DSG (first lipid-anchored polymer) and DSPE (second lipid-anchored polymer), DSPE (first lipid-anchored polymer) and DSG (second lipid-anchored polymer), DODA (first lipid-anchored polymer) and DSPE (second lipid-anchored polymer), DPG (first lipid-anchored polymer) and DSPE (second lipid-anchored polymer), DMG (first lipid-anchored polymer) and DSPE (second lipid-anchored polymer), DODA (first lipid-anchored polymer) and DSG (second lipid-anchored polymer), DPG (first lipid-anchored polymer) and DSG (second lipid-anchored polymer), DMG (first lipid-anchored polymer) and DSG (second lipid-anchored polymer), DPG (first lipid-anchored polymer) and DODA (second lipid-anchored polymer), DMG (first lipid-anchored polymer) and DODA (second lipid-anchored polymer), or DMG (first lipid-anchored polymer) and DPG (second lipid-anchored polymer).

[0046] In some embodiments, the first lipid anchor polymer and the second lipid anchor polymer are the same lipid anchor polymer, and the first lipid anchor polymer and the second lipid anchor polymer are selected from the following combinations: DSG (first lipid-anchored polymer) and DSG (second lipid-anchored polymer), DSPE (first lipid anchor polymer) and DSPE (second lipid anchor polymer), DODA (first lipid-anchored polymer) and DODA (second lipid-anchored polymer), or DPG (first lipid-anchored polymer) and DPG (second lipid-anchored polymer).

[0047] In some embodiments, the targeting moiety is conjugated to a DSPE anchor polymer. In some embodiments, the DSPE anchor polymer is DSPE-PEG or a derivative thereof. In some embodiments, the targeting moiety is conjugated to a DSG anchor polymer. In some embodiments, the DSG anchor polymer is DSG-PEG or a derivative thereof.

[0048] In some embodiments, the targeting moiety is capable of binding to liver cells. In some embodiments, the liver cells are hepatocytes. In some embodiments, the targeting moiety is N-acetylgalactosamine (GalNAc) or a GalNAc derivative. In some embodiments, the targeting moiety is a triantennary GalNAc conjugate or a tetraantennary GalNAc conjugate. 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, a fragment thereof, and a derivative of any of the foregoing. In some embodiments, the targeting moiety is selected from the group consisting of an ApoE protein conjugate, an ApoE polypeptide conjugate, an ApoB protein conjugate, and an ApoB polypeptide conjugate. In one embodiment, the targeting moiety is a modified ApoE protein conjugate.

[0049] In one embodiment, the ionizable lipid in the LNP of the present disclosure according to any of the preceding embodiments is ionizable lipid 81: [ka] or a pharmaceutically acceptable salt thereof.

[0050] In one embodiment, the ionizable lipid in the LNP of the present disclosure according to any of the preceding embodiments is ionizable lipid 89: [ka] or a pharmaceutically acceptable salt thereof.

[0051] In one embodiment, the ionizable lipid in the LNP of the present disclosure according to any of the preceding embodiments is ionizable lipid 87: [ka] or a pharmaceutically acceptable salt thereof.

[0052] In some embodiments, the ionizable lipids are present in the LNPs provided herein in an amount of about 35 mol % to about 60 mol % of the total lipids present in the LNP, hi some embodiments, the ionizable lipids are present in the LNPs in an amount of about 20 mol % to about 50 mol % of the total lipids present in the LNP.

[0053] In some embodiments, the sterol is present in the LNP in an amount of about 20 mol % to about 45 mol % of the total lipid present in the LNP, hi some embodiments, the sterol is present in the LNP in an amount of about 30 mol % to about 40 mol % of the total lipid present in the LNP.

[0054] In some embodiments, the first lipid-anchored polymer is present in the LNP in an amount of about 0.5 mol % to about 5 mol % of the total lipid present in the LNP. In some embodiments, the second lipid-anchored polymer is present in the LNP in an amount of about 0.005 mol % to about 5 mol % of the total lipid present in the LNP. In some embodiments, the first lipid-anchored polymer is present in the LNP in an amount of about 0.05 mol % to about 2 mol % of the total lipid present in the LNP.

[0055] 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 lipid present in the LNP. In some embodiments, the second lipid-anchored polymer is present in the LNP in an amount of about 0.5 mol % of the total lipid present in the LNP. In some embodiments, the first lipid-anchored polymer and the second lipid-anchored polymer are present in the LNP in an amount of about 2.5 mol % and 0.5 mol %, respectively, of the total lipid present in the LNP.

[0056] In some embodiments, the helper lipid represented by Formula (I), Formula (II), Formula (III), or Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present in the LNP in an amount of about 2 mol% to about 40 mol% of the total lipid present in the LNP. In some embodiments, the helper lipid represented by Formula (I), Formula (II), Formula (III), or Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present in the LNP in an amount of about 5 mol% to about 30 mol% of the total lipid present in the LNP. In some embodiments, the helper lipid represented by Formula (I), Formula (II), Formula (III), or Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present in the LNP in an amount of about 10 mol% to about 20 mol% of the total lipid present in the LNP. In some embodiments, the helper lipid represented by Formula (I), Formula (II), Formula (III), or Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present in the LNP in an amount of about 10 mol% of the total lipid present in the LNP. In some embodiments, the helper lipid represented by Formula (I), Formula (II), Formula (III), or Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present in the LNP in an amount of about 15 mol% of the total lipid present in the LNP. In some embodiments, the helper lipid represented by Formula (I), Formula (II), Formula (III), or Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present in the LNP in an amount of about 20 mol% of the total lipid present in the LNP.

[0057] In some embodiments, a helper lipid selected from the group consisting of distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) is present in the LNP in an amount of about 2 mol% to about 40 mol%, or about 5 mol% to about 35 mol%, or about 5 mol% to about 30 mol%, or about 5 mol% to about 25 mol%, or about 5 mol% to about 20 mol%, or about 5 mol% to about 15 mol%, or about 5 mol% to about 10 mol%, or about 10 mol% to about 15 mol% of the total lipid present in the LNP.

[0058] In some embodiments, the LNPs provided by the present disclosure are suitable for intravenous administration.

[0059] In some embodiments, the LNP is less immunogenic than a reference LNP, wherein the reference LNP either (i) does not comprise a helper lipid represented by Formula (I), Formula (II), Formula (III), or Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, or (ii) comprises a helper lipid selected from the group consisting of distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and a reference lipid polymer comprising at least two hydrophobic tails, each containing 12 to 15 carbon atoms in a single aliphatic chain backbone. In one embodiment, the reference lipid anchor polymer is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG).

[0060] In some embodiments, the LNPs provide a lower level of uptake of TNA by blood cells than a reference LNP.

[0061] In some embodiments, the LNPs induce a lower pro-inflammatory cytokine response than a reference LNP.

[0062] In some embodiments, the LNPs result in a lower expression level of TNAs in blood cells than the expression level of TNAs in blood cells resulting from a reference LNP, in some embodiments, the blood cells are erythrocytes, macrophages, and peripheral blood mononuclear cells.

[0063] In some embodiments, the therapeutic nucleic acid (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 closed-end DNA (ceDNA), a single-stranded DNA (ssDNA), a ministring, a doggybone™, a protelomeric closed-end DNA, a dumbbell linear DNA, a Dicer substrate dsRNA, a small hairpin RNA (shRNA), an asymmetric interfering RNA (aiRNA), an mRNA, a tRNA, a rRNA, a gRNA, a DNA viral vector, a viral RNA vector, a non-viral vector, and any combination thereof.

[0064] In some embodiments, the TNA is more than about 200 bp or more than about 200 nt in length. In some embodiments, the TNA is more than about 500 bp or more than about 500 nt in length. In some embodiments, the TNA is more than about 1000 bp or more than about 1000 nt in length. In some embodiments, the TNA is more than about 4000 bp or more than about 4000 nt in length.

[0065] In some embodiments, the TNA is closed-ended DNA (ceDNA). In some embodiments, the TNA is messenger RNA (mRNA). In some embodiments, the TNA is a single-stranded nucleic acid. In some embodiments, the TNA is a double-stranded nucleic acid.

[0066] In some aspects, the present disclosure provides pharmaceutical compositions comprising an LNP of the present disclosure and a pharmaceutically acceptable carrier.

[0067] In some aspects, the present disclosure also provides a method of producing an LNP of the present disclosure, the method comprising combining a therapeutic nucleic acid (TNA), an ionizable lipid, a sterol, a first lipid-anchored polymer, a helper lipid represented by Formula (I), Formula (II), Formula (III), or Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, or a helper lipid selected from the group consisting of distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), optionally a second lipid-anchored polymer, and optionally a targeting moiety.

[0068] In some aspects, the present disclosure also provides a method of treating a genetic disorder in a subject, the method comprising administering to the subject an effective amount of an LNP of the present disclosure or a pharmaceutical composition of the present disclosure.

[0069] In some embodiments, the subject is a human.

[0070] In some embodiments, the genetic disorder is sickle cell anemia, melanoma, hemophilia A (clotting factor VIII (FVIII) deficiency), hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited liver metabolic disorders, Lesch-Nyhan syndrome, thalassemia, xeroderma pigmentosum, Fan et al. Koni anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharidosis, Niemann-Pick disease, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandhoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis (ML), sialidosis type II, glycogen storage disease (GSD), Gaucher disease, cystinosis, Batten disease, aspartylglucosamine Diarrhea, Salla disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (NCL), sphingolipidosis, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy (SMA), Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy ( BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber's congenital amaurosis, Stargardt disease, wet macular degeneration (wet AMD), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC), and cathepsin A deficiency.

[0071] In some embodiments, the genetic disorder is phenylketonuria (PKU). In some embodiments, the genetic disorder is hemophilia A (factor VIII deficiency). In some embodiments, the genetic disorder is Wilson's disease. In some embodiments, the genetic disorder is Gaucher disease. In some embodiments, the genetic disorder is Gaucher disease type I, Gaucher disease type II, or Gaucher disease type III. In some embodiments, the genetic disorder is Leber's congenital amaurosis (LCA). In some embodiments, the LCA is LCA10. In some embodiments, the genetic disorder is Stargardt disease. In some embodiments, the genetic disorder is wet macular degeneration (wet AMD).

[0072] In some aspects, the present disclosure also provides a method of providing anti-tumor immunity to a subject, the method comprising administering to the subject an effective amount of an LNP of the present disclosure or a pharmaceutical composition of the present disclosure. In some aspects, the present disclosure also provides 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 an LNP of the present disclosure or a pharmaceutical composition of the present disclosure. In some embodiments, the subject is a human. In some embodiments, the TNA is retained in the spleen for at least about 6 hours, or at least about 9 hours, or at least about 12 hours, or at least about 15 hours, or at least about 18 hours, or at least about 21 hours, or at least about 24 hours, or at least about 27 hours, or at least about 30 hours, or at least about 33 hours, or at least about 36 hours after administration. In some embodiments, the concentration of TNA at the beginning of a 12-, 18-, or 24-hour time frame and the concentration of TNA at the end of the time frame are within the same order of magnitude. In one embodiment, the TNA is a messenger RNA (mRNA).

[0073] In some aspects, the present disclosure further provides methods of treating a hematological disease, disorder, or condition in a subject, the method comprising administering to the subject an effective amount of an LNP of the present disclosure or a pharmaceutical composition of the present disclosure. In some embodiments, the hematological disease, disorder, or condition is selected from the group consisting of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), Hodgkin's lymphoma (HL), multiple myeloma, myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), adrenoleukodystrophy (ALD), Hurler's syndrome, Krabbe's disease (globoid cell leukodystrophy or GLD), dysplasia, and leukemia. In one embodiment, the TNA is selected from the group consisting of myelodysplastic leukodystrophy (MLD), severe aplastic anemia (SAA), severe combined immunodeficiency (SCID), sickle cell disease (SCD), thalassemia, Wiskott-Aldrich syndrome, Diamond-Blackfan anemia, essential thrombocytosis, Fanconi anemia, hemophagocytic lymphohistiocytosis (HLH), juvenile myelomonocytic leukemia (JMML), myelofibrosis, polycythemia vera, and combinations thereof. In another embodiment, the TNA is a messenger RNA (mRNA). [Brief explanation of the drawings]

[0074] Embodiments of the present disclosure, briefly summarized above and discussed in more detail below, can be understood by reference to illustrative embodiments of the disclosure that are depicted in the accompanying drawings. However, because the present disclosure may admit of other equally effective embodiments, the accompanying drawings illustrate only typical embodiments of the present disclosure and therefore should not be considered limiting in scope.

[0075] [Figure 1] (Figure 1A) In vivo expression of luciferase from LNP D and C2 ceramide-containing LNP1 in CD-1 mice on day 4 post-administration. (Figure 1B) In vivo expression of luciferase from the same LNP formulations described above in Figure 1A in mice on day 7 post-administration. (Figure 1C) Percent change in mouse body weight on day 1 post-administration. [Figure 2](Figure 2A) In vivo expression of luciferase from LNP D, C2 ceramide-containing LNP1, C8 ceramide-containing LNP35, and C2 sphingomyelin-containing LNP36 in CD-1 mice on day 4 post-administration. (Figure 2B) In vivo expression of luciferase from the same LNP formulations described above in Figure 2A on day 7 post-administration in mice. (Figure 2C) Percent change in body weight of mice on day 1 post-administration. [Figure 3A] 1 shows in vivo expression of luciferase from LNP C and C2 ceramide-containing LNP37 in CD-1 mice 4 days after administration. [Figure 3B] Shown is the in vivo expression of luciferase from the same LNP formulations described above in Figure 3A in mice 7 days after administration. [Figure 4] Serum levels of the cytokines IFN-alpha (Figure 4A), IL-6 (Figure 4B), IFN-gamma (Figure 4C), TNF-alpha (Figure 4D), IL-18 (Figure 4E), and IP-10 (Figure 4F) measured in CD-1 mice at 6 hours post-administration after injection of LNP D and C2-ceramide-containing LNP1 are shown. [Figure 5] Serum levels of the cytokines IFN-alpha (Figure 5A), IL-6 (Figure 5B), IFN-gamma (Figure 5C), TNF-alpha (Figure 5D), and IL-18 (Figure 5E) measured in CD-1 mice at 6 hours post-administration after injection of LNP D, C2-ceramide-containing LNP1, C8-ceramide-containing LNP35, and C2-sphingomyelin-containing LNP36 are shown. [Figure 6] Serum levels of cytokines IFN-alpha (Figure 6A), IL-6 (Figure 6B), IFN-gamma (Figure 6C), TNF-alpha (Figure 6D), IL-18 (Figure 6E), and IP-10 (Figure 6F) are shown in CD-1 mice at 6 hours post-administration after injection of LNP A and C2 ceramide-containing LNP23, LNP24, LNP25, LNP26, LNP27, and LNP28. [Figure 7]Serum levels of the cytokines IFN-alpha (Figure 7A), IL-6 (Figure 7B), IFN-gamma (Figure 7C), TNF-alpha (Figure 7D), IL-18 (Figure 7E), and IP-10 (Figure 7F) measured in CD-1 mice after injection of LNP C and C2-ceramide-containing LNP37 at 6 hours post-administration are shown. [Figure 8] 1 shows whole blood and plasma levels of ceDNA cargo in CD-1 mice after injection of LNP E and C2-ceramide-containing LNP1 at 1 hour, 3 hours, and 6 hours post-dose. [Figure 9A] 1 shows in vitro expression of luciferase in primary mouse hepatocytes treated with C2-ceramide-containing LNP40 carrying mRNA luciferase cargo. [Figure 9B] DiD signal indicating uptake of LNP40 into primary mouse hepatocytes is shown. [Figure 10] 1 compares the in vitro expression of luciferase in primary mouse hepatocytes treated with LNP F, C2-ceramide-containing LNP41, C4-ceramide-containing LNP42, C6-ceramide-containing LNP43, or C8-ceramide-containing LNP45 (each carrying an mRNA luciferase cargo). [Figure 11] 24 h total IVIS fluorescence in the liver of CD-1 mice treated with LNP101, LNP102, LNP103, LNP104, and LNP G (all carrying luciferase mRNA as nucleic acid cargo) is shown and compared. [Figure 12A] This is a curve quantified via qPCR of the luciferase mRNA concentration (μg / mL) in whole blood of CD-1 mice administered LNP101, LNP102, LNP103, LNP104, and LNP G at 2 minutes, 1 hour, 6 hours, and 24 hours after administration. [Figure 12B] This is a curve showing the quantification, via qPCR, of the number of luciferase mRNA copies in the liver of CD-1 mice administered with LNP101, LNP102, LNP103, LNP104, and LNP G at 6 and 24 hours after administration. [Figure 12C]This is a curve quantified via qPCR for the number of luciferase mRNA copies in the spleen of CD-1 mice administered with LNP101, LNP102, LNP103, LNP104, and LNP G at 6 and 24 hours after administration. [Figure 12D] This is a curve quantified via qPCR for luciferase mRNA copies in the bone marrow of CD-1 mice administered LNP101, LNP102, LNP103, LNP104, and LNP G at 6 and 24 hours after administration. [Figure 13] Quantification curves of ceDNA blood copies via qPCR are shown for groups of CD-1 mice treated with LNP201, LNP202, and LNP203 at 0, 1, 3, 6, and 24 hours post-administration. [Figure 14A] The HPLC-SEC readouts of LNP formulations with incremental mol% of the first lipid-anchored polymer (i.e., LNPs with 1.5 mol%, 2 mol%, 2.5 mol%, 3 mol%, 5 mol%, and 7 mol% DSG-PEG2000-OMe) show different retention times. [Figure 14B] Retention times for LNP formulations with 1.5 mol% lipid-anchored polymer (DSG-PEG2000-OMe) are shown (wavelength readings: 214 nm to track lipids and 260 nm to track nucleic acid cargo). [Figure 14C] Retention times for LNPs with 7 mol% lipid-anchored polymer (DSG-PEG2000-OMe) are shown (wavelength readings: 214 nm to track lipids and 260 nm to track nucleic acid cargo). DETAILED DESCRIPTION OF THE INVENTION

[0076] The present disclosure provides lipid nanoparticles (LNPs) and LNP compositions (e.g., pharmaceutical compositions) containing therapeutic nucleic acids (TNAs), e.g., gene expression vectors such as closed-end DNA (ceDNA), single-stranded DNA vectors, or messenger RNA (mRNA). The structural components of the LNPs provided by the present disclosure include ionizable lipids, "helper" lipids such as C2 ceramide or C2 sphingomyelin ("C2-C8 containing helper lipids"), structural lipids such as sterols (e.g., cholesterol or beta-sitosterol), and one or more types of lipid-anchoring polymers.

[0077] The LNPs and LNP compositions disclosed herein offer surprising and unexpected properties compared to known LNPs. For example, the helper lipids of the LNPs function to increase the membrane fusogenicity of the lipid bilayer of the LNPs and facilitate endosomal escape; the structural lipids of the LNPs contribute to the membrane integrity and stability of the LNPs; and the lipid-anchored polymers of the LNPs can inhibit LNP aggregation and provide steric stabilization (e.g., enhancing the stealth properties of the overall LNP characteristics in the blood compartment by minimizing any interactions between opsonins that may be present in the blood and the surface of the LNPs). Furthermore, the disclosed LNPs and LNP compositions surprisingly feature reduced LNP-associated toxicity, as evidenced by reduced serum levels of immune response markers (see Examples herein). The present disclosure is based, at least in part, on the surprising observation that certain helper lipids, when present in LNPs, together with lipid-anchored polymers having at least two hydrophobic tails, each of a certain length, e.g., each independently containing 16 to 22 carbon atoms, can contribute to reduced LNP-associated immunogenicity. Such helper lipids include ceramides, sphingomyelins, and fatty acids with a specific number of aliphatic carbon atoms in the fatty acid moiety of the helper lipid, e.g., C2-C8 ceramide. It has also been found that helpers such as DSPC can support the stability and enhanced stealth properties of the disclosed LNPs as measured by in vivo pharmacokinetics. Furthermore, the disclosed LNPs containing a specific molecular percentage of sterols (30%-45% of the total lipids) are characterized by average diameters of approximately 70-100 nm, 70-80 nm or less, making them particularly useful for therapeutic administration. Therefore, LNPs with desirable properties, such as enhanced stealth properties that can evade rapid cellular uptake by blood cells and enhanced tolerability, can be achieved by combining LNP components with the specific physical properties of the disclosed helper lipids and lipid-anchored polymers.

[0078] I. Definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by one of ordinary skill in the art to which this disclosure belongs. It is understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc., described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition (published by Merck Sharp & Dohme Corp.), 2011 (ISBN 978-0-911910-19-3), Robert S. Porter et al. (eds.), Fields Virology, 6th Edition (published by Lippincott Williams & Wilkins, Philadelphia, PA, USA) (2013), Knipe, DM and Howley, PM (ed.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine (published by Blackwell Science Ltd.), 1999-2012 (ISBN 9783527600908), and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference (published by VCH Publishers, Inc.), 1995 (ISBN 1-56081-569-8), Immunology by Werner Luttmann (published by Elsevier), 2006, Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305), Lewin's Genes Harbor, NY, USA (2012) (ISBN 1936113414), Davis et al. Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (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, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005, and Current Protocols in Immunology (CPI) (John E. Coligan, A.D. M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of all of which are incorporated herein by reference in their entireties.

[0079] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0080] The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example."

[0081] The use of the alternative (eg, "or") should be understood to mean either one or both of the alternatives, or any combination thereof.

[0082] As used herein, the term "about," when referring to a measurable value such as an amount, temporal duration, etc., is intended to encompass a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, depending on the accuracy and precision of the methods available for determining such measurable values ​​or as appropriate for performing the disclosed methods.

[0083] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and fractions thereof, where appropriate (such as integer tenths and hundredths).

[0084] As used herein, "comprise," "comprising," and "comprises" are intended to be synonymous with "include," "including," "includes," or "contain," "containing," "contains," and are, for example, inclusive or open-ended terms specifying the presence of following components, but do not exclude or preclude the presence of additional, unrecited components, features, elements, members, steps that are known in the art or disclosed therein.

[0085] The term "consisting of" refers to compositions, methods, processes, and their respective components described herein, excluding any element not recited in the description of the embodiment.

[0086] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic novel or functional characteristics of that embodiment of the present disclosure.

[0087] As used herein, terms such as "such as," "for example," and the like are intended to refer to example embodiments and are not intended to limit the scope of the present disclosure.

[0088] Unless otherwise defined, all scientific and technical terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein.

[0089] As used herein, the terms "administration," "administering," and variations thereof refer to the introduction of a composition or agent (e.g., nucleic acid, specifically ceDNA, ssDNA, and mRNA) into a subject, including simultaneous and sequential introduction of one or more compositions or agents. "Administration" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. "Administration" also encompasses in vitro and ex vivo treatments. Introduction of a composition or agent into a subject is by any suitable route, including oral, pulmonary, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intratumoral, or topical. Administration includes self-administration and administration by another. Administration can be by any suitable route. A suitable route of administration enables the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into the subject's vein.

[0090] 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 induce an unwanted immune response against the LNP and its components in a subject after the LNP or composition comprising an LNP of the present disclosure is administered to the subject. In some embodiments, the immune response can be measured, for example, by measuring the levels of one or more pro-inflammatory cytokines before and after administration of a composition comprising an LNP of the present disclosure. Exemplary pro-inflammatory cytokines that can be used to determine the immunogenicity of LNPs of the present disclosure or compositions comprising LNPs of the present disclosure include, but are not limited to, granulocyte colony-stimulating factor (G-CSF), interleukin 1 alpha (IL-1α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), interleukin 8 (IL-8 or CXCL8), interleukin 11 (IL-11), interleukin 17 (IL-17), interleukin 18 (IL-18), interferon alpha (IFN-α), interferon beta (IFN-β), interferon gamma (IFN-γ), C-X-C motif chemokine ligand 10 (CXCL10 or IP-10), monocyte chemoattractant protein 1 (MCP-1), CD40L, CCL2, CCL3, CCL4, CCL5, CCL11, tumor necrosis factor alpha (TNF-α), and combinations thereof. In some embodiments, for example, immune responses before and after administration of a composition comprising an LNP of the present disclosure can be measured by measuring levels of specific antibodies against the LNP and components of the LNP.

[0091] As used herein, the term "off-target delivery" refers to the delivery of LNPs to non-target cells. After administration to a subject, the LNPs may be delivered to the non-target cells, resulting in expression of the therapeutic nucleic acid (TNAs) in the non-target cells.

[0092] In some embodiments, the non-target cells may be liver sinusoidal endothelial cells (LSEC cells), splenocytes, or Kupffer cells.

[0093] After administration to a subject, the LNPs may be delivered to non-target cells, resulting in expression of therapeutic nucleic acids (TNAs) in the non-target cells, or may be degraded, for example, upon phagocytosis by macrophages. In some embodiments, the reference LNPs may be characterized by a higher rate of random delivery to or uptake by non-target cells, e.g., 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 uptake level of TNAs (e.g., ceDNA, ssDNA, or mRNA) in blood cells than the uptake level of the reference LNPs. In some embodiments, the reference LNP is (i) an LNP that does not include a helper lipid represented by Formula (I), Formula (II), Formula (III), or Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, or (ii) an LNP that includes a helper lipid selected from the group consisting of distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and a reference lipid polymer that includes at least two hydrophobic tails, each containing 12 to 15 carbon atoms in a single aliphatic chain backbone, such as 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG, also known as PEG-DMG).

[0094] As used herein, the term "aqueous solution" refers to a composition that comprises, in whole or in part, water.

[0095] As used herein, the term "base" includes purines and pyrimidines, which further include the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and naturally occurring analogues, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0096] As used herein, the terms "carrier" and "excipient" are intended to include any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce toxic, allergic, or similar untoward reactions when administered to a host.

[0097] As used herein, the term "ceDNA" refers to capsid-free, closed-end, linear, double-stranded (ds) duplex DNA, whether synthetic or otherwise, for non-viral gene transfer. According to some embodiments, the ceDNA is closed-end, linear, double-stranded (CELiD) CELiD DNA. According to some embodiments, the ceDNA is a DNA-based minicircle. According to some embodiments, the ceDNA is a minimal immunologically defined gene expression (MIDGE) vector. According to some embodiments, the ceDNA is ministering DNA. According to some embodiments, the ceDNA is a dumbbell-shaped, linear, double-stranded, closed-end DNA containing two hairpin structures of ITRs at the 5' and 3' ends of the expression cassette. According to some embodiments, the ceDNA is doggybone™ DNA. A detailed description of ceDNA is provided in International Patent Application No. PCT / US2017 / 020828, filed March 3, 2017, the entire contents of which are expressly incorporated herein by reference. Certain methods for the generation of ceDNA containing various inverted terminal repeat (ITR) sequences and configurations using cell-based methods are described in Example 1 of International Patent Application No. PCT / US18 / 49996, filed September 7, 2018, and International Patent Application No. PCT / US2018 / 064242, filed December 6, 2018, each of which is incorporated herein by reference in its entirety. Certain methods for the generation of synthetic ceDNA vectors containing various ITR sequences and configurations are described, for example, in International Patent Application No. PCT / US2019 / 14122, filed January 18, 2019, the entire contents of which are incorporated herein by reference.

[0098] As used herein, the term "closed-end DNA vector" refers to a capsid-free DNA vector having at least one covalently closed end, wherein at least a portion of the vector has an intramolecular double-stranded structure. The terms "ceDNA vector" and "ceDNA" are used interchangeably and refer to a closed-end DNA vector containing at least one terminal palindrome. In some embodiments, the ceDNA contains two covalently closed ends.

[0099] As used herein, the term "ceDNA genome" refers to an expression cassette that further incorporates at least one inverted terminal repeat (ITR) region. The ceDNA genome may further comprise one or more spacer regions. In some embodiments, the ceDNA genome is incorporated into a plasmid or viral genome as an intermolecular double-stranded polynucleotide of DNA.

[0100] As used herein, the terms "DNA regulatory sequence," "control element," and "regulatory element" are used interchangeably herein and are intended to refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, proteolysis signals, etc., that provide and / or regulate the transcription of a non-coding sequence (e.g., a DNA-targeting RNA) or a coding sequence (e.g., a site-directed modifying polypeptide or a Cas9 / Csn1 polypeptide) and / or regulate the translation of an encoded polypeptide.

[0101] As used herein, the term "inverted terminal repeat" or "ITR" is intended to refer to a nucleic acid sequence located at the 5' and / or 3' end of an ssDNA vector disclosed herein, comprising at least one stem-loop structure that is partially duplexed and includes at least one loop. According to some embodiments, the ITR may be an artificial sequence (e.g., does not contain sequences derived from a virus). The ITR may further comprise one stem-loop structure (e.g., a "hairpin") or two or more stem-loop structures. For example, the ITR may comprise two stem-loop structures (e.g., a "hammerhead," "dogbone," or "dumbbell"), three stem-loop structures (e.g., a "cruciform"), or a more complex structure. The ITR may comprise an aptamer sequence or one or more chemical modifications.

[0102] According to some embodiments, "ITRs" can be artificially synthesized using a set of oligonucleotides containing one or more desired functional sequences (e.g., palindromic sequences). The ITR sequences can be artificial AAV ITRs, artificial non-AAV ITRs, or ITRs physically derived from viral AAV ITRs (e.g., ITR fragments removed from the viral genome). For example, ITRs can be derived from the Parvoviridae family, which includes parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, and human parvovirus B-19), or the SV40 hairpin that functions as the origin of SV40 replication can be used as an ITR, which can be further modified by truncation, substitution, deletion, insertion, and / or addition. The Parvoviridae family of viruses consists of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. Dependoparvoviruses include the adeno-associated virus (AAV) family of viruses that can replicate in vertebrate hosts, including, but not limited to, humans, primates, bovine, canine, equine, and bovine species. Typically, ITR sequences can be derived from AAV, as well as parvoviruses, lentiviruses, goose viruses, and B19, in wild-type, "dogbone," and "dumbbell" configurations, symmetric, or even asymmetric ITR orientations. While ITRs are typically present at both the 5' and 3' ends of AAV vectors in single-stranded DNA (ssDNA) molecules, ITRs can be present at only one end of a linear vector. For example, ITRs can be present only at the 5' end. In some other cases, ITRs can be present only at the 3' end in single-stranded DNA (ssDNA) molecules. For convenience herein, an ITR located 5' to (upstream of) an expression cassette of a single-stranded DNA (ssDNA) molecule vector is referred to as the "5' ITR" or "left ITR," and an ITR located 3' to (downstream of) an expression cassette of a single-stranded DNA (ssDNA) molecule is referred to as the "3' ITR" or "right ITR."

[0103] As used herein, "wild-type ITR" or "WT-ITR" refers to the sequence of a naturally occurring ITR sequence in an AAV or other dependovirus that retains, for example, Rep binding activity and Rep nicking ability. The nucleic acid sequence of a WT-ITR from any AAV serotype may differ slightly from the naturally occurring reference sequence due to degeneracy or drift in the genetic code; therefore, WT-ITR sequences encompassed for use herein include WT-ITR sequences that result from naturally occurring variations (e.g., replication errors).

[0104] As used herein, the terms "substantially symmetric WT-ITR" or "substantially symmetric WT-ITR pair" refer to a pair of wild-type ITRs in a synthetic AAV vector, both of which have reverse-complementary sequences throughout their entire length. For example, an ITR can be considered a wild-type sequence even if it has one or more nucleotides that deviate from a naturally occurring reference sequence, as long as the changes do not affect the physical and functional properties and overall three-dimensional structure (secondary and tertiary) of the sequence. In some embodiments, the deviating nucleotides represent conservative sequence changes. As one non-limiting example, a sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity with a reference sequence (e.g., as measured using BLAST with default settings) and also has a symmetrical three-dimensional spatial configuration relative to the other WT-ITR, such that their three-dimensional structures have the same shape in geometric space. A substantially symmetric WT-ITR has the same A, C-C', and B-B' loops in three-dimensional space. A substantially symmetric WT ITR can be functionally confirmed as WT by determining that it has an operable Rep binding site (RBE or RBE') and terminal resolution site (TRS) that pairs with the appropriate Rep protein. One skilled in the art can optionally test for other functions, including transgene expression under permissive conditions.

[0105] As used herein, the phrases "modified ITR" or "mod-ITR" or "mutated ITR" are used interchangeably herein and refer to an ITR that has a mutation in at least one nucleotide compared to a wild-type ITR from the same serotype. The mutation can result in a change in one or more of the A, C, C', B, and B' regions in the ITR, and can result in a change in the three-dimensional spatial configuration (i.e., its three-dimensional structure in geometric space) compared to the three-dimensional spatial configuration of a wild-type ITR from the same serotype.

[0106] As used herein, the term "asymmetric ITR," also referred to as an "asymmetric ITR pair," refers to a pair of ITRs in a single-stranded synthetic AAV genome that are not reverse complements over their entire length. As one non-limiting example, an asymmetric ITR pair does not have a symmetrical three-dimensional spatial configuration relative to its cognate ITR, such that their three-dimensional structures are different shapes in geometric space. In other words, an asymmetric ITR pair has a different overall geometric structure, i.e., they have different configurations of their A, C-C', and B-B' loops in three-dimensional space (e.g., one ITR may have a shorter C-C' arm and / or a shorter B-B' arm compared to the cognate ITR). The sequence difference between the two ITRs may be due to one or more nucleotide additions, deletions, truncations, or point mutations. According to some embodiments, one ITR of the asymmetric ITR pair may be a wild-type AAV ITR sequence, and the other ITR may be a modified ITR (e.g., a non-wild-type or synthetic ITR sequence) as defined herein. In another embodiment, neither ITR of the asymmetric ITR pair is a wild-type AAV sequence, but rather the two ITRs are modified ITRs with different shapes in geometric space (i.e., different overall geometric structures). In some embodiments, one mod-ITR of the asymmetric ITR pair can have a short C-C' arm, and the other ITR can have a different modification (e.g., a single arm or a short B-B' arm) such that it has a different three-dimensional spatial configuration compared to the cognate asymmetric mod-ITR.

[0107] As used herein, the term "symmetric ITRs" refers to a pair of ITRs in a single-stranded AAV genome that are mutated or modified compared to the wild-type depend virus ITR sequences and are reverse-complementary across their entire length. Neither ITR is the wild-type ITR AAV2 sequence (i.e., they are modified ITRs, also referred to as mutant ITRs), and may have a sequence that differs from the wild-type ITR due to nucleotide additions, deletions, substitutions, truncations, or point mutations. For convenience herein, the ITR located 5' to (upstream of) the expression cassette of a synthetic AAV vector is referred to as the "5' ITR" or "left ITR," and the ITR located 3' to (downstream of) the expression cassette of a synthetic AAV vector is referred to as the "3' ITR" or "right ITR."

[0108] As used herein, the term "substantially symmetric modified ITR" or "substantially symmetric mod-ITR pair" refers to a pair of modified ITRs in a synthetic AAV that both have reverse-complementary sequences throughout their entire length. For example, modified ITRs can be considered substantially symmetric even if they have some nucleotide sequence deviations from their reverse-complementary sequences, as long as the changes do not affect their properties and overall shape. As one non-limiting example, a sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a reference sequence (as measured using BLAST with default settings) and also has a symmetrical three-dimensional spatial organization relative to its cognate modified ITRs such that their three-dimensional structures have the same shape in geometric space. In other words, a substantially symmetric modified ITR pair has the same A, C-C', and B-B' loops organized in three-dimensional space. In some embodiments, the ITRs from a mod-ITR pair can have different reverse-complementary nucleotide sequences but still have the same symmetrical three-dimensional spatial organization, i.e., both ITRs have mutations that result in the same overall three-dimensional shape. For example, one ITR (e.g., the 5' ITR) of a mod-ITR pair can be from one serotype, and the other ITR (e.g., the 3' ITR) can be from a different serotype, but both can have the same corresponding mutations such that the modified ITR pair has the same symmetrical three-dimensional spatial organization (e.g., if the 5' ITR has a deletion in the C region, the cognate modified 3' ITR from the different serotype has a deletion at a corresponding position in the C' region). In such embodiments, each ITR of a modified ITR pair can be from a different serotype (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), e.g., a combination of AAV2 and AAV6, with modifications in one ITR mirrored at the corresponding position in the cognate ITR from the different serotype. According to some embodiments, a substantially symmetric modified ITR pair refers to a pair of modified ITRs (mod-ITRs) so long as the nucleotide sequence differences between the ITRs do not affect their properties or overall shape and they have substantially the same shape in three-dimensional space.Non-limiting examples include mod-ITRs that have at least 95%, 96%, 97%, 98%, or 99% sequence identity with a reference mod-ITR, as determined by standard means known in the art, such as BLAST (Basic Local Alignment Search Tool) or BLASTN with default settings, and also have symmetrical three-dimensional spatial configurations such that their three-dimensional structures are the same shape in geometric space. A substantially symmetric mod-ITR pair will have the same A, C-C', and B-B' loops in three-dimensional space; for example, if the modified ITR of a substantially symmetric mod-ITR pair has a deletion of the C-C' arm, the cognate mod-ITR will have a corresponding deletion of the C-C' loop and also have a similar three-dimensional structure of the remaining A and B-B' loops that are the same shape in the geometric space of the cognate mod-ITR.

[0109] As used herein, the term "flanking" refers to the relative position of one nucleic acid sequence with respect to another. Generally, in the sequence ABC, B is flanked by A and C. Similarly for the arrangement AxBxC. Thus, a flanking sequence precedes or follows the flanked sequence, but need not be contiguous with or immediately adjacent to it. According to some embodiments, the term flanking refers to the terminal repeat sequences at each end of a linear, single-stranded synthetic AAV vector.

[0110] As used herein, the term "spacer region" refers to an intervening sequence that separates functional elements within a vector or genome. In some embodiments, an AAV spacer region maintains two functional elements at a desired distance for optimal functionality. In some embodiments, the spacer region provides or adds to the genetic stability of the vector or genome. In some embodiments, the spacer region facilitates easy genetic manipulation of the genome by providing conveniently located cloning sites and gaps of a designed number of base pairs. For example, in certain aspects, oligonucleotide "polylinkers" or "polycloning sites" containing several restriction endonuclease sites, or non-open reading frame sequences designed to lack known protein (e.g., transcription factor) binding sites, can be positioned in the vector or genome to separate cis-acting elements, similar to an AAV vector or genome, for example, by inserting a 6-mer, 12-mer, 18-mer, 24-mer, 48-mer, 86-mer, 176-mer, etc., between the terminal resolution site and the upstream transcriptional regulatory element.

[0111] As used herein, the terms "Rep binding site" ("RBS") and "Rep binding element" ("RBE") are used interchangeably and refer to a binding site for a Rep protein (e.g., AAV Rep 78 or AAV Rep 68) that, upon binding by the Rep protein, enables the Rep protein to perform its site-specific endonuclease activity on a sequence incorporating the RBS. An RBS sequence and its reverse complement together form a single RBS. RBS sequences are well known in the art and include, for example, the RBS sequence specified in AAV2, 5'-GCGCGCTCGCTCGCTC-3'.

[0112] As used herein, the terms "terminal resolution site" and "TRS" are used interchangeably herein and refer to the region where Rep forms a tyrosine-phosphodiester bond with 5' thymidine, generating a 3'-OH that serves as a substrate for DNA elongation via a cellular DNA polymerase, e.g., DNA pol delta or DNA pol epsilon. Alternatively, the Rep-thymidine complex may participate in a coordinate ligation reaction.

[0113] As used herein, the terms "sense" and "antisense" refer to the orientation of structural elements on a polynucleotide. The sense and antisense versions of an element are the reverse complements of each other.

[0114] As used herein, the terms "synthetic AAV vector" and "synthetic production of AAV vector" refer to AAV vectors and methods for their synthetic production in a completely cell-free environment.

[0115] As used herein, the phrase "effective amount" or "therapeutically effective amount" of a therapeutic agent, such as an active agent or therapeutic nucleic acid, is an amount sufficient to produce a desired effect, e.g., inhibition of expression of a target sequence, compared to expression levels detected in the absence of the therapeutic nucleic acid. Suitable assays for measuring expression of a target gene or target sequence include, for example, examination of protein or RNA levels using techniques known to those of skill in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those of skill in the art.

[0116] As used herein, the term "expression" refers to the cellular processes involved in the production of RNA and proteins, and optionally secreted proteins, including, for example, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing, where applicable. As used herein, the phrase "expression product" includes RNA transcribed from a gene (e.g., a transgene) and polypeptides obtained by translation of mRNA transcribed from a gene.

[0117] As used herein, the term "expression vector" refers to a vector that directs the expression of RNA or polypeptides from sequences linked to transcriptional regulatory sequences on the vector. The expressed sequences are often, but not necessarily, heterologous to the host cell. An expression vector may contain additional elements; for example, an expression vector may have two replication systems, thus allowing it to be maintained in two organisms, e.g., human cells in the case of expression and prokaryotic hosts in the case of cloning and amplification. An expression vector may be a recombinant vector.

[0118] As used herein, it refers to a ceramide represented by Formula (I), Formula (II), Formula (III), or Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, or (ii) comprises a helper lipid selected from the group consisting of distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0119] As used herein, the terms "expression cassette" and "expression unit" are used interchangeably and are intended to refer to a heterologous DNA sequence operably linked to a promoter or other DNA regulatory sequence sufficient to direct transcription of a transgene in a DNA vector, e.g., a synthetic AAV vector. Suitable promoters include, for example, tissue-specific promoters. The promoter can also be of AAV origin.

[0120] As used herein, the phrase "genetic disease" or "genetic disorder" refers to a disease caused, directly or indirectly, in part or in whole by one or more abnormalities in the genome, including, and particularly, conditions present at birth. The abnormality may be a mutation, insertion, or deletion of a gene. The abnormality may affect the coding sequence of a gene or its regulatory sequence.

[0121] As used herein, the term "polypeptide" refers to a polymeric sequence of amino acids. In some embodiments, the polypeptide of the present disclosure is an ApoE polypeptide or an ApoB polypeptide. In some embodiments, the ApoE polypeptide is a functional fragment (or functional portion) of a full-length ApoE polypeptide. In some embodiments, the ApoE The polypeptide is a functional fragment (or functional portion) of a full-length ApoB polypeptide. In some embodiments, the ApoE polypeptide is 30 amino acids or less in length. In some embodiments, the ApoB polypeptide is 30 amino acids or less in length.

[0122] As used herein, the term "lipid" refers to a group of organic compounds, including, but not limited to, esters of fatty acids, which are characterized by being insoluble in water but soluble in many organic solvents. Lipids are generally divided into at least three classes: (1) "simple lipids," which include fats and oils and waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0123] Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine.Other compounds that lack phosphorus, such as sphingolipids, glycosphingolipid family, diacylglycerol, and β-acyloxyacid, are also included in the group called amphipathic lipid.In addition, the above amphipathic lipids can be mixed with other lipids, including triglycerides and sterols.

[0124] The term "lipid-anchored polymer" or "lipid polymer" or "lipid conjugate" refers to a conjugated lipid that inhibits lipid particle aggregation. Such lipid conjugates include, but are not limited to, PEG-lipid conjugates such as PEG conjugated to DSG (e.g., PEG-DSG conjugate), PEG conjugated to DSPE (e.g., PEG-DSPE conjugate), and PEG conjugated to ceramide (see, e.g., U.S. Pat. No. 5,885,613), polyglycerol (PG)-lipid conjugates such as DODA-PG, and mixtures thereof. An example of a PG-lipid conjugate is DODA-PG45. Further examples of POZ-lipid conjugates are described in PCT Publication No. 2010 / 006282. PEG, PG, or POZ can be directly conjugated to the lipid or can be linked to the lipid via a linker moiety. For example, any linker moiety suitable for linking PEG, PG, or POZ to the lipid can be used, including non-ester-containing and ester-containing linker moieties. In certain preferred embodiments, non-ester containing linker moieties such as amides or carbamates are used. The disclosures of each of the above patent documents are incorporated herein by reference in their entirety for all purposes.

[0125] As used herein, the term "lipid-anchored polymer," which may be used interchangeably with the terms "lipid conjugate" or "lipid polymer," refers to a molecule comprising a lipid moiety covalently attached to a hydrophilic polymer via a linker. Without wishing to be bound by any particular theory, it is believed that the lipid-anchored polymer inhibits aggregation of LNPs and provides in vivo steric stabilization and a prolonged blood half-life (t 1 / 2Lipid moieties ("lipid linkers" or "linker lipids") conjugated to hydrophilic polymers (e.g., PEG, PG, or POZ) include 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-dielide 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (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), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), derivatives thereof, and combinations of any of the foregoing. In one embodiment, the lipid-anchored polymer comprises a linker lipid moiety selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, and derivatives thereof, as well as any combination of the foregoing. For example, PEG2000 bound to DSG is the lipid-anchored polymer PEG2000-DSG (or DSG-PEG2000). PEG bound to DSPE is the lipid-anchored polymer PEG-DSPE (or DSPE-PEG2000 or DSPE-PEG500).An example of a lipid-anchored PG polymer can include DODA-PG, where the PG can be a multi-unit ranging from about 5 to about 50 PG units.

[0126] As used herein, the term "encapsulated lipid" refers to a lipid particle that provides an active or therapeutic agent, such as a nucleic acid (e.g., ceDNA, ssDNA, or mRNA), with complete encapsulation, partial encapsulation, or both. In preferred embodiments, the nucleic acid is completely encapsulated in the lipid particle (e.g., to form a nucleic acid-containing lipid particle).

[0127] As used herein, the term "lipid particle" or "lipid nanoparticle" refers to a lipid formulation that can be used to deliver a therapeutic agent, such as a nucleic acid therapeutic, to a desired target site (e.g., a cell, tissue, organ, etc.). In one embodiment, the lipid particle of the present disclosure is a nucleic acid-containing lipid particle formed from a cationic lipid, a non-cationic lipid, and optionally conjugated lipids, which typically prevent particle aggregation. In other preferred embodiments, a therapeutic agent, such as a therapeutic nucleic acid, may be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation. In one embodiment, the lipid particle comprises a nucleic acid (e.g., ceDNA) and a lipid comprising one or more tertiary amino groups, one or more phenyl ester bonds, and a disulfide bond.

[0128] According to some embodiments, lipid particles of the present disclosure typically have a size of about 20 nm to about 75 nm, about 20 nm to about 70 nm, about 25 nm to about 75 nm, about 25 nm to about 70 nm, about 30 nm to about 75 nm, about 30 nm to about 70 nm, about 35 nm to about 75 nm, about 35 nm to about 70 nm, about 40 nm to about 75 nm, about 40 nm to about 70 nm, about 45 nm to about 75 nm, about 50 nm to about 75 nm, about 50 nm to about 70 nm, about 60 nm to about 75 ...0 nm, about 75 nm to about 75 nm, about 70 nm to about 70 nm, about 75 nm to about 75 nm, about 70 nm to about 75 nm, about 7 nm to about 75nm, about 60nm to about 70nm, about 65nm to about 75nm, about 65nm to about 70nm, about 65nm to about 80nm, about 65nm to about 80nm, about 60nm to about 80nm, about 65nm to about 85nm, or Approximately 20nm, approximately 25nm, approximately 30nm, approximately 35nm, approximately 40nm, approximately 45nm, approximately 50nm, approximately 51nm, approximately 52nm, approximately 53nm, approximately 54nm, approximately 55nm, approximately 56nm, approximately 57nm, approximately 58nm, approximately 59nm and having an average diameter of about 60 nm, about 61 nm, about 62 nm, about 63 nm, about 64 nm, about 65 nm, about 66 nm, about 67 nm, about 68 nm, about 69 nm, about 70 nm, about 71 nm, about 72 nm, about 73 nm, about 74 nm, about 75 nm, about 76 nm, about 77 nm, about 78 nm, about 79 nm, about 80 nm, about 81 nm, about 82 nm, about 83 nm, about 84 nm, or about 85 nm (±3 nm) in size.

[0129] Generally, the LNPs of the present disclosure have an average diameter selected to produce an intended therapeutic effect. For example, the LNPs of the present disclosure have an average diameter compatible with delivery to a target organ (e.g., the liver) so that the LNPs of the present disclosure can diffuse through fenestrations in the target organ (e.g., liver) or target cell subpopulation (e.g., hepatocytes).

[0130] According to some embodiments, the lipid particles of the present disclosure typically have an average diameter of less than about 85 nm, less than about 80 nm, less than about 75 nm, less than about 70 nm, less than about 65 nm, less than about 60 nm, less than about 55 nm, less than about 50 nm, less than about 45 nm, less than about 40 nm, less than about 35 nm, less than about 30 nm, less than about 25 nm, or less than about 20 nm in size.

[0131] As used herein, the term "cationic lipid" refers to any lipid that is positively charged at physiological pH.The cationic lipid in lipid particles can include one or more cationic lipids, such as, for example, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), "SS-cleavable lipid", or a mixture thereof.In some embodiments, the cationic lipid can also be an ionizable lipid, i.e., an ionizable cationic lipid. The term "cationic lipid" also encompasses lipids that are positively charged at any pH, such as lipids containing quaternary amine groups, i.e., quaternary lipids. Any cationic lipid described herein that contains a primary, secondary, or tertiary amine group can be converted to the corresponding quaternary lipid, for example, by treatment with a solution of chloromethane (CHCl) in acetonitrile (CHCN) and chloroform (CHCl).

[0132] As used herein, the term "ionizable lipid" refers to a lipid, e.g., a cationic lipid, that has at least one protonatable or deprotonatable group such that the lipid is positively charged at a pH below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably above physiological pH. It will be understood by those skilled in the art that the addition or removal of protons as a function of pH is an equilibrium process, and reference to a charged or neutral lipid refers to the nature of the predominant species and does not require that all lipids exist in a charged or neutral form. Generally, ionizable lipids have a pKa of the protonatable group in the range of about 4 to about 7. In some embodiments, ionizable lipids may include "cleavable lipids" or "SS-cleavable lipids."

[0133] As used herein, the term "neutral lipid" refers to any of a number of lipid species that exist in either an uncharged form or a neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0134] As used herein, the term "cleavable lipid" or "SS-cleavable lipid" refers to an ionizable lipid containing a disulfide bond-cleavable unit. The cleavable lipid may contain a cleavable disulfide bond (SS) containing pH-sensitive amine, e.g., a tertiary amine, and a self-degrading phenyl ester-containing lipid-like material. For example, the SS-cleavable lipid can be ss-OP lipid (COATSOME® SS-OP), ss-M lipid (COATSOME® SS-M), ss-E lipid (COATSOME® SS-E), ss-EC lipid (COATSOME® SS-EC), ss-LC lipid (COATSOME® SS-LC), ss-OC lipid (COATSOME® SS-OC), and ss-PalmE lipid (see, e.g., Formulas I-IV), or lipids described in Togashi et al., (2018) Journal of Controlled Release “A hepatic pDNA delivery system based on an intracellular environment sensitive vitamin E-scaffold lipid-like material with the aid of an anti-inflammatory drug” 279:262-270. Further examples of cleavable lipids are described in U.S. Patent No. 9,708,628 and U.S. Patent No. 10,385,030, the entire contents of which are incorporated herein by reference. In one embodiment, the cleavable lipid comprises a tertiary amine that responds to disulfide bonds, which can be cleaved in a reducing environment, such as an acidic compartment, for example, an endosome or lysosome for membrane destabilization, and the cytoplasm. In one embodiment, the cleavable lipid is a cationic lipid. In one embodiment, the cleavable lipid is an ionizable cationic lipid. Cleavable lipids are described in more detail herein.

[0135] As used herein, the term "organic lipid solution" refers to a composition that comprises, in whole or in part, an organic solvent with a lipid.

[0136] As used herein, the term "liposome" refers to lipid molecules assembled in a spherical configuration that encapsulates an aqueous interior volume separated from an aqueous exterior. Liposomes are vesicles with at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic drug delivery in the context of pharmaceutical development. They function by fusing with cell membranes and rearranging their lipid structure to deliver the drug or active pharmaceutical ingredient (API). Liposomal compositions for such delivery are typically composed of phospholipids, particularly compounds with phosphatidylcholine groups, although these compositions may also contain other lipids.

[0137] As used herein, the term "nucleic acid" refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form, including DNA, RNA, and hybrids thereof. DNA can be in the form of, for example, an antisense molecule, a plasmid DNA, a DNA-DNA duplex, a pre-condensed DNA, a PCR product, a vector (P1, PAC, BAC, YAC, artificial chromosome), an expression cassette, a chimeric sequence, chromosomal DNA, or derivatives and combinations thereof. DNA can be in the form of a minicircle, a plasmid, a bacmid, a minigene, a ministring DNA (a covalently closed linear DNA vector), a closed-end linear double-stranded DNA (CELiD or ceDNA), a single-stranded DNA (ssDNA), doggybone™ DNA, a dumbbell-shaped DNA, a minimal immunologically defined gene expression (MIDGE) vector, a viral vector, or a non-viral vector. RNA can be in the form of small interfering RNA (siRNA), Dicer substrate dsRNA, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), messenger RNA (mRNA), rRNA, tRNA, gRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and have similar binding properties to the reference nucleic acid. Examples of such analogs and / or modified residues include, but are not limited to, phosphorothioates, phosphorodiamidate morpholino oligomers (morpholinos), phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, locked nucleic acids (LNA™), and peptide nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid.Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated.

[0138] As used herein, the phrases "nucleic acid therapeutic agent," "therapeutic nucleic acid," and "TNA" are used interchangeably and refer to any therapeutic modality that uses a nucleic acid as the active ingredient of a therapeutic agent to treat a disease or disorder. As used herein, these terms refer to RNA-based therapeutic agents and DNA-based therapeutic agents. Non-limiting examples of RNA-based therapeutic agents include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), Dicer substrate dsRNA, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), or guide RNA (gRNA). Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigenes, viral DNA (e.g., lentivirus or AAV genome) or non-viral synthetic DNA vectors, closed-ended linear double-stranded DNA (ceDNA / CELiD), single-stranded DNA (ssDNA), plasmids, bacmids, DOGGYBONE™ DNA vectors, minimal immunologically defined gene expression (MIDGE) vectors, non-viral ministring DNA vectors (covalently closed linear DNA vectors), or dumbbell-shaped DNA minimal vectors ("dumbbell DNA").

[0139] As used herein, a "nucleotide" comprises the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate group.

[0140] As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions such as oil / water or water / oil, and various types of wetting agents. The term also encompasses any of the agents approved by a U.S. federal regulatory agency or listed in the U.S. Pharmacopoeia for use in animals, including humans, as well as any carrier or diluent that does not cause significant irritation to the subject and does not destroy the biological activity and properties of the administered compound.

[0141] As used herein, the terms "gap" and "nick" are used interchangeably and refer to an interrupted portion of the synthetic DNA vector of the present disclosure that creates a continuous single-stranded DNA segment in an otherwise double-stranded ceDNA. A gap can be 1 nucleotide (nt) to 100 nucleotides (nt) in length in one strand of the duplex DNA. Exemplary gaps designed and generated by the methods described herein, and synthetic vectors generated thereby, can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 bp in length. Exemplary gaps in the present disclosure can be 1 nt to 10 nt, 1 to 20 nt, or 1 to 30 nt in length.

[0142] As used herein, the term "nick" refers to a discontinuity in a double-stranded DNA molecule, typically due to damage or enzymatic action, in which a phosphodiester bond is absent between adjacent nucleotides on one strand. It is understood that one or more nicks allow for twist release within the strand during DNA replication, and that nicks are also thought to play a role in facilitating the binding of the transcription machinery.

[0143] "Receptor" refers to a polypeptide or portion thereof present on a cell membrane that selectively binds to one or more ligands. As used herein, the term "receptor" is intended to encompass the entire receptor or its ligand-binding portion. These portions of the receptor specifically include a region sufficient for specific binding of the ligand to occur.

[0144] As used herein, the term "subject" refers to a human or animal to which treatment, including prophylactic treatment, using a therapeutic nucleic acid according to the present disclosure is provided. Typically, the animal is a vertebrate, such as, but not limited to, a primate, a rodent, a domestic animal, or a game animal. Primates include, but are not limited to, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include, but are not limited to, cattle, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cats, canine species, e.g., dogs, foxes, wolves, avian species, e.g., chickens, emus, ostriches, and fish, e.g., trout, catfish, and salmon. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate or a human. The subject can be male or female. Additionally, the subject can be an infant or a child. In some embodiments, the subject can be a newborn or unborn subject, e.g., the subject is present in utero. Preferably, the subject is a mammal. The mammal can be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals can be advantageously used as subjects that represent animal models of diseases and disorders. Additionally, the methods and compositions described herein can be used with domestic animals and / or pets. Human subjects can be of any age, sex, race, or ethnic group, e.g., Caucasian (white), Asian, African, Black, African-American, African-European, Hispanic, Middle Eastern, etc. In some embodiments, the subject can be a patient or another subject in a clinical setting. In some embodiments, the subject has already received treatment. In some embodiments, the subject is an embryo, fetus, newborn, infant, child, adolescent, or adult. In some embodiments, the subject is a human fetus, a human newborn, a human infant, a human child, a human adolescent, or a human adult. In some embodiments, the subject is an animal embryo, or a non-human embryo, or a non-human primate embryo.In some embodiments, the subject is a human embryo.

[0145] As used herein, unless the context and use of the phrase dictate otherwise, the phrase "subject in need" refers to a subject: (i) who is to be administered ceDNA-lipid particles (or a pharmaceutical composition comprising ceDNA-lipid particles) according to the described disclosure, (ii) who has received ceDNA-lipid particles (or a pharmaceutical composition comprising ceDNA-lipid particles) according to the described disclosure, or (iii) who has received ceDNA-lipid particles (or a pharmaceutical composition comprising ceDNA-lipid particles) according to the described disclosure.

[0146] As used herein, the terms "suppress," "reduce," "interfere," "inhibit," and / or "decrease" (and similar terms) generally refer to the act of decreasing, either directly or indirectly, a concentration, level, function, activity, or behavior compared to native, expected, or average conditions, or compared to a control condition.

[0147] As used herein, the term "systemic delivery" refers to the delivery of lipid particles that result in widespread biodistribution of an active agent, such as interfering RNA (e.g., siRNA), mRNA, ceDNA, or ssDNA, within an organism. Some administration techniques can result in systemic delivery of a particular agent, while others cannot. Systemic delivery means that a useful, preferably therapeutic, amount of the agent is exposed to most parts of the body. To achieve widespread biodistribution, a blood lifetime is generally required such that the agent is not rapidly degraded or eliminated (e.g., by first-pass organs (liver, lung, etc.) or rapid nonspecific cellular binding) before reaching disease sites distal to the administration site. Systemic delivery of LNPs can be by any means known in the art, including, for example, intravenous, subcutaneous, and intraperitoneal. In a preferred embodiment, systemic delivery of LNPs is by intravenous delivery.

[0148] As used herein, the term "effective amount," which may be used interchangeably with the terms "therapeutic amount," "therapeutically effective amount," "effective amount," or "pharmaceutically effective amount" of an active agent (e.g., a ceDNA described herein), refers to an amount sufficient to provide the intended benefit of treatment or effect, e.g., expression or inhibition of expression of a target sequence compared to the expression level detected in the absence of the therapeutic nucleic acid. Suitable assays for measuring the expression of a target gene or target sequence include, for example, testing protein or RNA levels using techniques known to those skilled in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those skilled in the art. Dosage levels are based on a variety of factors, including the type of injury, the patient's age, weight, sex, medical condition, severity of the condition, route of administration, and the particular active agent used. Thus, dosing regimens can vary widely but can be routinely determined by a physician using standard methods. Additionally, the terms "effective amount," "therapeutic amount," "therapeutically effective amount," and "pharmaceutically effective amount" include prophylactic or preventative amounts of the described compositions of the invention. In the described prophylactic or preventative uses of the invention, a sufficient amount is administered to a patient susceptible to or otherwise at risk of a disease, disorder, or condition, including the biochemical, histological, and / or behavioral symptoms of the disease, disorder, or condition, its complications, and intermediate pathological phenotypes manifesting during the development of the disease, disorder, or condition, to eliminate or reduce the risk of, lessen the severity of, or delay the onset of, the disease, disorder, or condition. It is generally preferred to use a maximum dose, i.e., the highest safe dose according to medical judgment. The terms "dose" and "dosage" are used interchangeably herein. In one aspect of any of the aspects or embodiments herein, the terms "therapeutic amount," "therapeutically effective amount," and "pharmaceutically effective amount" refer to non-prophylactic or non-preventative uses.

[0149] As used herein, the term "therapeutic effect" refers to an outcome of treatment, which outcome is deemed desirable and beneficial. A therapeutic effect can include, directly or indirectly, the arrest, reduction, or elimination of disease symptoms. A therapeutic effect can also include, directly or indirectly, the arrest, reduction, or elimination of the progression of disease symptoms.

[0150] For any therapeutic agent described herein, the therapeutically effective amount can be initially determined from preliminary in vitro studies and / or animal models. The therapeutically effective dose may also be determined from human data. The applied dose can be adjusted based on the relative bioavailability and efficacy of the administered compound. Adjusting the dose to achieve maximum efficacy based on the above and other well-known methods is within the ability of a person skilled in the art. The general principles for determining therapeutic efficacy, which can be found in Chapter 1 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001) (incorporated herein by reference), are summarized below.

[0151] Pharmacokinetic principles provide a basis for modifying dosing regimens to achieve a desired degree of therapeutic efficacy with minimal unacceptable adverse effects. Additional guidance for dosage modifications can be obtained in situations where the plasma concentration of the drug is measured and can be related to a therapeutic window.

[0152] As used herein, the terms "treat," "treating," and / or "treatment" include arresting, inhibiting, slowing, or reversing the progression of a condition, ameliorating clinical symptoms of a condition, or preventing the appearance of clinical symptoms to achieve a beneficial or desired clinical result. Treating further refers to achieving one or more of: (a) reducing the severity of the disorder; (b) limiting the onset of symptoms characteristic of the disorder being treated; (c) limiting the worsening of symptoms characteristic of the disorder being treated; (d) limiting the recurrence of the disorder in patients who previously had the disorder; and (e) limiting the recurrence of symptoms in patients who did not previously exhibit symptoms of the disorder. In one aspect of any of the aspects or embodiments herein, the terms "treat," "treating," and / or "treatment" include arresting, inhibiting, slowing, or reversing the progression of a condition, or ameliorating clinical symptoms of a condition.

[0153] Beneficial or desired clinical results, e.g., pharmacological and / or physiological effects, include, but are not limited to, preventing a disease, disorder, or condition from occurring in a subject who may be predisposed to the disease, disorder, or condition but who has not yet experienced or exhibited symptoms of the disease (prophylactic treatment), alleviating the disease, disorder, or condition, reducing the severity of the disease, disorder, or condition, stabilizing (i.e., not worsening) the disease, disorder, or condition, preventing the spread of the disease, disorder, or condition, delaying or slowing the progression of the disease, disorder, or condition, ameliorating or alleviating the disease, disorder, or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.

[0154] As used herein, the term "combination therapy" refers to a treatment regimen for a clinical indication that includes two or more therapeutic agents. Thus, the term refers to a treatment regimen in which a first therapy containing a first composition (e.g., active ingredient) is administered to a patient in conjunction with a second therapy containing a second composition (active ingredient) intended to treat the same or overlapping disease or clinical condition. Both the first and second compositions may act on the same or distinct cellular targets. The phrase "in conjunction with" in the context of combination therapy means that the therapeutic effect of the first therapy overlaps temporally and / or spatially with the therapeutic effect of the second therapy in a subject receiving the combination therapy. Thus, combination therapy can be formulated as a single formulation for simultaneous administration of the therapies or as separate formulations for sequential administration of the therapies.

[0155] As used herein, the term "alkyl" refers to a saturated monovalent hydrocarbon radical of 1 to 20 carbon atoms (i.e., C 1-20 "Monovalent" means that the alkyl has one point of attachment to the rest of the molecule. In one embodiment, an alkyl has 1 to 12 carbon atoms (i.e., C 1-12 alkyl) or 1 to 10 carbon atoms (i.e., C 1-10 In one embodiment, alkyl has 1 to 8 carbon atoms (i.e., C 1-8 alkyl), 1 to 7 carbon atoms (i.e., C 1-7 alkyl), 1 to 6 carbon atoms (i.e., C 1-6 alkyl), 1 to 4 carbon atoms (i.e., C 1-4 alkyl), or 1 to 3 carbon atoms (i.e., C 1-3Examples include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and the like. 1-6 Alkyl," "linear or branched C 1-4 Alkyl" or "Straight or branched C 1-3 Straight-chain or branched alkyl, such as "alkyl," means that the saturated monovalent hydrocarbon radical is straight-chained or branched. As used herein, the term "straight-chain" referring to an aliphatic hydrocarbon chain means that the chain is unbranched.

[0156] As used herein, the term "alkylene" refers to a saturated divalent hydrocarbon radical of 1 to 20 carbon atoms (i.e., C 1-20 "Alkylene" refers to an alkylene group, examples of which include, but are not limited to, those having the same core structure as the alkyl groups exemplified above. "Divalent" means that the alkylene has two points of attachment to the rest of the molecule. In one embodiment, alkylene is an alkylene group having 1 to 12 carbon atoms (i.e., C 1-12 alkylene) or 1 to 10 carbon atoms (i.e., C 1-10 In one embodiment, the alkylene has 1 to 8 carbon atoms (i.e., C 1-8 alkylene), 1 to 7 carbon atoms (i.e., C 1-7 alkylene), 1 to 6 carbon atoms (i.e., C 1-6 alkylene), 1 to 4 carbon atoms (i.e., C 1-4 alkylene), 1 to 3 carbon atoms (i.e., C 1-3alkylene), ethylene, or methylene. 1-6 alkylene," "linear or branched C 1-4 Alkylene" or "linear or branched C 1-3 Linear or branched alkylene, such as "alkylene," means that the saturated divalent hydrocarbon radical is linear or branched.

[0157] The term "alkenyl" refers to a straight-chain or branched aliphatic hydrocarbon radical having one or more (e.g., 1 or 2) carbon-carbon double bonds, and alkenyl radicals include radicals having "cis" and "trans" configurations, or, alternatively, "E" and "Z" configurations.

[0158] As used herein, "alkenylene" refers to an aliphatic divalent hydrocarbon radical of 2 to 20 carbon atoms having one or two carbon-carbon double bonds (i.e., C 2-20 "Alkenylene" refers to an alkylene group having 2 to 12 carbon atoms (i.e., C 2-16 alkenylene), 2 to 10 carbon atoms (i.e., C 2-10 In one embodiment, the alkenylene has 2 to 4 carbon atoms (C 2-4 Examples include, but are not limited to, ethylene or vinylene (-CH=CH-), allyl (-CHCH=CH-), etc. 2-6 Alkenylene," "linear or branched C 2-4 Alkenylene" or "linear or branched C 2-3 Linear or branched alkenylene, such as "alkenylene," means that the unsaturated divalent hydrocarbon radical is linear or branched.

[0159] As used herein, "cycloalkylene" refers to a divalent saturated carbocyclic ring radical having 3 to 12 carbon atoms as a monocyclic ring or 7 to 12 carbon atoms as a bicyclic ring. "Divalent" means that the cycloalkylene has two points of attachment to the rest of the molecule. In one embodiment, the cycloalkylene is a 3- to 7-membered monocyclic or a 3- to 6-membered monocyclic ring. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, cyclononylene, cyclodecylene, cycloundecylene, cyclododecylene, and the like. In one embodiment, the cycloalkylene is cyclopropylene.

[0160] The terms "heterocycle," "heterocyclyl," heterocyclic, and "heterocyclic ring" are used interchangeably herein and refer to a cyclic group containing at least one N atom, a heteroatom selected from N and S, and optionally 1 to 3 additional heteroatoms, that is non-aromatic (i.e., partially saturated or fully saturated). It can be monocyclic or bicyclic (bridged or fused). Examples of heterocyclic rings include, but are not limited to, aziridinyl, diaziridinyl, thiaaziridinyl, azetidinyl, diazetidinyl, triazetidinyl, thiadiazetidinyl, thiazetidinyl, pyrrolidinyl, pyrazolidinyl, imidazolinyl, isothiazolidinyl, thiazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl, azepanyl, azocanyl, and the like. A heterocycle contains 1 to 4 heteroatoms, which may be the same or different, selected from N and S. In one embodiment, the heterocycle contains 1 to 3 N atoms. In another embodiment, the heterocycle contains 1 or 2 N atoms. In another embodiment, the heterocycle contains 1 N atom. "4- to 8-membered heterocyclyl" refers to a radical having 4 to 8 atoms (including 1 to 4 heteroatoms selected from N and S, or 1 to 3 N atoms, or 1 or 2 N atoms, or 1 N atom) arranged in a monocyclic ring. "5- or 6-membered heterocyclyl" refers to a radical having 5 or 6 atoms (including 1 to 4 heteroatoms selected from N and S, or 1 to 3 N atoms, or 1 or 2 N atoms, or 1 N atom) arranged in a monocyclic ring. The term "heterocycle" is intended to include all possible isomeric forms.Heterocycles are described in Paquette, Leo A., Principles of Modern Heterocyclic Chemistry (WA Benjamin, New York, 1968), especially Chapters 1, 3, 4, 6, 7, and 9; The Chemistry of Heterocyclic Compounds, A Series of Monographs (John Wiley & Sons, New York, 1950 to present), especially Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. Heterocyclyl groups may be carbon (carbon-linked) or nitrogen (nitrogen-linked) attached to the remainder of the molecule, where such is possible.

[0161] When a group is described as "optionally substituted," the group can be either (1) unsubstituted or (2) substituted. When a carbon of a group is described as optionally substituted with one or more of a list of substituents, one or more of the hydrogen atoms on the carbon (to the extent present) can be replaced separately and / or together with any independently selected substituents.

[0162] Suitable substituents for alkyl, alkylene, alkenylene, cycloalkylene, and heterocyclyl are those that do not significantly adversely affect the biological activity of the molecule. Unless otherwise specified, exemplary substituents for these groups include linear, branched, or cyclic alkyl, alkenyl, or alkynyl having 1 to 10 carbon atoms; aryl; heteroaryl; heterocyclyl; halogen; guanidinium [—NH(C═NH)NH]; —OR 100 ;NR 101 R 102 ;-NO2;--NR 101 COR 102 ;-SR 100 ;--SOR 101 Sulfoxides represented by -SO2R 101 Sulfonic acid - SO3M; sulfuric acid - OSO3M; --SO2NR 101 R102 Sulfonamide; cyano; azide; -COR 101 ;-OCOR 101 ;--OCONR 101 R 102 and polyethylene glycol units (-OCH2CH2) n R 101 wherein M is H or a cation (Na + or K + ) etc.), and R 101 , R 102 , and R 103 are each independently H- a linear, branched, or cyclic alkyl, alkenyl, or alkynyl having 1 to 10 carbon atoms; a polyethylene glycol unit (-OCH2CH2) n -R 104 wherein n is an integer from 1 to 24; an aryl having 6 to 10 carbon atoms; a heterocyclic ring having 3 to 10 carbon atoms; and a heteroaryl having 5 to 10 carbon atoms; and R 104 is H or a linear or branched alkyl having 1 to 4 carbon atoms, and R 100 , R 101 , R 102 , R 103 , and R 104 The alkyl, alkenyl, alkynyl, aryl, heteroaryl, and heterocyclyl in the group represented by the formula (I) are optionally substituted with one or more (e.g., 2, 3, 4, 5, 6, or more) substituents independently selected from halogen, —OH, —CN, —NO2, and unsubstituted linear or branched alkyl having 1 to 4 carbon atoms. Preferably, the substituents of the above optionally substituted alkyl, alkylene, alkenylene, cycloalkylene, and heterocyclyl are halogen, —CN, —NR 101 R 102 , -CF3, -OR 100 , aryl, heteroaryl, heterocyclyl, -SR 101 , -SOR 101 , -SO2R 101 Alternatively, suitable substituents are selected from the group consisting of halogen, —OH, —NO, —CN, C1-4 Alkyl, -OR 100 , N.R. 101 R 102 , --NR 101 COR 102 , -SR 100 , -SO2R 101 , -SO2NR 101 R 102 , --COR 101 , -OCOR 101 , and -OCONR 101 R 102 wherein R 100 , R 101 , and R 102 are each independently -H or C 1-4 It is alkyl.

[0163] As used herein, "halogen" refers to F, Cl, Br, or I. "Cyano" is --CN.

[0164] "Amine" or "amino," as used interchangeably herein, refers to a functional group containing a basic nitrogen atom bearing a lone pair of electrons.

[0165] As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of an ionizable lipid of the present disclosure. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, acid tartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate "mesylate", ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. In cases where multiple charged atoms are part of a pharmaceutically acceptable salt, it may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0166] Grouping of alternative elements or embodiments of the present disclosure disclosed herein should not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. For reasons of convenience and / or patentability, one or more members of a group may be included in, or deleted from, a group. When any such inclusion or deletion occurs, the specification herein is deemed to include the group as modified and, therefore, to satisfy the specification of all Markush groups used in the appended claims.

[0167] In some embodiments of any of its aspects, the disclosure described herein does not relate to processes for cloning humans, processes for modifying the germline genetic identity of humans, the use of human embryos for industrial or commercial purposes, or processes for modifying the genetic identity of animals that are likely to cause suffering to humans or animals without providing any substantial medical benefit to them, and similarly processes for modifying the genetic identity of animals resulting from such processes.

[0168] Other terms are defined herein within the description of various aspects of the disclosure.

[0169] All patents and other publications, including literature references, issued patents, published patent applications, and copending patent applications, cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodology described in such publications, which may be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicant and do not constitute an admission as to the correctness of the dates or contents of these documents.

[0170] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While certain specific embodiments and examples of the present disclosure are described herein for illustrative purposes, those skilled in the art will recognize that various equivalent modifications are possible within the scope of the present disclosure. For example, while method steps or functions are presented in a given order, alternative embodiments may perform the functions in a different order, or may perform the functions substantially simultaneously. The teachings of the present disclosure provided herein can be applied to other procedures or methods, where appropriate. The various embodiments described herein can be combined to provide further embodiments. Where necessary, aspects of the present disclosure can be modified to employ compositions, functions, and concepts from the above references and applications to provide still further embodiments of the present disclosure. Furthermore, due to considerations of biological functional equivalence, some changes can be made to protein structure without affecting biological or chemical activity in terms of type or amount. These and other modifications can be made to the present disclosure in light of the Detailed Description. All such modifications are intended to be within the scope of the appended claims.

[0171] Specific elements of any of the foregoing embodiments can be combined with or substituted for elements of other embodiments. Furthermore, although advantages associated with certain embodiments of the present disclosure are described in connection with those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily exhibit such advantages to fall within the scope of the present disclosure.

[0172] The technology described herein is further illustrated by the following examples, which should not be construed as further limiting in any way. It is understood that the disclosure is not limited in any way to the particular methodology, protocols, and reagents, etc., described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the disclosure, which is defined solely by the claims.

[0173] II. Lipid Nanoparticles (LNPs) Provided herein are lipid nanoparticles (LNPs) comprising a therapeutic nucleic acid (TNA), an ionizable lipid, a structured lipid (e.g., a sterol), one or more lipid-anchored polymers, e.g., a first lipid-anchored polymer and a second lipid-anchored polymer, and a ceramide or other helper lipid. Also provided herein are LNPs consisting essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a structured lipid (e.g., a sterol), one or more lipid-anchored polymers, e.g., a first lipid-anchored polymer and a second lipid-anchored polymer, and a ceramide or other helper lipid. Also provided herein are LNPs consisting of a therapeutic nucleic acid (TNA), an ionizable lipid, a structured lipid (e.g., a sterol), one or more lipid-anchored polymers, e.g., a first lipid-anchored polymer and a second lipid-anchored polymer, and a ceramide or other helper lipid.

[0174] As used herein, the terms "lipid particle" or "lipid nanoparticle" (LNP) refer to lipid formulations that can be used to deliver therapeutic agents, such as therapeutic nucleic acids, to desired target sites (e.g., cells, tissues, organs, etc.). In some embodiments, the lipid nanoparticles of the present disclosure are typically formed from ionizable lipids (e.g., cationic lipids), sterols (e.g., cholesterol), conjugate lipids (e.g., lipid-anchored polymers) that prevent particle aggregation, and optionally helper lipids (e.g., non-cationic lipids). In some other embodiments, a therapeutic agent, such as a therapeutic nucleic acid (TNA), may be encapsulated in the lipid particle, thereby protecting it from degradation. In yet other embodiments, an immunosuppressant may optionally be included in the nucleic acid comprising the lipid nanoparticle. In one embodiment, the lipid particle comprises a nucleic acid (e.g., ceDNA, ssDNA, and / or mRNA). The present disclosure provides LNPs in which at least one of the lipids in the lipid-anchored polymer contains 16, 18, or 20 aliphatic carbons to more firmly anchor the lipid-anchored polymer to the LNP. In some embodiments, at least one lipid in a lipid-anchored polymer having at least 18 aliphatic carbons is useful for generating stealth LNPs. In other embodiments, at least one lipid in a lipid-anchored polymer having at least 20 aliphatic carbons is useful for generating stealth LNPs.

[0175] According to some embodiments, the lipid nanoparticles of the present disclosure typically have a size of about 20 nm to about 90 nm, about 25 nm to about 80 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 55 nm, about 55 nm to about 50 ... The average diameter is about 0 nm to about 75 nm, about 50 nm to about 70 nm, about 60 nm to about 75 nm, about 60 nm to about 70 nm, about 65 nm to about 75 nm, about 65 nm to about 70 nm, or about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 51 nm, about 52 nm, about 53 nm, about 54 nm, about 55 nm, about 56 nm, about 57 nm, about 58 nm, about 59 nm, about 60 nm, about 61 nm, about 62 nm, about 63 nm, about 64 nm, about 65 nm, about 66 nm, about 67 nm, about 68 nm, about 69 nm, about 70 nm, about 71 nm, about 72 nm, about 73 nm, about 74 nm, or about 75 nm (±3 nm).

[0176] Generally, the LNPs of the present disclosure have an average diameter selected to produce an intended therapeutic effect. For example, the LNPs of the present disclosure have an average diameter that is compatible with a target organ (e.g., the liver) so that the LNPs of the present disclosure can diffuse through fenestrations in the target organ (e.g., liver) or target cell subpopulation (e.g., hepatocytes).

[0177] According to some embodiments, the lipid particles of the present disclosure typically have an average diameter of less than about 100 nm, less than about 90 nm, less than about 80 nm, less than about 75 nm, less than about 70 nm, less than about 65 nm, less than about 60 nm, less than about 55 nm, less than about 50 nm, less than about 45 nm, less than about 40 nm, less than about 35 nm, less than about 30 nm, less than about 25 nm, or less than about 20 nm in size.

[0178] In some embodiments, the LNPs of the present disclosure do not include distearoylphosphatidylcholine (DSPC), provided that a helper lipid represented by (I), (II), (III), or (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present. In some embodiments, the LNPs of the present disclosure do not include 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), provided that a helper lipid represented by (I), (II), (III), or (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present.

[0179] A. Ionizable lipids In some embodiments, the ionizable lipids are present in an LNP provided by the present disclosure at about 20 mol% to about 70 mol%, about 20 mol% to about 65 mol%, about 20 mol% to about 60 mol%, about 20 mol% to about 55 mol%, about 20 mol% to about 50 mol%, about 25 mol% to about 70 mol%, about 25 mol% to about 65 mol%, about 25 mol% to about 60 mol%, about 25 mol% to about 55 mol%, about 25 mol% to about 50 mol%, about 30 mol% to about 70 mol% of the total lipids present in the LNP. %, about 30 mol% to about 65 mol%, about 30 mol% to about 60 mol%, about 30 mol% to about 55 mol%, about 30 mol% to about 50 mol%, about 35 mol% to about 70 mol%, about 35 mol% to about 65 mol%, about 35 mol% to about 60 mol%, about 35 mol% to about 55 mol%, about 35 mol% to about 50 mol%, 40 mol% to about 70 mol%, about 40 mol% to about 65 mol%, about 40 mol% to about 60 mol%, about 40 mol% to about 55 mol%, or about 40 mol% to about 50 mol%.

[0180] In some embodiments, the LNPs provided by the present disclosure comprise an ionizable lipid. Exemplary ionizable lipids in the LNPs of the present disclosure are listed in International Patent Application Publication Nos. 2015 / 095340, 2015 / 199952, 2018 / 011633, 2017 / 049245, 2015 / 061467, 2012 / 040184, 2012 / 000104, 2015 / 074085, 2016 / 081029, 2017 / 004143, and 2017 / 0755. 31, 2017 / 117528, 2011 / 022460, 2013 / 148541, 2013 / 116126, 2011 / 153120, 2012 / 044638, 201 2 / 054365, 2011 / 090965, 2013 / 016058, 2012 / 162210, 2008 / 042973, 2010 / 129709, 2010 / 144740 , 2012 / 099755, 2013 / 049328, 2013 / 086322, 2013 / 086373, 2011 / 071860 , same No. 2009 / 132131, same No. 2010 / 048536, same No. 2010 / 088537, same No. 2010 / 054401, same No. 2010 / 054406 , 2010 / 054405, 2010 / 054384, 2012 / 016184, 2009 / 086558, 2010 / 042877, 2011 / 000106, 2011 / 000107, 2005 / 1201 52, 2011 / 141705, 2013 / 126803, 2006 / 007712, 2011 / 038160, 2005 / 121348, 2011 / 066651, 2009 / 127060, 2011 / 141 704, 2006 / 069782, 2012 / 031043, 2013 / 006825, 2013 / 033563, 2013 / 089151, 2017 / 099823, 2015 / 095346, and 2013 / 086354, and U.S. Patent Application Publication Nos. 2016 / 0311759, 2015 / 0376115, 2016 / 0151284, 2017 / 0210697, 2015 / 0140070, and 2013 / 0178541,Same No. 2013 / 0303587, Same No. 2015 / 0141678, Same No. 2015 / 0239926, same No. 2016 / 0376224, same No. 2017 / 0119904, same No. 2012 / 014989 4, Same No. 2015 / 0057373, Same No. 2013 / 0090372, Same No. 2013 / 0274523, same No. 2013 / 0274504, same No. 2013 / 0274504, same No. 2009 / 0023 673, same No. 2012 / 0128760, same No. 2010 / 0324120, same No. 2014 / 0200257, same No. 2015 / 0203446, same No. 2018 / 0005363, same No. 2014 / 03 08304, 2013 / 0338210, 2012 / 0101148, 2012 / 0027796, 2012 / 0058144, 2013 / 0323269, 2011 / 0 117125, 2011 / 0256175, 2012 / 0202871, 2011 / 0076335, 2006 / 0083780, 2013 / 0123338, 2015 / 0064242, 2006 / 0051405, 2013 / 0065939, 2006 / 0008910, 2003 / 0022649, 2010 / 0130588, 201 Nos. 3 / 0116307, 2010 / 0062967, 2013 / 0202684, 2014 / 0141070, 2014 / 0255472, 2014 / 0039032, 2018 / 0028664, 2016 / 0317458, and 2013 / 0195920, the contents of all of which are incorporated herein by reference in their entireties.

[0181] Formula (A) In some embodiments, the ionizable lipid in the LNPs of the present disclosure has formula (A): [ka] (A) (In the formula, R 1 and R 1’ are each independently, C 1-3 is alkylene, R 2 and R2’ are each independently a linear or branched C 1-6 Alkylene, or C 3-6 is cycloalkylene, R 3 and R 3’ each independently represents an optionally substituted C 1-6 Alkyl or optionally substituted C 3-6 is cycloalkyl, or Alternatively, R 2 Branched C 1-6 alkylene, and R 3 C 1-6 If it is alkyl, R 2 and R 3 together with the intervening N atom to form a 4- to 8-membered heterocyclyl, or Alternatively, R 2’ Branched C 1-6 alkylene, and R 3’ C 1-6 If it is alkyl, R 2’ and R 3’ together with the intervening N atom to form a 4- to 8-membered heterocyclyl; R 4 and R 4’ are each independently -CH, -CHCH, or -(CH)CH; R 5 and R 5’ are each independently hydrogen, C 1-20 Alkylene or C 2-20 is alkenylene, R 6 and R 6’ But for each occurrence, independently, C 1-20 Alkylene, C 3-20 Cycloalkylene or C 2-20 is alkenylene, m and n are each independently an integer selected from 1, 2, 3, 4, and 5, or a pharmaceutically acceptable salt thereof.

[0182] In some embodiments, R 2 and R 2’are each independently, C 1-3 It is alkylene.

[0183] In some embodiments, R 1 or R 1’ Linear or branched C represented by 1-3 Alkylene, R 2 or R 2’ Linear or branched C represented by 1-6 Alkylene, and optionally substituted linear or branched C 1-6 Each alkyl is optionally substituted with one or more halo and cyano groups.

[0184] In some embodiments, R 1 and R 2 Together, C 1-3 alkylene, and R 1’ and R 2’ Together, C 1-3 Alkylene, for example, ethylene.

[0185] In some embodiments, R 3 and R 3’ each independently represents an optionally substituted C 1-3 Alkyl, for example, methyl.

[0186] In some embodiments, R 4 and R 4’ are each -CH.

[0187] In some embodiments, R 2 is an optionally substituted branched C 1-6 alkylene, and R 2 and R 3 taken together with their intervening N atom form a 5- or 6-membered heterocyclyl. In some embodiments, R 2’ is an optionally substituted branched C 1-6 alkylene, and R 2’ and R 3’taken together with their intervening N atom form a 5- or 6-membered heterocyclyl such as pyrrolidinyl or piperidinyl.

[0188] In some embodiments, R 4 is -C(R a )2CR a or -[C(R a )2]2CR a and R a is C 1-3 alkyl, and R 3 and R 4 taken together with their intervening N atom form a 5- or 6-membered heterocyclyl. In some embodiments, R 4’ is -C(R a )2CR a or -[C(R a )2]2CR a and R a is C 1-3 alkyl, and R 3’ and R 4’ taken together with their intervening N atom form a 5- or 6-membered heterocyclyl such as pyrrolidinyl or piperidinyl.

[0189] In some embodiments, R 5 and R 5’ are each independently, C 1-10 Alkylene or C 2-10 In one embodiment, R is alkenylene. 5 and R 5’ are each independently, C 1-8 Alkylene or C 1-6 It is alkylene.

[0190] In some embodiments, R 6 and R 6’ is independently generated for each occurrence of C 1-10 Alkylene, C 3-10 Cycloalkylene, or C 2-10 In one embodiment, C is an alkenylene. 1-6 Alkylene, C 3-6 Cycloalkylene, or C 2-6In one embodiment, C is an alkenylene. 3-10 Cycloalkylene or C 3-6 In some embodiments, m and n are each 3.

[0191] In some embodiments, the ionizable lipid in the LNPs of the present disclosure can be selected from any one of the lipids listed in Table 1 below, or a pharmaceutically acceptable salt thereof. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13]

[0192] Formula (B) In some embodiments, the ionizable lipid in the LNPs of the present disclosure has formula (B): [ka] (B) (In the formula, a is an integer ranging from 1 to 20 (e.g., a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); b is an integer ranging from 2 to 10 (e.g., b is 2, 3, 4, 5, 6, 7, 8, 9, or 10); R 1 is absent or (C2-C 20 ) alkenyl, -C(O)O(C2-C 20 ) alkyl, and (C2-C 20 ) cyclopropyl substituted with alkyl; R 2 However, (C2-C 20 ) alkyl) or a pharmaceutically acceptable salt thereof.

[0193] In a second embodiment of formula (B), the ionizable lipid of formula (B) has the formula (B-1): [ka] (B-1) wherein c and d are each independently an integer ranging from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and the remaining variables are as described in formula (B), or a pharmaceutically acceptable salt thereof.

[0194] In a third embodiment of Formula (B), c and d in Formula (B-1) are each independently an integer ranging from 2 to 8, from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5, from 4 to 8, from 4 to 7, from 4 to 6, from 5 to 8, from 5 to 7, or from 6 to 8, and the remaining variables are as described for Formula (B-1).

[0195] In a fourth embodiment of Formula (B), c in Formula (B-1) is 2, 3, 4, 5, 6, 7, or 8, and the remaining variables are as described for Formula (B), or the second or third embodiment of Formula (B). Alternatively, c and d in Formula (B-1) are each independently 1, 3, 5, or 7, and the remaining variables are as described for Formula (B), or the second or third embodiment of Formula (B).

[0196] In a fifth embodiment of Formula (B), d in the cationic lipid of Formula (B-1) is 2, 3, 4, 5, 6, 7, or 8, with the remaining variables being as described for Formula (B), or the second, third, or fourth embodiment of Formula (B). Alternatively, at least one of c and d in Formula (B-1) is 7, with the remaining variables being as described for Formula (B), or the second, third, or fourth embodiment of Formula (B).

[0197] In a sixth embodiment of formula (B), the ionizable lipid of formula (B) or formula (B-1) is of formula (B-2): [ka] (B-2) wherein the remaining variables are as described for Formula (B) or Formula (B-1), or a pharmaceutically acceptable salt thereof.

[0198] In a seventh embodiment of Formula (B), b in Formula (B), Formula (B-1), or Formula (B-2) is an integer ranging from 3 to 9, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, or sixth embodiment of Formula (B). Alternatively, b in Formula (B), Formula (B-1), or Formula (B-2) is an integer ranging from 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 5 to 9, 5 to 8, 5 to 7, 6 to 9, 6 to 8, or 7 to 9, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, or sixth embodiment of Formula (B). Alternatively, b in Formula (B), Formula (B-1), or Formula (B-2) is 3, 4, 5, 6, 7, 8, or 9, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, or sixth embodiment of Formula (B).

[0199] In an eighth embodiment of Formula (B), a in Formula (B), Formula (B-1), or Formula (B-2) is an integer ranging from 2 to 18, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, or seventh embodiment of Formula (B). Alternatively, a in formula (B), formula (B-1), or formula (B-2) is 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 18, 4 to 17, 4 to 16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 25-8, 5-7, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 7-18, 7-17, 7-16, 7 ~15, 7~14, 7~13, 7~12, 7~11, 7~10, 7~9, 8~18, 8~17, 8~16, 8~15, 8~14, 8~13, 8~12, 8~11, 8~10, 9~18, 9~17, 9~16, 9~15, 9~14, 9~13, 9~12, 9~11, 10~18, 10~17, 10~16, 10~15, 10~14, 10~13, 11~18, 11~17, 11~16, 11~1 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 124, 125, 136, 136, 137, 146, 147, 148, 1518, 1517, or 1618, and the remainder of the variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, or seventh embodiment of Formula (B). Alternatively, a in Formula (B), Formula (B-1), or Formula (B-2) is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, or seventh embodiment of Formula (B).

[0200] In a ninth embodiment of formula (B), R in formula (B), formula (B-1), or formula (B-2) 1 is absent or (C5-C 15 ) alkenyl, -C(O)O(C4-C 18 ) alkyl, and (C4-C 16 ) cyclopropyl substituted with alkyl, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B). Alternatively, R in Formula (B), Formula (B-1), or Formula (B-2) is selected from 1 is absent or (C5-C 15 ) alkenyl, -C(O)O(C4-C 16 ) alkyl, and (C4-C 16 ) cyclopropyl substituted with alkyl, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B). Alternatively, R in Formula (B), Formula (B-1), or Formula (B-2) is selected from 1 is absent or (C5-C 12 ) alkenyl, -C(O)O(C4-C 12 ) alkyl, and (C4-C 12 ) cyclopropyl substituted with alkyl, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B). In another alternative, R in the cationic lipid of Formula (B), Formula (B-1), or Formula (B-2) is selected from: 1 is absent or (C5-C 10 ) alkenyl, -C(O)O(C4-C 10 ) alkyl, and (C4-C 10 ) cyclopropyl substituted with alkyl, and the remainder of the variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B).

[0201] In a tenth embodiment of formula (B), R 1 is C 10alkenyl, and the remainder of the variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B).

[0202] In an eleventh embodiment of formula (B), R in formula (B), formula (B-1), or formula (B-2) 1 C(O)O(C2-C 20 ) alkyl, -C(O)O(C4-C 18 ) alkyl, -C(O)O(C4-C 12 ) alkyl, or -C(O)O(C4-C 10 ) alkyl is an unbranched alkyl, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh, eighth, or ninth embodiment of Formula (B). In one embodiment, R 1 is —C(O)O(C alkyl). Alternatively, R in formula (B), formula (B-1), or formula (B-2) 1 -C(O)O(C4-C 18 ) alkyl, -C(O)O(C4-C 12 ) alkyl, or -C(O)O(C4-C 10 The alkyl in alkyl is a branched alkyl, and the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, eighth, or ninth embodiment of Formula (B). In one embodiment, R 1 is -C(O)O(C 17 alkyl), and the remainder of the variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, eighth, or ninth embodiment of Formula (B).

[0203] In a twelfth embodiment of formula (B), R in formula (B), formula (B-1), or formula (B-2) 1is selected from any group listed in Table 2 below, where the wavy bond in each group indicates the point of attachment of the group to the remainder of the ionizable lipid molecule, and the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B). The present disclosure provides a method for treating R 1 and any one of the groups R in Table 3 of formula (B) 2 Further contemplated are combinations with any one of the groups, wherein the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B). [Table 2]

[0204] In a thirteenth embodiment, R in formula (B) 2 or a pharmaceutically acceptable salt thereof, selected from any of the groups listed in Table 3 below, where the wavy bond in each group indicates the point of attachment of the group to the remainder of the ionizable lipid molecule, and the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, or twelfth embodiment of Formula (B). [Table 3]

[0205] Table 4 below provides specific examples of ionizable lipids of Formula (B), including pharmaceutically acceptable salts, as well as ionized and neutral forms. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6]

[0206] Formula (C) In some embodiments, the ionizable lipid in the LNPs of the present disclosure has formula (C): [ka] (C) (In the formula, R 1 and R 1’ are each independently R a and (C1-C6) alkylene optionally substituted with one or more groups selected from R 2 and R 2’ are each independently (C1-C2) alkylene; R 3 and R 3’ are each independently R b or (C1-C6) alkyl optionally substituted with one or more groups selected from Alternatively, R 2 and R 3 and / or R 2’ and R 3’ together with the intervening N atom to form a 4- to 7-membered heterocyclyl; R 4 and R 4 each ' is (C2-C6)alkylene interrupted by -C(O)O-; R 5 and R 5 each independently being optionally interrupted by —C(O)O— or (C3-C6)cycloalkyl, (C2-C 30 ) alkyl or (C2-C 30 ) alkenyl, Ra and R b wherein each is halo or cyano, or a pharmaceutically acceptable salt thereof.

[0207] In a second embodiment of formula (C), R 1 and R 1 are each independently (C-C) alkylene, and the remaining variables are as described above for formula (C). Alternatively, R 1 and R 1’ is independently at each occurrence (C1-C3) alkylene, and the remainder of the variables are as described above for formula (C).

[0208] In a third embodiment of formula (C), the ionizable lipid of formula (C) has the formula (C-1): [ka] (C-1) (In the formula, R 2 and R 2’ , R 3 and R 3’ , R 4 and R 4 ', as well as R 5 and R 5 ' is represented by Formula (C), or as described above for the second embodiment of Formula (C), or a pharmaceutically acceptable salt thereof.

[0209] In a fourth embodiment, the ionizable lipid of formula (C) has formula (C-2) or formula (C-3): [ka] (C-2), or [ka] (C-3) (In the formula, R 4 and R 4 ' and R 5 and R 5' is as described above for formula (C)), or a pharmaceutically acceptable salt thereof.

[0210] In a fifth embodiment of formula (C), the ionizable lipid of formula (C) is of formula (C-4) or formula (C-5): [ka] (C-4), or [ka] (C-5) (In the formula, R 5 and R 5 ' is as described above for formula (C)), or a pharmaceutically acceptable salt thereof.

[0211] In a sixth embodiment of Formula (C), the ionizable lipid of Formula (C) is Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9): [ka] (C-6), [ka] (C-7), [ka] (C-8), or [ka] (C-9) (In the formula, R 5 and R 5 ' is as described above for formula (XV)), or a pharmaceutically acceptable salt thereof.

[0212] In a seventh embodiment of formula (C), R in formula (C), formula (C-1), formula (C-2), formula (C-3), formula (C-4), formula (C-5), formula (C-6), formula (C-7), formula (C-8), or formula (C-9) 5 and R 5’ is a branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 and R 5’ is a branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 is branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5’ is branched alkyl or branched alkenyl, and the remainder of the variables are as described above for Formula (C), or the second embodiment of Formula (C).

[0213] In an eighth embodiment of formula (C), R in formula (C), formula (C-1), formula (C-2), formula (C-3), formula (C-4), formula (C-5), formula (C-6), formula (C-7), formula (C-8), or formula (C-9) 5 are each optionally interrupted by —C(O)O— or (C-C)cycloalkyl, (C-C 26 ) alkyl or (C6-C 26) alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 are each optionally interrupted by —C(O)O— or (C3-C5)cycloalkyl, (C6-C 26 ) alkyl or (C6-C 26 ) alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 are each optionally interrupted by —C(O)O— or (C3-C5)cycloalkyl, (C7-C 26 ) alkyl or (C7-C 26 ) alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 are each optionally interrupted by —C(O)O— or (C3-C5)cycloalkyl, (C8-C 26 ) alkyl or (C8-C 26 ) alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 are each optionally interrupted by —C(O)O— or cyclopropyl, (C-C 24 ) alkyl or (C6-C 24) alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 is (C8-C 24 ) alkyl or (C8-C 24 ) alkenyl, and the (C8-C 24 )alkyl is optionally interrupted by -C(O)O- or cyclopropyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 is (C8-C 10 ) alkyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 is interrupted by cyclopropyl (C 14 -C 16 ) alkyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 is interrupted by -C(O)O- (C 10 -C 24 ) alkyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 is (C 16 -C 18) alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R5 in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) is —(CH2)3C(O)O(CH2)8CH3, —(CH2)5C(O)O(CH2)8CH 3, -(CH2)7C(O)O(CH2)8CH3,-(CH2)7C(O)OCH[(CH2)7CH3]2,-(CH2)7-C3H6-(CH2)7CH3,-(CH2)7CH3,-(CH2)9CH 3, -(CH2) 16 CH3, -(CH2)7CH=CH(CH2)7CH3, or -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, and the remainder of the variables are as described above for Formula (C), or the second embodiment of Formula (C).

[0214] In the ninth embodiment, R in formula (C), formula (C-1), formula (C-2), formula (C-3), formula (C-4), formula (C-5), formula (C-6), formula (C-7), formula (C-8), or formula (C-9) 5’ is interrupted by -C(O)O- (C 15 -C 28 ) alkyl, and the remaining variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5’ is interrupted by -C(O)O- (C 17 -C 28 ) alkyl, and the remaining variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5’ is interrupted by -C(O)O- (C 19 -C 28) alkyl, and the remaining variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5’ is interrupted by -C(O)O- (C 17 -C 26 ) alkyl, and the remaining variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5’ is interrupted by -C(O)O- (C 19 -C 26 ) alkyl, and the remaining variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5’ is interrupted by -C(O)O- (C 20 -C 26 ) alkyl, and the remaining variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C). Alternatively, R 5’ is interrupted by -C(O)O- (C 22 -C 24 ) alkyl, and the remaining variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C). Alternatively, R 5’ is —(CH2)5C(O)OCH[(CH2)7CH3]2, —(CH2)7C(O)OCH[(CH2)7CH3]2, —(CH2)5C(O)OCH(CH2)2[(CH2)7CH3]2, or —(CH2)7C(O)OCH(CH2)2[(CH2)7CH3]2, and the remainder of the variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C).

[0215] In some embodiments, the ionizable lipid of Formula (C), Formula (C-1), Formula (C-3), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-7), Formula (C-8), or Formula (C-9) may be selected from any of the lipids listed in Table 5 below, or a pharmaceutically acceptable salt thereof. [Table 5-1] [Table 5-2] [Table 5-3]

[0216] Formula (D) In some embodiments, the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure has formula (D): [ka] (D) (In the formula, R' is absent, hydrogen, or C1-C6 alkyl, provided that when R' is hydrogen or C1-C6 alkyl, R', R 1 , and R 2 provided that all nitrogen atoms to which they are attached are positively charged, R 1 and R 2 are each independently hydrogen, C1-C6 alkyl, or C2-C6 alkenyl; R 3 But C1-C 12 Alkylene or C2-C 12 is alkenylene, R 4 But C1-C 18 Unbranched alkyl, C2-C 18 unbranched alkenyl, or [ka] wherein: R 4a and R4b are each independently, C1-C 16 Unbranched alkyl or C2-C 16 is an unbranched alkenyl, R 5 is absent, C1-C8 alkylene, or C2-C8 alkenylene; R 6a and R 6b are each independently, C7-C 16 Alkyl or C7-C 16 alkenyl, provided that R 6a and R 6b the total number of carbon atoms in the X 1 and X 2 are each independently -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)-; R a is independently at each occurrence hydrogen or C1-C6 alkyl; and n is an integer selected from 1, 2, 3, 4, 5, and 6, or a pharmaceutically acceptable salt thereof.

[0217] In a second embodiment of formula (D), X 1 and X 2 is the same, and all other remaining variables are as described for formula (C).

[0218] In a 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 is each independently -C(=O)O- or -SS-, and all other remaining variables are as described for Formula (D), or the second embodiment of Formula (D).

[0219] In a fourth embodiment of Formula (D), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure has the formula (D-1): [ka] (D-1) wherein n is an integer selected from 1, 2, 3, and 4, and all other remaining variables are as described for Formula (D), or the second or third embodiment of Formula (D), or a pharmaceutically acceptable salt thereof.

[0220] In a fifth embodiment of Formula (D), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure is represented by Formula (D-2): [ka] (D-2) wherein n is an integer selected from 1, 2, and 3, and all other remaining variables are as described for Formula (D), or the second or third embodiment of Formula (D), or a pharmaceutically acceptable salt thereof.

[0221] In a sixth embodiment of Formula (D), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure is represented by Formula (D-3): [ka] (D-3) wherein all other remaining variables are as described for Formula (D), or the second or third embodiment of Formula (D), or a pharmaceutically acceptable salt thereof.

[0222] In a seventh embodiment of Formula (D), in an ionizable lipid, e.g., a cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), or the second or third embodiment of Formula (D), R 1 and R 2 are each independently hydrogen, C1-C6 alkyl or C2-C6 alkenyl, or C1-C5 alkyl or C2-C5 alkenyl, or C1-C4 alkyl or C2-C4 alkenyl, or C6 alkyl, or C5 alkyl, or C4 alkyl, or C3 alkyl, or C2 alkyl, or C1 alkyl, or C6 alkenyl, or C5 alkenyl, or C4 alkenyl, or C3 alkenyl, or C2 alkenyl, and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), or the second or third embodiment of Formula (D).

[0223] In an eighth embodiment of Formula (D), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure is represented by Formula (D-4): [ka] (D-4) wherein all other remaining variables are as described for the second, third, or seventh embodiment of Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), or Formula (D), or a pharmaceutically acceptable salt thereof.

[0224] In a ninth embodiment of Formula (D), in an ionizable lipid, e.g., a cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, or seventh embodiment of Formula (D), R3 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 is C 12 Alkylene, C 11 Alkylene, C 10 Alkylene, C9 alkylene, or C8 alkylene, or C7 alkylene, or C6 alkylene, or C5 alkylene, or C4 alkylene, or C3 alkylene, or C2 alkylene, or C1 alkylene, or C 12 Alkenylene, C 11 Alkenylene, C 10 alkenylene, C9 alkenylene, or C8 alkenylene, or C7 alkenylene, or C6 alkenylene, or C5 alkenylene, or C4 alkenylene, or C3 alkenylene, or C2 alkenylene, and all other remaining variables are as described for the second, third, or seventh embodiment of Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or Formula (D).

[0225] In a tenth embodiment of Formula (D), in an ionizable lipid, e.g., a cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, or seventh embodiment of Formula (D), R 5 is absent, C-C alkylene, or C-C alkenylene, or R 5 is absent, C-C alkylene, or C-C alkenylene, or R 5 is absent or R 5is C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, or C alkenylene, and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, or ninth embodiment of Formula (D).

[0226] In an eleventh embodiment of Formula (D), in an ionizable lipid, e.g., a cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, or tenth embodiment of Formula (D), R 4 is C1-C 14 Unbranched alkyl, C2-C 14 unbranched alkenyl, or [ka] (In the formula, R 4a and R 4b are each independently, C1-C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl), or R 4 is C2-C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl, or R 4 is a C5-C7 unbranched alkyl or a C5-C7 unbranched alkenyl, or R 4 is C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C 13 Unbranched alkyl, C 12 Unbranched alkyl, C 11 Unbranched alkyl, C 10Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 unbranched alkyl, C1 unbranched alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C 14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl, or R 4 teeth, [ka] (In the formula, R 4a and R 4b are each independently, C2-C 10 Unbranched alkyl or C2-C 10 unbranched alkenyl), or R 4 teeth, [ka] (In the formula, R 4a and R 4b are each independently, C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C 13 Unbranched alkyl, C 12 Unbranched alkyl, C 11 Unbranched alkyl, C 10 Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 alkyl, C1 alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl), and all other remaining variables are as described for the second, third, seventh, ninth, or tenth embodiment of Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or Formula (D).

[0227] In a twelfth embodiment, in an ionizable lipid, e.g., a cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth, or eleventh embodiment of Formula (D), R 6a and R 6b are each independently C6-C 14 Alkyl or C6-C 14 alkenyl, or R 6a and R 6b are each independently C8-C 12 Alkyl or C8-C 12 alkenyl, or R 6a and R 6b are each independently, C 16 Alkyl, C 15 Alkyl, C 14 Alkyl, C 13 Alkyl, C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 16 Alkenyl, C 15 Alkenyl, C 14 Alkenyl, C 13 Alkenyl, C 12 Alkenyl, C 11 Alkenyl, C 10alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, provided that R 6a and R 6b is greater than 15, and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth, or eleventh embodiment of Formula (D).

[0228] In a thirteenth embodiment of Formula (D), in an ionizable lipid, e.g., a cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth, eleventh, or twelfth embodiment of Formula (D), or a pharmaceutically acceptable salt thereof, R 6a and R 6b contain an equal number of carbon atoms, or R 6a and R 6b are the same or R 6a and R 6b are both C 16 Alkyl, C 15 Alkyl, C 14 Alkyl, C 13 Alkyl, C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 16 Alkenyl, C 15 Alkenyl, C 14 Alkenyl, C 13 Alkenyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, provided that R 6a and R 6b is greater than 15, and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth, eleventh, or twelfth embodiment of Formula (D).

[0229] In a fourteenth embodiment of Formula (D), in an ionizable lipid, e.g., a cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth, eleventh, twelfth, or thirteenth embodiment of Formula (D), R as defined in any one of the preceding embodiments. 6a and R 6b each contain a different number of carbon atoms, or R 6a and R 6b The number of carbon atoms in R differs by one or two carbon atoms, or 6a and R 6b The number of carbon atoms in R differs by one carbon atom, or 6a is a C7 alkyl, and R 6a is C8 alkyl or R 6a is C8 alkyl, and R 6a is a C7 alkyl or R 6a is C8 alkyl, and R 6a is a C9 alkyl or R 6a is a C9 alkyl, and R 6a is C8 alkyl or R 6a is a C9 alkyl, and R 6a C 10 alkyl or R 6a C 10 alkyl, and R 6a is a C9 alkyl or R 6a C 10 alkyl, and R 6a C 11 alkyl or R 6a C 11 alkyl, and R 6a C 10 alkyl or R 6a C 11 alkyl, and R 6a C 12 alkyl or R 6a C 12 alkyl, and R 6a C 11 alkyl or R 6a is a C7 alkyl, and R6a is a C9 alkyl or R 6a is a C9 alkyl, and R 6a is a C7 alkyl or R 6a is C8 alkyl, and R 6a C 10 alkyl or R 6a C 10 alkyl, and R 6a is C8 alkyl or R 6a is a C9 alkyl, and R 6a C 11 alkyl or R 6a C 11 alkyl, and R 6a is a C9 alkyl or R 6a C 10 alkyl, and R 6a C 12 alkyl or R 6a C 12 alkyl, and R 6a C 10 alkyl or R 6a C 11 alkyl, and R 6a C 13 alkyl or R 6a C 13 alkyl, and R 6a C 11 is alkyl, and all other remaining variables are as described for the second, third, seventh, ninth, tenth, eleventh, twelfth, or thirteenth embodiment of Formula I, Formula II, Formula III, Formula IV, Formula V, or Formula (D).

[0230] In a fifteenth embodiment of formula (D), R 4 is C1-C 16 Unbranched alkyl, C2-C 16 unbranched alkenyl, or [ka] (In the formula, R 4a and R 4bis as described above for the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, or fourteenth embodiment of Formula (D).

[0231] In one embodiment, the ionizable lipid of the present disclosure, e.g., a cationic lipid, or an ionizable lipid of Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), or Formula (D-4), is any one lipid selected from the lipids listed in Table 6 below, or a pharmaceutically acceptable salt thereof. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5]

[0232] In one embodiment, the ionizable lipid in the LNPs of the present disclosure is lipid number 87: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0233] Formula (E) In some embodiments, the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure has the formula (E): [ka] (E) (In the formula, R' is absent, hydrogen, or C1-C3 alkyl, provided that when R' is hydrogen or C1-C3 alkyl, R', R1 , and R 2 provided that all nitrogen atoms to which they are attached are positively charged, R 1 and R 2 are each independently hydrogen or C1-C3 alkyl; R 3 But C3-C 10 Alkylene or C3-C 10 is alkenylene, R 4 But C1-C 16 Unbranched alkyl, C2-C 16 unbranched alkenyl, or [ka] wherein: R 4a and R 4b are each independently, C1-C 16 Unbranched alkyl or C2-C 16 is an unbranched alkenyl, R 5 is absent, C1-C6 alkylene, or C2-C6 alkenylene; R 6a and R 6b are each independently, C7-C 14 Alkyl or C7-C 14 is alkenyl, X is -OC(=O)-,-SC(=O)-,-OC(=S)-,-C(=O)O-,-C(=O)S-,-SS-,-C(R a )=N-, -N=C(R a )-,-C(R a )=NO-,-ON=C(R a )-,-C(=O)NR a -,-NR a C(=O)-, -NR a C(=O)NR a -, -OC(=O)O-,-OSi(R a )2O-,-C(=O)(CR a 2) C(=O)O-, or OC(=O)(CR a2) C(=O)-; R a is independently at each occurrence hydrogen or C1-C6 alkyl; and n is an integer selected from 1, 2, 3, 4, 5, and 6, or a pharmaceutically acceptable salt thereof.

[0234] In a second embodiment of Formula (E), in an ionizable lipid, e.g., a cationic lipid according to the first embodiment, or a pharmaceutically acceptable salt thereof, X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, or -SS-, and all other remaining variables are as described for Formula I or the first embodiment.

[0235] In a third embodiment of Formula (E), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure has the formula (E-1): [ka] (E-1) wherein n is an integer selected from 1, 2, 3, and 4, and all other remaining variables are as described for Formula (E), or the second embodiment of Formula (E), or a pharmaceutically acceptable salt thereof. Alternatively, n is an integer selected from 1, 2, and 3, and all other remaining variables are as described for Formula (E), or the second embodiment of Formula (E).

[0236] In a fourth embodiment of Formula (E), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure has the formula (E-2): [ka] (E-2) wherein all other remaining variables are as described for Formula (E), Formula (E-1), or the second embodiment of Formula (E), or a pharmaceutically acceptable salt thereof.

[0237] In a fifth embodiment of Formula (E), in an ionizable lipid, e.g., a cationic lipid, in an LNP of the present disclosure, R 1 and R 2 are each independently hydrogen or C1-C2 alkyl or C2-C3 alkenyl, or R', R 1 , and R 2 are each independently hydrogen, C1-C2 alkyl, and all other remaining variables are as described for Formula (E), Formula (E-1), or the second embodiment of Formula (E).

[0238] In a sixth embodiment of Formula (E), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure is represented by Formula (E-3): [ka] (E-3) wherein all other remaining variables are as described for the second or fifth embodiment of Formula (E), Formula (E-1), Formula (E-2), or Formula (E), or a pharmaceutically acceptable salt thereof.

[0239] In a seventh embodiment of Formula (E), in an ionizable lipid, e.g., a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), or the second or fifth embodiment of Formula (E), R 5 is absent or C1-C8 alkylene, or R 5 is absent, C-C alkylene, or C-C alkenylene, or R 5 is absent, C-C alkylene, or C-C alkenylene, or R 5 is absent or R 5is C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, or C alkenylene, and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), or the second or fifth embodiment of Formula (E).

[0240] In an eighth embodiment of Formula (E), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure has the formula (E-4): [ka] (E-4) wherein all other remaining variables are as described for the second, fifth, or seventh embodiment of Formula (E), Formula (E-1), Formula (E-2), (E-3), or Formula (E), or a pharmaceutically acceptable salt thereof.

[0241] In a ninth embodiment, in an ionizable lipid, e.g., a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, or seventh embodiment of Formula (E), or a pharmaceutically acceptable salt thereof, R 4 is C1-C 14 Unbranched alkyl, C2-C 14 unbranched alkenyl, or [ka] (In the formula, R 4a and R 4b are each independently, C1-C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl), or R 4 is C2-C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl, or R 4 is C5-C12 Unbranched alkyl or C5-C 12 unbranched alkenyl, or R 4 is C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C 13 Unbranched alkyl, C 12 Unbranched alkyl, C 11 Unbranched alkyl, C 10 Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 unbranched alkyl, C1 unbranched alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C 14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl, or R 4 teeth, [ka] (In the formula, R 4a and R 4b are each independently, C2-C 10 Unbranched alkyl or C2-C 10 unbranched alkenyl), or R 4 teeth, [ka] (In the formula, R 4a and R 4b are each independently, C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C 13Unbranched alkyl, C 12 Unbranched alkyl, C 11 Unbranched alkyl, C 10 Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 alkyl, C1 alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C 14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl), and all other remaining variables are as described for the second, fifth, or seventh embodiment of Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or Formula (E).

[0242] In a tenth embodiment, in an ionizable lipid, e.g., a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, seventh, or ninth embodiment of Formula (E), R 3 is C3-C8 alkylene or alkenylene, C3-C7 alkylene or alkenylene, or C3-C5 alkylene or alkenylene, or R 3is C alkylene, or C alkylene, or C alkylene, or C alkylene, or C alkylene, or C alkylene, or C alkylene, or C alkylene, or C alkenylene, or C alkenylene, or C alkenylene, or C alkenylene, or C alkenylene, and all other remaining variables are as described for the second, fifth, seventh, or ninth embodiment of Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or Formula (E).

[0243] In an eleventh embodiment, in an ionizable lipid, e.g., a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, seventh, ninth, or tenth embodiment of Formula (E), R 6a and R 6b are each independently C7-C 12 Alkyl or C7-C 12 alkenyl, or R 6a and R 6b are each independently C8-C 10 Alkyl or C8-C 10 alkenyl, or R 6a and R 6b are each independently, C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, and all other remaining variables are as described for the second, fifth, seventh, ninth, or tenth embodiment of Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or Formula (E).

[0244] In a twelfth embodiment, in an ionizable lipid, e.g., a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, seventh, ninth, tenth, or eleventh embodiment of Formula (E), R 6a and R 6b contain an equal number of carbon atoms, or R 6a and R 6b are the same or R 6a and R 6b are both C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, and all other remaining variables are as described for the second, fifth, seventh, ninth, tenth, or eleventh embodiment of Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or Formula (E).

[0245] In a thirteenth embodiment, in an ionizable lipid, e.g., a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), R as defined in any one of the preceding embodiments is 6a and R 6b each contain a different number of carbon atoms, or R 6a and R 6b The number of carbon atoms in R differs by one or two carbon atoms, or 6a and R 6b The number of carbon atoms in R differs by one carbon atom, or 6a is a C7 alkyl, and R 6a is C8 alkyl, R 6a is C8 alkyl, and R 6a is C7 alkyl, R 6a is C8 alkyl, and R 6a is C9 alkyl, R 6a is a C9 alkyl, and R 6ais C8 alkyl, R 6a is a C9 alkyl, and R 6a C 10 R is alkyl 6a C 10 alkyl, and R 6a is C9 alkyl, R 6a C 10 alkyl, and R 6a C 11 R is alkyl 6a C 11 alkyl, and R 6a C 10 R is alkyl 6a C 11 alkyl, and R 6a C 12 R is alkyl 6a C 12 alkyl, and R 6a C 11 R is alkyl 6a is a C7 alkyl, and R 6a is C9 alkyl, R 6a is a C9 alkyl, and R 6a is C7 alkyl, R 6a is C8 alkyl, and R 6a C 10 R is alkyl 6a C 10 alkyl, and R 6a is C8 alkyl, R 6a is a C9 alkyl, and R 6a C 11 R is alkyl 6a C 11 alkyl, and R 6a is C9 alkyl, R 6a C 10 alkyl, and R 6a C 12 R is alkyl 6a C 12 alkyl, and R 6a C 10is alkyl, and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, seventh, ninth, tenth, eleventh, or twelfth embodiment of Formula (E).

[0246] In a fourteenth embodiment, in an ionizable lipid, e.g., a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, seventh, ninth, tenth, eleventh, twelfth, or thirteenth embodiment of Formula (E), R' is absent, and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, seventh, ninth, tenth, eleventh, twelfth, or thirteenth embodiment of Formula (E).

[0247] In one embodiment, the ionizable lipid, e.g., cationic lipid, or cationic lipid of Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), in the LNP of the present disclosure is any one lipid selected from the lipids in Table 7, or a pharmaceutically acceptable salt thereof. [Table 7-1] [Table 7-2] [Table 7-3]

[0248] Specific examples are provided in the Exemplification section below and are included as part of the cationic or ionizable lipids described herein. Pharmaceutically acceptable salts as well as neutral forms are also included.

[0249] Cleavable lipids In some embodiments, the LNPs provided by the present disclosure comprise an ionizable lipid that is also a cleavable lipid. As used herein, the term "cleavable lipid," which may be used interchangeably with the term "SS-cleavable lipid," refers to an ionizable lipid that contains a disulfide bond (SS). The SS in the cleavable lipid is the cleavable unit. In one embodiment, the cleavable lipid comprises an amine, e.g., a tertiary amine, and a disulfide bond. In this cleavable lipid, the amine can become protonated in an acidic compartment (e.g., an endosome or lysosome), resulting in LNP destabilization, and the cleavable lipid can become cleaved in a reducing environment (e.g., the cytoplasm). Cleavable lipids also include pH-activated lipid-like materials, such as ss-OP lipids, ssPalm lipids, ss-M lipids, ss-E lipids, ss-EC lipids, ss-LC lipids, and ss-OC lipids.

[0250] According to some embodiments, the SS-cleavable lipids are described in International Patent Application Publication No. WO 2019188867, which is incorporated herein by reference in its entirety.

[0251] In one embodiment, the cleavable lipid may comprise three components: an amine head group, a linker group, and a hydrophobic tail. In one embodiment, the cleavable lipid comprises one or more phenyl ester bonds, one or more tertiary amino groups, and a disulfide bond. The tertiary amine groups provide pH responsiveness and induce endosomal escape, the phenyl ester bonds enhance the degradability (autolysis) of the structure, and the disulfide bonds become cleaved in a reducing environment.

[0252] In one embodiment, the cleavable lipid is a ss-OP lipid. In one embodiment, the ss-OP lipid comprises the structure of lipid A shown below:

[0253] Lipid A [ka] . In one embodiment, the SS-cleavable lipid is SS-cleavable pH-activated lipid-like material (ssPalm). ssPalm lipid is well known in the art. For example, see Togashi et al., Journal of Controlled Release, 279 (2018) 262-270, the entire contents of which are incorporated herein by reference. In one embodiment, the ssPalm is ssPalmM lipid, comprising the structure of lipid B shown below.

[0254] lipid B [ka] . In one embodiment, the ssPalmE lipid is a ssPalmE-P4-C2 lipid comprising the structure of lipid C below:

[0255] lipid C [ka] . In one embodiment, the ssPalmE lipid is a ssPalmE-Paz4-C2 lipid comprising the structure of lipid D below:

[0256] lipid D [ka] In one embodiment, the cleavable lipid is a ss-M lipid. In one embodiment, the ss-M lipid comprises the structure shown in lipid E ​​below.

[0257] Lipid E [ka] In one embodiment, the cleavable lipid is an ss-E lipid. In one embodiment, the ss-E lipid comprises the structure shown in lipid F below.

[0258] lipid F [ka] . In one embodiment, the cleavable lipid is a ss-EC lipid. In one embodiment, the ss-EC lipid comprises the structure shown for lipid G below.

[0259] lipid G [ka] . 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.

[0260] lipid H [ka] In one embodiment, the cleavable lipid is a ss-OC lipid. In one embodiment, the ss-OC lipid comprises the structure shown for lipid J below.

[0261] lipid J [ka] . Other lipids In some embodiments, the ionizable lipid in the LNPs of the present disclosure is N-[1-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DL EPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)aminolbutylcarboxamidoethyl 1-3,4-di[oleyloxy]-benzamide (MVL5), dioctadecylamido-glycylspermine (DOGS), 3b-[N-( N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol), dioctadecyldimethylammonium bromide (DDAB), Saint lipids (e.g., SAINT-2, N-methyl-4-(dioleyl)methylpyridinium), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), 1,2-dioleoyl-3-dimethylhydroxyethylammonium bromide (DORIE), 1,2-dioleoyloxypropyl-3-dimethylhydroxyethylammonium chloride (DORI), dialkylated amino acids (DILA2) (e.g., C18:1-norArg-C16), dioleyldimethylammonium chloride (DODAC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC), and 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC).In some variations, the condensing agent, e.g., the cationic lipid, is, for example, dioctadecyldimethylammonium bromide (DDAB), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (Dlin-MC3-DMA), 1,2-dioleoyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2-dioleoyloxy-3-dimethylaminopropane (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA)], ...3-DMA)], 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin These lipids include diethylaminopropane (DODAP), 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA), morpholinocholesterol (Mo-CHOL), (R)-5-(dimethylamino)pentane-1,2-diyldioleate hydrochloride (DODAPen-C1), (R)-5-guanidinopentane-1,2-diyldioleate hydrochloride (DOPen-G), and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-aminium chloride (DOTAPen).

[0262] In some embodiments, the ionizable lipid in the LNPs of the present disclosure has the following structure: [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , or [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0263] B. Structured lipids In some embodiments, LNPs provided by the present disclosure comprise structured lipids. Without wishing to be bound by any particular theory, it is believed that structured lipids, when present in LNPs, contribute to the membrane integrity and stability of the LNPs.

[0264] In some embodiments, the structured lipid is a sterol, such as cholesterol, or a derivative thereof. In one embodiment, the structured lipid is cholesterol. In another embodiment, the structured lipid is a cholesterol derivative. Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholestanol, 5β-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol, non-polar analogs such as 5α-cholestan, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate, and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether. In some embodiments, the cholesterol derivative is cholestryl hemisuccinate (CHEMS).

[0265] Exemplary cholesterol derivatives are described in International Patent Application Publication No. 2009 / 127060 and US Patent Application Publication No. 2010 / 0130588, the contents of both of which are incorporated herein by reference in their entireties.

[0266] In some embodiments, the sterol in the LNPs of the present disclosure is selected from the group consisting of cholesterol, beta-sitosterol, stigmasterol, beta-sitostanol, campesterol, brassicasterol, and derivatives thereof, and any combination thereof. In one embodiment, the sterol is cholesterol. In another embodiment, the sterol is beta-sitosterol.

[0267] In some embodiments, structured lipids comprise about 20 mol% to about 45 mol% of the total lipids present in the LNP. In some embodiments, structured lipids comprise about 25 mol% to about 45 mol% of the total lipid content of the LNP. In some embodiments, structured lipids comprise about 30 mol% to about 45% of the total lipids present in the LNP. In some embodiments, structured lipids comprise about 30 mol% to about 40 mol% of the total lipids present in the LNP. In some embodiments, such components are about 40 mol% of the total lipids present in the LNP. In some embodiments, structured lipids, e.g., sterols, comprise about 20 mol% to about 45 mol% of the total lipids present in the LNP. In some embodiments, structured lipids, e.g., sterols, comprise about 30 mol% to about 40 mol% of the total lipids present in the LNP.

[0268] In some embodiments, the structured lipid is cholesterol and comprises about 30 mol% to about 45 mol% of the total lipids present in the LNP. In some embodiments, the structured lipid is cholesterol and comprises about 35 mol% to about 45 mol% of the total lipids present in the LNP. In some embodiments, the structured lipid is cholesterol and comprises about 40 mol% to about 45 mol% of the total lipids present in the LNP. In some embodiments, the structured lipid is cholesterol and comprises about 40 mol% of the total lipids present in the LNP. In some embodiments, the structured lipid is cholesterol and comprises about 45 mol% of the total lipids present in the LNP. In some embodiments, the structured lipid is cholesterol and comprises about 40 mol% to about 45 mol% of the total lipids present in the LNP, and the encapsulation efficiency ("Enc. Eff.") of the TNA is greater than 95%, and / or the average size of the LNP is in the range of about 70 nm to 90 nm in diameter.

[0269] In some embodiments, the structured lipid is dexamethasone or dexamethasone palmitate.

[0270] C. Helper lipids The LNPs provided by the present disclosure comprise a helper lipid. In some embodiments, the helper lipid is ceramide or sphingomyelin. Both ceramide and sphingomyelin are sphingolipids, a class of cell membrane lipids. Structurally, both ceramide and sphingomyelin comprise an N-acetylsphingosine (i.e., (E)-N-(1,3-dihydroxyoctadec-4-en-2-yl)acetamide) backbone and a fatty acid linked to an amide group. In sphingomyelin, the N-acetylsphingosine backbone is further linked to a phosphocholine group or a phosphoethanolamine group. In some embodiments, the LNPs provided by the present disclosure comprise ceramide or sphingomyelin or a combination thereof, wherein the fatty acid moiety of the ceramide or sphingomyelin is a fatty acid of a certain length or a certain number of carbon atoms, as described below. As used herein, the term "helper lipid" refers to an amphipathic lipid comprising at least one nonpolar chain and at least one polar moiety. Without wishing to be bound by any particular theory, it is believed that the helper lipids function to avoid off-targeting of LNPs to the blood compartment, increase the fusogenicity of the LNP lipid bilayer, stabilize the LNP structure, and facilitate endosomal escape.

[0271] In some embodiments, the ceramide or sphingomyelin in the LNPs of the present disclosure as a helper lipid has formula (I): [ka] Formula (I) (In the formula, [ka] is a single bond or a double bond, A is hydrogen, [ka] , or [ka] and R 1 But C1-C 17 Alkyl or C2-C 17 is alkenyl, R 2 But C1-C 22 Alkyl or C2-C 22 is alkenyl, R 3 is hydrogen or C1-C2 alkyl, R 4 is hydrogen or C1-C2 alkyl), or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0272] In some embodiments of Formula (I), R 1 is C1-C 10 Alkyl or C2-C 10 It is alkenyl.

[0273] In some embodiments of Formula (I), R 1 is C1-C 10 Alkyl or C2-C 10 is alkenyl, R 2 is C1-C 22 Alkyl or C2-C 22 is alkenyl, R 3 is hydrogen or C1-C2 alkyl, R 4 is hydrogen or C1-C2 alkyl.

[0274] In some embodiments of Formula (I), R 3 and R 4 are both hydrogen. In some embodiments of Formula (I), R 3 and R 4 are independently hydrogen or C1 alkyl.

[0275] In some embodiments of Formula (I), R 1is C1-C7 alkyl or C2-C7 alkenyl. 1 is C1-C7 alkyl. In one embodiment, R 1 is a C1 alkyl.

[0276] In some embodiments, the helper lipid is not distearoylphosphatidylcholine (DSPC), provided that a helper lipid represented by (I), (II), (III), or (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present.

[0277] In some embodiments, the helper lipid is not 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), provided that a helper lipid represented by (I), (II), (III), or (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present.

[0278] In some embodiments, the helper lipid is not DOPE, provided that a helper lipid represented by (I), (II), (III), or (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present.

[0279] In some embodiments, the helper lipid has the formula (II): [ka] Formula (II) (In the formula, R 1 , R 2 , R 3 , and R 4 is as defined in formula (I) above), or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0280] In some embodiments of Formula (II), R 3 and R 4are both hydrogen.

[0281] In some embodiments of Formula (II), R 3 and R 4 are independently hydrogen or C1 alkyl.

[0282] In some embodiments of Formula (II), R 1 is C1-C7 alkyl or C2-C7 alkenyl. 1 is C1-C7 alkyl. In one embodiment, R 1 is a C1 alkyl.

[0283] In some embodiments, the helper lipid has formula (III): [ka] Formula (III) (In the formula, R 1 , R 2 , R 3 , and R 4 is as defined in formula (I) above), or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0284] In some embodiments of Formula (III), R 3 and R 4 are both hydrogen.

[0285] In some embodiments of Formula (III), R 1 is C1-C 10 Alkyl or C2-C 10 In one embodiment, R is an alkenyl. 1 is C1-C 10 It is alkyl.

[0286] In some embodiments, the helper lipid has formula (IV): [ka] Formula (IV) (In the formula, R1 , R 2 , R 3 , and R 4 is as defined in formula (I) above), or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0287] As used herein, the term "salt" refers to a helper lipid represented by formula (I), formula (II), formula (III), or formula (IV), and refers to a pharmaceutically acceptable salt of the helper lipid represented by formula (I), formula (II), formula (III), or formula (IV), including both acid addition salts and base addition salts. The salts of the helper lipid represented by formula (I), formula (II), formula (III), or formula (IV) are not biologically or otherwise undesirable and retain the biological effectiveness and properties of the free acid or free base form of the helper lipid represented by formula (I), formula (II), formula (III), or formula (IV), formed with an inorganic or organic acid or base. Examples of inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Examples of organic acids include acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, and the like. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts.Salts derived from organic bases include, but are not limited to, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0288] As used herein, the term "ester" refers to a helper lipid represented by formula (I), formula (II), formula (III), or formula (IV), and refers to an ester of the helper lipid represented by formula (I), formula (II), formula (III), or formula (IV). As a non-limiting example, the hydroxyl group of the helper lipid represented by formula (I), formula (II), formula (III), or formula (IV) can be linked to an organic acid such as phosphoric acid or carboxylic acid through an esterification process to form an ester (e.g., a carboxylic acid ester or a phosphate ester) of the helper lipid represented by formula (I), formula (II), formula (III), or formula (IV).

[0289] As used herein, "deuterated analog," when referring to a helper lipid represented by formula (I), formula (II), formula (III), or formula (IV), means an analog of a helper lipid represented by formula (I), formula (II), formula (III), or formula (IV) in which any one or more hydrogen atoms of the lipid are replaced with deuterium, an isotope of hydrogen.

[0290] In some embodiments, the LNPs of the present disclosure do not contain or are free of distearoylphosphatidylcholine (DSPC), provided that a helper lipid represented by (I), (II), (III), or (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present. In some embodiments, the LNPs of the present disclosure do not contain or are free of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), provided that a helper lipid represented by (I), (II), (III), or (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present. In some embodiments, the LNPs of the present disclosure do not contain or include 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), provided that a helper lipid represented by (I), (II), (III), or (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present.

[0291] In one embodiment of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, [ka] is a double bond and R 1 , R 2 , R 3 , and R 4 In alternative embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, [ka] is a single bond, and R 1 , R 2 , R 3 , and R 4 is as defined above.

[0292] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is C1-C 15 Alkyl or C2-C 15 It is alkenyl.

[0293] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is C1-C 15 Alkyl or C2-C 15 is alkenyl, R 2 is C1-C 22 Alkyl or C2-C 22 is alkenyl, R 3 is hydrogen or C1-C2 alkyl, R 4 is hydrogen or C1-C2 alkyl.

[0294] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is C1-C 10 Alkyl or C2-C 10 It is alkenyl.

[0295] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or salts or esters thereof, R 1 is C1-C 10 Alkyl or C2-C 10 is alkenyl, R 2 is C1-C 22 Alkyl or C2-C 22 is alkenyl, R 3 is hydrogen or C1-C2 alkyl, R 4is hydrogen or C1-C2 alkyl.

[0296] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is C1-C8 alkyl or C2-C8 alkenyl. 1 is C1-C8 alkyl. In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is C1-C7 alkyl or C2-C7 alkenyl. 1 is C1-C7 alkyl. In one embodiment of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is C1-C7 alkyl, R 2 is C1-C 22 Alkyl or C2-C 22 is alkenyl, R 3 is hydrogen or C1-C2 alkyl, R 4 is hydrogen or C1-C2 alkyl.

[0297] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is a C1 alkyl, a C2 alkyl, a C3 alkyl, a C4 alkyl, a C5 alkyl, a C6 alkyl, or a C7 alkyl. In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1is a C1 alkyl, a C2 alkyl, a C3 alkyl, a C4 alkyl, a C5 alkyl, a C6 alkyl, or a C7 alkyl. In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is a C1 alkyl, a C3 alkyl, a C5 alkyl, or a C7 alkyl. In one embodiment of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 In one embodiment of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 In one embodiment of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is a C5 alkyl. In one embodiment of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 1 is a C7 alkyl.

[0298] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 2 is C3-C 15 Alkyl or C3-C 15 alkenyl, and R 1 , R 3 , and R 4 is as defined above. In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 2 is C5-C 15 Alkyl or C3-C 15 alkenyl, and R 1 , R 3 , and R 4is as defined above. In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 2 is C7-C 15 Alkyl or C3-C 15 alkenyl, and R 1 , R 3 , and R 4 is as defined above. In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 2 is C9-C 15 Alkyl or C9-C 15 alkenyl, and R 1 , R 3 , and R 4 is as defined above. In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 2 is C9 alkyl, C 10 Alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 14 Alkyl or C 15 alkyl, and R 1 , R 3 , and R 4 is as defined above. In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 2 is a C9 alkyl, and R 1 , R 3 , and R 4 is as defined above. In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 2 is C 11 alkyl, and R 1 , R 3 , and R4 is as defined above. In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 2 is C 13 alkyl, and R 1 , R 3 , and R 4 is as defined above.

[0299] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 3 is hydrogen or C1 alkyl, and R 1 , R 2、及び R 4 is as defined above. In one embodiment of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 3 is hydrogen and R 1 , R 2、及び R 4 is as defined above. In one embodiment of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 3 is a C1 alkyl, and R 1 , R 2、及び R 4 is as defined above.

[0300] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), 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、及び R 3 is as defined above. In one embodiment of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 4 is hydrogen and R1 , R 2、及び R 3 is as defined above. In one embodiment of Formula (I), Formula (II), Formula (III), and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, R 4 is a C1 alkyl, and R 1 , R 2、及び R 3 is as defined above.

[0301] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), R 1 is C1-C7 alkyl or C2-C7 alkenyl.

[0302] In some embodiments, R 1 is a C alkyl, a C alkyl, a C alkyl, or a C alkyl. In some embodiments, R 1 is a C1 alkyl.

[0303] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), R 2 is C3-C 15 Alkyl or C3-C 15 In some embodiments, R is alkenyl. 2 is C 10 Alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 14 Alkyl or C 15 In some embodiments, R 2 is C 12 Alkyl, C 13 Alkyl or C 14 In some embodiments, R 2 is C 13 In some embodiments, R 2 is C 12 In some embodiments, R 2 is C 11 It is alkyl.

[0304] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), R 1 and R 2 are both hydrogen, [ka] is a double bond.

[0305] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), R 1 and R 2 are both hydrogen, [ka] is a double bond and R 1 is a C alkyl, a C alkyl, a C alkyl, or a C alkyl. In one embodiment, R 1 is C alkyl. In another embodiment, R 1 is C alkyl. In yet another embodiment, R 1 is C5 alkyl. In yet another embodiment, R 1 is a C7 alkyl.

[0306] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), R 1 and R 2 are both hydrogen, [ka] is a double bond and R 1 is a C1 alkyl, a C3 alkyl, a C5 alkyl, or a C7 alkyl, and R 2 is C9 alkyl, C 11 , or C 13 In one embodiment, R 2 is C alkyl. In one embodiment, R 2 is C 11 In another embodiment, R 2 is C13 It is alkyl.

[0307] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), R 3 is hydrogen. In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), R 3 is a C1 alkyl.

[0308] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), R 4 is hydrogen. In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), R 4 is a C1 alkyl.

[0309] In some embodiments, the helper lipid (e.g., ceramide or sphingomyelin) represented by Formula (I), Formula (II), Formula (III), or Formula (IV) in the LNPs of the present disclosure is as in Table 8 below, or a salt or ester thereof, or a deuterated analog of any of the foregoing. [Table 8-1] [Table 8-2]

[0310] In some embodiments, the helper lipid is DSPC, a salt or ester thereof, or a deuterated analog of any of the foregoing. In some embodiments, the helper lipid is DOPE, a salt or ester thereof, or a deuterated analog of any of the foregoing. In some embodiments, the helper lipid is ceramide, a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0311] As used herein, the term "salt" refers to pharmaceutically acceptable salts of helper lipids, including both acid and base addition salts, which retain the biological effectiveness and properties of the free acid or base form of the helper lipid.

[0312] As used herein, the term "ester" refers to an ester of a helper lipid. As a non-limiting example, a hydroxyl group of a helper lipid can be linked to an organic acid such as a phosphoric acid or a carboxylic acid through an esterification process to form an ester (e.g., a carboxylate or a phosphate) of the helper lipid.

[0313] As used herein, "deuterated analog," when referring to a helper lipid, means an analog of a helper lipid in which any one or more hydrogen atoms of the helper lipid have been replaced with deuterium.

[0314] In some embodiments, the LNPs of the present disclosure do not contain or include a helper lipid (e.g., distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0315] In some embodiments, the helper lipid (e.g., a ceramide of the present disclosure) represents about 2 mol% to about 40 mol%, or about 5 mol% to about 40 mol%, or about 5 mol% to about 35 mol%, or about 5 mol% to about 30 mol%, or about 5 mol% to about 25 mol%, or about 5 mol% to about 20 mol%, or about 5 mol% to about 15 mol%, or 10 mol% to about 40 mol%, or about 10 mol% to about 35 mol%, or about 10 mol% to about 30 mol%, or about 10 mol% to about 25 mol%, or about 10 mol% to about 20 mol%, or 15 mol% to about 40 mol%, or about 15 mol% to about 35 mol%, or about 15 mol% to about 30 mol%, or about 15 mol% to about 25 mol%, or about 15 mol% to about 20 mol%, or 20 mol% to about 40 mol%, or about 20 mol% to about 35 mol%, or about 20 mol% to about 30 mol%, or about 20 mol% to about In some embodiments, the helper lipid (e.g., DSPC, DOPE, ceramide, etc.) comprises about 10 mol% to about 20 mol% of the total lipid present in the LNP, and such LNPs having about 10 mol% to about 20 mol% of the total lipid present in the LNP exhibit an overall increase in tolerability (e.g., as shown by a reduced weight loss profile and cytokine response in subjects) compared to LNPs containing less than 10% of the same helper lipid.

[0316] D. Lipid-anchored polymers In some embodiments, the LNPs provided by the present disclosure comprise at least one type of lipid-anchored polymer, e.g., a first lipid-anchored polymer. As used herein, the term "lipid-anchored polymer" refers to a molecule comprising a lipid moiety covalently attached to a polymer, optionally via a linker. Without wishing to be bound by theory, it is believed that the lipid-anchored polymer can inhibit aggregation of the LNP and provide steric stabilization. In some embodiments, the LNPs provided by the present disclosure comprise two lipid-anchored polymers, i.e., a first lipid-anchored polymer and a second lipid-anchored polymer.

[0317] Lipid moieties in lipid-anchored polymers More specifically, in one embodiment, the lipid-anchored polymer, e.g., the first lipid-anchored polymer according to the present disclosure, comprises: (i) a polymer; (ii) a lipid moiety comprising at least one hydrophobic tail, which may be linear or branched; (iii) optionally, a linker connecting the polymer to the lipid moiety; At least one hydrophobic tail (which may be linear or branched) comprises 16 to 22 carbon atoms in the single aliphatic chain backbone, i.e., 16, 17, 18, 19, 20, 21, or 22 carbon atoms in the single aliphatic chain backbone. In one embodiment, a lipid-anchored polymer, e.g., a first lipid-anchored polymer, comprises a lipid moiety comprising a single or two hydrophobic tails, each of which comprises 16 to 22 carbon atoms in the single aliphatic chain backbone, i.e., 16, 17, 18, 19, 20, 21, or 22 carbon atoms in the single aliphatic chain backbone. In one embodiment, each of the single or two hydrophobic tails comprises 18 to 22 carbon atoms in the single aliphatic chain backbone. In another embodiment, each of the single or two hydrophobic tails comprises 18 to 20 carbon atoms in the single aliphatic chain backbone. In a particular embodiment, each of the single or two hydrophobic tails comprises 18 carbon atoms in the single aliphatic chain backbone. In another embodiment, the single or two hydrophobic tails each contain at least 18 carbon atoms in a single aliphatic chain backbone.

[0318] As used herein, the term "linker lipid moiety" refers to a lipid moiety comprising at least two hydrophobic tails, e.g., two hydrophobic tails, covalently attached to a linker. In some embodiments, the linker lipid moiety can be part of a lipid-anchored polymer.

[0319] In one embodiment, at least one (e.g., single or two) hydrophobic tail is a fatty acid. Non-limiting examples of at least one (e.g., single or two) hydrophobic tail containing 16 to 22 carbon atoms in a single aliphatic chain backbone include octadecylamine, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, and derivatives thereof.

[0320] The term "derivative," as used herein with respect to a hydrophobic tail in a lipid-anchored polymer, refers to a hydrophobic tail that has been modified compared to the original or native hydrophobic tail. In some embodiments, a derivative comprises one or more of the following modifications compared to the original or native hydrophobic tail: a) a carboxylate group has been replaced with an amine, amide, ether, or carbonate group; b) one or more saturation points, e.g., double bonds, have been introduced into the hydrophobic tail (e.g., via dehydrogenation); c) one or more saturation points, e.g., double bonds, have been removed from the hydrophobic tail (e.g., via hydrogenation); or d) if present, the configuration of one or more double bonds has been changed, e.g., from a cis configuration to a trans configuration or from a trans configuration to a cis configuration. A derivative comprises the same number of carbon atoms as its original or native hydrophobic tail.

[0321] As used herein, the term "single aliphatic chain backbone" refers to the main linear aliphatic chain or carbon chain, i.e., the longest continuous linear aliphatic chain or carbon chain, when referring to the hydrophobic tail in a lipid-anchored polymer. For example, the following alkyl chain with several branches contains 18 carbon atoms in the single aliphatic chain backbone, i.e., the longest continuous linear alkyl chain contains 18 carbon atoms. Note that one or two carbon atoms at some branch points (all indicated by *) are not included in the number of carbon atoms in the single aliphatic chain backbone. [ka]

[0322] In one embodiment, the lipid-anchored polymer or first lipid-anchored polymer according to the present disclosure comprises: (i) a polymer; (ii) a lipid moiety comprising at least two hydrophobic tails, which may be linear or branched; (iii) optionally, a linker connecting the polymer to the lipid moiety; At least two hydrophobic tails (which may be linear or branched) comprise 16 to 22 carbon atoms in the single aliphatic chain backbone, i.e., 16, 17, 18, 19, 20, 21, or 22 carbon atoms in the single aliphatic chain backbone. In one embodiment, the lipid-anchored polymer or first lipid-anchored polymer comprises a lipid moiety comprising two hydrophobic tails, wherein the two hydrophobic tails each independently comprise 16 to 22 carbon atoms in the single aliphatic chain backbone, i.e., 16, 17, 18, 19, 20, 21, or 22 carbon atoms in the single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 16 to 21 carbon atoms in the single aliphatic chain backbone, i.e., 16, 17, 18, 19, 20, or 21 carbon atoms in the single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently contain 16 to 20 carbon atoms in the single aliphatic chain backbone, i.e., 16, 17, 18, 19, or 20 carbon atoms in the single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently contain 16 to 19 carbon atoms in the single aliphatic chain backbone, i.e., 16, 17, 18, or 19 carbon atoms in the single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently contain 16 to 18 carbon atoms in the single aliphatic chain backbone, i.e., 16, 17, or 18 carbon atoms in the single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently contain 16 or 18 carbon atoms in the single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently contain 16 or 20 carbon atoms in the single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently contain 18 or 20 carbon atoms in the single aliphatic chain backbone. In one embodiment, each of the two hydrophobic tails comprises 16 carbon atoms in the single aliphatic chain backbone. In one embodiment, each of the two hydrophobic tails comprises 17 carbon atoms in the single aliphatic chain backbone. In one embodiment, each of the two hydrophobic tails comprises 18 carbon atoms in the single aliphatic chain backbone. In one embodiment, each of the two hydrophobic tails comprises 19 carbon atoms in the single aliphatic chain backbone. In one embodiment, each of the two hydrophobic tails comprises 20 carbon atoms in the single aliphatic chain backbone.

[0323] In one embodiment, each of the at least two (e.g., two) hydrophobic tails is a fatty acid. Non-limiting examples of at least two hydrophobic tails containing 16 to 22 carbon atoms in a single aliphatic chain backbone include octadecylamine, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, and derivatives thereof.

[0324] In one embodiment, the lipid-anchored polymer or first lipid-anchored polymer according to the present disclosure comprises: (i) a polymer; (ii) a lipid moiety comprising at least two hydrophobic tails, which may be linear or branched; (iii) optionally, a linker connecting the polymer to the lipid moiety; At least two hydrophobic tails (which may be linear or branched) comprise 12 to 15 carbon atoms in the single aliphatic chain backbone, i.e., 12, 13, 14, or 15 carbon atoms in the single aliphatic chain backbone. In one embodiment, the lipid-anchored polymer or first lipid-anchored polymer comprises a lipid moiety comprising two hydrophobic tails, wherein the two hydrophobic tails each independently comprise 12 to 15 carbon atoms in the single aliphatic chain backbone, i.e., 12, 13, 14, or 15 carbon atoms in the single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 12 to 14 carbon atoms in the single aliphatic chain backbone, i.e., 12, 13, or 14 carbon atoms in the single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 12 or 14 carbon atoms in the single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 12 carbon atoms in the single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each contain 13 carbon atoms in the single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each contain 14 carbon atoms in the single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each contain 15 carbon atoms in the single aliphatic chain backbone.

[0325] In one embodiment, one of the two hydrophobic tails is a fatty acid. Non-limiting examples of at least two hydrophobic tails containing 12 to 15 carbon atoms in a single aliphatic chain backbone include lauric acid, myristic acid, myristoleic acid, and derivatives thereof.

[0326] In one embodiment, the lipid-anchored polymer or first lipid-anchored polymer according to the present disclosure comprises: (i) a polymer; (ii) a lipid moiety comprising a single hydrophobic tail (which may be linear or branched); optionally, (iii) a linker attaching the polymer to the lipid moiety; The single hydrophobic tail (which may be linear or branched) comprises 12 to 22 carbon atoms in the single aliphatic chain backbone, i.e., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon atoms in the single aliphatic chain backbone. In one embodiment, the lipid-anchored polymer or first lipid-anchored polymer comprises a lipid moiety comprising a single hydrophobic tail, and the single hydrophobic tail comprises 12 to 22 carbon atoms in the single aliphatic chain backbone, i.e., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon atoms in the single aliphatic chain backbone. In one embodiment, the single hydrophobic tail comprises 12, 14, 16, 18, 20, or 22 carbon atoms in the single aliphatic chain backbone. In one embodiment, the single hydrophobic tail comprises 16 to 20 carbon atoms in the single aliphatic chain backbone, i.e., 16, 17, 18, 19, or 20 carbon atoms in the single aliphatic chain backbone. In one embodiment, the single hydrophobic tail comprises 16 to 19 carbon atoms in the single aliphatic chain backbone, i.e., 16, 17, 18, or 19 carbon atoms in the single aliphatic chain backbone. In one embodiment, the single hydrophobic tail comprises 16 to 18 carbon atoms in the single aliphatic chain backbone, i.e., 16, 17, or 18 carbon atoms in the single aliphatic chain backbone. In one embodiment, the single hydrophobic tail comprises 12 carbon atoms in the single aliphatic chain backbone. In one embodiment, the single aliphatic chain backbone comprises 13 carbon atoms in the single aliphatic chain backbone. In one embodiment, the single aliphatic chain backbone comprises 14 carbon atoms in the single aliphatic chain backbone. In one embodiment, the single aliphatic chain backbone comprises 15 carbon atoms in the single aliphatic chain backbone. In one embodiment, the single aliphatic chain backbone comprises 16 carbon atoms in the single aliphatic chain backbone. In one embodiment, the single aliphatic chain backbone comprises 17 carbon atoms in the single aliphatic chain backbone. In one embodiment, the single aliphatic chain backbone contains 18 carbon atoms. In one embodiment, the single aliphatic chain backbone contains 19 carbon atoms. In one embodiment, the single aliphatic chain backbone contains 20 carbon atoms. In one embodiment, the single aliphatic chain backbone contains 21 carbon atoms. In one embodiment, the single aliphatic chain backbone contains 22 carbon atoms.

[0327] In one embodiment, the single hydrophobic tail is a fatty acid. Non-limiting examples of single hydrophobic tails containing 12 to 22 carbon atoms in a single aliphatic chain backbone include lauric acid, myristic acid, myristoleic acid, octadecylamine, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, and derivatives thereof.

[0328] Linkers in lipid-anchored polymers In some embodiments, in the lipid-anchored polymers of the present disclosure, the lipid moiety is covalently attached to the polymer, either directly or, optionally, via a linker. In some embodiments, the linker in the lipid-anchored polymers of the present disclosure is a glycerol linker, a phosphate linker, an ether linker, an amide linker, an amine linker, a peptide linker, a phosphoethanolamine linker, a phosphocholine linker, or any combination thereof. In some embodiments, the linker in the lipid-anchored polymers of the present disclosure is a glycerol linker. Thus, in some embodiments, the lipid-anchored polymer in the LNPs of the present disclosure is a glycerolipid, which comprises glycerol as a linker and one or more of two lipid moieties described above, e.g., distearoyl-rac-glycerol (DSG).

[0329] In some embodiments, the linker in the lipid-anchored polymer in the LNPs of the present disclosure is a phosphate linker. Thus, in some embodiments, the lipid-anchored polymer in the LNPs of the present disclosure is a phospholipid, which comprises a phosphate group as a linker and one or more lipid moieties as described above.

[0330] In some embodiments, the lipid-anchored polymer in the LNPs of the present disclosure is both a glycerolipid and a phospholipid, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DSPE).

[0331] In some embodiments, the first lipid-anchored polymer is selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielideyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl-2-oleo ...-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (DSPE and a linker lipid moiety (i.e., having one or more hydrophobic tails containing 16 to 22 carbon atoms in a single aliphatic chain) selected from the group consisting of dioctadecylamine (DODA), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), and dioctadecylamine (DODA), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), derivatives thereof, and combinations of any of the foregoing.

[0332] As used herein, the term "derivative," when used in reference to a linker lipid moiety, means a linker lipid moiety that contains one or more of the following modifications: a) if present, the phosphatidylethanolamine (PE) head group has been modified to convert the amino group to a methylamino or dimethylamino group; b) the modified linker lipid moiety contains one or more additional functional groups or moieties, e.g., -OH, -OCH3, -NH2, maleimide, azide, or cyclooctyne, such as dibenzocyclooctyne (DBCO).

[0333] In one embodiment, the first lipid-anchored polymer comprises a linker lipid moiety (i.e., having one or more hydrophobic tails comprising 16 to 22 carbon atoms in a single aliphatic chain) selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, derivatives thereof, and combinations of any of the foregoing.

[0334] In some embodiments, the first lipid-anchored polymer comprises a linker lipid moiety (i.e., having one or more hydrophobic tails comprising 12 to 15 carbon atoms in a single aliphatic chain) selected from the group consisting of 1,2-dimyristoyl-rac-glycero-3-methoxy (DMG), R-3-[(ω-methoxycarbamoyl)]-1,2-dimyristyloxyl-propyl-3-amine, derivatives thereof, and combinations of any of the foregoing. In one embodiment, the first lipid-anchored polymer comprises DMG.

[0335] Polymers in lipid-anchored polymers In some embodiments, the polymer 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), and combinations thereof.

[0336] In one embodiment, the polymer is polyethylene glycol (PEG), hi another embodiment, the polymer is polyglycerol (PG).

[0337] In some embodiments, the polymer in the lipid-anchored polymer has a molecular weight of about 5000 Da or less, e.g., about 4500 Da or less, about 4000 Da or less, about 3500 Da or less, about 3200 Da or less, about 3000 Da or less, about 2500 Da or less, about 2000 Da or less, about 1500 Da or less, about 1000 Da or less, about 500 Da or less, about 100 Da or less, or about 50 Da or less. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 20 Da to about 100 Da, about 50 Da to about 500 Da, about 500 Da to about 2000 Da, about 1000 Da to about 5000 Da, e.g., about 2000 Da to about 5000 Da, about 1000 Da to about 3000 Da, about 1500 Da to about 2500 Da, about 2000 Da to about 4000 Da, or about 2000 Da to about 5000 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 1000 Da, about 1500 Da, about 2000 Da, about 2500 Da, about 3000 Da, about 3200 Da, about 3300 Da, about 3350 Da, about 3400 Da, about 3500 Da, about 4000 Da, about 4500 Da, or about 5000 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 2000 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 2000 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 3200 Da to about 3500 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 3300 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 3350 Da. In some embodiments, the polymers in the lipid-anchored polymer have an average molecular weight of about 3400 Da. In some embodiments, the polymers in the lipid-anchored polymer have an average molecular weight of about 3500 Da.

[0338] Targeting Moiety and Second Lipid-Anchor Polymer In some embodiments, the LNPs of the present disclosure further comprise one or more targeting moieties. The targeting moiety targets the LNP for delivery to a specific cell type or tissue in a subject, such as the liver, bone marrow, spleen, or blood. In some embodiments, the targeting moiety can bind to a specific cell type, such as hepatocytes, T cells, B cells, NK cells, dendritic cells, or the like. In some embodiments, the one or more targeting moieties are conjugated to a second lipid-anchored polymer. In some embodiments, the one or more targeting moieties conjugated to the second lipid-anchored polymer can be an antibody.

[0339] The antibody may be an intact monoclonal or polyclonal antibody, as well as an immunologically active fragment (e.g., Fab or (Fab)2 fragment), an antibody heavy chain, an antibody light chain, a humanized antibody, a genetically engineered single-chain Fv (scFv) molecule, or a chimeric antibody, e.g., an antibody that contains the binding specificity of a murine antibody, but the remainder of which is human in origin. Antibodies, including monoclonal and polyclonal antibodies, fragments, and chimeras, can be prepared using methods known to those skilled in the art. In one embodiment, the targeting moiety is an antibody or antibody fragment, e.g., an antibody or antibody fragment capable of specifically binding to an antigen present on the surface of a cell. In one embodiment, the antibody or antibody fragment is a monoclonal antibody (mAb), a single-chain variable fragment (scFv), a heavy-chain antibody (hcAb), a nanobody (Nb), a heavy-chain-only immunoglobulin (HC1g), an immunoglobulin neoantigen receptor (IgNAR), a variable domain of an immunoglobulin neoantigen receptor (VNAR), a single-domain antibody, or a variable heavy-chain-only antibody (VHH). In one embodiment, the antibody targeting moiety is an scFv. In another embodiment, the antibody targeting moiety is an IgG. In yet another embodiment, the antibody targeting moiety is a VHH (e.g., a nanobody). In some embodiments, the targeting moiety is an antibody directed against an epitope present on a target cell. In some embodiments, the target cell is selected from the group consisting of a T cell, a B cell, a NK cell, a dendritic cell, a hematopoietic cell, a neuronal cell, and a hepatocyte. In some embodiments, the target cell is a T cell. In some embodiments, the antibody targeting moiety binds to an epitope of the T cell receptor (TCR), CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, CD19, CD21, CD28, or PD-1.

[0340] In some embodiments, the LNPs of the present disclosure further comprise one or more targeting moieties capable of binding to specific liver cells, such as hepatocytes. In one embodiment, the targeting moiety is capable of binding to the asialoglycoprotein 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 linked to a lipid-anchored polymer, e.g., as defined herein. In one embodiment, the targeting moiety is a triantennary or trivalent GalNAc conjugate (i.e., GalNAc3), which is a ligand conjugate having three GalNAc molecules or three GalNAc derivatives. In one embodiment, the targeting moiety is a triantennary GalNAc represented by the following structural formula: [ka]

[0341] In one embodiment, the targeting moiety is a tetraantennary GalNAc conjugate. In one embodiment, the targeting moiety is a tetraantennary or tetravalent GalNAc conjugate (i.e., GalNAc4), which is a ligand having four GalNAc molecules or four GalNAc derivatives.

[0342] In one embodiment, the targeting moiety can bind to a low-density lipoprotein receptor (LDLR), e.g., a hepatocyte-specific LDLR. In one embodiment, the targeting moiety comprises an apolipoprotein E (ApoE) protein, an ApoE polypeptide (or peptide), an apolipoprotein B (ApoB) protein, an ApoB polypeptide (or peptide), a fragment of any of the foregoing, or a derivative of any of the foregoing. In one embodiment, the ApoE polypeptide, ApoB polypeptide, or fragment thereof is an ApoE polypeptide, ApoB polypeptide, or fragment thereof disclosed in International Patent Application Publication No. WO 2022 / 261101, the entire contents of which are incorporated herein by reference. In one embodiment, the ApoE protein is a modified ApoE protein, and the ApoB protein is a modified ApoB protein. In one embodiment, the ApoE protein has the following amino acid sequence: MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVR (SEQ ID NO: 1). In one embodiment, the ApoE protein comprises or consists of the amino acid sequence set forth in SEQ ID NO:1.In one embodiment, the ApoE protein has the following amino acid sequence:MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRHHHHHH The ApoE protein has an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:2. In one embodiment, the ApoE protein comprises the amino acid sequence set forth in SEQ ID NO:2. In one embodiment, the ApoE protein consists of the amino acid sequence set forth in SEQ ID NO:2. In one embodiment, the ApoE protein has the following amino acid sequence: It has an amino acid sequence that has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98, or at least about 99% sequence identity to MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVSGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVR (SEQ ID NO: 3). In one embodiment, the ApoE protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3. In one embodiment, the ApoE protein comprises the following amino acid sequence: MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVSGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRHHHHHHGGSSGSGC (SEQ ID NO: 4) The ApoE protein has an amino acid sequence that shares about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the ApoE protein. In one embodiment, the ApoE protein comprises the amino acid sequence set forth in SEQ ID NO:4. In one embodiment, the ApoE protein consists of the amino acid sequence set forth in SEQ ID NO:4.

[0343] As used herein, the term "sequence identity" refers to the ratio of the number of identical amino acids between two aligned sequences over the aligned length, expressed as a percentage. In some embodiments, the two aligned sequences are identical in length, i.e., have the same number of amino acids.

[0344] In one embodiment, the targeting moiety in the LNPs of the present disclosure is an ApoE protein conjugate in an ApoB protein conjugate, e.g., a conjugate of one or more ApoE and / or ApoB protein molecules (native or modified) or fragments thereof covalently linked to a lipid-anchored polymer as defined herein. In one embodiment, the targeting moiety in the LNPs of the present disclosure is an ApoE polypeptide conjugate in an ApoB polypeptide conjugate, e.g., a conjugate of one or more ApoE and / or ApoB polypeptide molecules or fragments thereof covalently linked to a lipid-anchored polymer as defined herein.

[0345] Thus, an important embodiment of the LNPs of the present disclosure is one in which the LNP comprises a second lipid-anchored polymer, and a targeting moiety (including GalNAc, ApoE protein, ApoB protein, ApoE polypeptide, or ApoB polypeptide) as defined herein is conjugated to the second lipid-anchored polymer. The second lipid-anchored polymer is structurally similar to the first lipid-anchored polymer described herein in that the second lipid-anchored polymer also comprises a lipid moiety covalently attached to the polymer via a linker. In one embodiment, the second lipid-anchored polymer is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielideyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl-2- The lipid moiety comprises a linker lipid moiety selected from the group consisting of 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), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), derivatives thereof, and combinations of any of the foregoing. In one embodiment, the second lipid-anchored polymer comprises a linker lipid moiety selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, and derivatives thereof, and combinations of any of the foregoing.

[0346] In one embodiment, the ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide, or fragment thereof is covalently linked to a lipid-anchored polymer (e.g., a second lipid-anchored polymer) or an LNP of the disclosure via strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry, for example, via an azide-modified lipid-anchored polymer (e.g., DSG-PEG2000-azide, DSPE-PEG2000-azide, DSG-PEG3400-azide, DSPE-PEG3400-azide, DSG-PEG5000-azide, DSPE-PEG5000-azide), and a dibenzocyclooctyne (DBCO)-functionalized ApoE protein, ApoB protein, ApoE protein, ApoB protein, or fragment thereof.

[0347] In some embodiments, LNPs of the present disclosure can include a first lipid-anchored polymer and a second lipid-anchored polymer. For example, LNPs of the present disclosure can include a first lipid-anchored polymer that does not include a targeting moiety and a second type of lipid-anchored polymer that includes a targeting moiety, such as GalNAc. For example, LNPs of the present disclosure can include DSG-PEG2000 modified to include an additional OCH3 group (DSG-PEG2000-OMe) as the first lipid-anchored polymer and DSPE-PEG2000-GalNAc3 as the second lipid-anchored polymer.

[0348] In some embodiments, an LNP of the present disclosure can comprise a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the second lipid-anchored polymer comprises a targeting moiety. In some embodiments, the second lipid-anchored polymer comprises a lipid moiety selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, and derivatives thereof. In some embodiments, the first lipid-anchored polymer is any lipid-anchored polymer described above.

[0349] In one embodiment, an LNP of the present disclosure comprises a second lipid-anchored polymer, wherein a targeting moiety (e.g., mAb, IgG, scFv, VHH, GalNAc, ApoE protein or peptide, ApoB protein or peptide) as defined herein is conjugated to the second lipid-anchored polymer. The second lipid-anchored polymer comprises a C -linker covalently attached to the polymer via a linker. 18 -C 22The first lipid-anchored polymer is structurally similar to the first lipid-anchored polymer in that it also contains a lipid moiety comprising a hydrophobic fatty acid tail having a single aliphatic chain backbone of In one embodiment, the second lipid-anchored polymer is selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielideyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl-2-oleoyl-sn -glycero-3-phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 18-1-trans PE, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), dioctadecylamine (DODA), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), derivatives thereof, and combinations of any of the foregoing. In one embodiment, the second lipid-anchored polymer comprises a lipid linker moiety (linker lipid moiety) selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, and derivatives thereof, and combinations of any of the foregoing.

[0350] The lipid-anchored polymer of the present disclosure may also contain a reactive species. In some embodiments, the reactive species is conjugated to the polymer in the lipid-anchored polymer. The reactive species present in the lipid-anchored polymer of the present disclosure can be used, for example, for conjugation to a targeting moiety functionalized with a complementary reactive species, i.e., a reactive species capable of reacting with the reactive species contained in the lipid-anchored polymer of the present disclosure. In some embodiments, the reactive species conjugated to the lipid-anchored polymer of the present disclosure can be a reagent selected from the group consisting of a thiol reagent, a maleimide reagent, or a click chemistry reagent, for example, an alkyne reagent such as a dibenzocyclooctyne (DBCO) reagent, a transcyclooctene (TCO) reagent, a tetrazine (TZ) reagent, and an azide (AZ) reagent.

[0351] In one embodiment, an antibody or fragment thereof, e.g., an IgG, scFv, VHH, is covalently linked to a lipid-anchored polymer (e.g., a second lipid-anchored polymer) via strain-promoted alkyne azide cycloaddition (SPAAC) chemistry, for example, via an azide-modified lipid-anchored polymer (e.g., DSG-PEG2000-azide, DSPE-PEG2000-azide, DSG-PEG3400-azide, DSPE-PEG3400-azide, DSG-PEG5000-azide, DSPE-PEG5000-azide, DODA-PG46-azide), and a dibenzocyclooctyne (DBCO)-functionalized scFv, VHH, IgG, or fragment thereof.

[0352] In an exemplary embodiment, the second lipid-anchored polymer conjugated to the targeting moiety is represented by the following structure: [ka] .

[0353] In another exemplary embodiment, the second lipid-anchored polymer conjugated to the targeting moiety is represented by the following structure: [ka] .

[0354] In one embodiment, the ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide, or fragment thereof is covalently linked to a lipid-anchored polymer (e.g., a second lipid-anchored polymer) via strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry, for example, via an azide-modified lipid-anchored polymer (e.g., DSG-PEG2000-azide, DSPE-PEG2000-azide, DSG-PEG3400-azide, DSPE-PEG3400-azide, DSG-PEG5000-azide, DSPE-PEG5000-azide, DODA-PG-azide), and a dibenzocyclooctyne (DBCO)-functionalized ApoE protein, ApoB protein, ApoE protein, ApoB protein, or fragment thereof.

[0355] In some embodiments, LNPs of the present disclosure can include a first lipid-anchored polymer and a second lipid-anchored polymer. For example, LNPs of the present disclosure can include a first lipid-anchored polymer that does not include a targeting moiety and a second type of lipid-anchored polymer that includes a targeting moiety, such as an scFv, VHH, GalNAc, ApoE protein / peptide, or ApoB protein / peptide. For example, LNPs of the present disclosure can include DSG-PEG2000 modified to include an additional OCH3 group (DSG-PEG2000-OMe) as the first lipid-anchored polymer and DSPE-PEG2000-scFv as the second lipid-anchored polymer.

[0356] In one particular embodiment, the first lipid-anchored polymer is a polymer-conjugated lipid of the present disclosure, e.g., DODA-PG34, DODA-PG45, DODA-PG46, or DODA-PG58. For example, an LNP of the present disclosure may include DODA-PG45 as the first lipid-anchored polymer and DSPE-PEG2000-scFv as the second lipid-anchored polymer.

[0357] In some embodiments, an LNP of the present disclosure can comprise a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the second lipid-anchored polymer comprises a targeting moiety. In some embodiments, the second lipid-anchored polymer comprises a lipid linker moiety (linker lipid moiety) selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, and derivatives thereof. In some embodiments, the first lipid-anchored polymer is any lipid-anchored polymer described above.

[0358] In some embodiments, an LNP of the present disclosure can comprise a first lipid-anchored polymer and a second lipid-anchored polymer, where the second lipid-anchored polymer comprises a targeting moiety, and the first lipid-anchored polymer and the second lipid-anchored polymer have the same lipid linker but different hydrophilic polymers.

[0359] In some embodiments, an LNP of the present disclosure can comprise a first lipid-anchored polymer and a second lipid-anchored polymer, where the second lipid-anchored polymer comprises a targeting moiety, and the first lipid-anchored polymer and the second lipid-anchored polymer differ in their lipid linkers (linker-lipid moieties), as shown below: DSG-PEG (first lipid-anchored polymer) and DSPE-PEG (second lipid-anchored polymer), DSPE-PEG (first lipid-anchored polymer) and DSG-PEG (second lipid-anchored polymer), DODA-PG (first lipid-anchored polymer) and DSPE-PEG (second lipid-anchored polymer), DPG-PEG (first lipid-anchored polymer) and DSPE-PEG (second lipid-anchored polymer), DODA-PG (first lipid-anchored polymer) and DSG-PEG (second lipid-anchored polymer), DPG-PEG (first lipid-anchored polymer) and DSG-PEG (second lipid-anchored polymer), and DPG-PEG (first lipid-anchored polymer) and DODA-PG (second lipid-anchored polymer).

[0360] In some embodiments, an LNP 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, and wherein the first lipid-anchored polymer and the second lipid-anchored polymer are the same lipid-anchored polymer and are selected from one of the following combinations: DSG-PEG (first lipid-anchored polymer) and DSG-PEG (second lipid-anchored polymer), DSPE-PEG (first lipid-anchored polymer) and DSPE-PEG (second lipid-anchored polymer), DODA-PG (first lipid-anchored polymer) and DODA-PG (second lipid-anchored polymer), and DPG-PEG (first lipid-anchored polymer) and DPG-PEG (second lipid-anchored polymer).

[0361] In some embodiments, the targeting moiety is conjugated to a DSPE anchor polymer, hi some embodiments, the DSPE anchor polymer is DSPE-PEG or a derivative thereof.

[0362] In some embodiments, the targeting moiety is conjugated to a DSG anchor polymer, hi some embodiments, the DSG anchor polymer is DSG-PEG or a derivative thereof.

[0363] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DSPE-PEG-IgG. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DSPE-PEG-IgG. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DSPE-PEG-IgG.

[0364] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DSPE-PEG-VHH. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DSPE-PEG-VHH. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DSPE-PEG-VHH.

[0365] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DODA-PG-scFv. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DODA-PG-scFv. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DODA-PG-scFv.

[0366] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DODA-PG-VHH. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, and DODA-PG, and DODA-PG-VHH. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DODA-PG-VHH.

[0367] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, and DODA-PG46 (i.e., a polyglycerol with an average of 46 glycerol repeat units). In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, and bis-DODA-PG46 (e.g., d18:1 / 2:0 or d14:1 / 2:0). In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, and DODA-PG46.

[0368] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, and DODA-PG34 (i.e., a polyglycerol with an average of 34 glycerol units). In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, and DODA-PG34. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, and DODA-PG34.

[0369] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG46, and DODA-PG46-VHH. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG46, and DODA-PG46-VHH. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG46, and DODA-PG46-VHH.

[0370] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG46, and DODA-PG46-scFv. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG46, and DODA-PG46-scFv. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG46, and DODA-PG46-scFv.

[0371] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OMe, and DODA-PG-VHH. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OMe, and DODA-PG-VHH. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OMe, and DODA-PG-VHH.

[0372] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OH, and DODA-PG-VHH. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OH, and DODA-PG-VHH. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OH, and DODA-PG-VHH.

[0373] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, and DSG-PEG2000-OMe. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, and DSG-PEG2000-OMe. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OMe, and DSPE-PEG2000-VHH.

[0374] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, and DSG-PEG2000-OH. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OH, and DSPE-PEG2000-VHH. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OH, and DSPE-PEG2000-VHH.

[0375] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OMe, and DSPE-PEG2000-scFv. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OMe, and DSPE-PEG2000-scFv. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OMe, and DSPE-PEG2000-scFv.

[0376] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OH, and DSPE-PEG2000-scFv. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OH, and DSPE-PEG2000-scFv. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OH, and DSPE-PEG2000-scFv.

[0377] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, bis-DSG-PEG2000, and DSPE-PEG2000-scFv. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, bis-DSG-PEG2000, and DSPE-PEG2000-scFv. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, bis-DSG-PEG2000, and DSPE-PEG2000-scFv.

[0378] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DSPE-PEG-scFv. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG45, and DSPE-PEG2000-scFv. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG45, and DSPE-PEG2000-scFv.

[0379] In some embodiments, the lipid-anchored polymer (the first lipid-anchored polymer and the second lipid-anchored polymer in combination) comprises about 0.1 mol % to about 20 mol % of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 0.5 mol % to about 10 mol % of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 1 mol % to about 10 mol % of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 2 mol % to about 10 mol % of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymer comprises more than about 2 mol % (e.g., 2.1 mol %, 2.2 mol %, 2.3 mol %, 2.4 mol %, 2.5 mol %, 2.6 mol %, 2.7 mol %, 2.8 mol %, 2.9 mol %, 3.0 mol %) to about 10 mol % of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 3 mol % to about 8 mol % of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 3 mol% to about 7 mol% present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 3 mol% to about 5 mol% present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 2 mol% to about 4 mol% present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 2% to about 3% present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 2 mol% present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 2.5 mol% present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 3 mol% present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 3.5 mol% present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 4 mol% present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 5 mol% present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 6 mol% present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 7 mol% present in the LNP.In some embodiments, the lipid-anchored polymer comprises about 8 mol% present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 9 mol% present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 10 mol% present in the LNP.

[0380] In some embodiments, the first lipid-anchored polymer comprises from about 0.1 mol% to about 10 mol%, or from about 0.2 mol% to about 8 mol%, or from about 0.2 mol% to about 7 mol%, or from about 0.2% mol% to about 5 mol%, or from about 0.3 mol% to about 4 mol%, or from about 0.4 mol% to about 4 mol%, or from about 0.5 mol% to about 5 mol%, or from about 0.5 mol% to about 5 mol%, of the total lipid present in the LNP. about 4 mol%, or about 0.5 mol% to about 3.5 mol%, or about 0.5 mol% to about 3 mol%, or about 0.7 mol% to about 5 mol%, or about 0.7 mol% to about 4 mol%, or about 0.7 mol% to about 3.5 mol%, or about 0.7 mol% to about 3 mol%, or about 1 mol% to about 5 mol%, or about 1 mol% to about 4 mol%, or about 1 mol% to about 3.5 mol%, or about 1 mol% to about 3 mol% %, or about 1.5 mol % to about 5 mol %, or about 1.5 mol % to about 4 mol %, or about 1.5 mol % to about 3.5 mol %, or about 1.5 mol % to about 3 mol %, or about 2 mol % to about 5 mol %, or about 2 mol % to about 4 mol %, or about 2 mol % to about 3.5 mol %, or about 2 mol % to about 3 mol %, or about 2.5 mol % to about 5 mol %, or about 2.5 mol % to about 4 mol %, or It is present at about 2.5 mol% to about 3.5 mol%, or about 2.5 mol% to about 3 mol%, or about 3 mol% to about 5 mol%, or about 3 mol% to about 4.5 mol%, or about 3 mol% to about 4 mol%, or about 3 mol% to about 3.5 mol%, or about 3.5 mol% to about 5 mol%, or about 3.5 mol% to about 4.5 mol%, or about 3.5 mol% to about 4 mol%, or about 3 mol% to about 7 mol%.

[0381] In some embodiments, the second lipid-anchored polymer, if present, comprises from about 0.005 mol % to about 5 mol %, or from about 0.005 mol % to about 3 mol %, or from about 0.005 mol % to about 2 mol %, or from about 0.005 mol % to about 1 mol %, or from about 0.005 mol % to about 0.5 mol %, or from about 0.01 mol % to about 3 mol %, or about 0.01 mol % to about 1 mol % of the total lipid present in the LNP. % to about 2 mol%, or about 0.01 mol% to about 1 mol%, or about 0.01 mol% to about 0.5 mol%, or about 0.025 mol% to about 3 mol%, or about 0.025 mol% to about 2 mol%, or about 0.025 mol% to about 1 mol%, or about 0.025 mol% to about 0.5 mol%, or about 0.05 mol% to about 3 mol%, or about 0.05 mol% to about 2 mol%, or about 0.05 mol% % to about 1 mol%, or about 0.05 mol% to about 0.5 mol%, or about 0.01 mol% to about 0.4 mol%, or about 0.01 mol% to about 0.3 mol%, or about 0.01 mol% to about 0.25 mol%, or about 0.01 mol% to about 0.2 mol%, or about 0.01 mol% to about 0.1 mol%, or about 0.025 mol% to about 0.4 mol%, or about 0.025 mol% to about 0.3 mol% In some embodiments, the second lipid-anchored polymer is present at about 0.5 mol %, or from about 0.025 mol % to about 0.25 mol %, or from about 0.025 mol % to about 0.2 mol %, or from about 0.025 mol % to about 0.1 mol %, or from about 0.05 mol % to about 0.4 mol %, or from about 0.05 mol % to about 0.3 mol %, or from about 0.05 mol % to about 0.25 mol %, or from about 0.05 mol % to about 0.2 mol %, or from about 0.05 mol % to about 0.1 mol %.

[0382] In some embodiments, the targeting moiety is conjugated to a DSPE anchor polymer, hi some embodiments, the DSPE anchor polymer is DSPE-PEG or a derivative thereof.

[0383] In some embodiments, the targeting moiety is conjugated to a DSG anchor polymer, hi some embodiments, the DSG anchor polymer is DSG-PEG or a derivative thereof.

[0384] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0), cholesterol, and DSG-PEG2000-OMe. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0), cholesterol, and DSG-PEG2000-OMe. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0), cholesterol, and DSG-PEG2000-OMe.

[0385] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0), cholesterol, and DSG-PEG2000-OH. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0), cholesterol, and DSG-PEG2000-OH. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0), cholesterol, and DSG-PEG2000-OH.

[0386] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0), cholesterol, and bis-DSG-PEG2000-OMe. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0), cholesterol, and bis-DSG-PEG2000-OMe. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0), cholesterol, and bis-DSG-PEG2000-OMe.

[0387] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, C2-ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0), cholesterol, and DODA-PG46 (i.e., a polyglycerol having an average of 46 glycerol repeat units). In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, C2-ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0), cholesterol, and bis-DODA-PG46 (e.g., d18:1 / 2:0 or d14:1 / 2:0). In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, C2-ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0), cholesterol, and DODA-PG46.

[0388] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, C2-ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0), cholesterol, and DODA-PG34 (i.e., a polyglycerol having an average of 34 glycerol units). In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, C2-ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0), cholesterol, and bis-DODA-PG34. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, C2-ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0), cholesterol, and DODA-PG34.

[0389] In some embodiments, the LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, DSPC, cholesterol, and DSG-PEG2000-OMe. In some embodiments, the LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, DSPC, cholesterol, and DSG-PEG2000-OMe. In some embodiments, the LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, DSPC, cholesterol, and DSG-PEG2000-OMe.

[0390] In some embodiments, the LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, DSPC, cholesterol, and DSG-PEG2000-OH. In some embodiments, the LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, DSPC, cholesterol, and DSG-PEG2000-OH. In some embodiments, the LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, DSPC, cholesterol, and DSG-PEG2000-OH.

[0391] In some embodiments, the LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, DOPE, cholesterol, and DSG-PEG2000-OMe. In some embodiments, the LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, DOPE, cholesterol, and DSG-PEG2000-OMe. In some embodiments, the LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, DOPE, cholesterol, and DSG-PEG2000-OMe.

[0392] In some embodiments, the LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, DSPC, cholesterol, and DSPE-PEG2000-OH. In some embodiments, the LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, DSPC, cholesterol, and DSPE-PEG2000-OH. In some embodiments, the LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, DSPC, cholesterol, and DSPE-PEG2000-OH.

[0393] In some embodiments, the LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, DOPE, cholesterol, and DSG-PEG2000-OMe. In some embodiments, the LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, DOPE, cholesterol, and DSG-PEG2000-OMe. In some embodiments, the LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, DOPE, cholesterol, and DSG-PEG2000-OMe.

[0394] In some embodiments, the LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, DOPE, cholesterol, and DSPE-PEG2000-OH. In some embodiments, the LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, DOPE, cholesterol, and DSPE-PEG2000-OH. In some embodiments, the LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, DOPE, cholesterol, and DSPE-PEG2000-OH.

[0395] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, DSPC, cholesterol, DSG-PEG2000-OMe, and DSPE-PEG2000-GalNAc3. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, DSPC, cholesterol, DSG-PEG2000-OMe, and DSPE-PEG2000-GalNAc3. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, DSPC, cholesterol, DSG-PEG2000-OMe, and DSPE-PEG2000-GalNAc3.

[0396] In some embodiments, the LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0), cholesterol, DSG-PEG2000-OH, and DSPE-PEG2000-GalNAc3. In some embodiments, the LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0), cholesterol, DSG-PEG2000-OH, and DSPE-PEG2000-GalNAc3. In some embodiments, the LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNAP), an ionizable lipid, C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0), cholesterol, DSG-PEG2000-OH, and DSPE-PEG2000-GalNAc3.

[0397] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, DSPC, cholesterol, bis-DSG-PEG2000, and DSPE-PEG2000. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, DSPC, cholesterol, bis-DSG-PEG2000, and DSPE-PEG2000. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, DSPC, cholesterol, bis-DSG-PEG2000, and DSPE-PEG2000.

[0398] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, DSPC, cholesterol, DODA-PG46, and DSPE-PEG2000-GalNAc3. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, DSPC, cholesterol, DODA-PG46, and DSPE-PEG2000-GalNAc3. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, DSPC, cholesterol, DODA-PG46, and DSPE-PEG2000-GalNAc3.

[0399] In some embodiments, the lipid-anchored polymer (the first lipid-anchored polymer and the second lipid-anchored polymer in combination) comprises about 0.1 mol % to about 20 mol % of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 0.5 mol % to about 10 mol % of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 1 mol % to about 10 mol % of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 2 mol % to about 10 mol % of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymer comprises more than about 2 mol % (e.g., 2.1 mol %, 2.2 mol %, 2.3 mol %, 2.4 mol %, 2.5 mol %, 2.6 mol %, 2.7 mol %, 2.8 mol %, 2.9 mol %) to about 10 mol % of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 3 mol % to about 8 mol % of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 3 mol % to about 7 mol % of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 3 mol% to about 5 mol% present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 2 mol% to about 4 mol% present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 2 mol% to about 3% present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 2 mol% present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 2.5 mol% present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 3 mol% present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 3.5 mol% present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 4 mol% present in the LNP.

[0400] In some embodiments, the first lipid anchor polymer represents from about 0.1 mol% to about 10 mol%, or from about 0.2 mol% to about 8 mol%, or from about 0.2 mol% to about 7 mol%, or from about 0.2% mol% to about 5 mol%, or from about 0.3 mol% to about 4 mol%, or from about 0.4 mol% to about 4 mol%, or from about 0.5 mol% to about 5 mol%, or from about 0.5 mol% to about 4 mol%, or from about 0.5 mol% to about 3.5 mol%, or from about 0.5 mol% to about 3 mol%, or from about 0.7 mol% to about 5 mol%, or from about 0.7 mol% to about 4 mol%, or from about 0.7 mol% to about 3.5 mol%, or from about 0.7 mol% to about 3 mol%, or from about 1 mol% to about 5 mol%, or from about 1 mol% to about 4 mol%, or from about 1 mol% to about 3.5 mol%, or ... mol% to about 3 mol%, or about 1.5 mol% to about 5 mol%, or about 1.5 mol% to about 4 mol%, or about 1.5 mol% to about 3.5 mol%, or about 1.5 mol% to about 3 mol%, or about 2 mol% to about 5 mol%, or about 2 mol% to about 4 mol%, or about 2 mol% to about 3.5 mol%, or about 2 mol% to about 3 mol%, or about 2.5 mol% to about 5 mol%, or about 2.5 mol% % to about 4 mol%, or about 2.5 mol% to about 3.5 mol%, or about 2.5 mol% to about 3 mol%, or about 3 mol% to about 5 mol%, or about 3 mol% to about 4.5 mol%, or about 3 mol% to about 4 mol%, or about 3 mol% to about 3.5 mol%, or about 3.5 mol% to about 5 mol%, or about 3.5 mol% to about 4.5 mol%, or about 3.5 mol% to about 4 mol%.

[0401] In some embodiments, the second lipid-anchored polymer, if present, comprises from about 0.005 mol % to about 5 mol %, or from about 0.005 mol % to about 3 mol %, or from about 0.005 mol % to about 2 mol %, or from about 0.005 mol % to about 1 mol %, or from about 0.005 mol % to about 0.5 mol %, or from about 0.01 mol % to about 3 mol %, or about 0.01 mol % to about 1 mol % of the total lipid present in the LNP. % to about 2 mol%, or about 0.01 mol% to about 1 mol%, or about 0.01 mol% to about 0.5 mol%, or about 0.025 mol% to about 3 mol%, or about 0.025 mol% to about 2 mol%, or about 0.025 mol% to about 1 mol%, or about 0.025 mol% to about 0.5 mol%, or about 0.05 mol% to about 3 mol%, or about 0.05 mol% to about 2 mol%, or about 0.05 mol% % to about 1 mol%, or about 0.05 mol% to about 0.5 mol%, or about 0.01 mol% to about 0.4 mol%, or about 0.01 mol% to about 0.3 mol%, or about 0.01 mol% to about 0.25 mol%, or about 0.01 mol% to about 0.2 mol%, or about 0.01 mol% to about 0.1 mol%, or about 0.025 mol% to about 0.4 mol%, or about 0.025 mol% to about 0.3 mol% , or from about 0.025 mol % to about 0.25 mol %, or from about 0.025 mol % to about 0.2 mol %, or from about 0.025 mol % to about 0.1 mol %, or from about 0.05 mol % to about 0.4 mol %, or from about 0.05 mol % to about 0.3 mol %, or from about 0.05 mol % to about 0.25 mol %, or from about 0.05 mol % to about 0.2 mol %, or from about 0.05 mol % to about 0.1 mol %.

[0402] Lipid nanoparticles (LNPs) containing ceDNA are disclosed in International Patent Application No. PCT / US2018 / 050042, filed September 7, 2018, which is incorporated herein in its entirety and are contemplated for use in the methods and compositions disclosed herein.

[0403] The size of the LNPs can be determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK). In some embodiments, the LNPs of the present disclosure have an average diameter, as determined by light scattering, of less than about 90 nm, e.g., less than about 80 nm or less than about 75 nm. According to some embodiments, the LNPs of the present disclosure have an average diameter, as determined by light scattering, of between about 50 nm and about 75 nm, or between about 50 nm and about 70 nm.

[0404] The pKa of formulated cationic lipids can correlate with the effectiveness of LNPs for delivery of nucleic acids (see Jayaraman et al., Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al., Nature Biotechnology 28, 172-176 (2010), both of which are incorporated by reference in their entirety). In one embodiment, the pKa of each cationic lipid is determined in lipid nanoparticles using a fluorescence-based assay of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). LNPs in PBS at a concentration of 0.4 mM total lipid can be prepared using the in-line process described herein and elsewhere. TNS can be prepared as a 100 mM stock solution in distilled water. Vesicles can be diluted to 24 mM lipid in 2 mL of buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, with a pH ranging from 2.5 to 11. An aliquot of TNS solution can be added to obtain a final concentration of 1 mM. After vortex mixing, fluorescence intensity is measured at room temperature on an SLM Aminco Series 2 luminescence spectrophotometer using excitation and emission wavelengths of 321 nm and 445 nm. A sigmoidal best-fit analysis can be applied to the fluorescence data, and the pKa is determined when the pH yielded half-maximal fluorescence intensity.

[0405] In one embodiment, relative activity can be determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection. Activity is compared at 0.3 mg and 1.0 mg doses of ceDNA / kg and expressed as ng luciferase / g liver measured 4 hours after administration.

[0406] Without limitation, LNPs of the present disclosure include lipid formulations that can be used to deliver capsid-free, non-viral DNA vectors to a desired target site (e.g., a cell, tissue, organ, specific cell type, etc.) Generally, LNPs comprise a capsid-free, non-viral DNA vector and a cationic lipid or a salt thereof.

[0407] Further exemplary lipid-anchored polymers include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEGylated lipid, such as a (methoxypolyethylene glycol)-conjugated lipid. PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEGylated phosphatidylethanolamine (PEG-PE), PEG diacylglycerol succinate (PEGS-DAG) (such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbamate, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or a mixture thereof.Additional exemplary PEG-lipid conjugates are described, for example, in International Patent Application Publication Nos. 1996 / 010392, 1998 / 051278, 02002 / 087541, 02005 / 026372, 2008 / 147438, 02009 / 086558, 02012 / 000104, Nos. 2017 / 117528, 2017 / 099823, 2015 / 199952, 2017 / 004143, 2015 / 095346, 2012 / 000104, 2012 / 000104, and 2010 / 006282, U.S. Patent Application Publication No. 2003 / 0077829 , 2005 / 0175682, 2008 / 0020058, 2011 / 0117125, 2013 / 0303587, 2018 / 0028664, 2015 / 0376115, 2016 / 0376224, 2016 / 0317458, 2013 / 0303587, 2013 / 0303587, and 20110123453, as well as U.S. Patent Nos. 5,885,613, 6,287,591, 6,320,017, and 6,586,559, the contents of all of which are incorporated herein by reference in their entireties.

[0408] Further examples of PEG-DAA PEGylated lipids include, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. PEG-lipids include PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol(1-[8'-(cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaotanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol) ), PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In one embodiment, the PEG-lipid is one or more of PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], [ka] , [ka] , [ka] , and The structure may be selected from the group consisting of: [ka] .

[0409] Still further exemplary lipid anchor polymers include N-(carbonyl-methoxypolyethylene glycol n)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEGn (wherein n is 350, 500, 750, 1000, or 2000)), N-(carbonyl-methoxypolyethylene glycol) n )-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG n (wherein n is 350, 500, 750, 1000, or 2000), DSPE-polyglycerin-cyclohexyl-carboxylic acid, DSPE-polyglycerin-2-methylglutaric-carboxylic acid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated polyethylene glycol...

Claims

1. A lipid nanoparticle (LNP), a therapeutic nucleic acid (TNA); an ionizable lipid; Sterols and A first lipid-anchored polymer, the first lipid-anchored polymer comprising: i) a polymer; and ii) a lipid moiety comprising at least one hydrophobic tail; iii) optionally a linker connecting the polymer to the lipid moiety; a first lipid-anchored polymer, wherein the at least one hydrophobic tail comprises 12 to 22 carbon atoms in a single aliphatic chain backbone; Formula (I): 【Chemical 95】 Formula (I) (In the formula, 【Chemistry 96】 is a single bond or a double bond, A is hydrogen, 【Chemistry 97-1】 , or 【Chemistry 97-2】 and R １ But C １ -C １７ Alkyl or C ２ -C １７ is alkenyl, R ２ But C １ -C ２２ Alkyl or C ２ -C ２２ is alkenyl, R ３ is hydrogen or C １ -C ２ is alkyl, R ４ is hydrogen or C １ -C ２ and a helper lipid represented by the formula (I), or a salt or ester thereof, wherein I is alkyl.

2. A lipid nanoparticle (LNP), a therapeutic nucleic acid (TNA); an ionizable lipid; Sterols and A first lipid-anchored polymer, the first lipid-anchored polymer comprising: i) a polymer; and ii) a lipid moiety comprising at least two hydrophobic tails; iii) optionally a linker connecting the polymer to the lipid moiety; a first lipid-anchored polymer, wherein the at least two hydrophobic tails each comprise 16 to 22 carbon atoms in a single aliphatic chain backbone; Formula (I): 【Chem.98】 Formula (I) (In the formula, 【Chem.99】 is a single bond or a double bond, A is hydrogen, 【Chemistry 100-1】 , or 【Chemistry 100-2】 and R １ But C １ -C １７ Alkyl or C ２ -C １７ is alkenyl, R ２ But C １ -C ２２ Alkyl or C ２ -C ２２ is alkenyl, R ３ is hydrogen or C １ -C ２ is alkyl, R ４ is hydrogen or C １ -C ２ and a helper lipid represented by the formula (I), or a salt or ester thereof, wherein I is alkyl.

3. A lipid nanoparticle (LNP), a therapeutic nucleic acid (TNA); an ionizable lipid; Sterols and A first lipid-anchored polymer, the first lipid-anchored polymer comprising: i) a polymer; and ii) a lipid moiety comprising at least two hydrophobic tails; iii) optionally a linker connecting the polymer to the lipid moiety; a first lipid-anchored polymer, wherein the at least two hydrophobic tails each comprise 12 to 15 carbon atoms in a single aliphatic chain backbone; Formula (I): 【Chemistry 101】 Formula (I) (In the formula, 【Chemical Engineering 102】 is a single bond or a double bond, A is hydrogen, 【Chemistry 103-1】 , or 【Chemistry 103-2】 and R １ But C １ -C １７ Alkyl or C ２ -C １７ is alkenyl, R ２ But C １ -C ２２ Alkyl or C ２ -C ２２ is alkenyl, R ３ is hydrogen or C １ -C ２ is alkyl, R ４ is hydrogen or C １ -C ２ and a helper lipid represented by the formula (I), or a salt or ester thereof, wherein I is alkyl.

4. A lipid nanoparticle (LNP), a therapeutic nucleic acid (TNA); an ionizable lipid; Sterols and A first lipid-anchored polymer, the first lipid-anchored polymer comprising: i) a polymer; and ii) a lipid moiety comprising a single hydrophobic tail; iii) optionally a linker connecting the polymer to the lipid moiety; a first lipid-anchored polymer, wherein the single hydrophobic tail comprises 12 to 22 carbon atoms in a single aliphatic chain backbone; Formula (I): 【Chemical 104】 Formula (I) (In the formula, 【Chemistry 105】 is a single bond or a double bond, A is hydrogen, 【Chemistry 106-1】 , or 【Chemistry 106-2】 and R １ But C １ -C １７ Alkyl or C ２ -C １７ is alkenyl, R ２ But C １ -C ２２ Alkyl or C ２ -C ２２ is alkenyl, R ３ is hydrogen or C １ -C ２ is alkyl, R ４ is hydrogen or C １ -C ２ and a helper lipid represented by the formula (I) wherein R is an alkyl group, or a salt or ester thereof, or a deuterated analog of any of the foregoing.

5. The helper lipid has the formula (II): 【Chemistry 107】 Formula (II) 5. The lipid nanoparticle (LNP) of any one of claims 1 to 4, wherein the LNP is represented by: or a salt or ester thereof, or a deuterated analogue of any of the foregoing.

6. The helper lipid has the formula (III): 【Chemistry 108】 Formula (III) 5. The lipid nanoparticle (LNP) of any one of claims 1 to 4, wherein the LNP is represented by: or a salt or ester thereof, or a deuterated analogue of any of the foregoing.

7. The helper lipid has the formula (IV): 【Chemistry 109】 Formula (IV) 5. The lipid nanoparticle (LNP) of any one of claims 1 to 4, wherein the LNP is represented by: or a salt or ester thereof, or a deuterated analogue of any of the foregoing.

8. 8. The lipid nanoparticle (LNP) of any one of claims 1 to 7, wherein the LNP does not contain distearoylphosphatidylcholine (DSPC), provided that a helper lipid represented by (I), (II), (III), or (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present.

9. 9. The lipid nanoparticle (LNP) of any one of claims 1 to 8, wherein the LNP does not comprise 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), provided that a helper lipid represented by (I), (II), (III), or (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present.

10. The lipid nanoparticle (LNP) of any one of claims 1 to 9, wherein the LNP does not contain phosphatidylcholine that is not conjugated to a polymer.

11. R １ But C １ -C １０ Alkyl or C ２ -C １０ The lipid nanoparticle (LNP) of any one of claims 1 to 7, which is alkenyl. 【Request Item 12】 【Chemistry 110】 The lipid nanoparticle (LNP) according to any one of claims 1 to 11, wherein is a double bond.

13. R １ But C １ -C ８ Alkyl or C ２ -C ８ The lipid nanoparticle (LNP) of any one of claims 1 to 11, which is alkenyl.

14. R １ But C １ -C ７ Alkyl or C ２ -C ７ The lipid nanoparticle (LNP) of claim 13, which is alkenyl.

15. R １ But C １ Alkyl, C ３ Alkyl, C ５ Alkyl, or C ７ The lipid nanoparticle (LNP) of claim 14, which is alkyl.

16. R １ But C １ The lipid nanoparticle (LNP) of claim 15, which is alkyl.

17. R ２ But C ３ -C １５ Alkyl or C ３ -C １５ A lipid nanoparticle (LNP) according to any one of claims 1 to 16, which is alkenyl.

18. R ２ But C ９ Alkyl, C １１ Alkyl, C １２ Alkyl, C １３ Alkyl, C １４ Alkyl, or C １５ The lipid nanoparticle (LNP) of claim 17, which is alkyl.

19. R ２ But C １２ Alkyl, C １３ Alkyl, or C １４ The lipid nanoparticle (LNP) of claim 18, which is alkyl.

20. R ２ But C １３ The lipid nanoparticle (LNP) of claim 19, which is alkyl.

21. R ３ The lipid nanoparticle (LNP) of any one of claims 1 to 20, wherein is hydrogen.

22. R ３ But C １ The lipid nanoparticle (LNP) of any one of claims 1 to 20, which is alkyl.

23. R ４ The lipid nanoparticle (LNP) of any one of claims 1 to 22, wherein is hydrogen.

24. R ４ But C １ The lipid nanoparticle (LNP) of any one of claims 1 to 22, which is alkyl.

25. The helper lipid represented by formula (I) is selected from any of the helper lipids listed in Table 8, or a salt or ester thereof, or a deuterated analogue of any of the foregoing, or the helper lipid represented by formula (I) is 【Chemistry 111-1】 ； 【Chemistry 111-2】 ； 【Chemistry 111-3】 ； 【Chemistry 111-4】 ； 【Chemistry 111-5】 , and Selected from the structure or 【Chemistry 112】 ； or a salt or ester thereof, or a deuterated analogue of any of the foregoing.

26. The helper lipid represented by formula (I) has the structure: 【Chemistry 113】 ； or a salt or ester thereof, or a deuterated analogue of any of the foregoing.

27. The lipid represented by formula (I) has the structure: 【Chemistry 114】 ； or a salt or ester thereof, or a deuterated analogue of any of the foregoing.

28. A lipid nanoparticle (LNP), a therapeutic nucleic acid (TNA); an ionizable lipid; Sterols and A first lipid-anchored polymer, the first lipid-anchored polymer comprising: i) a polymer; and ii) a lipid moiety comprising at least two hydrophobic tails; iii) optionally a linker connecting the polymer to the lipid moiety; a first lipid-anchored polymer, wherein the at least two hydrophobic tails each comprise 16 to 22 carbon atoms in a single aliphatic chain backbone; a helper lipid selected from distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), The LNP has a whole blood half-life (t １／２ ) lipid nanoparticles (LNPs).

29. The LNP has a whole blood half-life (t) of about 3 hours to about 8 hours, or about 3 hours to about 7.5 hours, or about 3 hours to about 7 hours, or about 3 hours to about 6.5 hours, or about 3 hours to about 6 hours, or about 3 hours to about 9 hours, or about 3 hours to about 10 hours, or about 3 hours to about 11 hours, or about 3 hours to about 12 hours, or about 3 hours to about 13 hours, or about 3 hours to about 14 hours, or about 3 hours to about 15 hours, or about 3 hours to about 16 hours, or about 3 hours to about 24 hours. １／２ 29. The lipid nanoparticle (LNP) of claim 28, having a

30. The LNP has a whole blood half-life (t) of about 3 hours to about 3.5 hours, or about 3 hours to about 4 hours, or about 3 hours to about 4.5 hours, or about 3 hours to about 5 hours, or about 3 hours to about 5.5 hours, or about 3.5 hours to about 4 hours, or about 3.5 hours to about 4.5 hours, or about 3.5 hours to about 5 hours, or about 3.5 hours to about 5.5 hours, or about 4 hours to about 4.5 hours, or about 4 hours to about 5 hours, or about 4 hours to about 5.5 hours, or about 4.5 hours to about 5 hours, or about 4.5 hours to about 5.5 hours, or about 5 hours to about 5.5 hours. １／２ 30. The lipid nanoparticle (LNP) of claim 29, having a

31. The lipid nanoparticle (LNP) of any one of claims 28 to 30, wherein the LNP has a whole blood clearance rate (Cl) of about 10 mL / min / kg to about 50 mL / min / kg, or about 10 mL / min / kg to about 45 mL / min / kg, or about 10 mL / min / kg to about 40 mL / min / kg.

32. 32. The lipid nanoparticle (LNP) of claim 31, wherein the LNP has a whole blood clearance rate (Cl) of about 30 mL / min / kg to about 40 mL / min / kg, or about 35 mL / min / kg to about 40 mL / min / kg, or about 10 mL / min / kg to about 20 mL / min / kg, or about 10 mL / min / kg to about 18 mL / min / kg, or about 10 mL / min / kg to about 15 mL / min / kg.

33. The LNPs have a whole blood end-point exposure (AUC) that is at least 5-fold higher than that of a reference LNP having a C14-15 lipid polymer. ｌａｓｔ 33. The lipid nanoparticle (LNP) of any one of claims 28 to 32, having a

34. The lipid nanoparticle (LNP) of claim 33, wherein the C14-15 lipid polymer is DMA PEG.

35. [0023] The LNPs have a whole blood end time point exposure (AUC) of about 200hr*ng / mL to about 250hr*ng / mL, or about 200hr*ng / mL to about 300hr*ng / mL, or about 500hr*ng / mL to about 700hr*ng / mL, or about 500hr*ng / mL to about 550hr*ng / mL, or about 500hr*ng / mL to about 600hr*ng / mL, or about 550hr*ng / mL to about 600hr*ng / mL, or about 600hr*ng / mL to about 700hr*ng / mL, or about 600hr*ng / mL to about 650hr*ng / mL, or about 650hr*ng / mL to about 700hr*ng / mL. ｌａｓｔ 34. The lipid nanoparticle (LNP) of claim 33, having a

36. The lipid nanoparticle (LNP) of claim 34 or 35, wherein the final time point is 24 hours.

37. The lipid nanoparticle (LNP) of any one of claims 1 to 27, wherein the first lipid-anchored polymer comprises a lipid moiety comprising a single or two hydrophobic tails.

38. The lipid nanoparticle (LNP) of any one of claims 28 to 30, wherein the first lipid-anchored polymer comprises a lipid moiety comprising two hydrophobic tails.

39. 39. The lipid nanoparticle (LNP) of claim 38, wherein each of the two hydrophobic tails is a fatty acid.

40. 40. The lipid nanoparticle (LNP) of any one of claims 27-36, 38, and 39, wherein the two hydrophobic tails each independently contain 16, 17, 18, 19, 20, 21, or 22 carbon atoms.

41. 41. The lipid nanoparticle (LNP) of claim 40, wherein each of the two hydrophobic tails independently contains 16, 17, 18, 19, 20, or 21 carbon atoms.

42. 42. The lipid nanoparticle (LNP) of claim 41, wherein each of the two hydrophobic tails independently contains 16, 17, 18, 19, or 20 carbon atoms.

43. 43. The lipid nanoparticle (LNP) of claim 42, wherein the two hydrophobic tails each independently contain 16, 17, 18, or 19 carbon atoms.

44. 44. The lipid nanoparticle (LNP) of claim 43, wherein the two hydrophobic tails each independently contain 16, 17, or 18 carbon atoms.

45. 45. The lipid nanoparticle (LNP) of claim 44, wherein each of the two hydrophobic tails contains 16 carbon atoms.

46. 45. The lipid nanoparticle (LNP) of claim 44, wherein each of the two hydrophobic tails contains 18 carbon atoms.

47. 43. The lipid nanoparticle (LNP) of claim 42, wherein each of the two hydrophobic tails contains 20 carbon atoms.

48. 48. The lipid nanoparticle (LNP) of any one of claims 38 to 47, wherein the two hydrophobic tails are each independently selected from the group consisting of octadecylamine, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, and derivatives thereof.

49. 39. The lipid nanoparticle (LNP) of claim 38, wherein one of the two hydrophobic tails contains 12, 13, 14, or 15 carbon atoms.

50. 50. The lipid nanoparticle (LNP) of claim 49, wherein one of the two hydrophobic tails contains 12, 13, or 14 carbon atoms.

51. 51. The lipid nanoparticle (LNP) of claim 50, wherein one of the two hydrophobic tails contains 12 carbon atoms.

52. 51. The lipid nanoparticle (LNP) of claim 50, wherein one of the two hydrophobic tails each contains 14 carbon atoms.

53. 53. The lipid nanoparticle (LNP) of any one of claims 49 to 52, wherein one of the two hydrophobic tails is selected from the group consisting of lauric acid, myristic acid, myristoleic acid, and derivatives thereof.

54. 38. The lipid nanoparticle (LNP) of claim 37, wherein the first lipid-anchored polymer comprises a lipid moiety that includes a single hydrophobic tail.

55. 55. The lipid nanoparticle (LNP) of claim 54, wherein the single hydrophobic tail is a fatty acid.

56. 56. The lipid nanoparticle (LNP) of claim 55, wherein the single hydrophobic tail comprises 16, 17, 18, 19, 20, 21, or 22 carbon atoms.

57. 57. The lipid nanoparticle (LNP) of claim 56, wherein the single hydrophobic tail comprises 16 or 18 carbon atoms.

58. 57. The lipid nanoparticle (LNP) of claim 56, wherein the single hydrophobic tail comprises 20 carbon atoms.

59. 57. The lipid nanoparticle (LNP) of claim 56, wherein the single hydrophobic tail comprises 18 carbon atoms.

60. 57. The lipid nanoparticle (LNP) of claim 56, wherein the single hydrophobic tail comprises 16 carbon atoms.

61. 56. The lipid nanoparticle (LNP) of claim 55, wherein the single hydrophobic tail is selected from the group consisting of octadecylamine, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, and derivatives thereof.

62. The lipid nanoparticle (LNP) of any one of claims 1 to 61, wherein the first lipid-anchored polymer is a glycerolipid.

63. The lipid nanoparticle (LNP) of any one of claims 1 to 61, wherein the first lipid-anchored polymer is a phospholipid.

64. The first lipid-anchored polymer may be selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielideyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phospho ...DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielideyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (DEPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (DEPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielideyl-sn-phosphatidylethanolamine 64. The lipid nanoparticle (LNP) of any one of claims 1-48 and 62-63, comprising a linker lipid moiety selected from the group consisting of dioctadecylamine (DODA), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-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), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), derivatives thereof, and combinations of any of the foregoing.

65. 65. The lipid nanoparticle (LNP) of claim 64, wherein the first lipid anchor polymer comprises a linker lipid moiety selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, derivatives thereof, and combinations of any of the foregoing.

66. 69. The lipid nanoparticle (LNP) of any one of claims 1-38, 49-53, 62, and 68, wherein the first lipid anchor polymer comprises a linker lipid moiety selected from the group consisting of 1,2-dimyristoyl-rac-glycero-3-methoxy (DMG), R-3-[(ω-methoxycarbamoyl)]-1,2-dimyristyloxyl-propyl-3-amine, derivatives thereof, and combinations of any of the foregoing.

67. 67. The lipid nanoparticle (LNP) of claim 66, wherein the first lipid-anchored polymer comprises DMG.

68. 68. The lipid nanoparticle (LNP) of any one of claims 1 to 67, wherein the 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.

69. 69. The lipid nanoparticle (LNP) of claim 68, wherein the polymer is selected from the group consisting of polyethylene glycol (PEG), polyglycerol (PG), polysarcosine (pSar), and combinations thereof.

70. The lipid nanoparticle (LNP) of any one of claims 1 to 69, wherein the polymer has an average molecular weight of about 1000 Da to about 5000 Da.

71. The lipid nanoparticle (LNP) of claim 70, wherein the polymer has an average molecular weight of about 2000 Da to about 5000 Da.

72. 72. The lipid nanoparticle (LNP) of claim 71, wherein the polymer has an average molecular weight of about 2000 Da.

73. The lipid nanoparticle (LNP) of claim 71, wherein the polymer has an average molecular weight of about 3200 Da to about 3500 Da.

74. The lipid nanoparticle (LNP) of any one of claims 69 to 73, wherein the polymer is polyethylene glycol (PEG).

75. 75. The lipid nanoparticle (LNP) of any one of claims 1 to 74, wherein the sterol is selected from the group consisting of cholesterol, beta-sitosterol, stigmasterol, beta-sitostanol, campesterol, brassicasterol, and derivatives thereof, and combinations thereof.

76. 76. The lipid nanoparticle (LNP) of claim 75, wherein the sterol is cholesterol.

77. The lipid nanoparticle (LNP) of claim 75, wherein the sterol is beta-sitosterol.

78. The ionizable lipid is a) Formula (A): 【Chemical 115】 Formula (A) (In the formula, R １ and R １’ each independently represents an optionally substituted linear or branched C １－３ is alkylene, R ２ and R ２’ each independently represents an optionally substituted linear or branched C １－６ is alkylene, R ３ and R ３’ each independently represents an optionally substituted linear or branched C １－６ Is it alkyl? Or, R ２ optionally substituted branched C １－６ When R is alkylene, ２ and R ３ together with the intervening N atom form a 4- to 8-membered heterocyclyl; or Or, R ２’ optionally substituted branched C １－６ When R is alkylene, ２’ and R ３’ together with the intervening N atom form a 4- to 8-membered heterocyclyl; R ４ and R ４’ each independently represents -CR ａ , -C(R ａ ) ２ CR ａ , or -[C(R ａ ) ２ ] ２ CR ａ and R ａ is, for each occurrence, independently H or C １－３ Is it alkyl? Or, R ４ -C(R ａ ) ２ CR ａ or −[C(R ａ ) ２ ] ２ CR ａ and R ａ is C １－３ When R is alkyl, ３ and R ４ together with the intervening N atom form a 4- to 8-membered heterocyclyl; or Or, R ４’ -C(R ａ ) ２ CR ａ or −[C(R ａ ) ２ ] ２ CR ａ and R ａ is C １－３ When R is alkyl, ３’ and R ４’ together with the intervening N atom form a 4- to 8-membered heterocyclyl; R ５ and R ５’ are each independently hydrogen, C １－２０ Alkylene or C ２－２０ is alkenylene, R ６ and R ６’ But for each occurrence, independently, C １－２０ Alkylene, C ３－２０ Cycloalkylene, or C ２－２０ is alkenylene, m and n are each independently an integer selected from 1, 2, 3, 4, and 5, or a pharmaceutically acceptable salt thereof; b) Formula (B): 【Chemistry 116】 Formula (B) (In the formula, a is an integer ranging from 1 to 20; b is an integer ranging from 2 to 10; R １ is absent, or (C ２ -C ２０ ) alkenyl, —C(O)O(C ２ -C ２０ ) alkyl, and (C ２ -C ２０ ) cyclopropyl substituted with alkyl; R ２ However, (C ２ -C ２０ ) alkyl), or a pharmaceutically acceptable salt thereof; or c) Formula (C): 【Chemistry 117】 Formula (C) (In the formula, R １ and R １’ are each independently R ａ (C １ -C ６ ) alkylene; R ２ and R ２’ are each independently １ -C ２ ) alkylene; R ３ and R ３’ are each independently R ｂ (C １ -C ６ ) alkyl or Or, R ２ and R ３ and / or R ２’ and R ３’ together with the intervening N atom form a 4- to 7-membered heterocyclyl; R ４ and R ４ ' are each interrupted by -C(O)O- (C ２ -C ６ ) alkylene; R ５ and R ５ Each ' is independently -C(O)O- or (C ３ -C ６ ) each optionally interrupted by cycloalkyl, (C ２ -C ３０ ) alkyl or (C ２ -C ３０ ) alkenyl, R ａ and R ｂ wherein each is halo or cyano, or a pharmaceutically acceptable salt thereof; d) Formula (D): 【Chemistry 118】 Formula (D) (In the formula, R' is absent, hydrogen, or C １ -C ６ alkyl, provided that R' is hydrogen or C １ -C ６ When R′, R １ , and R ２ are all positively charged nitrogen atoms, R １ and R ２ are each independently hydrogen, C １ -C ６ Alkyl, or C ２ -C ６ is alkenyl, R ３ But C １ -C １２ Alkylene or C ２ -C １２ is alkenylene, R ４ But C １ -C １８ Unbranched alkyl, C ２ -C １８ unbranched alkenyl, or 【Chemical 119】 wherein: R ４ａ and R ４ｂ are each independently C １ -C １６ Unbranched alkyl or C ２ -C １６ is an unbranched alkenyl; R ５ But, absent, C １ -C ８ Alkylene, or C ２ -C ８ is alkenylene, R ６ａ and R ６ｂ are each independently C ７ -C １６ Alkyl or C ７ -C １６ alkenyl, provided that R ６ａ and R ６ｂ the total number of carbon atoms in the X １ and X ２ each independently represents -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-、-S-S-、-C(R ａ )=N-、-N=C(R ａ )-、-C(R ａ )=NO-、-O-N=C(R ａ )-、-C(=O)NR ａ -、 -NR ａ C(=O)-, -NR ａ C(=O)NR ａ -, -OC(=O)O-, -OSi(R ａ ) ２ O-, -C(=O)(CR ａ ２ ) C(=O)O-, or OC(=O)(CR ａ ２ )C(=O)-, wherein R ａ is, at each occurrence, independently hydrogen or C １ -C ６ is alkyl, and n is an integer selected from 1, 2, 3, 4, 5, and 6, or a pharmaceutically acceptable salt thereof. e) Formula (E): 【Chemical 120】 Formula (E) (In the formula, R' is absent, hydrogen, or C １ -C ３ alkyl, provided that R' is hydrogen or C １ -C ３ When R′, R １ , and R ２ provided that all nitrogen atoms to which they are attached are positively charged, R １ and R ２ are each independently hydrogen or C １ -C ３ is alkyl, R ３ But C ３ -C １０ Alkylene or C ３ -C １０ is alkenylene, R ４ But C １ -C １６ Unbranched alkyl, C ２ -C １６ unbranched alkenyl, or 【Chemistry 121】 wherein: R ４ａ and R ４ｂ are each independently C １ -C １６ Unbranched alkyl or C ２ -C １６ is an unbranched alkenyl; R ５ But, absent, C １ -C ６ Alkylene, or C ２ -C ６ is alkenylene, R ６ａ and R ６ｂ are each independently C ７ -C １４ Alkyl or C ７ -C １４ is alkenyl, Xが、-OC(=O)-、-SC(=O)-、-OC(=S)-、-C(=O)O-、-C(=O)S-、-S-S-、-C(R ａ )=N-、 -N=C(R ａ )-、-C(R ａ )=NO-、-O-N=C(R ａ )-、-C(=O)NR ａ -、-NR ａ C(=O)-、-NR ａ C(=O)NR ａ -、 -OC(=O)O-, -OSi(R ａ ) ２ O-, -C(=O)(CR ａ ２ ) C(=O)O-, or OC(=O)(CR ａ ２ )C(=O)-, wherein R ａ is, at each occurrence, independently hydrogen or C １ -C ６ is alkyl, n is an integer selected from 1, 2, 3, 4, 5, and 6, or a pharmaceutically acceptable salt thereof; f) A lipid nanoparticle (LNP) described in any one of claims 1 to 77, wherein the lipid is an ionizable lipid selected from any of the ionizable lipids of Table 1, Table 4, Table 5, Table 6, or Table 7.

79. The lipid nanoparticle (LNP) of any one of claims 1 to 78, wherein the LNP further comprises a targeting moiety.

80. 80. The lipid nanoparticle (LNP) of claim 79, wherein the LNP comprises a second lipid-anchored polymer, and the targeting moiety is conjugated to the second lipid-anchored polymer.

81. The second lipid-anchored polymer may be selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielideyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl-2-oleoyl-sn-glycero- 81. The lipid nanoparticle (LNP) of claim 80, comprising a linker lipid moiety selected from the group consisting of 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), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), derivatives thereof, and combinations of any of the foregoing.

82. 82. The lipid nanoparticle (LNP) of claim 81, wherein the second lipid anchor polymer comprises a linker lipid moiety selected from the group consisting of DSPE, DSG, DODA, DPG, derivatives thereof, and combinations of any of the foregoing.

83. The first lipid-anchored polymer and the second lipid-anchored polymer are different lipid-anchored polymers, and the linker lipids of the first lipid-anchored polymer and the second lipid-anchored polymer are selected from the following combinations: DSG (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DSPE (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DODA (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DPG (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DMG (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DODA (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DPG (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DMG (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DPG (the first lipid-anchored polymer) and DODA (the second lipid-anchored polymer); DMG (the first lipid-anchored polymer) and DODA (the second lipid-anchored polymer), or 83. The lipid nanoparticle (LNP) of claim 82, comprising one of DMG (the first lipid-anchored polymer) and DPG (the second lipid-anchored polymer).

84. The first lipid-anchored polymer and the second lipid-anchored polymer are the same lipid-anchored polymer, and the linker lipids of the first lipid-anchored polymer and the second lipid-anchored polymer are selected from the following combinations: DSG (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer); DSPE (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer); DODA (the first lipid-anchored polymer) and DODA (the second lipid-anchored polymer), or 83. The lipid nanoparticle (LNP) of claim 82, comprising one of DPG (the first lipid-anchored polymer) and DPG (the second lipid-anchored polymer).

85. 81. The lipid nanoparticle (LNP) of claim 80, wherein the targeting moiety is conjugated to a DSPE anchor polymer.

86. The lipid nanoparticle (LNP) of claim 85, wherein the DSPE anchor polymer is DSPE-PEG or a derivative thereof.

87. 81. The lipid nanoparticle (LNP) of claim 80, wherein the targeting moiety is conjugated to a DSG anchor polymer.

88. The lipid nanoparticle (LNP) of claim 87, wherein the DSG anchor polymer is DSG-PEG or a derivative thereof.

89. The lipid nanoparticle (LNP) of claim 80, wherein the targeting moiety is capable of binding to liver cells.

90. The lipid nanoparticle (LNP) of claim 89, wherein the liver cell is a hepatocyte.

91. 81. The lipid nanoparticle (LNP) of claim 80, wherein the targeting moiety is N-acetylgalactosamine (GalNAc) or a GalNAc derivative.

92. The lipid nanoparticle (LNP) of claim 91, wherein the targeting moiety is a triantennary GalNAc conjugate or a tetraantennary GalNAc conjugate.

93. 81. The lipid nanoparticle (LNP) of claim 80, wherein the targeting moiety is selected from the group consisting of an ApoE protein, an ApoE polypeptide, an ApoB protein, an ApoB polypeptide, a fragment thereof, and a derivative of any of the foregoing.

94. 94. The lipid nanoparticle (LNP) of claim 93, wherein the targeting moiety is selected from the group consisting of an ApoE protein conjugate, an ApoE peptide conjugate, an ApoB protein conjugate, and an ApoB peptide conjugate.

95. The lipid nanoparticle (LNP) of claim 94, wherein the targeting moiety is an ApoE protein conjugate.

96. The ionizable lipid is ionizable lipid 81: 【Chemistry 122】 or a pharmaceutically acceptable salt thereof.

97. The ionizable lipid is ionizable lipid 89: 【Chemical 123】 or a pharmaceutically acceptable salt thereof.

98. The ionizable lipid is ionizable lipid 87: 【Chemistry 124】 or a pharmaceutically acceptable salt thereof.

99. A lipid nanoparticle (LNP) described in any one of claims 1 to 98, wherein the ionizable lipid is present in the LNP in an amount of about 30 mol% to about 60 mol% of the total lipid present in the LNP.

100. A lipid nanoparticle (LNP) described in any one of claims 1 to 99, 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.

101. A lipid nanoparticle (LNP) described in any one of claims 1 to 100, wherein the sterol is present in the LNP in an amount of about 20 mol% to about 45 mol% of the total lipids present in the LNP.

102. The lipid nanoparticle (LNP) of claim 101, wherein the sterol is present in the LNP in an amount of about 30 mol% to about 40 mol% of the total lipids present in the LNP.

103. A lipid nanoparticle (LNP) according to any one of claims 1 to 102, wherein the first lipid-anchored polymer is present in the LNP in an amount of about 2 mol% to about 5 mol% of the total lipid present in the LNP.

104. The lipid nanoparticle (LNP) of any one of claims 80 to 102, wherein the second lipid-anchored polymer is present in the LNP in an amount of from about 0.005 mol% to about 5 mol% of the total lipid present in the LNP.

105. The lipid nanoparticle (LNP) of claim 104, wherein the second lipid-anchored polymer is present in the LNP in an amount of about 0.05 mol% to about 2 mol% of the total lipid present in the LNP.

106. The lipid nanoparticle (LNP) of claim 105, wherein the second lipid-anchored polymer is present in the LNP in an amount of about 0.1 mol% to about 1 mol% of the total lipid present in the LNP.

107. The lipid nanoparticle (LNP) of claim 106, wherein the second lipid anchor polymer is present in the LNP in an amount of about 0.5 mol% of the total lipid present in the LNP.

108. The lipid nanoparticle (LNP) of any one of claims 103 to 107, wherein the first lipid-anchored polymer and the second lipid-anchored polymer are present in the LNP in amounts of about 2.5 mol% and 0.5 mol%, respectively, of the total lipid present in the LNP.

109. The lipid nanoparticle (LNP) of any one of claims 1 to 27 and 37 to 108, wherein the helper lipid represented by formula (I), formula (II), formula (III), or formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present in the LNP in an amount of about 2 mol% to about 40 mol% of the total lipid present in the LNP.

110. The lipid nanoparticle (LNP) of claim 109, wherein the helper lipid represented by formula (I), formula (II), formula (III), or formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present in the LNP in an amount of about 5 mol% to about 30 mol% of the total lipid present in the LNP.

111. The lipid nanoparticle (LNP) of claim 110, wherein the helper lipid represented by formula (I), formula (II), formula (III), or formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present in the LNP in an amount of about 10 mol% to about 20 mol% of the total lipid present in the LNP.

112. The lipid nanoparticle (LNP) of claim 111, wherein the helper lipid represented by formula (I), formula (II), formula (III), or formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present in the LNP in an amount of about 10 mol% of the total lipid present in the LNP.

113. The lipid nanoparticle (LNP) of claim 111, wherein the helper lipid represented by formula (I), formula (II), formula (III), or formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present in the LNP in an amount of about 15 mol% of the total lipid present in the LNP.

114. The lipid nanoparticle (LNP) of claim 111, wherein the helper lipid represented by formula (I), formula (II), formula (III), or formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, is present in the LNP in an amount of about 20 mol% of the total lipid present in the LNP.

115. The lipid nanoparticle (LNP) of any one of claims 1 to 114, wherein the helper lipid is present in the LNP in an amount of about 2 mol% to about 40 mol%, or about 5 mol% to about 35 mol%, or about 5 mol% to about 30 mol%, or about 5 mol% to about 25 mol%, or about 5 mol% to about 20 mol%, or about 5 mol% to about 15 mol%, or about 5 mol% to about 10 mol%, or about 10 mol% to about 15 mol% of the total lipid present in the LNP.

116. A lipid nanoparticle (LNP) according to any one of claims 1 to 115, wherein the LNP is suitable for intravenous administration.

117. The lipid nanoparticle (LNP) of claim 116, wherein the LNP is less immunogenic than a reference LNP, wherein the reference LNP either (i) does not comprise the helper lipid represented by Formula (I), Formula (II), Formula (III), or Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, or (ii) comprises a helper lipid selected from the group consisting of distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and a reference lipid polymer comprising at least two hydrophobic tails, each containing 12 to 15 carbon atoms in a single aliphatic chain backbone.

118. The lipid nanoparticle (LNP) of claim 117, wherein the reference lipid polymer is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG).

119. The lipid nanoparticle (LNP) of claim 116 or 118, wherein the LNP results in an expression level of TNA in target cells that is equivalent to or higher than that of the reference LNP.

120. The lipid nanoparticle (LNP) of any one of claims 116 to 119, wherein the LNP induces a lower pro-inflammatory cytokine response than the reference LNP.

121. The lipid nanoparticle (LNP) of claim 119 or 120, wherein the LNP results in lower uptake of the TNA by blood cells than the reference LNP.

122. The lipid nanoparticle (LNP) of claim 121, wherein the blood cells are red blood cells.

123. 123. The lipid nanoparticle (LNP) of any one of claims 1 to 122, wherein the therapeutic nucleic acid (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 closed-end DNA (ceDNA), a single-stranded DNA (ssDNA), a ministring, doggybone™, a protelomeric closed-end DNA, a dumbbell-shaped linear DNA, a dicer substrate dsRNA, a small hairpin RNA (shRNA), an asymmetric interfering RNA (aiRNA), an mRNA, a tRNA, a rRNA, a gRNA, a DNA viral vector, a viral RNA vector, a non-viral vector, and any combination thereof.

124. The lipid nanoparticle (LNP) of any one of claims 1 to 123, wherein the TNA is greater than about 200 bp in length or greater than about 200 nt in length.

125. The lipid nanoparticle (LNP) of claim 124, wherein the TNA is greater than about 500 bp in length or greater than about 500 nt in length.

126. The lipid nanoparticle (LNP) of claim 125, wherein the TNA is greater than about 1000 bp in length or greater than about 1000 nt in length.

127. The lipid nanoparticle (LNP) of claim 126, wherein the TNA is greater than about 4000 bp in length or greater than about 4000 nt in length.

128. A lipid nanoparticle (LNP) according to any one of claims 1 to 127, wherein the TNA is closed-end DNA (ceDNA).

129. A lipid nanoparticle (LNP) according to any one of claims 1 to 127, wherein the TNA is messenger RNA (mRNA).

130. A lipid nanoparticle (LNP) according to any one of claims 1 to 129, wherein the TNA is a single-stranded nucleic acid.

131. A lipid nanoparticle (LNP) described in any one of claims 1 to 129, wherein the TNA is a double-stranded nucleic acid.

132. A pharmaceutical composition comprising a lipid nanoparticle (LNP) according to any one of claims 1 to 131 and a pharmaceutically acceptable carrier.

133. 132. A method for producing a lipid nanoparticle (LNP) according to any one of claims 1 to 131, comprising: the therapeutic nucleic acid (TNA); the ionizable lipid; The sterol; the first lipid-anchored polymer; and a helper lipid represented by formula (I), formula (II), formula (III), or formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing, or a helper lipid selected from the group consisting of distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); Optionally, the second lipid-anchored polymer; and Optionally, combining said targeting moiety with

134. 132. A method of treating a genetic disorder in a subject, said method comprising administering to said subject an effective amount of a lipid nanoparticle (LNP) of any one of claims 1 to 131 or a pharmaceutical composition of claim 132.

135. 135. The method of claim 134, wherein the subject is a human.

136. The genetic disorder is sickle cell anemia, melanoma, hemophilia A (clotting factor VIII (FVIII) deficiency), hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited liver metabolic disorders, Lesch-Nyhan syndrome, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa , ataxia-telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharidosis, Niemann-Pick disease, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandhoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis (ML), sialidosis type II, glycogen storage disease (GSD), Gaucher disease, cystinosis, Batten disease, aspartylglucosaminuria, Salla disease, Danon disease (L AMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (NCL), sphingolipidosis, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy (SMA), Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa ( 136. The method of claim 134 or 135, wherein the inflammatory bowel disease is selected from the group consisting of: ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber's congenital amaurosis, Stargardt's disease, wet macular degeneration (wet AMD), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC), and cathepsin A deficiency.

137. 137. The method of claim 136, wherein the genetic disorder is phenylketonuria (PKU).

138. 137. The method of claim 136, wherein the genetic disorder is hemophilia A (factor VIII deficiency).

139. 137. The method of claim 136, wherein the genetic disorder is Wilson's disease.

140. 137. The method of claim 136, wherein the genetic disorder is Gaucher disease.

141. 137. The method of claim 136, wherein the genetic disorder is Gaucher disease type I, Gaucher disease type II, or Gaucher disease type III.

142. 137. The method of claim 136, wherein the genetic disorder is Leber congenital amaurosis (LCA).

143. 137. The method of claim 136, wherein the LCA is LCA10.

144. 137. The method of claim 136, wherein the genetic disorder is Stargardt's disease.

145. 137. The method of claim 136, wherein the genetic disorder is wet macular degeneration (wet AMD).

146. A therapeutic method for providing anti-tumor immunity to a subject, the method comprising administering to the subject an effective amount of the LNP of any one of claims 1 to 131 or the pharmaceutical composition of claim 132.

147. 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 1 to 131 or the pharmaceutical composition of claim 132.

148. 148. The method of claim 146 or 147, wherein the TNA is retained in the spleen for at least about 6 hours, or at least about 9 hours, or at least about 12 hours, or at least about 15 hours, or at least about 18 hours, or at least about 21 hours, or at least about 24 hours, or at least about 27 hours, or at least about 30 hours, or at least about 33 hours, or at least about 36 hours after administration.

149. 149. The method of claim 148, wherein the amount of TNA at the beginning of a 12, 18, or 24 hour time frame after administration and the amount of TNA at the end of the time frame are within the same order of magnitude.

150. 132. A method of treating a blood disease, disorder, or condition in a subject, said method comprising administering to said subject an effective amount of the LNP of any one of claims 1 to 131 or the pharmaceutical composition of claim 132.

151. The blood disease, disorder, or condition is selected from the group consisting of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), Hodgkin's lymphoma (HL), multiple myeloma, myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), adrenoleukodystrophy (ALD), Hurler syndrome, Krabbe disease (globoid cell leukodystrophy or GLD), metachromatic leukodystrophy ( 151. The method of claim 150, wherein the erythrocyte proliferation / degeneration syndrome (PDS) is selected from the group consisting of myeloproliferative disorders (MYCs), ...

152. 152. The method of any one of claims 146 to 151, wherein the TNA is messenger RNA (mRNA).

153. 152. The method of any one of claims 146 to 151, wherein the TNA is single-stranded DNA (ssDNA).

154. A lipid nanoparticle (LNP), a therapeutic nucleic acid (TNA); an ionizable lipid; Helper lipids and Sterols and A lipid-anchored polymer, comprising: i) a polymer, ii) a lipid moiety comprising at least one hydrophobic tail; the polymer is linked to the lipid moiety; a lipid-anchored polymer, wherein the at least one hydrophobic tail comprises 18 to 22 carbon atoms in a single aliphatic chain backbone; the sterol is present in the LNP in an amount of about 30 mol% to about 40 mol% of the total lipid present in the LNP; the lipid-anchored polymer is present in the LNP in an amount of about 2 mol % to about 7 mol % of the total lipid present in the LNP; Lipid nanoparticles (LNPs), wherein the LNPs have an average particle size of 50 to 100 nm in diameter.

155. The lipid nanoparticle (LNP) of claim 154, wherein the LNP has an average particle size of 60 to 80 nm in diameter.

156. The lipid nanoparticle (LNP) of claim 155, wherein the LNP further comprises a second lipid-anchored polymer.

157. The lipid nanoparticle (LNP) of claim 156, wherein the second lipid-anchored polymer comprises a conjugation reactive moiety.

158. The lipid nanoparticle (LNP) of claim 156, wherein the second lipid-anchored polymer comprises a targeting moiety.

159. The lipid nanoparticle (LNP) of claim 158, wherein the targeting moiety is selected from the group consisting of IgG, Fab, VHH, scFv, peptide ligands, and sugar ligands.

160. The lipid nanoparticle (LNP) of claim 154, wherein the lipid-anchored polymer is present in the LNP in an amount of about 2 mol% to about 5 mol% of the total lipid present in the LNP.

161. A lipid nanoparticle (LNP), a therapeutic nucleic acid (TNA); an ionizable lipid; Helper lipids and Sterols and A first lipid-anchored polymer, the first lipid-anchored polymer comprising: i) a polymer; and ii) a lipid moiety comprising at least one hydrophobic tail; a first lipid-anchored polymer, wherein the polymer is linked to the lipid moiety; and A second lipid-anchored polymer, the second lipid-anchored polymer comprising: i) a polymer; and ii) a reactive moiety for conjugation to a targeting moiety; and iii) a lipid moiety comprising at least one hydrophobic tail; a second lipid-anchored polymer, wherein the polymer is linked to the lipid moiety; the first lipid-anchored polymer comprises at least one hydrophobic tail comprising 18 to 22 carbon atoms in a single aliphatic chain backbone; the second lipid-anchored polymer comprises at least one hydrophobic tail comprising 18 to 22 carbon atoms in a single aliphatic chain backbone; the sterol is present in the LNP in an amount of about 30 mol% to about 40 mol% of the total lipid present in the LNP; the first lipid-anchored polymer is present in the LNP in an amount of about 2 mol % to about 7 mol % of the total lipid present in the LNP; the second lipid-anchored polymer is present in the LNP in an amount of about 0.2 mol % to about 2 mol % of the total lipid present in the LNP; Lipid nanoparticles (LNPs), wherein the LNPs have an average particle size of 50 to 100 nm in diameter.

162. A lipid nanoparticle (LNP), an ionizable lipid; Helper lipids and Sterols and A first lipid-anchored polymer, the first lipid-anchored polymer comprising: i) a polymer; and ii) a lipid moiety comprising at least one hydrophobic tail; a first lipid-anchored polymer, wherein the polymer is linked to the lipid moiety; and A second lipid-anchored polymer, the second lipid-anchored polymer comprising: i) a polymer; and ii) a reactive moiety for conjugation to a targeting moiety; and iii) a lipid moiety comprising at least one hydrophobic tail; a second lipid-anchored polymer, wherein the polymer is linked to the lipid moiety; the first lipid-anchored polymer comprises at least one hydrophobic tail comprising 18 to 22 carbon atoms in a single aliphatic chain backbone; the second lipid-anchored polymer comprises at least one hydrophobic tail comprising 18 to 22 carbon atoms in a single aliphatic chain backbone; the sterol is present in the LNP in an amount of about 30 mol% to about 40 mol% of the total lipid present in the LNP; the first lipid-anchored polymer is present in the LNP in an amount of about 2 mol % to about 7 mol % of the total lipid present in the LNP; the second lipid-anchored polymer is present in the LNP in an amount of about 0.2 mol % to about 2 mol % of the total lipid present in the LNP; Lipid nanoparticles (LNPs), wherein the LNPs have an average particle size of 50 to 100 nm in diameter.

163. A lipid nanoparticle (LNP), a therapeutic nucleic acid (TNA); an ionizable lipid; Helper lipids and Sterols and A first lipid-anchored polymer, the first lipid-anchored polymer comprising: i) a polymer; and ii) a lipid moiety comprising at least one hydrophobic tail; a first lipid-anchored polymer, wherein the polymer is linked to the lipid moiety; and A second lipid-anchored polymer, the second lipid-anchored polymer comprising: i) a polymer; and ii) a lipid moiety comprising at least one hydrophobic tail; iii) optionally a reactive moiety or a targeting moiety for conjugation to a targeting moiety; a second lipid-anchored polymer, wherein the polymer is linked to the lipid moiety; the first lipid-anchored polymer comprises at least one hydrophobic tail comprising 18 to 22 carbon atoms in a single aliphatic chain backbone; the second lipid-anchored polymer comprises at least one hydrophobic tail comprising 18 to 22 carbon atoms in a single aliphatic chain backbone; the sterol is present in the LNP in an amount of about 30 mol% to about 40 mol% of the total lipid present in the LNP; the first lipid-anchored polymer is present in the LNP in an amount of about 2 mol % to about 7 mol % of the total lipid present in the LNP; the second lipid-anchored polymer is present in the LNP in an amount of about 0.2 mol % to about 2 mol % of the total lipid present in the LNP; Lipid nanoparticles (LNPs), wherein the LNPs have an average particle size of 50 to 100 nm in diameter.

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