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

Abstract

Provided herein are lipid nanoparticle (LNP) compositions (e.g., pharmaceutical compositions) comprising a therapeutic nucleic acid (TNA) wherein the LNP comprises an ionizable lipid; a "helper" lipid, such as ceramide or distearoyl phosphatidylcholine (DSPC); a structural lipid, e.g., a sterol; and one or more types of lipid anchoring polymers; and uses thereof.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 429,267, filed December 1, 2022, U.S. Provisional Application No. 63 / 449,617, filed March 3, 2023, U.S. Provisional Application No. 63 / 452,077, filed March 14, 2023, and U.S. Provisional 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 Art

[0003] Lipid-based nanoparticles have been used in COVID-19 vaccines and many other nanomedicines (e.g. and ) and is therefore considered a leader in nanoscale drug delivery systems. However, the effective targeted delivery of bioactive substances (such as therapeutic nucleic acids) is an ongoing medical challenge. This severely limits the widespread application of nucleic acids (such as mRNA and DNA) in non-viral gene replacement therapy, gene therapy, gene editing, and vaccination.

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

[0005] In addition, the viral delivery of nucleic acid therapeutics to targeted cells is greatly hindered by the activation of innate and / or adaptive immune responses. Although RNA sensing of myeloid dendritic cells (MDCs) can be avoided by chemically modifying RNA goods (e.g., with 1mΨ, 2'OMe, etc.), there is currently no known chemical modification of DNA goods that can limit pattern recognition receptor (PRR) sensing and still maintain transcriptional activity. An alternative approach to gene therapy is a recombinant adeno-associated virus (rAAV) vector platform, which packages heterologous DNA in viral capsids. However, there are several major drawbacks using rAAV vectors as gene delivery vectors. A major drawback associated with rAAV is that the viral packaging capacity of its approximately 4.5kb heterologous DNA is limited. Another major drawback is capsid immunogenicity, which prevents re-application to patients.

[0006] Therefore, there remains a need for effective delivery vehicles that enable safe and efficient non-viral delivery of nucleic acid therapeutics to desired cell populations. Summary of the Invention

[0007] The present disclosure provides lipid nanoparticles (LNPs) and LNP compositions (e.g., pharmaceutical compositions), the LNP compositions comprising therapeutic nucleic acids (TNAs), e.g., gene expression vectors, such as end-blocked DNA (ceDNA), single-stranded DNA (ssDNA) vectors, or messenger RNA (mRNA). The LNPs of the present disclosure comprise structural LNP components comprising ionizable lipids; "helper" lipids, e.g., ceramide or distearoylphosphatidylcholine (DSPC); structural lipids, e.g., sterols; and one or more types of lipid-anchored polymers. Compared to known LNPs, the LNPs disclosed herein provide surprising and unexpected properties. For example, the helper lipids of the LNPs act to increase the fusogenicity of the lipid bilayer of the LNPs and promote endosome 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 the aggregation of the LNPs and provide steric stability (e.g., by minimizing the interaction between the opsonins present in the blood and the LNP surface, enhancing the stealth properties of the overall LNP properties in the circulation (e.g., blood compartment)). Furthermore, the disclosed LNP compositions are characterized by reduced LNP-related toxicity, as demonstrated by serum levels of immune response markers (see Examples herein). Additionally, the disclosed LNPs having a certain molecular percentage of sterols (e.g., 30%-45% molecular percentage of total lipids) are characterized by a diameter of about 80 nm or less, making them particularly suitable for therapeutic administration specifically targeted to certain tissues / organs that have size limitations for effective delivery.

[0008] According to one aspect, the present disclosure provides a lipid nanoparticle (LNP) comprising:

[0009] Therapeutic nucleic acids (TNA);

[0010] ionizable lipids;

[0011] sterols;

[0012] a first lipid-anchored polymer; wherein the lipid-anchored polymer comprises:

[0013] i) polymers;

[0014] ii) a lipid portion comprising at least one hydrophobic tail; and

[0015] iii) optionally a linker, which connects the polymer to the lipid moiety;

[0016] wherein the at least one hydrophobic tail comprises 12 to 22 carbon atoms in a single aliphatic chain backbone; and

[0017] Helper lipid represented by formula (I):

[0018]

[0019] or a salt or ester thereof, or a deuterated analog of any of the foregoing, wherein:

[0020] Is a single bond or a double bond;

[0021] A is hydrogen, And among them

[0022] R 1 It is C1-C 17 Alkyl or C2-C 17 alkenyl;

[0023] R 2 It is C1-C 22 Alkyl or C2-C 22 alkenyl;

[0024] R 3 is hydrogen or C1-C2 alkyl; and

[0025] R 4 is hydrogen or C1-C2 alkyl.

[0026] According to another aspect, the present disclosure provides a lipid nanoparticle (LNP) comprising:

[0027] Therapeutic nucleic acids (TNA);

[0028] ionizable lipids;

[0029] sterols;

[0030] a first lipid-anchored polymer; wherein the lipid-anchored polymer comprises:

[0031] i) polymers;

[0032] ii) a lipid portion comprising at least two hydrophobic tails; and

[0033] iii) a linker connecting the polymer to the lipid moiety;

[0034] wherein the at least two hydrophobic tails each comprise from 16 to 22 carbon atoms in a single aliphatic chain backbone; and

[0035] Helper lipid represented by formula (I):

[0036]

[0037] or a salt or ester thereof, or a deuterated analog of any of the foregoing, wherein:

[0038] Is a single bond or a double bond;

[0039] A is hydrogen, And among them

[0040] R 1 It is C1-C 17 Alkyl or C2-C 17 alkenyl;

[0041] R 2 It is C1-C 22 Alkyl or C2-C 22 alkenyl;

[0042] R 3 is hydrogen or C1-C2 alkyl; and

[0043] R 4 is hydrogen or C1-C2 alkyl.

[0044] According to yet another aspect, the present disclosure provides a lipid nanoparticle (LNP) comprising:

[0045] Therapeutic nucleic acids (TNA);

[0046] ionizable lipids;

[0047] sterols;

[0048] a first lipid-anchored polymer; wherein the lipid-anchored polymer comprises:

[0049] i) polymers;

[0050] ii) a lipid portion comprising at least two hydrophobic tails; and

[0051] iii) a linker connecting the polymer to the lipid moiety;

[0052] wherein the at least two hydrophobic tails each comprise 12 to 15 carbon atoms in a single aliphatic chain backbone; and

[0053] Helper lipid represented by formula (I):

[0054]

[0055] or a salt or ester thereof, or a deuterated analog of any of the foregoing, wherein:

[0056] Is a single bond or a double bond;

[0057] A is hydrogen,

[0058] R 1 It is C1-C 17 Alkyl or C2-C 17 alkenyl;

[0059] R 2 It is C1-C 22 Alkyl or C2-C 22 alkenyl;

[0060] R 3 is hydrogen or C1-C2 alkyl; and

[0061] R 4 is hydrogen or C1-C2 alkyl.

[0062] According to a further aspect, the present disclosure provides a lipid nanoparticle (LNP) comprising:

[0063] Therapeutic nucleic acids (TNA);

[0064] ionizable lipids;

[0065] sterols;

[0066] a first lipid-anchored polymer; wherein the lipid-anchored polymer comprises:

[0067] i) polymers;

[0068] ii) a lipid portion comprising a single hydrophobic tail; and

[0069] iii) a linker connecting the polymer to the lipid moiety;

[0070] wherein the single hydrophobic tail comprises 18 to 22 carbon atoms in a single aliphatic chain backbone; and

[0071] Helper lipid represented by formula (I):

[0072]

[0073] or a salt or ester thereof, or a deuterated analog of any of the foregoing, wherein: Is a single bond or a double bond;

[0074] A is hydrogen,

[0075] R 1 It is C1-C 17Alkyl or C2-C 17 alkenyl;

[0076] R 2 It is C1-C 22 Alkyl or C2-C 22 alkenyl;

[0077] R 3 is hydrogen or C1-C2 alkyl; and

[0078] R 4 is hydrogen or C1-C2 alkyl.

[0079] In some embodiments, the helper lipid in the LNP provided herein is represented by formula (II):

[0080]

[0081] or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0082] In some embodiments, the helper lipid is represented by formula (III):

[0083]

[0084] or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0085] In some embodiments, the helper lipid is represented by formula (IV):

[0086]

[0087] or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0088] In some embodiments, the LNPs of the present disclosure do not include distearoylphosphatidylcholine (DSPC), provided that there is a helper lipid represented by (I), (II), (III) or (IV), or a salt or ester thereof, or a deuterated analogue of any of the foregoing. In some embodiments, the LNPs of the present disclosure do not include 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), provided that there is a helper lipid represented by (I), (II), (III) or (IV), or a salt or ester thereof, or a deuterated analogue of any of the foregoing. In some embodiments, the LNPs of the present disclosure do not include 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), provided that there is a helper lipid represented by (I), (II), (III) or (IV), or a salt or ester thereof, or a deuterated analogue of any of the foregoing.

[0089] In some embodiments, R 1is C1-C in formula (I), (II), (III) or (IV) 10 Alkyl or C2-C 10 alkenyl; wherein R 2 、R 3 and R 4 As defined above.

[0090] In some embodiments, is a double bond in formula (I), (II), (III) or (IV); wherein R 1 、R 2 、R 3 and R 4 As defined above.

[0091] In some embodiments, R 1 is a C1-C8 alkyl or C2-C8 alkenyl group in formula (I), (II), (III) or (IV); wherein R 2 、R 3 and R 4 As defined above. In some embodiments, R 1 is a C1-C7 alkyl or C2-C7 alkenyl group in formula (I), (II), (III) or (IV); wherein R 2 、R 3 and R 4 As defined above. In some embodiments, R 1 is a C1 alkyl, C3 alkyl, C5 alkyl or C7 alkyl group in formula (I), (II), (III) or (IV); wherein R 2 、R 3 and R 4 As defined above. In some embodiments, R 1 is a C1 alkyl group in formula (I), (II), (III) or (IV), wherein R 2 、R 3 and R 4 As defined above.

[0092] In some embodiments, R 2 is C3-C in formula (I), (II), (III) or (IV) 15 Alkyl or C3-C 15 alkenyl; wherein R 1 、R 3 and R 4 As defined above. In some embodiments, R 2 is a C9 alkyl, C 11 Alkyl, C 12 Alkyl, C 13Alkyl or C 15 Alkyl; wherein R 1 、R 3 and R 4 As defined above. In some embodiments, R 2 It is C 12 Alkyl, C 13 Alkyl or C 14 Alkyl; wherein R 1 、R 3 and R 4 As defined above. In some embodiments, R 2 is C in formula (I), (II), (III) or (IV) 13 Alkyl, where R 1 、R 3 and R 4 As defined above.

[0093] In some embodiments, R 3 is hydrogen in formula (I), (II), (III) or (IV); wherein R 1 、R 2 and R 4 As defined above. In some embodiments, R 3 is a C1 alkyl group in formula (I), (II), (III) or (IV), wherein R 1 、R 2 and R 4 As defined above.

[0094] In some embodiments, R 4 is hydrogen in formula (I), (II), (III) or (IV); wherein R 1 、R 2 and R 3 As defined above. In some embodiments, R 4 is a C1 alkyl group; wherein R 1 、R 2 and R 3 As defined above.

[0095] 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.

[0096] In some embodiments, the helper lipid represented by formula (I) is selected from:

[0097]

[0098]

[0099] as well as

[0100]

[0101] or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0102] In some embodiments, the helper lipid represented by formula (I) or formula (II) is:

[0103]

[0104] or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0105] In some embodiments, the lipid represented by formula (I), formula (III) or formula (IV) is:

[0106]

[0107] or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0108] According to another aspect, the present disclosure provides a lipid nanoparticle (LNP) comprising:

[0109] Therapeutic nucleic acids (TNA);

[0110] ionizable lipids;

[0111] sterols;

[0112] a first lipid-anchored polymer; wherein the first lipid-anchored polymer comprises:

[0113] i) polymers;

[0114] ii) a lipid portion comprising at least two hydrophobic tails; and

[0115] iii) a linker connecting the polymer to the lipid moiety;

[0116] wherein the at least two hydrophobic tails each comprise from 16 to 22 carbon atoms in a single aliphatic chain backbone; and

[0117] 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);

[0118] The whole blood half-life of the LNP (t 1 / 2 ) is at least about 3 hours.

[0119] In some embodiments, each of the at least two hydrophobic tails of the first lipid-anchored polymer has 18 to 22 carbon atoms in a single aliphatic chain backbone. In some embodiments, each of the at least two hydrophobic tails of the first lipid-anchored polymer has 18 to 20 carbon atoms in a single aliphatic chain backbone. In some embodiments, each of the at least two hydrophobic tails of the first lipid-anchored polymer has 18 carbon atoms in a single aliphatic chain backbone.

[0120] In some embodiments, the 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 lipids present in the LNP. In some embodiments, the 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%.

[0121] In some embodiments, the blood half-life (t 1 / 2 ) is from about 3 hours to about 24 hours, or from about 3 hours to about 18 hours, or from about 3 hours to about 15 hours, or from about 3 hours to about 12 hours, or from about 3 hours to about 10 hours, or from about 3 hours to about 9 hours, or from about 3 hours to about 8 hours, or from about 3 hours to about 7.5 hours, or from about 3 hours to about 6.5 hours, or from about 3 hours to about 6 hours. In some embodiments, the blood half-life (t 1 / 2) is from about 3 hours to about 3.5 hours, or from about 3 hours to about 4 hours, or from about 3 hours to about 4.5 hours, or from about 3 hours to about 5 hours, or from about 3 hours to about 5.5 hours, or from about 3.5 hours to about 4 hours, or from about 3.5 hours to about 4.5 hours, or from about 3.5 hours to about 5 hours, or from about 3.5 hours to about 5.5 hours, or from about 4 hours to about 4.5 hours, or from about 4 hours to about 5 hours, or from about 4 hours to about 5.5 hours, or from about 4.5 hours to about 5 hours, or from about 4.5 hours to about 5 hours, or from about 5 hours to about 5.5 hours. In some embodiments, by comparison, the whole blood half-life (t 1 / 2 ) does not exceed about 3 hours, for example, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 1 hour, about 2 hours or about 2.5 hours. The reference LNP does not comprise a first lipid-anchored polymer having at least two hydrophobic tails with 16 to 22 carbon atoms in a single aliphatic chain backbone. In one embodiment, the reference LNP comprises a first lipid-anchored polymer comprising at least two hydrophobic tails, each of which comprises 12 to 15 carbon atoms 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, or also referred to as PEG-DMG). In one embodiment, the reference LNP comprises DMG-PEG and a helper lipid selected from the group consisting of DSPC, DOPC and DOPE.

[0122] In some embodiments, the LNP comprising a helper lipid selected from the group consisting of DSPC, DOPC, and DOPE has a blood clearance (CI) 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, the LNP has a blood clearance (CI) 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, by comparison, the whole blood clearance (CI) of the reference LNP is at least twice the whole blood clearance of the LNP comprising a helper lipid, 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, wherein the helper lipid is selected from the group consisting of DSPC, DOPC, and DOPE, as described above. In one embodiment, the reference LNP does not comprise a first lipid-anchored polymer having at least two hydrophobic tails having 16 to 22 carbon atoms in a single aliphatic chain backbone. In one embodiment, the reference LNP comprises a lipid-anchored polymer comprising at least two hydrophobic tails, each comprising 12 to 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 comprises DMG-PEG and a helper lipid selected from the group consisting of DSPC, DOPC, and DOPE.

[0123] In some embodiments, the whole blood terminal time point exposure (AUC) of the LNP comprising a helper lipid selected from the group consisting of DSPC, DOPC, and DOPE last) is at least 50 hours*ng / mL. In one embodiment, the terminal time point is 24 hours. In other embodiments, the terminal 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 whole blood terminal time point exposure (AUC) of the LNP is last ) is about 50 hours*ng / mL to about hours*ng / mL, or about 100 hours*ng / mL to about 750 hours*ng / mL, or about 150 hours*ng / mL to about 750 hours*ng / mL, or about 200 hours*ng / mL to about 700 hours*ng / mL.

[0124] In some embodiments, the whole blood terminal time point exposure (AUC) of the LNP comprising a helper lipid selected from the group consisting of DSPC, DOPC, and DOPE last ) is from about 200 hours*ng / mL to about 250 hours*ng / mL, or from about 200 hours*ng / mL to about 300 hours*ng / mL, or from about 500 hours*ng / mL to about 700 hours*ng / mL, or from about 500 hours*ng / mL to about 550 hours*ng / mL, or from about 500 hours*ng / mL to about 600 hours*ng / mL, or from about 550 hours*ng / mL to about 600 hours*ng / mL, or from about 600 hours*ng / mL to about 700 hours*ng / mL, or from about 600 hours*ng / mL to about 650 hours*ng / mL, or from about 650 hours*ng / mL to about 700 hours*ng / mL. In some embodiments, by comparison, the whole blood terminal time point exposure (AUC last) or no more than 50 hours*ng / mL, for example, about 40-45 hours*ng / mL, or about 35-40 hours*ng / mL, or about 30-35 hours*ng / mL, or about 25-30 hours*ng / mL, or about 20-25 hours*ng / mL, or about 15-20 hours*ng / mL, or about 10-15 hours*ng / mL, or about 5-10 hours*ng / mL. In one embodiment, the reference LNP does not comprise a first lipid-anchored polymer having at least two hydrophobic tails having 16 to 22 carbon atoms in a single aliphatic chain backbone. In one embodiment, the reference LNP comprises a reference lipid-anchored polymer comprising at least two hydrophobic tails, each hydrophobic tail comprising 12 to 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.

[0125] In some embodiments, the first lipid-anchored polymer in the LNP of the present disclosure comprises a lipid portion comprising one or two hydrophobic tails. In one embodiment, the first lipid-anchored polymer in the LNP of the present disclosure comprises a lipid portion comprising two hydrophobic tails. In one embodiment, each of the two hydrophobic tails is a fatty acid. In some embodiments, each of the two hydrophobic tails independently comprises 16, 17, 18, 19, 20, 21, or 22 carbon atoms. In some embodiments, each of the two hydrophobic tails independently comprises 16, 17, 18, 19, 20, or 21 carbon atoms. In some embodiments, each of the two hydrophobic tails independently comprises 16, 17, 18, 19, or 20 carbon atoms. In some embodiments, each of the two hydrophobic tails independently comprises 16, 17, 18, 19, or 20 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, homosapiens acid, oleic acid, elaidic acid, vaccinic acid, linoleic acid, trans-linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid and derivatives thereof.

[0126] 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.

[0127] In one embodiment, the first lipid-anchored polymer in the LNP of the present disclosure comprises a lipid portion 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, homosapiens acid, oleic acid, elaidic acid, vaccinic acid, linoleic acid, trans-linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, and derivatives thereof.

[0128] In some embodiments, the first lipid-anchoring polymer is a glycerolipid. In some embodiments, the first lipid-anchoring polymer is a phospholipid. In some embodiments, the first lipid-anchoring polymer does not comprise distearoylphosphatidylcholine (DSPC), provided that a helper lipid represented by (I), (II), (III), or (IV) is present, or a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0129] In some embodiments, the first lipid-anchoring polymer comprises a linker lipid moiety selected from the group consisting of: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-phosphatidylethanolamine (PPE), 1,2-dioleoyl-sn-phosphatidylethanolamine (DSPE), 1,2-dioleoyl-sn-phosphatidylethanolamine (PPE), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-phosphatidylethanolamine (PPE ... 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 one of the foregoing.

[0130] In some embodiments, the linker lipid moiety in the first lipid-anchoring polymer is selected from the group consisting of: 1,2-dimyristoyl-rac-glycero-3-methoxy (DMG), R-3-[(ω-methoxycarbamoyl)]-1,2-dimyristyloxy-propyl-3-amine, derivatives thereof, and combinations of any one of the foregoing. In some embodiments, the first lipid-anchoring polymer comprises DMG.

[0131] 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.

[0132] In some embodiments, the molecular weight of the polymer is between about 1000Da and about 5000Da. In some embodiments, the molecular weight of the polymer is between about 2000Da and about 5000Da. In some embodiments, the molecular weight of the polymer is about 2000Da. In some embodiments, the molecular weight of the polymer is about 3200Da to about 3500Da.

[0133] In some embodiments, the polymer is polyethylene glycol (PEG).

[0134] In some embodiments, the sterol is selected from the group consisting of cholesterol, β-sitosterol, stigmasterol, β-sitostanol, campesterol, brassicasterol and derivatives thereof, and combinations thereof. In some embodiments, the sterol is cholesterol. In some embodiments, the sterol is β-sitosterol.

[0135] In some embodiments, the ionizable lipid is a lipid represented by:

[0136] a) Formula (A):

[0137]

[0138] or a pharmaceutically acceptable salt thereof, wherein:

[0139] R 1 and R 1' Each independently is an optionally substituted linear or branched C 1-3 alkylene;

[0140] R 2 and R 2' Each independently is an optionally substituted linear or branched C 1-6 alkylene;

[0141] R 3 and R 3' Each independently is an optionally substituted linear or branched C 1-6 alkyl;

[0142] Or alternatively, when R 2 is an optionally substituted branched C 1-6 When alkylene, R 2 and R 3 Together with its central nitrogen atom, it forms a 4- to 8-membered heterocyclic group;

[0143] Or alternatively, when R 2' is an optionally substituted branched C 1-6 When alkylene, R 2' and R 3' Together with its central nitrogen atom, it forms a 4- to 8-membered heterocyclic group;

[0144] R 4 and R 4' Each independently is -CR a 、-C(R a )2CR a or -[C(R a )2]2CR a ;

[0145] R a Each occurrence is independently H or C 1-3 alkyl;

[0146] Or alternatively, R 4 Yes-C(R a )2CR a or -[C(R a )2]2CR a , and when R a It is C 1-3 When alkyl, R 3 and R 4 Together with its central nitrogen atom, it forms a 4- to 8-membered heterocyclic group;

[0147] Or alternatively, R 4' Yes-C(R a )2CR a or -[C(R a )2]2CR a , and when R a It is C 1-3 When alkyl, R 3' and R 4' Together with its central nitrogen atom, it forms a 4- to 8-membered heterocyclic group;

[0148] R 5 and R 5' are independently hydrogen, C 1-20 Alkylene or C 2-20 alkenylene;

[0149] R 6 and R 6' Each occurrence is independently C 1-20 Alkylene, C 3-20 Cycloalkylene or C 2-20 alkenylene; and

[0150] m and n are each independently an integer selected from 1, 2, 3, 4 and 5; or

[0151] b) Formula (B):

[0152]

[0153] or a pharmaceutically acceptable salt thereof, wherein:

[0154] a is an integer ranging from 1 to 20;

[0155] b is an integer ranging from 2 to 10;

[0156] R 1 Not present or selected from (C2-C 20 )alkenyl, -C(O)O(C2-C 20 ) alkyl and (C2-C 20 ) alkyl-substituted cyclopropyl; and

[0157] R 2 Yes (C2-C 20 )alkyl; or

[0158] c) Formula (C):

[0159]

[0160] or a pharmaceutically acceptable salt thereof, wherein:

[0161] R 1 and R 1' Each independently is optionally selected from one or more R a (C1-C6)alkylene substituted with a group;

[0162] R 2 and R 2' are each independently (C1-C2)alkylene;

[0163] R 3 and R 3' Each independently is optionally selected from one or more R b (C1-C6) alkyl substituted with a group;

[0164] Or alternatively, R 2 and R 3 and / or R 2' and R 3' Together with its central nitrogen atom, it forms a 4- to 7-membered heterocyclic group;

[0165] R 4 and R 4 'Each is a (C2-C6)alkylene group interrupted by -C(O)O-;

[0166] R 5 and R 5 'Each independently is (C2-C 30 )alkyl or (C2-C 30 )alkenyl, each of which is optionally interrupted by -C(O)O- or (C3-C6)cycloalkyl; and

[0167] R a and R b are each halo or cyano; or

[0168] d) Formula (D):

[0169]

[0170] or a pharmaceutically acceptable salt thereof, wherein:

[0171] R' is absent and is hydrogen or C1-C6 alkyl; provided that when R' is hydrogen or C1-C6 alkyl, R', R 1 and R 2 The nitrogen atoms they are all connected to have a positive charge;

[0172] R 1 and R 2 are each independently hydrogen, C1-C6 alkyl or C2-C6 alkenyl;

[0173] R 3 It is C1-C 12 Alkylene or C2-C 12 alkenylene;

[0174] R 4 It is C1-C 18 Unbranched alkyl, C2-C 18 Unbranched alkenyl or in:

[0175] R 4a and R 4b Each is independently C1-C 16 Unbranched alkyl or C2-C 16 unbranched alkenyl;

[0176] R 5 Not present, C1-C8 alkylene or C2-C8 alkenylene;

[0177] R 6a and R 6b Each independently is C7-C 16 Alkyl or C7-C 16 alkenyl; provided that the combined R 6a and R 6b The total number of carbon atoms in is greater than 15;

[0178] X 1 and X 2 Each is independently -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -SS-, -C(R a )=N-、-N=C(Ra )-、-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:

[0179] R a Each occurrence is independently hydrogen or C1-C6 alkyl; and

[0180] n is an integer selected from 1, 2, 3, 4, 5 and 6;

[0181] In some embodiments, R 4 It is C1-C 16 Unbranched alkyl, C2-C 16 Unbranched alkenyl or where R 4a and R 4b as defined above; or

[0182] e) Formula (E):

[0183]

[0184] or a pharmaceutically acceptable salt thereof, wherein:

[0185] R' is absent and is hydrogen or C1-C3 alkyl; provided that when R' is hydrogen or C1-C3 alkyl, R', R 1 and R 2 The nitrogen atoms they are all connected to have a positive charge;

[0186] R 1 and R 2 are each independently hydrogen or C1-C3 alkyl;

[0187] R 3 It is C3-C 10 Alkylene or C3-C 10 alkenylene;

[0188] R 4 It is C1-C 16 Unbranched alkyl, C2-C 16 Unbranched alkenyl or in:

[0189] R 4a and R 4b Each is independently C1-C 16 Unbranched alkyl or C2-C 16 unbranched alkenyl;

[0190] R 5 Not present, C1-C6 alkylene or C2-C6 alkenylene;

[0191] R 6a and R 6b Each independently is C7-C 14 Alkyl or C7-C 14 alkenyl;

[0192] 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:

[0193] R a Each occurrence is independently hydrogen or C1-C6 alkyl; and

[0194] n is an integer selected from 1, 2, 3, 4, 5 and 6; or

[0195] f) an ionizable lipid selected from any one of the ionizable lipids in Table 1, 4, 5, 6 or 7.

[0196] In some embodiments, the LNPs of the present disclosure further comprise a targeting moiety.

[0197] In some embodiments, the LNP comprises a second lipid-anchored polymer, and the targeting moiety is conjugated to the second lipid-anchored polymer. In some embodiments, the second lipid-anchored polymer comprises a linker lipid moiety selected from the group consisting of: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-phosphatidylethanolamine (PTE), 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 one of the foregoing.

[0198] In some embodiments, the first lipid-anchoring polymer and the second lipid-anchoring polymer are different lipid-anchoring polymers; and the first lipid-anchoring polymer and the second lipid-anchoring polymer comprise one of the following combinations:

[0199] DSG (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer);

[0200] DSPE (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer);

[0201] DODA (the first lipid-anchoring polymer) and DSPE (the second lipid-anchoring polymer);

[0202] DPG (the first lipid-anchoring polymer) and DSPE (the second lipid-anchoring polymer);

[0203] DMG (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer);

[0204] DODA (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer);

[0205] DPG (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer);

[0206] DMG (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer);

[0207] DPG (the first lipid-anchoring polymer) and DODA (the second lipid-anchoring polymer);

[0208] DMG (the first lipid-anchored polymer) and DODA (the second lipid-anchored polymer); or

[0209] DMG (the first lipid-anchoring polymer) and DPG (the second lipid-anchoring polymer).

[0210] In some embodiments, the first lipid-anchoring polymer and the second lipid-anchoring polymer are the same lipid-anchoring polymer; and wherein the first lipid-anchoring polymer and the second lipid-anchoring polymer comprise one of the following combinations:

[0211] DSG (the first lipid-anchored polymer) and DSG (the second lipid-anchored polymer);

[0212] DSPE (the first lipid-anchored polymer) and DSPE (the second lipid-anchored polymer);

[0213] DODA (the first lipid-anchored polymer) and DODA (the second lipid-anchored polymer); or

[0214] DPG (the first lipid-anchoring polymer) and DPG (the second lipid-anchoring polymer).

[0215] In some embodiments, the targeting moiety is conjugated to a DSPE anchoring polymer. In some embodiments, the DSPE anchoring polymer is DSPE-PEG or a derivative thereof. In some embodiments, the targeting moiety is conjugated to a DSG anchoring polymer. In some embodiments, the DSG anchoring polymer is DSG-PEG or a derivative thereof.

[0216] In some embodiments, the targeting moiety is capable of binding to a hepatocyte. In some embodiments, the liver cell is a hepatocyte. In some embodiments, the targeting moiety is N-acetylgalactosamine (GalNAc) or a GalNAc derivative. In some embodiments, the targeting moiety is a triantennary GalNAc conjugate or a tetraantennary GalNAc conjugate. In some embodiments, the targeting moiety is selected from the group consisting of: ApoE protein, ApoE polypeptide, ApoB protein, 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: ApoE protein conjugate, ApoE polypeptide conjugate, ApoB protein conjugate, and ApoB polypeptide conjugate. In one embodiment, the targeting moiety is a modified ApoE protein conjugate.

[0217] In one embodiment, the ionizable lipid in the LNP of the present disclosure according to any one of the preceding embodiments is ionizable lipid 81:

[0218]

[0219] or a pharmaceutically acceptable salt thereof.

[0220] In one embodiment, the ionizable lipid in the LNP of the present disclosure according to any one of the preceding embodiments is ionizable lipid 89:

[0221]

[0222] or a pharmaceutically acceptable salt thereof.

[0223] In one embodiment, the ionizable lipid in the LNP of the present disclosure according to any one of the preceding embodiments is ionizable lipid 87:

[0224]

[0225] or a pharmaceutically acceptable salt thereof.

[0226] In some embodiments, the ionizable lipid is present in the LNP provided by the present disclosure in an amount of about 35mol% to about 60mol% of the total lipid present in the LNP. In some embodiments, the ionizable lipid is present in the LNP in an amount of about 20mol% to about 50mol% of the total lipid present in the LNP.

[0227] 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. In 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.

[0228] In some embodiments, the first lipid-anchored polymer is present in the LNP in an amount of about 0.5 mol % to about 5 mol % of the total lipids present in the LNP. In some embodiments, the second lipid-anchored polymer is present in the LNP in an amount of about 0.005 mol % to about 5 mol % of the total lipids present in the LNP. In some embodiments, the first lipid-anchored polymer is present in the LNP in an amount of about 0.05 mol % to about 2 mol % of the total lipids present in the LNP.

[0229] In some embodiments, the second lipid-anchored polymer is present in the LNP in an amount of about 0.1 mol % to about 1 mol % of the total lipids present in the LNP. In some embodiments, the second lipid-anchored polymer is present in the LNP in an amount of about 0.5 mol % of the total lipids 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 lipids present in the LNP.

[0230] In some embodiments, the helper lipid represented by formula (I), (II), (III) or (IV), or a salt or ester thereof, or a deuterated analogue 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), (II), (III) or (IV), or a salt or ester thereof, or a deuterated analogue 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), (II), (III) or (IV), or a salt or ester thereof, or a deuterated analogue 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), (II), (III) or (IV), or a salt or ester thereof, or a deuterated analogue of any one of the foregoing is present in the LNP in an amount of about 10mol% of the total lipid present in the LNP. In some embodiments, the helper lipid represented by formula (I), (II), (III) or (IV), or a salt or ester thereof, or a deuterated analogue of any one of the foregoing is present in the LNP in an amount of about 15mol% of the total lipid present in the LNP. In some embodiments, the helper lipid represented by formula (I), (II), (III) or (IV), or a salt or ester thereof, or a deuterated analogue of any one of the foregoing is present in the LNP in an amount of about 20mol% of the total lipid present in the LNP.

[0231] In some embodiments, the 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 lipids present in the LNP.

[0232] In some embodiments, the LNP dosage forms provided herein are suitable for intravenous administration.

[0233] In some embodiments, the LNP is less immunogenic than a reference LNP; wherein the reference LNP: (i) does not comprise the helper lipid represented by formula (I), (II), (III) or (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing; or (ii) comprises a helper lipid and a reference lipid polymer, wherein the helper lipid is 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 the reference lipid-anchored polymer comprises at least two hydrophobic tails, each hydrophobic tail comprising 12 to 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).

[0234] In some embodiments, the uptake of the TNA by blood cells induced by the LNP is lower than the uptake of the TNA by blood cells induced by the reference LNP.

[0235] In some embodiments, the LNP elicits a pro-inflammatory cytokine response that is lower than the pro-inflammatory cytokine response elicited by the reference LNP.

[0236] In some embodiments, the LNP induces the expression level of TNA in blood cells that is lower than the expression level of TNA in blood cells that is induced by a reference LNP. In some embodiments, the blood cells are red blood cells, macrophages, and peripheral blood mononuclear cells.

[0237] In some embodiments, the therapeutic nucleic acid (TNA) is selected from the group consisting of: minigene, plasmid, minicircle, small interfering RNA (siRNA), microRNA (miRNA), guide RNA (gRNA), antisense oligonucleotide (ASO), ribozyme, end-blocked DNA (ceDNA), single-stranded DNA (ssDNA), ministring, doggybone TM , protelomeric end-blocked DNA, dumbbell-shaped linear DNA, Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), mRNA, tRNA, rRNA, gRNA, DNA viral vectors, viral RNA vectors, non-viral vectors, and any combination thereof.

[0238] In some embodiments, the TNA is greater than about 200 bp or greater than about 200 nt in length. In some embodiments, the TNA is greater than about 500 bp or greater than about 500 nt in length. In some embodiments, the TNA is greater than about 1000 bp or greater than about 1000 nt in length. In some embodiments, the TNA is greater than about 4000 bp or greater than about 4000 nt in length.

[0239] In some embodiments, the TNA is end-blocked DNA (ceDNA). In some embodiments, the TNA is messenger RNA (mRNA). In some embodiments, the TNA is single-stranded nucleic acid. In some embodiments, the TNA is double-stranded nucleic acid.

[0240] In some aspects, the present disclosure provides a pharmaceutical composition comprising the LNPs of the present disclosure and a pharmaceutically acceptable carrier.

[0241] In some aspects, the present disclosure also provides a method for producing an LNP of the present disclosure, the method comprising combining the following: the therapeutic nucleic acid (TNA); the ionizable lipid; the sterol; the first lipid-anchored polymer; the helper lipid represented by formula (I), (II), (III) or (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, the targeting moiety.

[0242] 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 the LNP of the present disclosure or the pharmaceutical composition of the present disclosure.

[0243] In some embodiments, the subject is a human.

[0244] In some embodiments, the genetic disorder is selected from the group consisting of sickle cell anemia; melanoma; hemophilia A (factor VIII (FVIII) deficiency); hemophilia B (factor IX (FIX) deficiency); cystic fibrosis (CFTR); familial hypercholesterolemia (LDL receptor deficiency); hepatoblastoma; Wilson's disease; phenylketonuria (PKU); congenital hepatic porphyria; inherited liver metabolic disorders, Lesch-Nyhan syndrome; thalassemia; xeroderma pigmentosum, Fanconi's anemia; retinitis pigmentosa; ataxia-telangiectasia; Bloom's syndrome; retinoblastoma; mucopolysaccharidoses; Niemann-Pick Disease; Fabry disease; Schindler disease. 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 (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 disease); spinocerebellar ataxia; spinal muscular atrophy (SMA); Friedreich's ataxia; Duchenne muscular dystrophy (DMD); Becker muscular dystrophy (BMD); dystrophic epidermolysis bullosa (DEB); ectonucleotide pyrophosphatase 1 deficiency; systemic arterial calcification of infancy (GACI); Leber congenital amaurosis; Stargardt disease;Wet macular degeneration (wet AMD); ornithine transcarbamylase (OTC) deficiency; Usher syndrome; alpha-1 antitrypsin deficiency; progressive familial intrahepatic cholestasis (PFIC); and cathepsin A deficiency.

[0245] In some embodiments, the genetic condition is phenylketonuria (PKU). In some embodiments, the genetic condition is hemophilia A (factor VIII deficiency). In some embodiments, the genetic condition is Wilson's disease. In some embodiments, the genetic condition is Gaucher disease. In some embodiments, the genetic condition is Gaucher disease type I, Gaucher disease type II, or Gaucher disease type III. In some embodiments, the genetic condition is Leber congenital amaurosis (LCA). In some embodiments, the LCA is LCA10. In some embodiments, the genetic condition is Stargardt's disease. In some embodiments, the genetic condition is wet macular degeneration (wet AMD).

[0246] In some aspects, the present disclosure also provides a method of providing anti-tumor immunity in a subject, 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 suffering from a disease, disorder, or condition associated with elevated expression of a tumor antigen, 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 human. In some embodiments, after administration, 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. In some embodiments, the concentration of the TNA at the beginning of a 12-hour, 18-hour, or 24-hour time window after administration and the concentration of the TNA at the end of the time window are within the same order of magnitude. In one embodiment, the TNA is messenger RNA (mRNA).

[0247] In some aspects, the present disclosure further provides a method of treating a blood disease, disorder, or condition in a subject, the method comprising administering to the subject an effective amount of the LNP of the present disclosure or the pharmaceutical composition of the present disclosure. In some embodiments, 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 myeloid leukemia (CML), Hodgkin lymphoma (HL), multiple myeloma, myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), adrenoleukodystrophy (ALD), Hurler syndrome, Krabbe disease (globular cell leukodystrophy or GLD), metachromatic leukodystrophy (MLD), severe aplastic anemia (SAA), severe combined immunodeficiency (SCID), sickle cell disease (SCD), thalassemia, Wiskott-Aldrich syndrome, Diamond-Blackfan syndrome, anemia, essential thrombocythaemia, Fanconi anemia, hemophagocytic lymphohistiocytosis (HLH), juvenile myelomonocytic leukemia (JMML), myelofibrosis, polycythemia vera, and combinations thereof. In one embodiment, the TNA is messenger RNA (mRNA). BRIEF DESCRIPTION OF THE DRAWINGS

[0248] The embodiments of the present disclosure, briefly summarized above and discussed in more detail below, may be understood by reference to the illustrative embodiments of the disclosure depicted in the accompanying drawings. However, the drawings illustrate only typical embodiments of the disclosure and are therefore not to be considered limiting of its scope, as the disclosure may admit to other equally effective embodiments.

[0249] Figure 1A Shown is the in vivo expression of luciferase from LNP D and LNP1 containing C2 ceramide in CD-1 mice on day 4 after administration. Figure 1B The results show the expression of the above-described Figure 1A In vivo expression of luciferase from the same LNP formulation. Figure 1C The percentage change in body weight of mice on day 1 after administration is shown.

[0250] Figure 2A The graph shows the in vivo expression of luciferase by LNP D, LNP1 containing C2 ceramide, LNP35 containing C8 ceramide, and LNP36 containing C2 sphingomyelin in CD-1 mice on day 4 after administration. Figure 2B The results show the changes in the mice at day 7 after administration from the Figure 2A In vivo expression of luciferase from the same LNP formulation. Figure 2C The percentage change in body weight of mice on day 1 after administration is shown.

[0251] Figure 3A Shown is the in vivo expression of luciferase from LNP C and LNP37 containing C2 ceramide in CD-1 mice on day 4 after administration. Figure 3B The results show the expression of the above-described Figure 3A In vivo expression of luciferase from the same LNP formulation.

[0252] Figures 4A-4F Figure 3. Expression of IFN-α in CD-1 mice 6 hours after injection of LNP D and LNP1 containing C2 ceramide. Figure 4A ), IL-6( Figure 4B ), IFN-γ( Figure 4C ), TNF-α( Figure 4D )、IL-18( Figure 4E ) and IP-10( Figure 4F ) serum levels.

[0253] Figures 5A-5E Figure 3 shows the expression of cytokine IFN-α ( Figure 5A ), IL-6( Figure 5B ), IFN-γ( Figure 5C ), TNF-α( Figure 5D ) and IL-18( Figure 5E ) serum levels.

[0254] Figures 6A-6F Figure 3 shows the expression of IFN-α in CD-1 mice 6 hours after injection of LNP A, LNP23, LNP24, LNP25, LNP26, LNP27, and LNP28 containing C2 ceramide. Figure 6A ), IL-6( Figure 6B ), IFN-γ( Figure 6C ), TNF-α( Figure 6D )、IL-18( Figure 6E ) and IP-10( Figure 6F ) serum levels.

[0255] Figures 7A-7FFigure 3 shows the expression of IFN-α in CD-1 mice 6 hours after injection of LNP C and LNP37 containing C2 ceramide. Figure 7A ), IL-6( Figure 7B ), IFN-γ( Figure 7C ), TNF-α( Figure 7D )、IL-18( Figure 7E ) and IP-10( Figure 7F ) serum levels.

[0256] Figure 8 Depicted are whole blood and plasma levels of ceDNA cargo in CD-1 mice injected with LNP E and LNP1 containing C2 ceramide at 1, 3, and 6 hours post-dose.

[0257] Figure 9A Shown is the in vitro expression of luciferase in primary mouse hepatocytes treated with C2-ceramide-containing LNP40 carrying an mRNA luciferase cargo. Figure 9B Shown is a DiD signal indicating uptake of LNP40 into primary mouse hepatocytes.

[0258] Figure 10 The in vitro expression of luciferase in primary mouse hepatocytes treated with LNP F, LNP41 containing C2 ceramide, LNP42 containing C4 ceramide, LNP43 containing C6 ceramide, or LNP45 containing C8 ceramide, each carrying an mRNA luciferase cargo, was compared.

[0259] Figure 11 Shown and compared are 24-hour total IVIS fluorescence in the livers of groups of CD-1 mice dosed with LNP101, LNP102, LNP103, LNP104, and LNP G, all of which carried luciferase mRNA as the nucleic acid cargo.

[0260] Figure 12A The graph shows the concentration (μg / mL) of luciferase mRNA in whole blood of the CD-1 mouse groups to which LNP101, LNP102, LNP103, LNP104, and LNP G were quantified by qPCR at 2 minutes, 1 hour, 6 hours, and 24 hours after administration.

[0261] Figure 12B Figure 3 is a graph showing quantification of luciferase mRNA copies in the liver of the CD-1 mouse groups administered with LNP101, LNP102, LNP103, LNP104, and LNP G by qPCR at 6 hours and 24 hours after administration.

[0262] Figure 12CFigure 3 is a graph showing quantification of luciferase mRNA copies in the spleen of the CD-1 mouse groups administered with LNP101, LNP102, LNP103, LNP104, and LNP G by qPCR at 6 hours and 24 hours after administration.

[0263] Figure 12D Figure 3 is a graph showing quantification of luciferase mRNA copies in the bone marrow of CD-1 mouse groups administered with LNP101, LNP102, LNP103, LNP104, and LNP G by qPCR at 6 hours and 24 hours after administration.

[0264] Figure 13 Figure 3 is a curve of quantification of ceDNA blood copies of CD-1 mouse groups treated with LNP201, LNP202, and LNP203 at 0, 1, 3, 6, and 24 hours after administration by qPCR.

[0265] Figure 14A Depicted are the different retention times from HPLC-SEC readings of LNP formulations with increasing mol% of the first lipid-anchoring polymer (i.e., LNPs with DSG-PEG2000-OMe at 1.5 mol%, 2 mol%, 2.5 mol%, 3 mol%, 5 mol% and 7 mol%).

[0266] Figure 14B Retention times of LNP formulations with mol % of lipid-anchoring polymer (DSG-PEG2000-OMe) at 1.5 mol % are depicted (wavelength reading: 214 nm for tracking lipids and 260 nm for tracking nucleic acid cargo).

[0267] Figure 14C Retention times of LNPs with mol% of lipid-anchored polymer (DSG-PEG2000-OMe) at 7 mol% are depicted (wavelength reading: 214 nm for tracking lipids and 260 nm for tracking nucleic acid cargo). DETAILED DESCRIPTION

[0268] The present disclosure provides lipid nanoparticles (LNPs) and LNP compositions (e.g., pharmaceutical compositions), wherein the LNP compositions include therapeutic nucleic acids (TNAs), e.g., gene expression vectors, such as end-blocked 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, e.g., C2 ceramides or C2 sphingomyelins ("helper lipids containing C2-C8"); structural lipids, e.g., sterols (e.g., cholesterol or β-sitosterol); and one or more types of lipid-anchored polymers.

[0269] Compared to known LNPs, the LNPs and LNP compositions disclosed herein provide surprising and unexpected properties. For example, the role of the helper lipids of the LNPs is to increase the fusogenicity of the LNP lipid bilayer and promote 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 the aggregation of the LNPs and provide steric stability (e.g., by minimizing any interaction between opsonins present in the blood and the LNP surface, enhancing the stealth properties of the overall LNP properties in the blood compartment). In addition, the disclosed LNPs and LNP compositions are characterized by surprisingly reduced LNP-related toxicity, as demonstrated 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 which has a certain length, e.g., each independently comprising 16 to 22 carbon atoms), may help mitigate LNP-related immunogenicity. Such helper lipids include ceramides, sphingomyelins, and fatty acids having a certain number of aliphatic carbon atoms in the fatty acid portion of the helper lipid (e.g., in C2-C8 ceramides). It has also been found that adjuvants such as DSPC can support the stability and extended stealth of the LNPs of the present disclosure, as measured by in vivo pharmacokinetics. In addition, the disclosed LNPs comprising a certain molecular percentage of sterols (30%-45% molecular percentage of total lipids) are characterized by an average diameter of about 70-100 nm, 70-80 nm or less, making them particularly suitable for therapeutic administration. Therefore, by combining LNP components having specific physical properties of helper lipids with lipid-anchored polymers disclosed herein, LNPs with desired properties (such as improved stealth properties, which can avoid rapid cellular uptake by blood cells and enhance tolerance) can be achieved.

[0270] I. Definition

[0271] Unless otherwise defined herein, scientific and technical terms used in conjunction with this application shall have the meanings commonly understood by those of ordinary skill in the art of the present disclosure. It should be understood that the present disclosure is not limited to the specific methods, protocols, and reagents described herein and may vary accordingly. The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present disclosure, which is defined solely by the claims. Definitions of commonly used terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th ed., Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), Fields Virology, 6th ed., Lippincott Williams & Wilkins, Philadelphia, PA, USA (2013); Knipe, DM and Howley, PM (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc.), 1995 (ISBN 1-56081-569-8); Werner Luttmann, Immunology, Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, Jones & Bartlett Publishers, 2014 (ISBN 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring 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, 2013 (ISBN 0124199542); Sons), 2014 (ISBN047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E.Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.), John Wiley and Sons, 2003 (ISBN 0471142735, 9780471142737), the contents of which are incorporated herein by reference in their entirety.

[0272] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0273] 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".

[0274] Use of alternatives (eg, "or") should be understood to mean either one, both, or any combination of the alternatives.

[0275] As used herein, when referring to a measurable value such as an amount, duration, etc., the term "about" is intended to encompass deviations of ±20% or ±10% (more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%) from the specified value, depending on the accuracy and precision of the method that can be used to determine such measurable value or because such deviations are appropriate for performing the disclosed methods.

[0276] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the range, and fractions thereof (e.g., tenths and hundredths of integers) where appropriate.

[0277] As used herein, "comprise," "comprising," and "comprises" are intended to be synonymous with "include," "including," "includes," or "contain," "containing," "contains," and are inclusive or open-ended terms that specify the presence of, for example, components, and do not preclude or exclude the presence of additional, unrecited components, features, elements, components, steps known in the art or disclosed herein.

[0278] The term "consisting of refers to compositions, methods, processes, and corresponding components thereof as described herein, excluding any elements not recited in the description of the embodiment.

[0279] As used herein, the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the disclosure.

[0280] As used herein, the terms "such as," "for example," and the like are intended to refer to exemplary embodiments without limiting the scope of the present disclosure.

[0281] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to any methods and materials described herein can be used for implementation to test the present disclosure, preferred materials and methods are described herein.

[0282] As used herein, the terms "administration," "administering," and variations thereof refer to the introduction of a composition or agent (e.g., nucleic acids, specifically ceDNA, ssDNA, and mRNA) into a subject and include the simultaneous and sequential introduction of one or more compositions or agents. "Administration" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, placebo, and experimental methods. "Administration" also encompasses in vitro and ex vivo treatments. The composition or agent is introduced into the subject by any suitable route, including oral, pulmonary, nasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intratumoral, or local. Administration includes self-administration and administration by another person. Administration can be performed by any suitable route. A suitable route of administration allows the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into the subject's vein.

[0283] 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 undesirable immune response to the LNP and its components in a subject after administration of the LNP of the present disclosure or a composition comprising the LNP of the present disclosure to the subject. In some embodiments, the immune response can be measured, for example, by measuring the levels of one or more proinflammatory cytokines before and after administration of a composition comprising an LNP of the present disclosure. Exemplary proinflammatory cytokines that can be used to determine the immunogenicity of the LNPs of the present disclosure or compositions comprising the LNPs of the present disclosure include, but are not limited to, granulocyte colony-stimulating factor (G-CSF), interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), interleukin 8 (IL-8 or CXCL8), interleukin 11 (IL-11), interleukin 17 (IL-17), interleukin 18 (IL-18), interferon alpha (IFN-alpha), interferon beta (IFN-beta), interferon gamma (IFN-gamma), CXC motif chemokine ligand 10 (CXCL10 or IP-10), monocyte chemoattractant protein 1 (MCP-1), CD40L, CCL2, CCL3, CCL4, CCL5, CCL11, tumor necrosis factor alpha (TNF-alpha), and combinations thereof. In some embodiments, the immune response can be measured, for example, by measuring the levels of specific antibodies against the LNPs and components of the LNPs before and after administration of a composition comprising the LNPs of the present disclosure.

[0284] As used herein, the term "off-target delivery" refers to the delivery of LNPs to non-target cells. After administration to a subject, LNPs can be delivered to non-target cells and may cause expression of therapeutic nucleic acids (TNAs) in non-target cells.

[0285] In some embodiments, the non-target cell may be a liver sinusoidal endothelial cell (LSEC cell), a spleen cell, or a Kupffer cell.

[0286] After administration to a subject, the LNPs can be delivered to non-target cells and may cause expression of therapeutic nucleic acids (TNAs) in non-target cells, or can be degraded once engulfed by, for example, macrophages. In some embodiments, the reference LNPs can be characterized by a higher rate of random delivery to non-target cells 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 cause a lower level of uptake of TNAs (e.g., ceDNA, ssDNA, or mRNA) in blood cells than the level of uptake of the reference LNPs. In some embodiments, the reference LNP is a LNP that (i) does not comprise the helper lipid represented by formula (I), (II), (III) or (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing; or (ii) comprises a helper lipid and a reference lipid polymer, wherein the helper lipid is 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 the reference lipid-anchored polymer comprises at least two hydrophobic tails, each hydrophobic tail comprising 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).

[0287] As used herein, the term "aqueous solution" refers to a composition that comprises in whole or in part water.

[0288] As used herein, the term "base" includes purines and pyrimidines, which further includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including but not limited to, modifications that place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0289] 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 may also be incorporated into the compositions. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce toxic, allergic, or similar adverse reactions when administered to a host.

[0290] As used herein, the term "ceDNA" refers to a capsid-free, end-blocked, linear double-stranded (ds) duplex DNA for non-viral gene transfer, synthetic or otherwise. According to some embodiments, the ceDNA is an end-blocked linear duplex (CELiD) CELiD DNA. According to some embodiments, the ceDNA is a DNA-based minicircle. According to some embodiments, the ceDNA is a minimalistic immunologically defined gene expression (MIDGE)-vector. According to some embodiments, the ceDNA is a helper DNA. According to some embodiments, the ceDNA is a dumbbell-shaped linear duplex end-blocked DNA containing two hairpin structures of the ITRs at the 5' and 3' ends of the expression cassette. According to some embodiments, the ceDNA is a doggybone TM DNA. A detailed description of ceDNA is described in International Patent Application No. PCT / US2017 / 020828, filed March 3, 2017, the entire contents of which are expressly incorporated herein by reference. Certain methods for producing ceDNA comprising various inverted terminal repeat (ITR) sequences and configurations using cell-based methods are described in Example 1 of International Patent Application No. PCT / US18 / 49996, filed September 7, 2018, and PCT / US2018 / 064242, filed December 6, 2018, each of which is incorporated herein by reference in its entirety. Certain methods for producing synthetic ceDNA vectors comprising various ITR sequences and configurations are described, for example, in International Application PCT / US2019 / 14122, filed January 18, 2019, the entire contents of which are incorporated herein by reference.

[0291] As used herein, the term "end-blocked DNA vector" refers to a capsid-free DNA vector having at least one covalently blocked end, wherein at least a portion of the vector has an intramolecular duplex structure. The terms "ceDNA vector" and "ceDNA" are used interchangeably and refer to an end-blocked DNA vector comprising at least one terminal palindrome. In some embodiments, the ceDNA comprises two covalently blocked ends.

[0292] As used herein, the term "ceDNA genome" refers to an expression cassette that also 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 duplex polynucleotide of DNA.

[0293] As used herein, the terms "DNA regulatory sequence," "control element," and "regulatory element" are used interchangeably herein and refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide and / or regulate the transcription of a non-coding sequence (e.g., a DNA-targeting RNA) or a coding sequence (e.g., a site-directed modifying polypeptide, or a Cas9 / Csn1 polypeptide) and / or regulate the translation of the encoded polypeptide.

[0294] As used herein, the term "inverted terminal repeat" or "ITR" refers to a nucleic acid sequence located at the 5' and / or 3' end of the ssDNA vector disclosed herein, which comprises at least one stem-loop structure comprising a partial duplex and at least one loop. According to some embodiments, the ITR can be an artificial sequence (e.g., does not contain sequences derived from a virus). The ITR can also comprise one stem-loop structure (e.g., a "hairpin") or more than one stem-loop structure. For example, the ITR can comprise two stem-loop structures (e.g., a "hammerhead," "dogbone," or "dumbbell"), three stem-loop structures (e.g., a "cross"), or a more complex structure. The ITR can comprise an aptamer sequence or one or more chemical modifications.

[0295] According to some embodiments, "ITR" can be artificially synthesized using a set of oligonucleotides comprising one or more desired functional sequences (e.g., palindromic sequences). The ITR sequence can be an artificial AAV ITR, an artificial non-AAV ITR, or an ITR physically derived from a viral AAV ITR (e.g., an ITR fragment removed from the viral genome). For example, ITR can be derived from the Parvoviridae family, which covers parvoviruses and dependent viruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19), or an SV40 hairpin acting as an SV40 origin of replication can be used as an ITR, which can be further modified by truncation, substitution, deletion, insertion, and / or addition. The Parvoviridae family of viruses consists of two subfamilies: the Parvovirinae family that infects vertebrates and the Densovirinae family that infects invertebrates. The genus Dependaviridae is a family of viruses that includes adeno-associated viruses (AAV), which are capable of replicating in vertebrate hosts, including but not limited to humans, primates, cattle, dogs, horses, and sheep species. Generally speaking, ITR sequences can be derived not only from AAV, but also from parvoviruses, lentiviruses, goose viruses, B19, in configurations of wild-type, "dog bone" and "dumbbell-shaped", symmetrical or even asymmetrical ITR orientations. Although ITRs are typically present at both the 5' and 3' ends of AAV vectors, in single-stranded DNA (ssDNA) molecules, ITRs can only be present at one end of a linear vector. For example, ITRs can only be present at the 5' end. In some other cases, ITRs can only be present at the 3' end in single-stranded DNA (ssDNA) molecules. For convenience herein, the ITR located 5' (upstream) of the expression cassette in the single-stranded DNA (ssDNA) molecule is referred to as the "5' ITR" or "left ITR", and the ITR located 3' (downstream) of the expression cassette in the single-stranded DNA (ssDNA) molecule is referred to as the "3' ITR" or "right ITR".

[0296] As used herein, "wild-type ITR" or "WT-ITR" refers to a sequence of an ITR sequence naturally occurring in the AAV genome or other dependent virus that retains, for example, Rep binding activity and Rep cleavage ability. Due to the degeneracy or drift of the genetic code, the nucleotide sequence of the WT-ITR from any AAV serotype may differ slightly from the typical naturally occurring sequence, and therefore, the WT-ITR sequences contemplated for use herein include those resulting from naturally occurring variations (e.g., replication errors).

[0297] As used herein, the term "substantially symmetrical WT-ITR" or "substantially symmetrical WT-ITR pair" refers to a pair of WT-ITRs within a synthetic AAV vector that are both wild-type ITRs with reverse complementary sequences over their entire length. For example, even if an ITR has one or more nucleotides that deviate from a canonical, naturally occurring canonical sequence, the ITR can be considered a wild-type sequence as long as these changes do not affect the physical and functional properties and overall three-dimensional structure (secondary and tertiary structure) of the sequence. In some aspects, the deviated nucleotides represent conservative sequence changes. As a non-limiting example, a sequence has at least 95%, 96%, 97%, 98% or 99% sequence identity to a typical sequence (as measured, for example, using BLAST under default settings) and also has a symmetrical three-dimensional spatial organization with another WT-ITR such that its 3D structure has the same shape in geometric space. Substantially symmetrical WT-ITRs have identical A, C-C' and B-B' loops in 3D space. Substantially symmetrical WT-ITRs can be functionally confirmed to be WT by confirming that they have an operable Rep binding site (RBE or RBE') and a terminal resolution site (TRS) that are paired with the appropriate Rep protein.Other functions can optionally be tested, including transgene expression under permissive conditions.

[0298] As used herein, the phrases "modified ITR" or "mod-ITR" or "mutant ITR" are used interchangeably and refer to an ITR that has a mutation in at least one or more nucleotides compared to a WT-ITR from the same serotype. The mutation can result in a change in one or more of the A, C, C', B, or B' regions of the ITR and can result in a change in the three-dimensional organization (i.e., its 3D structure in geometric space) compared to the 3D organization of the WT-ITR of the same serotype.

[0299] As used herein, the term "asymmetric ITR", also referred to as an "asymmetric ITR pair", refers to a pair of ITRs within a single synthetic AAV genome that are not reverse complementary over their full length. As a non-limiting example, an asymmetric ITR does not have a symmetrical three-dimensional spatial organization with its cognate ITR, such that its 3D structure has a different shape in geometric space. In other words, the asymmetric ITR pair has a different overall geometric structure, i.e., it has a different A, C-C' and B-B' loop organization in 3D space (e.g., one ITR may have a short C-C' arm and / or a short 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 in the asymmetric ITR pair may be a wild-type AAV ITR sequence, and the other ITR is a modified ITR as defined herein (e.g., a non-wild-type or synthetic ITR sequence). In another embodiment, neither ITR in the asymmetric ITR pair is a wild-type AAV sequence, and the two ITRs are modified ITRs having different shapes in geometric space (i.e., different overall geometric structures). In some embodiments, one mod-ITR in the asymmetric ITR pair can have a short C-C' arm, and the other ITR can have a different modification (e.g., a single arm or a short B-B' arm, etc.) such that they have a different three-dimensional spatial organization than the homologous asymmetric mod-ITR.

[0300] As used herein, the term "symmetrical ITR" refers to a pair of ITRs within a single-stranded AAV genome that are mutated or modified relative to the wild-type dependent viral ITR sequence and are reverse complementary over their entire length. Neither of these two ITRs is a wild-type ITR AAV2 sequence (i.e., it is a modified ITR, also referred to as a mutant ITR), and differs in sequence from the wild-type ITR due to the addition, deletion, substitution, truncation or point mutation of nucleotides. For convenience herein, the ITR located 5' (upstream) of the expression cassette in the synthetic AAV vector is referred to as the "5' ITR" or "left ITR", and the ITR located 3' (downstream) of the expression cassette in the synthetic AAV vector is referred to as the "3' ITR" or "right ITR".

[0301] As used herein, the term "substantially symmetrical modified ITR" or "substantially symmetrical mod-ITR pair" refers to a pair of modified ITRs within a synthetic AAV that have reverse complementary sequences throughout their entire length. For example, even if a modified ITR has some nucleotide sequence that deviates from the reverse complementary sequence, it can be considered to be substantially symmetrical as long as these changes do not affect the properties and overall shape. As a non-limiting example, a sequence has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a typical sequence (as measured using BLAST at default settings) and also has a symmetrical three-dimensional spatial organization with its homologous modified ITR, such that its 3D structure has the same shape in geometric space. In other words, a substantially symmetrical modified ITR pair has the same A, C-C' and B-B' loops organized in 3D space. In some embodiments, the ITRs from a mod-ITR pair can have different reverse complementary nucleotide sequences but still have the same symmetrical three-dimensional spatial organization, i.e., both ITRs have mutations that produce the same overall 3D shape. For example, one ITR in a mod-ITR pair (e.g., a 5' ITR) can be from one serotype, while the other ITR (e.g., a 3' ITR) can be from a different serotype, but both can have the same corresponding mutation (e.g., if the 5' ITR has a deletion in the C region, then the homologous modified 3' ITR from a different serotype also has a deletion at the corresponding position in the C' region) such that the modified ITR pair has the same symmetrical three-dimensional spatial organization. In such embodiments, each ITR in the modified ITR pair can be from a different serotype (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), such as a combination of AAV2 and AAV6, wherein the modification in one ITR is reflected in the corresponding position in the homologous ITR from the different serotype. According to some embodiments, a substantially symmetrical modified ITR pair refers to a pair of modified ITRs (mod-ITRs) as long as the difference in nucleotide sequence between the ITRs does not affect the properties or overall shape and they have substantially the same shape in 3D space. As non-limiting examples, the mod-ITR has at least 95%, 96%, 97%, 98% or 99% sequence identity with a typical mod-ITR as determined by standard methods well known in the art, such as BLAST (Basic Local Alignment Search Tool) or BLASTN under default settings, and also has a symmetrical three-dimensional organization such that their 3D structures have the same shape in geometric space.A substantially symmetric mod-ITR pair has identical A, C-C', and BB' loops in 3D space. For example, if a modified ITR in a substantially symmetric mod-ITR pair lacks the C-C' arm, then the corresponding homologous mod-ITR lacks the C-C' loop and also has a similar 3D structure with the remaining A and BB' loops being identically shaped in the geometric space of its cognate mod-ITR.

[0302] As used herein, the term "flanking" refers to the relative position of one nucleic acid sequence relative to another nucleic acid sequence. Typically, in the sequence ABC, B is flanked by A and C. The same is true for the arrangement AxBxC. Thus, a flanking sequence precedes or follows the flanking sequence, but is not necessarily adjacent to or immediately adjacent to the flanking sequence. According to some embodiments, the term flanking refers to the terminal repeats at each end of a linear single-stranded synthetic AAV vector.

[0303] As used herein, the term "spacer" refers to the intermediate sequence of the functional element in the separation vector or genome. In certain embodiments, the AAV spacer is maintained at a distance desired for optimal functionality for two functional elements. In certain embodiments, the spacer provides or increases the gene stability of the vector or genome. In certain embodiments, the spacer promotes the ready gene manipulation of the genome by providing the gap of the base pair of the appropriate position of the cloning site and the design number. For example, in some aspects, the oligonucleotide " polylinker " or " multiple cloning site " containing some restriction endonuclease sites or the non-open reading frame sequence designed to not have known protein (for example, transcription factor) binding site can be positioned in the vector or genome to separate cis-acting factors, for example, 6 aggressiveness, 12 aggressiveness, 18 aggressiveness, 24 aggressiveness, 48 ​​aggressiveness, 86 aggressiveness, 176 aggressiveness etc. are inserted into for example, between the end resolution site and the upstream transcriptional regulatory element, as in AAV vector or genome.

[0304] As used herein, the terms "Rep binding site" ("RBS") and "Rep binding element" ("RBE") are used interchangeably and refer to a binding site of a Rep protein (e.g., AAV Rep 78 or AAV Rep 68) that, upon binding of the Rep protein, allows the Rep protein to exert its site-specific endonuclease activity on the sequence into which the RBS is incorporated. The RBS sequence and its reverse complement together form a single RBS. RBS sequences are well known in the art and include, for example, 5'-GCGCGCTCGCTCGCTC-3', which is an RBS sequence identified in AAV2.

[0305] As used herein, the terms "terminal resolution site" and "TRS" are used interchangeably herein and refer to a region in which Rep forms a tyrosine-phosphodiester bond with a 5' thymidine, generating a 3'-OH group that serves as a substrate for DNA extension by a DNA polymerase, such as DNA pol δ or DNA pol ε. Alternatively, the Rep-thymidine complex can participate in a ligand-ligation reaction.

[0306] As used herein, the terms "sense" and "antisense" refer to the orientation of a structural element on a polynucleotide. The sense and antisense versions of the element are reverse complements of each other.

[0307] As used herein, the terms "synthetic AAV vector" and "synthetic production of AAV vectors" refer to AAV vectors and methods for their synthetic production in a completely cell-free environment.

[0308] As used herein, the phrase "effective amount" or "therapeutically effective amount" of an active agent or therapeutic agent (such as a therapeutic nucleic acid) is an amount sufficient to produce the desired effect (e.g., inhibiting expression of a target sequence as compared to the expression level detected in the absence of the therapeutic nucleic acid). Suitable assays for measuring expression of a target gene or target sequence include, for example, examining protein or RNA levels using techniques known to those skilled in the art (e.g., dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function), as well as phenotypic assays known to those skilled in the art.

[0309] As used herein, the term "expression" refers to the cellular processes involved in producing RNA and protein, and secreting protein when appropriate, including, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing, where applicable. As used herein, the phrase "expression product" includes RNA transcribed from a gene (e.g., a transgene), as well as polypeptides obtained by translating mRNA transcribed from a gene.

[0310] As used herein, the term "expression vector" refers to a vector that directs the expression of an RNA or polypeptide from a sequence linked to a transcriptional regulatory sequence on the vector. The expressed sequence is usually, but not necessarily, heterologous to the host cell. The expression vector may contain additional elements, for example, the expression vector may have two replication systems, thereby allowing it to be maintained in two organisms, for example, for expression in human cells and for cloning and amplification in a prokaryotic host. The expression vector may be a recombinant vector.

[0311] As used herein, the "helper lipid" refers to a ceramide represented by formula (I), (II), (III) or (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing; or (ii) 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).

[0312] As used herein, the terms "expression cassette" and "expression unit" are used interchangeably and refer to a heterologous DNA sequence operably linked to a promoter or other DNA regulatory sequence sufficient to direct transgene transcription of 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.

[0313] As used herein, the phrases "genetic disease" or "genetic disorder" refer to a disease that is caused, in part or in whole, directly or indirectly, by one or more abnormalities in the genome, including and especially conditions that present from birth. The abnormality can be a mutation, insertion, or deletion in a gene. The abnormality can affect the coding sequence of a gene or its regulatory sequences.

[0314] As used herein, the term "polypeptide" refers to a polymer sequence of amino acids. According to some embodiments, the polypeptide of the present disclosure is an ApoE or ApoB polypeptide. According to some embodiments, the ApoE polypeptide is a functional fragment (or functional portion) of a full-length ApoE polypeptide. According to some embodiments, the ApoE polypeptide is a functional fragment (or functional portion) of a full-length ApoB polypeptide. According to some embodiments, the ApoE polypeptide is 30 amino acids or less in length. According to some embodiments, the ApoB polypeptide is 30 amino acids or less in length.

[0315] As used herein, the term "lipid" refers to a group of organic compounds, including but not limited to fatty acid esters, that are characterized by being insoluble in water but soluble in many organic solvents. They are generally divided into at least three categories: (1) "simple lipids," which include fats and oils as well as waxes; (2) "compound lipids," which include phospholipids and glycolipids; and (3) "derivative lipids," such as steroids.

[0316] The representative examples of phospholipids include but are not limited to phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine and dilinoleoylphosphatidylcholine. Other compounds lacking phosphorus, such as sphingolipids, glycosphingolipid family, diacylglycerol and β-acyloxy acid, are also in the group referred to as amphipathic lipids. In addition, above-mentioned amphipathic lipids can be mixed with other lipids (comprising triglyceride and sterol).

[0317] The term "lipid anchor polymer" or "lipid polymer" or "lipid conjugate" refers to a conjugated lipid that suppresses the aggregation of lipid particles. Such lipid conjugates include, but are not limited to, PEG-lipid conjugates, such as PEG (e.g., PEG-DSG conjugate) coupled with DSG, PEG (e.g., PEG-DSPE conjugate) coupled with DSPE, and PEG (see, e.g., U.S. Patent No. 5,885,613), polyglycerol (PG)-lipid conjugates, such as DODA-PG and mixtures thereof, coupled with ceramide. Examples of PG-lipid conjugates include DODA-PG45. Other examples of POZ-lipid conjugates are described in PCT Publication No. WO 2010 / 006282. PEG, PG or POZ can be directly conjugated to lipids, or can be connected to lipids through a linker moiety. Any linker moiety suitable for coupling PEG, PG or POZ to lipids can be used, including, for example, non-ester-containing linker moieties and ester-containing linker moieties. In certain preferred embodiments, non-ester-containing linker moieties, such as amides or carbamates, are used. The disclosure of each of the above patent documents is incorporated herein by reference in its entirety for all purposes.

[0318] As used herein, the term "lipid-anchored polymer" can be used interchangeably with the term "lipid conjugate" or "lipopolymer" to refer to a molecule comprising a lipid portion covalently linked to a hydrophilic polymer via a linker. Without wishing to be bound by a particular theory, it is believed that the lipid-anchored polymer can inhibit aggregation of LNPs and provide steric stability and extended blood half-life (t 1 / 2). The lipid moiety having a linker conjugated to a hydrophilic polymer (e.g., PEG, PG, or POZ) ("lipid linker" or "linker lipid moiety") includes, but is not limited to, 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-ditrans Oleoyl-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 one of the foregoing. In one embodiment, the lipid-anchoring polymer comprises a linker lipid portion selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, derivatives thereof, and combinations of any one of the foregoing. For example, PEG2000 coupled to DSG is a lipid-anchored polymer PEG2000-DSG (or DSG-PEG2000). PEG coupled to DSPE is a lipid-anchored polymer PEG-DSPE (or DSPE-PEG2000 or DSPE-PEG500). Examples of lipid-anchored PG polymers can include DODA-PG, wherein PG can be a multiunit ranging from about 5 to about 50 PG units.

[0319] As used herein, "lipid encapsulation" refers to providing an active agent or therapeutic agent, such as a lipid particle of a nucleic acid (e.g., ceDNA, ssDNA, or mRNA), that is completely encapsulated, partially encapsulated, or both. In a preferred embodiment, the nucleic acid is completely encapsulated in the lipid particle (e.g., to form a lipid particle containing the nucleic acid).

[0320] 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 agent) to a target site of interest (e.g., a cell, tissue, organ, etc.). In one embodiment, lipid particles of the present disclosure are lipid particles containing nucleic acids, which are typically formed by cationic lipids, non-cationic lipids, and optionally conjugated lipids that prevent particle aggregation. In other preferred embodiments, therapeutic agents such as therapeutic nucleic acids can be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation. In one embodiment, the lipid particle comprises 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.

[0321] According to some embodiments, the average diameter of the lipid particles of the present disclosure is generally about 20 nm to about 75 nm, about 20 nm to about 70 nm, about 25 nm to about 75 nm, about 25 nm to about 70 nm, about 30 nm to about 75 nm, about 30 nm to about 70 nm, about 35 nm to about 75 nm, about 35 nm to about 70 nm, about 40 nm to about 75 nm, about 40 nm to about 70 nm, about 45 nm to about 75 nm, about 50 nm to about 75 nm, about 50 nm to about 70 nm, about 60 nm to about 75 nm, about 60 nm to about 70 nm, about 65 nm to about 75 nm, about 65 nm to about 70 nm, about 65 nm to about 80 nm, about 65 nm to about 80 nm, about 65 nm to about 80 nm, about 65 nm to about 80 nm. m to about 85 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, 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).

[0322] Typically, the LNPs of the present disclosure have an average diameter selected to provide the desired therapeutic effect. For example, the average diameter of the LNPs of the present disclosure is compatible with delivery to the target organ, such that the LNPs of the present disclosure are able to diffuse through the fenestrations of the target organ (e.g., liver) or target cell subpopulations (e.g., hepatocytes).

[0323] According to some embodiments, the lipid particles of the present disclosure generally 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.

[0324] As used herein, the term " cationic lipid " refers to any lipid with a positive charge at physiological pH. The cationic lipid in lipid granules can include, for example, one or more cationic lipids, such as 1,2- dilinoleyloxy-N, N- dimethylaminopropane (DLinDMA), 1,2- dihydrolinoleoyloxy-N, N- dimethylaminopropane (DLenDMA), 1,2- bis-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- can cut lipid " or its mixture. In certain embodiments, cationic lipid can also be ionizable lipid, that is, ionizable cationic lipid. The term " cationic lipid " also encompasses lipids with a positive charge at any pH, for example, comprising a lipid of a quaternary amine group, i.e., quaternary lipid. Any of the cationic lipids described herein containing primary, secondary, or tertiary amine groups can be converted to a quaternary lipid, for example, by treatment with chloromethane (CH 3 Cl) in acetonitrile (CH 3 CN) and chloroform (CHCl 3 ).

[0325] As used herein, the term "ionizable lipid" refers to a lipid having at least one protonable or deprotonable group, e.g., a cationic lipid, such that the lipid is positively charged at a pH equal to or lower than physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably equal to or higher than physiological pH. One of ordinary skill in the art will appreciate that the addition or removal of protons as a function of pH is a balancing process, and reference to charged or neutral lipids refers to the properties of the dominant species, without requiring that all lipids exist in a charged or neutral form. Typically, the pKa of the protonable group of an ionizable lipid is in the range of about 4 to about 7. In some embodiments, the ionizable lipid may include a "cleavable lipid" or "SS-cleavable lipid."

[0326] As used herein, the term "neutral lipid" refers to any number of lipid species that exist as uncharged or neutral zwitterionic species at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, cephalins, cholesterol, cerebrosides, and diacylglycerols.

[0327] As used herein, the term "cleavable lipid" or "SS-cleavable lipid" refers to an ionizable lipid comprising a disulfide cleavable unit. The cleavable lipid may include a cleavable disulfide bond ("ss") containing a lipid-like material comprising a pH-sensitive amine (e.g., a tertiary amine) and a self-degradable phenyl ester. For example, the SS-cleavable lipid may be a ss-OP lipid (

[0328] SS-OP), ss-M lipid ( SS-M), ss-E lipids ( SS-E), ss-EC lipids ( SS-EC), ss-LC lipids ( SS-LC), ss-OC lipids ( SS-OC) and ss-PalmE lipids (see, e.g., Formulas I-IV), or lipids described 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. Additional examples of cleavable lipids are described in U.S. Patent Nos. 9,708,628 and 10,385,030, the entire contents of which are incorporated herein by reference. In one embodiment, the cleavable lipid comprises a tertiary amine that responds to acidic compartments, e.g., endosomes or lysosomes for membrane destabilization, and a disulfide bond that can be cleaved in a reducing environment (e.g., 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.

[0329] As used herein, the term "organic lipid solution" refers to a composition comprising entirely or partially an organic solvent with lipids.

[0330] As used herein, the term "liposome" refers to a lipid molecule assembled into a spherical configuration that encapsulates an internal aqueous volume isolated from an aqueous exterior. A liposome is a vesicle having at least one lipid bilayer. In the context of pharmaceutical research and development, liposomes are typically used as carriers for drug / therapeutic agent delivery. Liposomes work by fusing with cell membranes and repositioning their lipid structure to deliver drugs or active pharmaceutical ingredients. The liposome compositions used for this type of delivery are often composed of phospholipids (specifically, compounds with phosphatidylcholine groups), but these compositions can also include other lipids.

[0331] As used herein, the term "nucleic acid" refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in single-stranded or double-stranded form and includes DNA, RNA, and hybrids thereof. DNA can be in the form of, for example, an antisense molecule, plasmid DNA, DNA-DNA duplex, precondensed DNA, PCR product, vector (P1, PAC, BAC, YAC, artificial chromosome), expression cassette, chimeric sequence, chromosomal DNA, or derivatives and combinations of these groups. DNA can be in the form of minicircles, plasmids, bacmids, minigenes, ministring DNA (linear covalently closed DNA vectors), end-blocked linear duplex DNA (CELiD or ceDNA), single-stranded DNA (ssDNA), doggybone TM DNA, dumbbell-shaped DNA, simple immunologically defined gene expression (MIDGE) vector, viral vector or non-viral vector. RNA can be in the form of small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), messenger RNA (mRNA), rRNA, tRNA, gRNA, viral RNA (vRNA) and combinations thereof. Nucleic acid includes nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring and non-naturally occurring, and have binding properties similar to reference nucleic acids. Examples of such analogs and / or modified residues include but are not limited to phosphorothioates, diaminophosphorothioate morpholino oligomers (morpholinos), phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methyl ribonucleotides, locked nucleic acids (LNAs). TM) and peptide nucleic acids (PNA). Unless otherwise limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequences explicitly indicated.

[0332] As used herein, the phrases "nucleic acid therapeutic," "therapeutic nucleic acid," and "TNA" are used interchangeably and refer to any modality of therapy that uses nucleic acids as the active ingredient of a therapeutic agent for treating a disease or condition. As used herein, these phrases refer to RNA-based therapeutics and DNA-based therapeutics. Non-limiting examples of RNA-based therapeutics include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), or guide RNA (gRNA). Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigenes, viral DNA (e.g., lentiviral or AAV genomes) or non-viral synthetic DNA vectors, end-blocked linear duplex DNA (ceDNA / CELiD), single-stranded DNA (ssDNA), plasmids, bacmids, DOGGYBONE TM DNA vectors, minimalist immunologically defined gene expression (MIDGE) vectors, non-viral ministring DNA vectors (linear covalently enclosed DNA vectors), or dumbbell-shaped minimal DNA vectors ("dumbbell DNA").

[0333] As used herein, a "nucleotide" contains the sugar deoxynucleoside (DNA) or ribose (RNA), a base, and a phosphate group. The nucleotides are linked together by the phosphate groups.

[0334] As used herein, the term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, 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 agent approved by U.S. federal regulatory agencies or listed in the U.S. Pharmacopoeia for use in animals (including humans), as well as any carrier or diluent that does not cause significant irritation to the subject and does not abrogate the biological activity and properties of the administered compound.

[0335] As used herein, the terms "gap" and "nick" are used interchangeably and refer to an interruption in the synthetic DNA vector of the present disclosure that produces an extension of the single-stranded DNA portion in an otherwise double-stranded ceDNA. In one strand of the duplex DNA, the length of the gap can be 1 nucleotide (nt) to 100 nucleotides (nt) long. Typical gaps designed and produced by the methods described herein and the length of the synthetic vectors produced by the methods can be, for example, 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 11 bp, 12 bp, 13 bp, 14 bp, 15 bp, 16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp, 26 bp, 27 bp, 28 bp, 29 bp, 30 bp, 31 bp, 32 bp, 33 bp, 34 bp, 35 bp, 36 bp, 37 bp, 38 bp, 39 bp, 40 bp, 41 bp, 42 bp, 43 bp, 44 bp, 45 bp, 46 bp, 47 bp, 48 bp, 49 bp, 50 bp, 51 bp, 52 bp, 53 bp, 54 bp, 55 bp, 56 bp, 57 bp, 58 bp, 59 bp, 60 bp, 61 bp, 62 bp, 63 bp, 64 bp, 65 bp, 66 bp, bp, 57bp, 58bp, 59bp or 60bp. The length of the gaps exemplified in the present disclosure can be 1 nt to 10 nt long, 1 nt to 20 nt long, or 1 nt to 30 nt long.

[0336] As used herein, the term "nick" refers to a discontinuity in a double-stranded DNA molecule in which the phosphodiester bond between adjacent nucleotides of one strand is absent, typically by damage or enzymatic action. It will be understood that one or more nicks allow for release of the torsion in the strand during DNA replication, and nicks are also believed to play a role in facilitating the binding of the transcription machinery.

[0337] "Receptor" means a polypeptide or portion thereof present on a cell membrane that selectively binds to one or more ligands. As used herein, the term "receptor" is intended to encompass the entire receptor or its ligand-binding portion. These portions of the receptor particularly include those regions sufficient for specific binding of the ligand to occur.

[0338] As used herein, the term "subject" refers to a human or animal to which treatment with a therapeutic nucleic acid according to the present disclosure is provided, including preventive and therapeutic treatment. Generally speaking, an animal is a vertebrate, such as, but not limited to, a primate, a rodent, livestock, or a wild animal. Primates include, but are not limited to, chimpanzees, crab-eating macaques, spider monkeys, and macaques, for example, rhesus macaques. Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Livestock and hunting animals include, but are not limited to, cattle, horses, pigs, deer, bison, buffaloes, cat 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, for example, a primate or a human. The subject can be male (male) or female (female). In addition, the subject can be an infant or a child. In certain embodiments, the subject can be a newborn or unborn subject, for example, while the subject is still in the womb. Preferably, the subject is a mammal. Mammal can be people, non-human primate, mouse, rat, dog, cat, horse or cattle, but is not limited to these examples.Mammals other than people can be advantageously used as the experimenter of the animal model representing disease and illness.In addition, method and composition described herein can be used for domestic animals and / or pets.Human experimenter can be any age, sex, race or ethnic group, for example, Caucasian (white race), Asian, African, black race, African American, African European, Hispanic, Middle Eastern etc.In certain embodiments, experimenter can be another experimenter in patient or clinical setting.In certain embodiments, experimenter has been treated.In certain embodiments, experimenter is embryo, fetus, newborn, baby, child, teenager or adult.In certain embodiments, experimenter is human fetus, human newborn, human baby, human child, human teenager or human adult.In certain embodiments, experimenter is animal embryo, or non-human embryo or non-human primate embryo.In certain embodiments, experimenter is human embryo.

[0339] As used herein, the phrase "a subject in need thereof" refers to a subject who (i) is to be administered a ceDNA lipid particle according to the disclosure as described (or a pharmaceutical composition comprising a ceDNA lipid particle), (ii) is currently receiving a ceDNA lipid particle according to the disclosure as described (or a pharmaceutical composition comprising a ceDNA lipid particle), or (iii) has already received a ceDNA lipid particle according to the disclosure as described (or a pharmaceutical composition comprising a ceDNA lipid particle), unless the context and usage of the phrase indicates otherwise.

[0340] As used herein, the terms "suppress," "reduce," "interfere with," "inhibit," and / or "reduce" (and similar terms) generally refer to the act of directly or indirectly decreasing the concentration, level, function, activity, or behavior relative to natural, expected, or average conditions, or relative to control conditions.

[0341] As used herein, the term "systemic delivery" refers to the delivery of lipid particles, which results in extensive biodistribution of active agents such as interfering RNA (e.g., siRNA) mRNA, ceDNA or ssDNA in vivo. Some administration techniques can result in the systemic delivery of certain medicaments rather than other medicaments. Systemic delivery means exposing a useful amount (preferably, a therapeutic amount) of the medicament to most parts of the body. In order to obtain extensive biodistribution, blood life is generally required so that the medicament will not be rapidly degraded or cleared (such as by first-pass organs (liver, lungs, etc.) or by fast, non-specific cell binding) before arriving at the disease site at the distal end of the administration site. The systemic delivery of LNP can be carried out by any method known in the art (including, for example, intravenous, intra-arterial, subcutaneous and intraperitoneal). In a preferred embodiment, the systemic delivery of LNP is by intravenous delivery.

[0342] As used herein, the term "effective amount" can be used interchangeably with the terms "therapeutic amount," "therapeutically effective amount," "effective amount," or "pharmaceutically effective amount" of an active agent (e.g., a ceDNA as described herein) to refer 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, protein or RNA levels using techniques known to those skilled in the art (e.g., 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 various factors, including the type of injury, the patient's age, weight, sex, medical condition, severity of the condition, route of administration, and the specific active agent employed. Thus, dosing regimens can vary widely but can be routinely determined by a physician using standard methods. Additionally, the terms "effective amount," "therapeutic amount," "therapeutically effective amount," and "pharmaceutically effective amount" include the described prophylactic or preventative amount of the composition of the present invention. In the described control or preventive applications of the present invention, a pharmaceutical composition or drug is administered to a patient susceptible to or otherwise at risk of developing a disease, disorder or condition in an amount sufficient to eliminate or reduce the risk of a disease, disorder or condition, reduce the severity of the disease, disorder or condition, or delay the onset of the disease, disorder or condition, including biochemical, histological and / or behavioral symptoms of the disease, disorder or condition; its complications; and intermediate pathological phenotypes presented during the development of the disease, disorder or condition. It is generally preferred to use the maximum dose, that is, the highest safe dose according to some medical judgment. The terms "dose / dosage" are used interchangeably herein. In one aspect of any one of the aspects or embodiments herein, "therapeutic amount," "therapeutically effective amount," and "pharmaceutically effective amount" refer to non-control or non-preventive applications.

[0343] As used herein, the term "therapeutic effect" refers to the result of treatment, which is judged to be desirable and beneficial. Therapeutic effect can directly or indirectly include the suppression, reduction or elimination of disease manifestations. Therapeutic effect can also directly or indirectly include the suppression, reduction or elimination of the progression of disease manifestations.

[0344] For any therapeutic agent described herein, a therapeutically effective amount can be initially determined based on preliminary in vitro studies and / or animal models. A therapeutically effective dose can also be determined based on human data. The dosage administered can be adjusted based on the relative bioavailability and the efficacy of the compound administered. It is within the capabilities of ordinary technicians to adjust the dosage to achieve maximum efficacy based on the above methods and other well-known methods. The general principles for determining therapeutic effectiveness are summarized below, which can be found in Chapter 1 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th edition, McGraw-Hill (New York) (2001), which is incorporated herein by reference.

[0345] Pharmacokinetic principles provide the basis for modifying the dosage regimen to obtain the desired degree of therapeutic efficacy with minimal unacceptable side effects. Additional guidance for dosage modifications can be obtained where the plasma concentration of the drug can be measured and related to the therapeutic window.

[0346] As used herein, the terms "treat," "treating," and / or "treatment" include eliminating, inhibiting, slowing, or reversing the progression of a condition, improving the clinical symptoms of a condition, or preventing the appearance of clinical symptoms of a condition, obtaining a beneficial or desired clinical outcome. Treatment further refers to achieving one or more of the following: (a) reducing the severity of the condition; (b) limiting the development of symptoms characteristic of the condition being treated; (c) limiting the worsening of symptoms characteristic of the condition being treated; (d) limiting the recurrence of the condition in patients who previously had the condition; and (e) limiting the recurrence of symptoms in patients who were previously asymptomatic for the condition. In one aspect of any of the aspects or embodiments herein, the terms "treat," "treating," and / or "treatment" include eliminating, inhibiting, slowing, or reversing the progression of a condition, or improving the clinical symptoms of a condition.

[0347] Beneficial or desired clinical results, such as pharmacological and / or physiological effects, include, but are not limited to: preventing the development of a disease, disorder or condition in a subject who may be susceptible to the disease, disorder or condition but who does not yet experience or exhibit symptoms of the disease (prophylactic or preventive treatment); alleviating the symptoms of the disease, disorder or condition; reducing the extent of the disease, disorder or condition; stabilizing the disease, disorder or condition (i.e., not worsening); preventing the spread of the disease, disorder or condition; delaying or slowing the progression of the disease, disorder or condition; ameliorating or palliating the disease, disorder or condition; and combinations thereof, and prolonging survival as compared to expected survival if not receiving treatment.

[0348] As used herein, the term "combination therapy" refers to a treatment regimen for a clinical indication comprising two or more therapeutic agents. Therefore, the term refers to a treatment regimen, in which a first therapy comprising a first composition (e.g., active ingredient) is combined with a second therapy comprising a second composition (active ingredient) and is administered to a patient, for the purpose of treating identical or overlapping diseases or clinical symptoms. Both the first composition and the second composition can act on identical cell targets or discrete cell targets. In the context of combination therapy, phrase "combination" means that in a subject receiving combination therapy, the therapeutic effect of the first therapy is temporarily and / or spatially overlapped with the therapeutic effect of the second therapy. Therefore, combination therapy can be formulated as a single formulation for simultaneous administration, or formulated as a separate preparation for continuous administration of therapy.

[0349] As used herein, the term "alkyl" refers to a saturated monovalent hydrocarbon group (i.e., C 1-20 Alkyl). "Monovalent" means that the alkyl group has one point of attachment to the rest of the molecule. In one embodiment, the alkyl group has 1 to 12 carbon atoms (i.e., C 1-12 Alkyl) or 1 to 10 carbon atoms (ie C 1-10 In one embodiment, the alkyl group has 1 to 8 carbon atoms (i.e., C 1-8 alkyl), 1 to 7 carbon atoms (i.e., C 1-7 alkyl), 1 to 6 carbon atoms (i.e., C 1-6 alkyl), 1 to 4 carbon atoms (i.e., C 1-4 Alkyl) or 1 to 3 carbon atoms (ie C 1-3Examples include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and the like. Straight or branched chain alkyl groups, such as “straight or branched chain C 1-6 Alkyl", "straight or branched chain C 1-4 Alkyl", or "straight or branched C 1-3 "Alkyl" means a saturated monovalent hydrocarbon group, which may be straight or branched. As used herein, the term "straight chain" refers to an aliphatic hydrocarbon chain, meaning that the chain is unbranched.

[0350] As used herein, the term "alkylene" refers to a saturated divalent hydrocarbon group (i.e., C 1-20 Alkylene), examples of which are not limited to saturated divalent hydrocarbon groups with the same core structure as the alkyl groups exemplified above. "Divalent" means that the alkylene group has two points of attachment to the rest of the molecule. In one embodiment, the alkylene group has 1 to 12 carbon atoms (i.e., C 1-12 Alkylene) or 1 to 10 carbon atoms (ie C 1-10 In one embodiment, the alkylene group has 1 to 8 carbon atoms (i.e., C 1-8 Alkylene), 1 to 7 carbon atoms (i.e., C 1-7 Alkylene), 1 to 6 carbon atoms (i.e., C 1-6 alkylene), 1 to 4 carbon atoms (i.e., C 1-4 Alkylene), 1 to 3 carbon atoms (i.e., C 1-3 Alkylene), ethylene or methylene. Straight chain or branched chain alkylene, such as "straight chain or branched chain C 1-6 Alkylene", "straight or branched chain C 1-4 Alkylene", or "straight or branched C 1-3 "Alkylene" means a saturated divalent hydrocarbon group which is straight or branched.

[0351] The term "alkenyl" refers to a straight or branched aliphatic hydrocarbon group having one or more (e.g., one or two) carbon-carbon double bonds, wherein the alkenyl group includes groups having "cis" and "trans" orientations, or by alternative nomenclature, "E" and "Z" orientations.

[0352] As used herein, "alkenylene" refers to an aliphatic divalent hydrocarbon group of 2 to 20 carbon atoms (i.e., C 2-20 "Alkenylene"), wherein the alkenylene includes groups having "cis" and "trans" orientations, or by alternative nomenclature, "E" and "Z" orientations. "Divalent" means that the alkenylene has two points of attachment to the rest of the molecule. In one embodiment, the alkenylene has 2 to 12 carbon atoms (i.e., C 2-16 Alkenyl), 2 to 10 carbon atoms (i.e., C 2-10 In one embodiment, the alkenylene group has 2 to 4 carbon atoms (C 2-4 Examples include, but are not limited to, ethylenylene or vinylene (-CH=CH-), allyl (-CH2CH=CH-), etc. Straight-chain or branched alkenylene, such as "straight-chain or branched C 2-6 Alkenylene", "straight or branched chain C 2-4 Alkenylene", or "straight or branched C 2-3 "Alkenylene" means an unsaturated divalent hydrocarbon group which is straight or branched.

[0353] As used herein, "cycloalkylene" refers to a divalent saturated carbocyclic group having 3 to 12 carbon atoms as a monocycle or 7 to 12 carbon atoms as a bicyclic ring. "Divalent" means that the cycloalkylene and the rest of the molecule have two points of attachment. In one embodiment, the cycloalkylene is a 3- to 7-membered monocycle or a 3- to 6-membered monocycle. Examples of monocyclic cycloalkyls include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, cyclononylene, cyclodecylene, cycloundecylene, cyclododecylene, etc. In one embodiment, the cycloalkylene is cyclopropylene.

[0354] The terms "heterocycle", "heterocyclyl", heterocyclic and "heterocyclic ring" are used interchangeably herein and refer to a cyclic group containing at least one N atom, having a heteroatom and optionally 1-3 additional heteroatoms selected from N and S, and being non-aromatic (i.e., partially or completely saturated). It can be monocyclic or bicyclic (bridged or fused). Examples of heterocycles include, but are not limited to, aziridinyl, diaziridinyl, thiaaziridinyl, azetidinyl, diazetidinyl, triazetidinyl, thiadiazetidinyl, thiazetidinyl, pyrrolidinyl, pyrazolidinyl, imidazolinyl, isothiazolidinyl, thiazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl, azepanyl, azooctanyl and the like. The heterocycle contains 1 to 4 heteroatoms selected from N and S that may be the same or different. 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" means a group having 4 to 8 atoms (including 1 to 4 heteroatoms selected from N and S, or 1 to 3 N atoms, or 1 or 2 N atoms, or 1 N atom) arranged in a monocyclic ring. "5- or 6-membered heterocyclyl" means a group having 5 or 6 atoms (including 1 to 4 heteroatoms selected from N and S, or 1 to 3 N atoms, or 1 or 2 N atoms, or 1 N atom) arranged in a monocyclic ring. The term "heterocycle" is intended to include all possible isomeric forms. Heterocycles are described in Paquette, Leo A., Principles of Modern Heterocyclic Chemistry (WA Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; The Chemistry of Heterocyclic Compounds, A Series of Monographs (John Wiley & Sons, New York, 1950-present), particularly Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82: 5566. Where possible, the heterocyclic group can be carbon (carbon-bonded) or nitrogen (nitrogen-bonded) attached to the rest of the molecule.

[0355] If a group is described as "optionally substituted," the group may be (1) unsubstituted or (2) substituted. If a carbon of a group is described as being optionally substituted with one or more of a list of substituents, one or more of the hydrogen atoms on the carbon (if any) may be substituted individually and / or collectively with independently selected optional substituents.

[0356] Suitable substituents for alkyl, alkylene, alkenylene, cycloalkylene and heterocyclyl groups 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 groups having 1 to 10 carbon atoms, aryl, heteroaryl, heterocyclyl, halogen, guanidinyl [-NH(C=NH)NH2], -OR 100 NR 101 R 102 、-NO2、-NR 101 COR 102 、-SR 100 、-SOR 101 Sulfoxide, -SO2R 101 Sulfone, sulfonate -SO3M, sulfate -OSO3M, -SO2NR 101 R 102 Sulfonamide, cyano, azide, -COR 101 、-OCOR 101 、-OCONR 101 R 102 and polyethylene glycol units (-OCH2CH2) n R 101 , where M is H or a cation (such as Na + or K + );R 101 、R 102 and R 103 Each independently selected from H, a linear, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms, a polyethylene glycol unit (-OCH2CH2) n -R 104 (wherein n is an integer from 1 to 24), an aryl group having 6 to 10 carbon atoms, a heterocyclic ring having 3 to 10 carbon atoms, and a heteroaryl group having 5 to 10 carbon atoms; and R 104 is H or a straight or branched chain alkyl group having 1 to 4 carbon atoms, wherein R 100 、R 101 、R 102 、R 103 and R 104The alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclic groups in the groups represented by are optionally substituted by one or more (e.g., 2, 3, 4, 5, 6 or more) substituents independently selected from halogen, -OH, -CN, -NO2 and unsubstituted straight or branched alkyl groups having 1 to 4 carbon atoms. Preferably, the substituents of the optionally substituted alkyl, alkylene, alkenylene, cycloalkylene and heterocyclic groups described above are selected from the group consisting of halogen, -CN, -NR 101 R 102 、-CF3、-OR 100 , aryl, heteroaryl, heterocyclic, -SR 101 、-SOR 101 、-SO2R 101 Alternatively, suitable substituents are selected from the group consisting of halogen, -OH, -NO2, -CN, C 1-4 Alkyl, -OR 100 NR 101 R 102 、-NR 101 COR 102 、-SR 100 、-SO2R 101 、-SO2NR 101 R 102 、-COR 101 、-OCOR 101 and-OCONR 101 R 102 , where R 100 、R 101 and R 102 Each independently is -H or C 1-4 alkyl.

[0357] As used herein, "halogen" refers to F, Cl, Br, or I. "Cyano" is -CN.

[0358] As used herein, "amine" or "amino" interchangeably refer to a functional group containing a basic nitrogen atom with a lone pair.

[0359] 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, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, gentisates, fumarates, gluconates, glucuronates, sucrose salts, formate, benzoates, glutamate, methanesulfonates (mesylate), ethanesulfonates, benzenesulfonates, p-toluenesulfonates, pamoates (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)), 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 can be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of a pharmaceutically acceptable salt may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0360] The grouping of the alternative elements of the present disclosure disclosed herein or embodiments should not be construed as limiting.Each group member can be mentioned and claimed individually or with any combination of other elements found by other members of the group or this paper.For convenience and / or patentability reasons, one or more members in a group may be included in a group or therefrom deleted.When any such inclusion or deletion occurs, description described in this article is considered to contain the group of modification, thereby meeting the written description of all Markush groups (Markush group) used in the appended claims.

[0361] In some embodiments of any aspect, the disclosure described herein does not relate to processes for cloning humans, processes for modifying the germline genetic identity of humans, the use of human embryos for industrial or commercial purposes, or processes for modifying the genetic identity of animals that may cause suffering to them without any substantial medical benefit to humans or animals, and animals resulting from such processes.

[0362] Additional terms are defined herein within the description of various aspects of the disclosure.

[0363] All patents and other publications cited in this application throughout, including references, granted patents, published patent applications, and co-pending patent applications, are expressly incorporated herein by reference to describe and disclose, for example, methods described in these publications that can be used in combination 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 inventor has no right to advance the disclosure by virtue of prior disclosure or for any other reason. All statements about the dates or contents of these documents are based on information available to the applicant and do not constitute any admission as to the correctness of the dates or contents of these documents.

[0364] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications can be made within the scope of the present disclosure as will be appreciated by those skilled in the relevant art. For example, although method steps or functions are presented in a given order, alternative embodiments can perform functions in a different order or can perform functions substantially simultaneously. The teachings of the present disclosure provided herein can be appropriately applied to other processes or methods. The various embodiments described herein can be combined to provide additional embodiments. If necessary, aspects of the present disclosure can be modified to provide another embodiment of the present disclosure using the compositions, functions, and concepts in the above-mentioned references and applications. In addition, due to considerations of biological functional equivalence, some changes can be made in the protein structure without affecting the type or quantity of the biological effect. These and other changes can be made to the present disclosure based on the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0365] The specific elements of any of the aforementioned embodiments may be combined with or replace elements in other embodiments. In addition, although the advantages associated with certain embodiments of the present disclosure have been described in the context of these embodiments, other embodiments may also exhibit these advantages and not all embodiments must exhibit these advantages to fall within the scope of the present disclosure.

[0366] The technology described herein is further illustrated by the following examples, but these examples should not be construed as further limiting. It should be understood that the present disclosure is not limited in any way to the specific methods, protocols, reagents, etc. described herein and can vary accordingly. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present disclosure, which is defined solely by the claims.

[0367] II. Lipid Nanoparticles (LNPs)

[0368] Provided herein are lipid nanoparticles (LNPs) comprising: a therapeutic nucleic acid (TNA); an ionizable lipid; a structural 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 structural 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 structural 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.

[0369] As used herein, the term "lipid particle" or "lipid nanoparticle" (LNP) refers to a lipid formulation that can be used to deliver a therapeutic agent (such as a therapeutic nucleic acid) to a target site of interest (e.g., a cell, tissue, organ, etc.). In some embodiments, lipid nanoparticles of the present disclosure are typically formed by ionizable lipids (e.g., cationic lipids), sterols (e.g., cholesterol), conjugated lipids (e.g., lipid-anchored polymers) that prevent particle aggregation, and optionally auxiliary lipids (e.g., non-cationic lipids). In some other embodiments, therapeutic agents such as therapeutic nucleic acids (TNAs) can be encapsulated in lipid particles to protect them from degradation. In yet other embodiments, immunosuppressants can be optionally included in nucleic acid-containing lipid nanoparticles. In one embodiment, the lipid particle comprises nucleic acid (e.g., ceDNA, ssDNA, and / or mRNA). The present disclosure provides LNPs, wherein at least one of the lipids in the lipid-anchored polymer contains 16, 18, or 20 aliphatic carbons to more securely anchor the lipid-anchored polymer to the LNP. In some embodiments, at least one lipid having a lipid-anchored polymer with at least 18 aliphatic carbons can be used to generate stealth LNPs. In another embodiment, at least one lipid having a lipid-anchored polymer with at least 20 aliphatic carbons can be used to generate stealth LNPs.

[0370] According to some embodiments, the lipid nanoparticles of the present disclosure generally have an average diameter 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 75 nm, about 50 nm to about 70 nm, about 60 nm to about 75 nm, about 60 nm to about 75 nm, about 65 nm to about 75 nm.

[0015] The present invention further comprises a size of about 15 nm, 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).

[0371] Typically, the LNPs of the present disclosure have an average diameter selected to provide the desired therapeutic effect. For example, the LNPs of the present disclosure have an average diameter that is compatible with the target organ, such that the LNPs of the present disclosure are able to diffuse through the fenestrations of the target organ (e.g., liver) or target cell subpopulations (e.g., hepatocytes).

[0372] According to some embodiments, the lipid particles of the present disclosure generally 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, less than about 20 nm.

[0373] 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) is present, or a salt or ester thereof, or a deuterated analogue of any of the foregoing. 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) is present, or a salt or ester thereof, or a deuterated analogue of any of the foregoing.

[0374] A. Ionizable lipids

[0375] In some embodiments, the ionizable lipids in the LNPs provided herein are present in an amount of 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%, about 30 ...0 mol% to about 50 mol%, about 25 mol% to about 70 mol%, about 30 mol% to about 55 mol%, about 25 mol% to about 50 mol%, about 25 mol% to about 70 mol%, about 30 mol% to about The invention also provides an amount of the present invention that is present in an amount of 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%.

[0376] In some embodiments, the LNPs provided by the present disclosure comprise ionizable lipids. Exemplary ionizable lipids in the LNPs disclosed herein are disclosed in International Patent Application Nos. WO2015 / 095340, WO2015 / 199952, WO2018 / 011633, WO2017 / 049245, WO2015 / 061467, WO2012 / 040184, WO2012 / 000104, WO2015 / 074085, WO2016 / 081029, WO2017 / 004143, WO2017 / 075531, WO2017 / 117528, WO2011 / 0224 60, WO2013 / 148541, WO2013 / 116126, WO2011 / 153120, WO2012 / 044638, WO2012 / 054365, WO2011 / 090965, WO2013 / 016058, WO2012 / 162210, WO2008 / 042973, WO2010 / 129709, WO2010 / 144740, WO2012 / 099755, WO2013 / 049328, WO2013 / 086322, WO 2013 / 086373, WO2011 / 071860, WO2009 / 132131, WO2010 / 048536, WO2010 / 088537, WO2010 / 054401, WO2010 / 054406, WO2010 / 054405, WO2010 / 054384, WO2012 / 016184, WO2009 / 086558, WO2010 / 042877, WO2011 / 000106, WO2011 / 000107, WO2005 / 1 20152, WO2011 / 141705, WO2013 / 126803, WO2006 / 007712, WO2011 / 038160, WO2005 / 121348, WO2011 / 066651, WO2009 / 127060, WO2011 / 141704, WO2006 / 069782, WO2012 / 031043, WO2013 / 006825, WO2013 / 033563, WO2013 / 089151, WO2017 / 099823,Nos. WO2015 / 095346 and WO2013 / 086354 and U.S. Patent Application Publication Nos. US2016 / 0311759, US2015 / 0376115, US2016 / 0151284, US2017 / 0210697, US2015 / 0140070, US2013 / 0178541, US2013 / 0303587, US2015 / 0141678, US2015 / 0239926, US2016 / 0376224, US2017 / 0119904, and US No. 2012 / 0149894, No. US2015 / 0057373, No. US2013 / 0090372, No. US2013 / 0274523, No. US2013 / 0274504, No. US2013 / 0274504, No. US2009 / 0023673, No. US2012 / 0128760, No. US2010 / 0324120, No. US2014 / 0200257, No. US2015 / 0203446, No. US2018 / 0005363, No. US2014 / 0308304, No. US2013 / 033821 No. 0, No. US2012 / 0101148, No. US2012 / 0027796, No. US2012 / 0058144, No. US2013 / 0323269, No. US2011 / 0117125, No. US2011 / 0256175, No. US2012 / 02 No. 02871, No. US2011 / 0076335, No. US2006 / 0083780, No. US2013 / 0123338, No. US2015 / 0064242, No. US2006 / 0051405, No. US2013 / 0065939, No. US2006 / 0008910, US2003 / 0022649, US2010 / 0130588, US2013 / 0116307, US2010 / 0062967, US2013 / 0202684, US2014 / 0141070, US2014 / 0255472, US2014 / 0039032, US2018 / 0028664, US2016 / 0317458, and US2013 / 0195920, all of which are incorporated herein by reference in their entirety.

[0377] Formula (A)

[0378] In some embodiments, the ionizable lipid in the LNPs of the present disclosure is represented by formula (A):

[0379]

[0380] or a pharmaceutically acceptable salt thereof, wherein:

[0381] R 1 and R 1 Each is independently C 1-3 alkylene;

[0382] R 2 and R 2' Each independently is a straight chain or branched C 1-6 Alkylene or C 3-6 cycloalkylene;

[0383] R 3 and R 3' are each independently an optionally substituted C 1-6 Alkyl or optionally substituted C 3-6 Cycloalkyl;

[0384] Or alternatively, when R 2 It is a branched chain C 1-6 Alkylene and when R 3 It is C 1-6 When alkyl, R 2 and R 3 Together with its central nitrogen atom, it forms a 4- to 8-membered heterocyclic group;

[0385] Or alternatively, when R 2' It is a branched chain C 1-6 Alkylene and when R 3' It is C 1-6 When alkyl, R 2' and R 3' Together with its central nitrogen atom, it forms a 4- to 8-membered heterocyclic group;

[0386] R 4 and R 4' are each independently -CH, -CH2CH or -(CH2)2CH;

[0387] R 5 and R 5' are independently hydrogen, C 1-20 Alkylene or C 2-20 alkenylene;

[0388] R 6 and R 6' Each occurrence is independently C 1-20 Alkylene, C 3-20 Cycloalkylene or C 2-20alkenylene; and

[0389] m and n are each independently an integer selected from 1, 2, 3, 4 and 5.

[0390] In some embodiments, R 2 and R 2' Each is independently C 1-3 Alkylene.

[0391] In some embodiments, R 1 or R 1' Indicates a straight or branched chain C 1-3 Alkylene, R 2 or R 2' Indicates a straight or branched chain C 1-6 Alkylene and optionally substituted straight chain and branched C 1-6 The alkyl groups are each optionally substituted with one or more halo and cyano groups.

[0392] In some embodiments, R 1 and R 2 Together is C 1-3 Alkylene, and R 1' and R 2' Together is C 1-3 Alkylene, for example, ethylene.

[0393] In some embodiments, R 3 and R 3' are each independently an optionally substituted C 1-3 Alkyl groups, for example, methyl groups.

[0394] In some embodiments, R 4 and R 4' Each is -CH.

[0395] In some embodiments, R 2 is an optionally substituted branched C 1-6 alkylene; and R 2 and R 3 Together with its central nitrogen atom, it forms a 5-membered or 6-membered heterocyclic group. 2' is an optionally substituted branched C 1-6 alkylene; and R 2' and R 3' Together with its central nitrogen atom, it forms a 5-membered or 6-membered heterocyclic group, such as pyrrolidinyl or piperidinyl.

[0396] In some embodiments, R 4 Yes-C(R a )2CR a or -[C(R a )2]2CR a, and R a It is C 1-3 alkyl; and R 3 and R 4 Together with its central nitrogen atom, it forms a 5-membered or 6-membered heterocyclic group. 4' Yes-C(R a )2CR a or -[C(R a )2]2CR a , and R a It is C 1-3 alkyl; and R 3' and R 4' Together with its central nitrogen atom, it forms a 5-membered or 6-membered heterocyclic group, such as pyrrolidinyl or piperidinyl.

[0397] In some embodiments, R 5 and R 5' Each is independently C 1-10 Alkylene or C 2-10 In one embodiment, R 5 and R 5' Each is independently C 1-8 Alkylene or C 1-6 Alkylene.

[0398] In some embodiments, R 6 and R 6' Each occurrence is independently C 1-10 Alkylene, C 3-10 Cycloalkylene or C 2-10 In one embodiment, C 1-6 Alkylene, C 3-6 Cycloalkylene or C 2-6 In one embodiment, C 3-10 Cycloalkylene or C 3-6 In some embodiments, m and n are each 3.

[0399] In some embodiments, the ionizable lipid in the LNP of the present disclosure can be selected from any one of the lipids listed in Table 1 below, or a pharmaceutically acceptable salt thereof.

[0400] Table 1. Exemplary ionizable lipids of formula (A)

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414] Formula (B)

[0415] In some embodiments, the ionizable lipid in the LNPs of the present disclosure is represented by formula (B):

[0416]

[0417] or a pharmaceutically acceptable salt thereof, wherein:

[0418] 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);

[0419] b is an integer ranging from 2 to 10 (e.g., b is 2, 3, 4, 5, 6, 7, 8, 9, or 10);

[0420] R 1 Not present or selected from (C2-C 20 )alkenyl, -C(O)O(C2-C 20 ) alkyl and (C2-C 20 ) alkyl-substituted cyclopropyl; and

[0421] R 2 Yes (C2-C 20 )alkyl.

[0422] In a second embodiment of formula (B), the ionizable lipid of formula (B) is represented by formula (B-1):

[0423]

[0424] or a pharmaceutically acceptable salt thereof, wherein c and d are each independently an integer ranging from 1 to 8 (eg, 1, 2, 3, 4, 5, 6, 7, or 8), and wherein the remaining variables are as described for Formula (B).

[0425] In a third embodiment of Formula (B), c and d in Formula (B-1) are each independently an integer ranging from 2 to 8, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 8, 4 to 7, 4 to 6, 5 to 8, 5 to 7, or 6 to 8, with the remaining variables being as described for Formula (B-1).

[0426] In a fourth embodiment of Formula (B), c in Formula (B-1) is 2, 3, 4, 5, 6, 7, or 8, with the remaining variables being as described for Formula (B) or the second or third embodiment of Formula (B). Alternatively, c and d in Formula (B-1) are each independently 1, 3, 5, or 7, with the remaining variables being as described for Formula (B) or the second or third embodiment of Formula (B).

[0427] In a fifth embodiment of Formula (B), d in the cationic lipid of Formula (B-1) is 2, 3, 4, 5, 6, 7 or 8, wherein the remaining variables are as described for Formula (B) or the second, third or fourth embodiment of Formula (B). Alternatively, at least one of c and d in Formula (B-1) is 7, wherein the remaining variables are as described for Formula (B) or the second, third or fourth embodiment of Formula (B).

[0428] In a sixth embodiment of formula (B), the ionizable lipid of formula (B) or formula (B-1) is represented by formula (B-2):

[0429]

[0430] or a pharmaceutically acceptable salt thereof, wherein the remaining variables are as described for Formula (B) or Formula (B-1).

[0431] In a seventh embodiment of Formula (B), b in Formula (B), (B-1) or (B-2) is an integer ranging from 3 to 9, with the remaining variables as described for Formula (B) or the second, third, fourth, fifth or sixth embodiment of Formula (B). Alternatively, b in Formula (B), (B-1) or (B-2) is an integer ranging from 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 5 to 9, 5 to 8, 5 to 7, 6 to 9, 6 to 8 or 7 to 9, with the remaining variables as described for Formula (B) or the second, third, fourth, fifth or sixth embodiment of Formula (B). Alternatively, b in Formula (B), (B-1) or (B-2) is 3, 4, 5, 6, 7, 8 or 9, with the remaining variables as described for Formula (B) or the second, third, fourth, fifth or sixth embodiment of Formula (B).

[0432] In an eighth embodiment of Formula (B), b in Formula (B), (B-1) or (B-2) is an integer ranging from 2 to 18, with the remaining variables being as described for Formula (B) or the second, third, fourth, fifth, sixth or seventh embodiment of Formula (B). Alternatively, a in Formula (B), (B-1) or (B-2) is an integer ranging from 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 18, 4 to 17, 4 to 16, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 25 to 8, 5 to 7, 6 to 18, 6 to 17, 6 to 16, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 6 to 9, 6 to 8, 7 to 18 , 7 to 17, 7 to 16, 7 to 15, 7 to 14, 7 to 13, 7 to 12, 7 to 11, 7 to 10, 7 to 9, 8 to 18, 8 to 17, 8 to 16, 8 to 15, 8 to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 9 to 18, 9 to 17, 9 to 16, 9 to 15, 9 to 14, 9 to 13, 9 to 12, 9 to 11, 10 to 18, 10 to 17, 10 to 16, 10 to 15, 10 to 14, 10 to 13, 11 to 17, 14 to 16, 15 to 18, 15 to 17, 14 to 16, 15 to 18, 15 to 17, or 16 to 18, wherein the remaining variables are as described for Formula (B) or the second, third, fourth, fifth, sixth, or seventh embodiment of Formula (B). Alternatively, a in Formula (B), (B-1) or (B-2) is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18, with the remaining variables being as described for Formula (B) or the second, third, fourth, fifth, sixth or seventh embodiment of Formula (B).

[0433] In the ninth embodiment of formula (B), R in formula (B), formula (B-1) or formula (B-2) 1 Not present or selected from (C5-C 15 )alkenyl, -C(O)O(C4-C 18) alkyl and (C4-C 16 )alkyl-substituted cyclopropyl, wherein the remaining variables are as described for Formula (B) or the second, third, fourth, fifth, sixth, seventh or eighth embodiment of Formula (B). Alternatively, R in Formula (B), Formula (B-1) or Formula (B-2) 1 Not present or selected from (C5-C 15 )alkenyl, -C(O)O(C4-C 16 ) alkyl and (C4-C 16 )alkyl-substituted cyclopropyl, wherein the remaining variables are as described for Formula (B) or the second, third, fourth, fifth, sixth, seventh or eighth embodiment of Formula (B). Alternatively, R in Formula (B), Formula (B-1) or Formula (B-2) 1 Not present or selected from (C5-C 12 )alkenyl, -C(O)O(C4-C 12 ) alkyl and (C4-C 12 )alkyl-substituted cyclopropyl, wherein the remaining variables are as described for Formula (B) or the second, third, fourth, fifth, sixth, seventh or eighth embodiment of Formula (B). In another alternative, R in the cationic lipid of Formula (B), Formula (B-1) or Formula (B-2) is 1 Not present or selected from (C5-C 10 )alkenyl, -C(O)O(C4-C 10 ) alkyl and (C4-C 10 )alkyl-substituted cyclopropyl, wherein the remaining variables are as described for Formula (B) or the second, third, fourth, fifth, sixth, seventh or eighth embodiment of Formula (B).

[0434] In the tenth embodiment of formula (B), R 1 It is C 10 alkenyl, wherein the remaining variables are as described for Formula (B) or the second, third, fourth, fifth, sixth, seventh or eighth embodiment of Formula (B).

[0435] In the eleventh embodiment of formula (B), R in formula (B), formula (B-1) or formula (B-2) 1 C(O)O(C2-C 20 )alkyl, -C(O)O(C4-C 18 )alkyl, -C(O)O(C4-C 12 )alkyl or -C(O)O(C4-C 10 )alkyl is an unbranched alkyl, wherein the remaining variables are as described for Formula (B) or the second, third, fourth, fifth, sixth, seventh, eighth or ninth embodiment of Formula (B). In one embodiment, R 1is -C(O)O(C9 alkyl). Alternatively, R in formula (B), formula (B-1) or formula (B-2) 1 -C(O)O(C4-C 18 )alkyl, -C(O)O(C4-C 12 )alkyl or -C(O)O(C4-C 10 )alkyl is a branched alkyl, wherein the remaining variables are as described for Formula (B), Formula (B-1) or Formula (B-2) or the second, third, fourth, fifth, sixth, seventh, eighth or ninth embodiment of Formula (B). In one embodiment, R 1 is -C(O)O(C 17 alkyl), wherein the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, eighth, or ninth embodiment of Formula (B).

[0436] In the twelfth embodiment of formula (B), R in formula (B), formula (B-1) or formula (B-2) 1 is selected from any of the groups listed in Table 2 below, wherein the wavy bond in each group indicates the point of attachment of the group to the rest of the ionizable lipid molecule, and wherein the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B). The present disclosure further contemplates R in Table 2 1 Any of the groups and R in Table 3 2 A combination of any of the groups in Formula (B), 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).

[0437] Table 2. Exemplary R in Formula (B), Formula (B-1), or Formula (B-2) 1 Group

[0438]

[0439] In the thirteenth embodiment, R in formula (B) or a pharmaceutically acceptable salt thereof 2 selected from any of the groups listed in Table 3 below, wherein the wavy bond in each of the groups indicates the point of attachment of the group to the rest of the ionizable lipid molecule, and wherein the remaining variables are as described for Formula (B), Formula (B-1) or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, or eighth, ninth, tenth, eleventh, or twelfth embodiment of Formula (B).

[0440] Table 3. Exemplary R in Formula (B) 2 Group

[0441]

[0442] Specific examples of ionizable lipids of formula (B) are provided in Table 4 below. Pharmaceutically acceptable salts as well as ionized and neutral forms are also included.

[0443] Table 4. Exemplary ionizable lipids of formula (B), (B-1), or (B-2)

[0444]

[0445]

[0446]

[0447]

[0448]

[0449] Formula (C) In some embodiments, the ionizable lipid in the LNPs of the present disclosure is represented by formula (C):

[0450]

[0451] or a pharmaceutically acceptable salt thereof, wherein:

[0452] R 1 and R 1' Each independently is optionally selected from one or more R a (C1-C6)alkylene substituted with a group;

[0453] R 2 and R 2' are each independently (C1-C2)alkylene;

[0454] R 3 and R 3' Each independently is optionally selected from one or more R b (C1-C6) alkyl substituted with a group;

[0455] Or alternatively, R 2 and R 3 and / or R 2' and R 3' Together with its central nitrogen atom, it forms a 4- to 7-membered heterocyclic group;

[0456] R 4 and R 4 'Each is a (C2-C6)alkylene group interrupted by -C(O)O-;

[0457] R 5 and R 5 'Each independently is (C2-C 30 )alkyl or (C2-C 30 )alkenyl, each of which is optionally interrupted by -C(O)O- or (C3-C6)cycloalkyl; and

[0458] R a and R b Each is halo or cyano.

[0459] In the second embodiment of formula (C), R 1 and R 1 are each independently (C1-C6)alkylene, wherein the remaining variables are as described above for formula (C). Alternatively, R 1 and R 1' is each independently (C1-C3)alkylene, wherein the remaining variables are as described above for Formula (C).

[0460] In a third embodiment of formula (C), the ionizable lipid of formula (C) is represented by formula (C-1):

[0461]

[0462] or a pharmaceutically acceptable salt thereof, wherein R 2 and R 2' 、R 3 and R 3' 、R 4 and R 4 ' and R 5 and R 5 'As described above for Formula (C) or the second embodiment of Formula (C).

[0463] In a fourth embodiment, the ionizable lipid of formula (C) is represented by formula (C-2) or formula (C-3):

[0464]

[0465] or a pharmaceutically acceptable salt thereof, wherein R 4 and R 4 ' and R 5 and R 5 'As described above for formula (C).

[0466] In a fifth embodiment of formula (C), the ionizable lipid of formula (C) is represented by formula (C-4) or (C-5):

[0467]

[0468] or a pharmaceutically acceptable salt thereof, wherein R5 and R 5 'As described above for formula (C).

[0469] In a sixth embodiment of formula (C), the ionizable lipid of formula (C) is represented by formula (C-6), (C-7), (C-8), or (C-9):

[0470]

[0471] or a pharmaceutically acceptable salt thereof, wherein R 5 and R 5 'As described above for formula (XV).

[0472] In the seventh embodiment of formula (C), R in formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8) or (C-9) 5 and R 5' At least one of R is a branched alkyl or alkenyl group, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is 5 and R 5' One of the R in Formula (C) is a branched alkyl or alkenyl group, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is 5 is a branched alkyl or alkenyl group, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is 5' is a branched alkyl or alkenyl group, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C).

[0473] In an eighth embodiment of formula (C), R in formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) 5 Yes (C6-C 26 )alkyl or (C6-C 26)alkenyl, each of which is optionally interrupted by -C(O)O- or (C3-C6)cycloalkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9 5 Yes (C6-C 26 )alkyl or (C6-C 26 )alkenyl, each of which is optionally interrupted by -C(O)O- or (C3-C5)cycloalkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9 5 Yes (C7-C 26 )alkyl or (C7-C 26 )alkenyl, each of which is optionally interrupted by -C(O)O- or (C3-C5)cycloalkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9 5 Yes (C8-C 26 )alkyl or (C8-C 26 )alkenyl, each of which is optionally interrupted by -C(O)O- or (C3-C5)cycloalkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9 5 Yes (C6-C 24 )alkyl or (C6-C 24 )alkenyl, each of which is optionally interrupted by -C(O)O- or cyclopropyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is 5 Yes (C8-C 24 )alkyl or (C8-C 24 ) alkenyl, wherein the (C8-C 24)alkyl is optionally interrupted by -C(O)O- or cyclopropyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) 5 Yes (C8-C 10 ) alkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) 5 is intercalated with cyclopropyl groups (C 14 -C 16 ) alkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) 5 is mixed with -C(O)O- 10 -C 24 ) alkyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) 5 Yes (C 16 -C 18 )alkenyl, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) is 5 is -(CH2)3C(O)O(CH2)8CH3, -(CH2)5C(O)O(CH2)8CH3, -(CH2)7C(O)O(CH2)8CH3, -(CH2 )7C(O)OCH[(CH2)7CH3]2, -(CH2)7-C3H6-(CH2)7CH3, -(CH2)7CH3, -(CH2)9CH3, -(CH2) 16 CH3, -(CH2)7CH=CH(CH2)7CH3 or

[0474] -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, and the remaining variables are as described above for Formula (C) or the second embodiment of Formula (C).

[0475] In a ninth embodiment, R in formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) 5 is mixed with -C(O)O- 15 -C 28 ) alkyl, and the remaining variables are as described above for Formula (C) or the second or eighth embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) 5' is mixed with -C(O)O- 17 -C 28 ) alkyl, and the remaining variables are as described above for Formula (C) or the second or eighth embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) 5' is mixed with -C(O)O- 19 -C 28 ) alkyl, and the remaining variables are as described above for Formula (C) or the second or eighth embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) 5' is mixed with -C(O)O- 17 -C 26 ) alkyl, and the remaining variables are as described above for Formula (C) or the second or eighth embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) 5' is mixed with -C(O)O- 19 -C 26 ) alkyl, and the remaining variables are as described above for Formula (C) or the second or eighth embodiment of Formula (C). Alternatively, R in Formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9) 5' is mixed with -C(O)O- 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 mixed with -C(O)O-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' yes

[0476] -(CH2)5C(O)OCH[(CH2)7CH3]2, -(CH2)7C(O)OCH[(CH2)7CH3]2,

[0477] -(CH2)5C(O)OCH(CH2)2[(CH2)7CH3]2 or -(CH2)7C(O)OCH(CH2)2[(CH2)7CH3]2, and the remaining variables are as described above for Formula (C) or the second or eighth embodiment of Formula (C).

[0478] In some embodiments, the ionizable lipid of Formula (C), (C-1), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8) or (C-9) can be selected from any one of the lipids listed in Table 5 below or a pharmaceutically acceptable salt thereof.

[0479] Table 5. Exemplary ionizable lipids of formula (C), (C-1), (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), or (C-9)

[0480]

[0481]

[0482] Formula (D)

[0483] In some embodiments, the ionizable lipid (e.g., cationic lipid) in the LNPs of the present disclosure is represented by formula (D):

[0484]

[0485] or a pharmaceutically acceptable salt thereof, wherein:

[0486] R' is absent and is hydrogen or C1-C6 alkyl; provided that when R' is hydrogen or C1-C6 alkyl, R', R 1 and R 2 The nitrogen atoms they are all connected to have a positive charge;

[0487] R 1 and R 2 are each independently hydrogen, C1-C6 alkyl or C2-C6 alkenyl;

[0488] R 3It is C1-C 12 Alkylene or C2-C 12 alkenylene;

[0489] R 4 It is C1-C 18 Unbranched alkyl, C2-C 18 Unbranched alkenyl or in:

[0490] R 4a and R 4b Each is independently C1-C 16 Unbranched alkyl or C2-C 16 unbranched alkenyl;

[0491] R 5 Not present, C1-C8 alkylene or C2-C8 alkenylene;

[0492] R 6a and R 6b Each independently is C7-C 16 Alkyl or C7-C 16 alkenyl; provided that the combined R 6a and R 6b The total number of carbon atoms in is greater than 15;

[0493] 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:

[0494] R a Each occurrence is independently hydrogen or C1-C6 alkyl; and

[0495] n is an integer selected from 1, 2, 3, 4, 5 and 6.

[0496] In the second embodiment of formula (D), X1 and X 2 are the same; and all other remaining variables are as described for Formula (C).

[0497] In the third embodiment of formula (D), X 1 and X 2 are each independently -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, or -SS-; or X 1 and X 2 are each independently -C(=O)O-, -C(=O)S- or -SS-; or X 1 and X 2 each is independently -C(=O)O- or -SS-; and all other remaining variables are as described for Formula (D) or the second embodiment of Formula (D).

[0498] In a fourth embodiment of Formula (D), the ionizable lipid (e.g., cationic lipid) in the LNP of the present disclosure is represented by Formula (D-1):

[0499]

[0500] or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 1, 2, 3, and 4; and all other remaining variables are as described for Formula (D) or the second or third embodiment of Formula (D).

[0501] 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):

[0502]

[0503] or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 1, 2, and 3; and all other remaining variables are as described for Formula (D) or the second or third embodiment of Formula (D).

[0504] 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):

[0505]

[0506] or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (D) or the second or third embodiment of Formula (D).

[0507] 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 each independently represents 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).

[0508] 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):

[0509]

[0510] or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3) or the second, third or seventh embodiment of Formula (D).

[0511] In a ninth embodiment of Formula (D), in an ionizable lipid (e.g., a cationic lipid), according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, or seventh embodiment of Formula (D), R 3 is C1-C9 alkylene or C2-C9 alkenylene, C1-C7 alkylene or C2-C7 alkenylene, C1-C5 alkylene or C2-C5 alkenylene, or C2-C8 alkylene or C2-C8 alkenylene, or C3-C7 alkylene or C3-C7 alkenylene, or C5-C7 alkylene or C5-C7 alkenylene; or R 3 It 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 10and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4) or the second, third or seventh embodiment of Formula (D).

[0512] 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 does not exist, is C1-C6 alkylene or C2-C6 alkenylene; or R 5 does not exist, is C1-C4 alkylene or C2-C4 alkenylene; or R 5 does not exist; or R 5 is C8 alkylene, C7 alkylene, C6 alkylene, C5 alkylene, C4 alkylene, C3 alkylene, C2 alkylene, C1 alkylene, C8 alkenylene, C7 alkenylene, C6 alkenylene, C5 alkenylene, C4 alkenylene, C3 alkenylene or C2 alkenylene; and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4) or the second, third, seventh or ninth embodiment of Formula (D).

[0513] 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 It is C1-C 14 Unbranched alkyl, C2-C 14 Unbranched alkenyl or where R 4a and R 4b Each is independently C1-C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl; or R 4 It is C2-C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl; or R 4 is a C5-C7 unbranched alkyl group or a C5-C7 unbranched alkenyl group; or R 4 It is C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C 13 Unbranched alkyl, C 12 Unbranched alkyl, C11 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 yes where R 4a and R 4b Each is independently C2-C 10 Unbranched alkyl or C2-C 10 unbranched alkenyl; or R 4 yes where R 4a and R 4b Each is 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, C 14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl or C2 alkenyl; and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4) or the second, third, seventh, ninth or tenth embodiment of Formula (D).

[0514] 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 Each independently is C6-C 14 Alkyl or C6-C 14 alkenyl; or R 6a and R 6b Each is independently C8-C 12 Alkyl or C8-C 12 alkenyl; or R 6a and R 6b Each is 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 10 alkenyl, C9 alkenyl, C8 alkenyl or C7 alkenyl; provided that the combined R 6a and R 6b the total number of carbon atoms in is greater than 15; and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4) or the second, third, seventh, ninth, tenth or eleventh embodiment of Formula (D).

[0515] In the thirteenth embodiment of Formula (D), in an ionizable lipid (e.g., a cationic lipid), according to the second, third, seventh, ninth, tenth, eleventh or twelfth embodiment of Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4) or 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 Both are C 16 Alkyl, C 15 Alkyl, C 14 Alkyl, C 13 Alkyl, C 12 Alkyl, C11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 16 Alkenyl, C 15 Alkenyl, C 14 Alkenyl, C 13 Alkenyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, C9 alkenyl, C8 alkenyl or C7 alkenyl; provided that the combined R 6a and R 6b the total number of carbon atoms in is greater than 15; and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth, eleventh or twelfth embodiment of Formula (D).

[0516] In the 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 of the preceding embodiments 6a and R 6b Each contains a different number of carbon atoms; or the number of carbon atoms R 6a and R 6b Differ by one or two carbon atoms; or the number of carbon atoms R 6a and R 6b Differ by one carbon atom; or R 6a is a C7 alkyl group and R 6a is a C8 alkyl group, R 6a is a C8 alkyl group and R 6a is a C7 alkyl group, R 6a is a C8 alkyl group and R 6a is a C9 alkyl group, R 6a is a C9 alkyl group and R 6a is a C8 alkyl group, R 6a is a C9 alkyl group and R 6a It is C 10 Alkyl, R 6a It is C 10 Alkyl and R 6a is a C9 alkyl group, R 6a It is C 10 Alkyl and R 6a It is C 11 Alkyl, R 6a It is C 11 Alkyl and R 6a It is C 10 Alkyl, R 6aIt is C 11 Alkyl and R 6a It is C 12 Alkyl, R 6a It is C 12 Alkyl and R 6a It is C 11 Alkyl, R 6a is a C7 alkyl group and R 6a is a C9 alkyl group, R 6a is a C9 alkyl group and R 6a is a C7 alkyl group, R 6a is a C8 alkyl group and R 6a It is C 10 Alkyl, R 6a It is C 10 Alkyl and R 6a is a C8 alkyl group, R 6a is a C9 alkyl group and R 6a It is C 11 Alkyl, R 6a It is C 11 Alkyl and R 6a is a C9 alkyl group, R 6a It is C 10 Alkyl and R 6a It is C 12 Alkyl, R 6a It is C 12 Alkyl and R 6a It is C 10 Alkyl, R 6a It is C 11 Alkyl and R 6a It is C 13 Alkyl, or R 6a It is C 13 Alkyl and R 6a It is C 11 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).

[0517] In the fifteenth embodiment of formula (D), R 4 It is C1-C 16 Unbranched alkyl, C2-C 16 Unbranched alkenyl or where R 4a and R 4b As described above for the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth or fourteenth embodiment of Formula (D).

[0518] In one embodiment, the ionizable lipid (e.g., cationic lipid) of the present disclosure or the 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:

[0519] Table 6. Exemplary lipids of formula (D), formula (D-1), formula (D-2), formula (D-3), or formula (D-4)

[0520]

[0521]

[0522]

[0523]

[0524]

[0525] In one embodiment, the ionizable lipid in the LNP of the present disclosure comprises lipid No. 87:

[0526]

[0527] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0528] Formula (E)

[0529] In some embodiments, the ionizable lipid (e.g., cationic lipid) in the LNPs of the present disclosure is represented by formula (E):

[0530]

[0531] or a pharmaceutically acceptable salt thereof, wherein:

[0532] R' is absent and is hydrogen or C1-C3 alkyl; provided that when R' is hydrogen or C1-C3 alkyl, R', R 1 and R 2 The nitrogen atoms they are all connected to have a positive charge;

[0533] R 1 and R 2 are each independently hydrogen or C1-C3 alkyl;

[0534] R 3 It is C3-C 10 Alkylene or C3-C 10 alkenylene;

[0535] R 4 It is C1-C16 Unbranched alkyl, C2-C 16 Unbranched alkenyl or in:

[0536] R 4a and R 4b Each is independently C1-C 16 Unbranched alkyl or C2-C 16 unbranched alkenyl;

[0537] R 5 Not present, C1-C6 alkylene or C2-C6 alkenylene;

[0538] R 6a and R 6b Each independently is C7-C 14 Alkyl or C7-C 14 alkenyl;

[0539] 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:

[0540] R a Each occurrence is independently hydrogen or C1-C6 alkyl; and

[0541] n is an integer selected from 1, 2, 3, 4, 5 and 6.

[0542] In a second embodiment of Formula (E), in the ionizable lipid (e.g., 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.

[0543] In a third embodiment of Formula (E), the ionizable lipid (e.g., cationic lipid) in the LNP of the present disclosure is represented by Formula (E-1):

[0544]

[0545] or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 1, 2, 3, and 4; and all other remaining variables are as described for Formula (E) or the second embodiment of Formula (E). Alternatively, n is an integer selected from 1, 2, and 3; and all other remaining variables are as described for Formula (E) or the second embodiment of Formula (E).

[0546] In a fourth embodiment of Formula (E), the ionizable lipid (e.g., cationic lipid) in the LNP of the present disclosure is represented by Formula (E-2):

[0547]

[0548] or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (E), Formula (E-1) or the second embodiment of Formula (E).

[0549] In a fifth embodiment of Formula (E), in the ionizable lipid (e.g., cationic lipid) in the LNP of the present disclosure, R 1 and R 2 are independently hydrogen or C1-C2 alkyl or C2-C3 alkenyl; or R', R 1 and R 2 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).

[0550] 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):

[0551]

[0552] or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2) or the second or fifth embodiment of Formula (E).

[0553] 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 Absent or C1-C8 alkylene; or R 5 does not exist, is C1-C6 alkylene or C2-C6 alkenylene; or R 5does not exist, is C1-C4 alkylene or C2-C4 alkenylene; or R 5 does not exist; or R 5 is C8 alkylene, C7 alkylene, C6 alkylene, C5 alkylene, C4 alkylene, C3 alkylene, C2 alkylene, C1 alkylene, C8 alkenylene, C7 alkenylene, C6 alkenylene, C5 alkenylene, C4 alkenylene, C3 alkenylene or C2 alkenylene; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3) or the second or fifth embodiment of Formula (E).

[0554] In an eighth embodiment of Formula (E), the ionizable lipid (e.g., cationic lipid) in the LNP of the present disclosure is represented by Formula (E-4):

[0555]

[0556] or a pharmaceutically acceptable salt thereof; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3) or the second, fifth or seventh embodiment of Formula (E).

[0557] 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 It is C1-C 14 Unbranched alkyl, C2-C 14 Unbranched alkenyl or where R 4a and R 4b Each is independently C1-C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl; or R 4 It is C2-C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl; or R 4 It is C5-C 12 Unbranched alkyl or C5-C 12 unbranched alkenyl; or R 4 It 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 yes where R 4a and R 4b Each is independently C2-C 10 Unbranched alkyl or C2-C 10 unbranched alkenyl; or R 4 yes where R 4a and R 4b Each is 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, C 14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl or C2 alkenyl; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth or seventh embodiment of Formula (E).

[0558] 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 C3-C8 alkenylene, C3-C7 alkylene or C3-C7 alkenylene, or C3-C5 alkylene or C3-C5 alkenylene; or R 3 is C8 alkylene or C7 alkylene or C6 alkylene or C5 alkylene or C4 alkylene or C3 alkylene or C1 alkylene or C8 alkenylene or C7 alkenylene or C6 alkenylene or C5 alkenylene or C4 alkenylene or C3 alkenylene; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh or ninth embodiment of Formula (E).

[0559] 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 Each independently is C7-C 12 Alkyl or C7-C 12 alkenyl; or R 6a and R 6b Each is independently C8-C 10 Alkyl or C8-C 10 alkenyl; or R 6a and R 6b Each is independently C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 Alkenyl, C 11 Alkenyl, C 10 and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, seventh, ninth, or tenth embodiment of Formula (E).

[0560] 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 R6b are the same; or R 6a and R 6b Both are C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 Alkenyl, C 11 Alkenyl, C 10 and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh, ninth, tenth or eleventh embodiment of Formula (E).

[0561] 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), or formula (E-4), R as defined in any one of the preceding embodiments 6a and R 6b Each contains a different number of carbon atoms; the number of carbon atoms R 6a and R 6b Differ by one or two carbon atoms; or the number of carbon atoms R 6a and R 6b Differ by one carbon atom; or R 6a is a C7 alkyl group and R 6a is a C8 alkyl group, R 6a is a C8 alkyl group and R 6a is a C7 alkyl group, R 6a is a C8 alkyl group and R 6a is a C9 alkyl group, R 6a is a C9 alkyl group and R 6a is a C8 alkyl group, R 6a is a C9 alkyl group and R 6a It is C 10 Alkyl, R 6a It is C 10 Alkyl and R 6a is a C9 alkyl group, R 6a It is C 10 Alkyl and R 6a It is C 11 Alkyl, R 6a It is C 11 Alkyl and R 6a It is C 10 Alkyl, R 6a It is C 11 Alkyl and R 6a It is C 12 Alkyl, R 6a It is C 12 Alkyl and R6a It is C 11 Alkyl, R 6a is a C7 alkyl group and R 6a is a C9 alkyl group, R 6a is a C9 alkyl group and R 6a is a C7 alkyl group, R 6a is a C8 alkyl group and R 6a It is C 10 Alkyl, R 6a It is C 10 Alkyl and R 6a is a C8 alkyl group, R 6a is a C9 alkyl group and R 6a It is C 11 Alkyl, R 6a It is C 11 Alkyl and R 6a is a C9 alkyl group, R 6a It is C 10 Alkyl and R 6a It is C 12 Alkyl, R 6a It is C 12 Alkyl and R 6a It is C 10 alkyl, etc.; and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4) or the second, fifth, seventh, ninth, tenth, eleventh or twelfth embodiment of Formula (E).

[0562] In a fourteenth embodiment, in an ionizable lipid (e.g., a cationic lipid), according to Formula (E), (E-1), (E-2), (E-3), (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), (E-1), (E-2), (E-3), (E-4), or the second, fifth, seventh, ninth, tenth, eleventh, twelfth, or thirteenth embodiment of Formula (E).

[0563] In one embodiment, the ionizable lipid (e.g., cationic lipid) or cationic lipid of Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), or Formula (E-4) in the LNP of the present disclosure is any one lipid selected from the lipids in Table 7 or a pharmaceutically acceptable salt thereof:

[0564] Table 7. Exemplary lipids of formula (E), formula (E-1), formula (E-2), formula (E-3), and formula (E-4)

[0565]

[0566]

[0567]

[0568] Specific examples are provided in the Exemplary section and are included as part of the cationic lipids or ionizable lipids described herein. Pharmaceutically acceptable salts and neutral forms are also included.

[0569] Cleavable lipids

[0570] In certain embodiments, the LNP provided by the present disclosure comprises ionizable lipids, and the ionizable lipids are also cleavable lipids. As used herein, the term "cleavable lipids" can be used interchangeably with the term "SS-cleavable lipids", and refers to the ionizable lipids comprising disulfide bonds (" SS "). The SS in the cleavable lipids is a cleavable unit. In one embodiment, the cleavable lipids comprise amines, for example, tertiary amines, for example, and disulfide bonds. In this cleavable lipid, amine can be protonated in acidic compartments (for example, endosomes or lysosomes), causing the LNP to destabilize, and cleavable lipids can be cut in reducing environments (for example, 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 etc.

[0571] According to some embodiments, the SS-cleavable lipids are described in International Patent Application Publication No. WO2019188867, which is herein incorporated by reference in its entirety.

[0572] In one embodiment, the cleavable lipid may comprise three components: an amine head group, a linker group, and a hydrophobic tail. In one embodiment, the cleavable lipid comprises one or more phenyl ester bonds, one of multiple tertiary amino groups, and a disulfide bond. The tertiary amino group provides pH responsiveness and induces endosomal escape, the phenyl ester bond enhances the degradability (self-degradability) of the structure, and the disulfide bond is cleaved in a reducing environment.

[0573] 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:

[0574] Lipid A

[0575]

[0576] In one embodiment, the SS-cleavable lipid is an SS-cleavable and pH-activated lipid-like material (ssPalm). ssPalm lipids are well known in the art. 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, ssPalm is an ssPalmM lipid comprising the structure of lipid B shown below:

[0577] Lipid B

[0578]

[0579] In one embodiment, the ssPalmE lipid is a ssPalmE-P4-C2 lipid, which comprises the following lipid C structure:

[0580] Lipid C

[0581]

[0582] In one embodiment, the ssPalmE lipid is a ssPalmE-Paz4-C2 lipid comprising the following lipid D structure:

[0583] Lipid D

[0584]

[0585] In one embodiment, the cleavable lipid is a ss-M lipid. In one embodiment, the ss-M lipid comprises the structure shown in the following lipid E:

[0586] Lipid E

[0587]

[0588] In one embodiment, the cleavable lipid is a ss-E lipid. In one embodiment, the ss-E lipid comprises the structure shown in the following lipid F:

[0589] Lipid F

[0590]

[0591] In one embodiment, the cleavable lipid is a ss-EC lipid. In one embodiment, the ss-EC lipid comprises the structure shown in the following lipid G:

[0592] Lipid G

[0593]

[0594] In one embodiment, the cleavable lipid is a ss-LC lipid. In one embodiment, the ss-LC lipid comprises the structure shown in the following lipid H:

[0595] Lipid H

[0596]

[0597] In one embodiment, the cleavable lipid is a ss-OC lipid. In one embodiment, the ss-OC lipid comprises the structure shown in the following lipid J:

[0598] Lipid J

[0599]

[0600] Other lipids

[0601] In some embodiments, the ionizable lipids in the LNPs of the present disclosure are selected from the group consisting of: N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-dilauroyl-sn-glycero-3-ethyl Phosphocholine (DLEPC); 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC); 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[bis(3-amino-propyl)amino]butylcarboxamidoethyl]-3,4-bis[oleyloxy]-benzamide (MVL5); dioctadecylamido- Glycyl spermine (DOGS); 3b-[N-(N',N'-dimethylaminoethyl)carbamoyl] cholesterol (DC-Chol); dioctadecyldimethylammonium bromide (DDAB); Saint lipids (e.g., SAINT-2, N-methyl-4-(dioleyl)methylpyridinium ion); 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE); 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide ammonium (DORIE); 1,2-dioleoyloxypropyl-3-dimethylhydroxyethylammonium chloride (DORI); dialkylated amino acids (DILA2) (e.g., C18:1-norArg-C16); dioleyldimethylammonium chloride (DODAC); 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC); and 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC).In some variations, the condensing agent, e.g., a cationic lipid, is a lipid, e.g., dioctadecyldimethylammonium bromide (DDAB), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptathriacontane-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (Dlin-MC3-DMA), 1,2-dioleoyloxy -3-dimethylaminopropane (DODAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), morpholinocholesterol (Mo-CHOL), (R)-5-(dimethylamino)pentane-1,2-diyl dioleate hydrochloride (DODAPen-C1), (R)-5-guanidinopentane-1,2-diyl dioleate hydrochloride (DOPen-G), and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentane-1-ammonium chloride (DOTAPen).

[0602] In some embodiments, the ionizable lipid in the LNP of the present disclosure is represented by the following structure:

[0603]

[0604]

[0605] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0606] B. Structural lipids

[0607] In some embodiments, the LNPs provided herein comprise structural lipids. Without wishing to be bound by a particular theory, it is believed that when structural lipids are present in LNPs, they contribute to the membrane integrity and stability of the LNPs.

[0608] In some embodiments, the structured lipid is a sterol, for example, cholesterol or a derivative thereof. In one embodiment, the structured lipid is cholesterol. In another embodiment, the structured lipid is a derivative of cholesterol. Non-limiting examples of cholesterol derivatives include: polar analogs such as 5α-cholestanol, 5β-coprolol, cholesterol-(2'-hydroxy)-ethyl ether, cholesterol-(4'-hydroxy)-butyl ether and 6-ketocholestanol; non-polar analogs such as 5α-cholestane, cholesterenone, 5α-cholestol, 5β-cholestol and cholesterol decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog, such as cholesterol-(4'-hydroxy)-butyl ether. In some embodiments, the cholesterol derivative is cholesterol hemisuccinate (CHEMS).

[0609] Exemplary cholesterol derivatives are described in International Patent Application Publication No. WO 2009 / 127060 and U.S. Patent Publication No. US 2010 / 0130588, the contents of both of which are incorporated herein by reference in their entirety.

[0610] In some embodiments, the sterol in the LNP of the present disclosure is selected from the group consisting of cholesterol, β-sitosterol, stigmasterol, β-sitostanol, campesterol, brassicasterol and derivatives thereof, and any combination thereof. In one embodiment, the sterol is cholesterol. In another embodiment, the sterol is β-sitosterol.

[0611] In certain embodiments, structural lipids constitute the approximately 20mol% to approximately 45mol% of the TL present in LNP. In certain embodiments, structural lipids constitute the approximately 25mol% to approximately 45mol% of the TL content of LNP. In certain embodiments, structural lipids constitute the approximately 30% to approximately 45% of the TL present in LNP. In certain embodiments, structural lipids constitute the approximately 30mol% to approximately 40mol% of the TL present in LNP. In certain embodiments, such components are the approximately 40mol% of the TL present in LNP. In certain embodiments, structural lipids (e.g., sterols) constitute the approximately 20mol% to approximately 45mol% of the TL present in LNP. In certain embodiments, structural lipids (e.g., sterols) constitute the approximately 30mol% to approximately 40mol% of the TL present in LNP.

[0612] In some embodiments, the structural lipid is cholesterol and constitutes about 30 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid is cholesterol and constitutes about 35 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid is cholesterol and constitutes about 40 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid is cholesterol and constitutes about 40 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid is cholesterol and constitutes about 40 mol% to about 45 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid is cholesterol and constitutes about 45 mol% of the total lipid present in the LNP. In some embodiments, the structural lipid is cholesterol and constitutes about 40 mol% to about 45 mol% of the total lipid present in the LNP, wherein the encapsulation efficiency ("Enc. Eff.") of the TNA is greater than 95% and / or the average size range of the LNP is about 70 nm to 90 nm in diameter.

[0613] In some embodiments, the structured lipid is dexamethasone or dexamethasone-palmitate.

[0614] C. Helper lipids

[0615] The LNP provided by the present disclosure comprises a helper lipid. In certain embodiments, the helper lipid is a ceramide or a sphingomyelin. Both ceramide and sphingomyelin are sphingolipids, which are a class of cell membrane lipids. Structurally, both ceramide and sphingomyelin contain an N-acetyl sphingosine (i.e., (E)-N-(1,3-dihydroxyoctadecane-4-ene-2-yl) acetamide) backbone and a fatty acid connected to an amide group. In sphingomyelin, the N-acetyl sphingosine backbone is further connected to a phosphorylcholine or phosphoethanolamine group. In certain embodiments, the LNP provided by the present disclosure comprises a ceramide or a sphingomyelin or a combination thereof, whereby the fatty acid moiety of the ceramide or sphingomyelin has a certain length, or is a fatty acid with a certain number of carbon atoms, as described below. As used herein, the term "helper lipid" refers to an amphiphilic lipid comprising at least one non-polar chain and at least one polar part. Without wishing to be bound by a particular theory, it is believed that the role of the helper lipid is to avoid LNP from being off-target to the blood compartment, to improve the fusogenicity of the lipid bilayer of the LNP, to stabilize the LNP structure, and to promote endosome escape.

[0616] In some embodiments, the ceramide or sphingomyelin in the LNPs of the present disclosure as a helper lipid is represented by a helper lipid represented by formula (I):

[0617]

[0618] or a salt or ester thereof, or a deuterated analog of any of the foregoing, wherein:

[0619] Is a single bond or a double bond;

[0620] A is hydrogen,

[0621] R 1 It is C1-C 17 Alkyl or C2-C 17 alkenyl;

[0622] R 2 It is C1-C 22 Alkyl or C2-C 22 alkenyl;

[0623] R 3 is hydrogen or C1-C2 alkyl; and

[0624] R 4 is hydrogen or C1-C2 alkyl.

[0625] In some embodiments of Formula (I), R 1 It is C1-C 10 Alkyl or C2-C 10 Alkenyl.

[0626] In some embodiments of Formula (I),

[0627] R 1 It is C1-C 10 Alkyl or C2-C 10 alkenyl;

[0628] R 2 It is C1-C 22 Alkyl or C2-C 22 alkenyl;

[0629] R 3 is hydrogen or C1-C2 alkyl; and

[0630] R 4 is hydrogen or C1-C2 alkyl.

[0631] In some embodiments of Formula (I), R 3 and R 4 Both are hydrogen. In some embodiments of formula (I), R 3 and R 4 are independently hydrogen or C1 alkyl.

[0632] In some embodiments of Formula (I), R 1 is C1-C7 alkyl or C2-C7 alkenyl. In one embodiment, R 1 is a C1-C7 alkyl group. In one embodiment, R 1 It is a C1 alkyl group.

[0633] 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.

[0634] 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.

[0635] 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.

[0636] In some embodiments, the helper lipid is represented by formula (II):

[0637]

[0638] or a salt or ester thereof, or a deuterated analog of any of the foregoing, wherein R 1 、R 2 、R 3 and R 4 As defined above in formula (I).

[0639] In some embodiments of Formula (II), R 3 and R 4 Both are hydrogen.

[0640] In some embodiments of Formula (II), R 3 and R 4 are independently hydrogen or C1 alkyl.

[0641] In some embodiments of Formula (II), R 1 is C1-C7 alkyl or C2-C7 alkenyl. In one embodiment, R 1 is a C1-C7 alkyl group. In one embodiment, R 1 It is a C1 alkyl group.

[0642] In some embodiments, the helper lipid is represented by formula (III):

[0643]

[0644] or a salt or ester thereof, or a deuterated analog of any of the foregoing, wherein R 1 、R 2 、R 3 and R 4 As defined above in formula (I).

[0645] In some embodiments of Formula (III), R 3 and R 4 Both are hydrogen.

[0646] In some embodiments of Formula (III), R 1 It is C1-C 10 Alkyl or C2-C 10 In one embodiment, R 1 It is C1-C 10 alkyl.

[0647] In some embodiments, the helper lipid is represented by formula (IV):

[0648]

[0649] or a salt or ester thereof, or a deuterated analog of any of the foregoing, wherein R 1 、R 2 、R 3 and R4 As defined above in formula (I).

[0650] As used herein, when referring to a helper lipid represented by Formula (I), (II), (III) or (IV), the term "salt" means a pharmaceutically acceptable salt of the helper lipid represented by Formula (I), (II), (III) or (IV), including both acid addition salts and base addition salts. Salts of the helper lipid represented by Formula (I), (II), (III) or (IV) retain the biological effectiveness and properties of the free acid form or free base form of the helper lipid represented by Formula (I), (II), (III) or (IV), which is biologically or otherwise undesirable and is formed from an inorganic acid or organic acid, or an inorganic base or organic base. Examples of inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; and examples of organic acids include, but are not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, hexanoic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, gluconic acid, gluconic acid , glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, etc. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines; substituted amines (including naturally occurring substituted amines); cyclic amines; and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, denanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, phenethylbenzylamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0651] As used herein, when referring to a helper lipid represented by Formula (I), Formula (II), Formula (III), or Formula (IV), the term "ester" means an ester of the helper lipid represented by Formula (I), Formula (II), Formula (III), or Formula (IV). As a non-limiting example, a hydroxyl group of a 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 carboxylate or phosphate) of the helper lipid represented by Formula (I), Formula (II), Formula (III), or Formula (IV).

[0652] As used herein, when referring to a helper lipid represented by Formula (I) (II), (III) (III) or (IV), "deuterated analog" means an analog of the helper lipid represented by Formula (I) (II), (III) or (IV) whereby any one or more hydrogen atoms of the lipid are replaced by deuterium, which is an isotope of hydrogen.

[0653] In some embodiments, the LNPs of the present disclosure do not contain or include distearoylphosphatidylcholine (DSPC), provided that there is a helper lipid represented by (I), (II), (III) or (IV), or a salt or ester thereof, or a deuterated analogue of any of the foregoing. In some embodiments, the LNPs of the present disclosure do not contain or include 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), provided that there is a helper lipid represented by (I), (II), (III) or (IV), or a salt or ester thereof, or a deuterated analogue of any of the foregoing. In some embodiments, the LNPs of the present disclosure do not contain or include 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), provided that there is a helper lipid represented by (I), (II), (III) or (IV), or a salt or ester thereof, or a deuterated analogue of any of the foregoing.

[0654] 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, is a double bond; R 1 、R 2 、R 3 and R 4 As defined above. In an alternative 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, is a single bond; R 1 、R 2 、R 3 and R 4 As defined above.

[0655] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or salts or esters thereof, or deuterated analogs of any of the foregoing, R 1 It is C1-C 15 Alkyl or C2-C 15 Alkenyl.

[0656] 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:

[0657] R 1 It is C1-C 15 Alkyl or C2-C 15 alkenyl;

[0658] R 2 It is C1-C 22 Alkyl or C2-C 22 alkenyl;

[0659] R 3 is hydrogen or C1-C2 alkyl; and

[0660] R 4 is hydrogen or C1-C2 alkyl.

[0661] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or salts or esters thereof, or deuterated analogs of any of the foregoing, R 1 It is C1-C 10 Alkyl or C2-C 10 Alkenyl.

[0662] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or salts or esters thereof:

[0663] R 1 It is C1-C 10 Alkyl or C2-C 10 alkenyl;

[0664] R 2 It is C1-C 22 Alkyl or C2-C 22 alkenyl;

[0665] R 3 is hydrogen or C1-C2 alkyl; and

[0666] R 4 is hydrogen or C1-C2 alkyl.

[0667] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or salts or esters thereof, or deuterated analogs of any of the foregoing, R 1 is C1-C8 alkyl or C2-C8 alkenyl. In one embodiment, R 1 is C1-C8 alkyl. In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or salts or esters thereof, or deuterated analogs of any of the foregoing, R 1 is C1-C7 alkyl or C2-C7 alkenyl. In one embodiment, R 1 is C1-C7 alkyl. In one embodiment of Formula (I), Formula (II), Formula (III) and Formula (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing:

[0668] R 1 is a C1-C7 alkyl group;

[0669] R 2 It is C1-C 22 Alkyl or C2-C 22 alkenyl;

[0670] R 3 is hydrogen or C1-C2 alkyl; and

[0671] R 4 is hydrogen or C1-C2 alkyl.

[0672] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or salts or esters thereof, or deuterated analogs of any of the foregoing, R 1 is C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl or C7 alkyl. In some embodiments of Formula (I), Formula (II), Formula (III) and Formula (IV), or salts or esters thereof, or deuterated analogs of any of the foregoing, R 1 is C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl or C7 alkyl. In some embodiments of Formula (I), Formula (II), Formula (III) and Formula (IV), or salts or esters thereof, or deuterated analogs of any of the foregoing, R 1 is C1 alkyl, C3 alkyl, C5 alkyl or 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 1In 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 It is a C7 alkyl group.

[0673] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or salts or esters thereof, or deuterated analogs of any of the foregoing, R 2 It is C3-C 15 Alkyl or C3-C 15 alkenyl; and R 1 、R 3 and R 4 As defined above. In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or salts or esters thereof, or deuterated analogs of any of the foregoing, R 2 It is C5-C 15 Alkyl or C3-C 15 alkenyl; and R 1 、R 3 and R 4 As defined above. In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or salts or esters thereof, or deuterated analogs of any of the foregoing, R 2 It is C7-C 15 Alkyl or C3-C 15 alkenyl; and R 1 、R 3 and R 4 As defined above. In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or salts or esters thereof, or deuterated analogs of any of the foregoing, R 2 It is C9-C 15 Alkyl or C9-C 15 alkenyl; and R 1 、R 3 and R 4 As defined above. In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or salts or esters thereof, or deuterated analogs of any of the foregoing, R 2 It is C9 alkyl, C 10 Alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 14 Alkyl or C15 alkyl; and R 1 、R 3 and R 4 As defined above. In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or salts or esters thereof, or deuterated analogs of any of the foregoing, R 2 is a C9 alkyl group; and R 1 、R 3 and R 4 As defined above. In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or salts or esters thereof, or deuterated analogs of any of the foregoing, R 2 It is C 11 alkyl; and R 1 、R 3 and R 4 As defined above. In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or salts or esters thereof, or deuterated analogs of any of the foregoing, R 2 It is C 13 alkyl; and R 1 、R 3 and R 4 As defined above.

[0674] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or salts or esters thereof, or deuterated analogs of any of the foregoing, R 3 is hydrogen or C1 alkyl; and R 1 、R 2 and R 4 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 and R 4 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 group; and R 1 、R 2 and R 4 As defined above.

[0675] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), or salts or esters thereof, or deuterated analogs of any of the foregoing, R 4 is hydrogen or C1 alkyl; and R 1、R 2 and R 3 As defined above. In one embodiment of Formula (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 R 1 、R 2 and R 3 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 group; and R 1 、R 2 and R 3 As defined above.

[0676] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), R 1 It is a C1-C7 alkyl group or a C2-C7 alkenyl group.

[0677] In some embodiments, R 1 is C1 alkyl, C3 alkyl, C5 alkyl or C7 alkyl. In some embodiments, R 1 It is a C1 alkyl group.

[0678] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), R 2 It is C3-C 15 Alkyl or C3-C 15 In some embodiments, R 2 It is C 10 Alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 14 Alkyl or C 15 In some embodiments, R 2 It is C 12 Alkyl, C 13 Alkyl or C 14 In some embodiments, R 2 It is C 13 In some embodiments, R 2 It is C 12 In some embodiments, R 2 It is C 11 alkyl.

[0679] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), R 1 and R 2Both are hydrogen; and It's a double bond.

[0680] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), R 1 and R 2 Both are hydrogen and is a double bond; and R 1 is C1 alkyl, C3 alkyl, C5 alkyl or C7 alkyl. In one embodiment, R 1 In another embodiment, R 1 In yet another embodiment, R 1 In yet another embodiment, R 1 It is a C7 alkyl group.

[0681] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), R 1 and R 2 Both are hydrogen and is a double bond; R 1 is C1 alkyl, C3 alkyl, C5 alkyl or C7 alkyl, and R 2 It is C9 alkyl, C 11 or C 13 In one embodiment, R 2 is a C9 alkyl group. In one embodiment, R 2 It is C 11 In another embodiment, R 2 It is C 13 alkyl.

[0682] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), R 3 In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), R 3 It is a C1 alkyl group.

[0683] In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), R 4 In some embodiments of Formula (I), Formula (II), Formula (III), and Formula (IV), R 4 It is a C1 alkyl group.

[0684] In some embodiments, the auxiliary lipid (e.g., ceramide or sphingomyelin) represented by formula (I), formula (II), formula (III), or formula (IV) in the LNP of the present disclosure is as shown in Table 8 below, or is a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0685] Table 8. Exemplary helper lipids (e.g., ceramide or sphingomyelin) in the LNPs of the present disclosure

[0686]

[0687]

[0688] 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 or 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.

[0689] As used herein, the term "salt" refers to pharmaceutically acceptable salts of the helper lipid, including both acid addition salts and base addition salts. Salts of the helper lipid retain the biological effectiveness and properties of the free acid form or free base form of the helper lipid.

[0690] As used herein, when referring to a helper lipid, the term "ester" means an ester of a helper lipid. As a non-limiting example, the hydroxyl group of a helper lipid can be connected to an organic acid (such as phosphoric acid or carboxylic acid) through an esterification process to form an ester (e.g., carboxylate or phosphate) of a helper lipid.

[0691] As used herein, "deuterated analog," when referring to a helper lipid, means an analog of the helper lipid in which any one or more hydrogen atoms of the helper lipid are replaced with deuterium.

[0692] 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).

[0693] In some embodiments, the helper lipid (e.g., a ceramide of the present disclosure) constitutes 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 % or 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 25 mol%, or 25 mol% to about 40 mol%, or about 25 mol% to about 35 mol%, or about 25 mol% to about 30 mol%, or 30 mol% to about 40 mol%, or about 30 mol% to about 35 mol%, or about 35 mol% to about 40 mol%, or about 5 mol%, or about 10 mol%, or about 15 mol%, or about 20%, or about 25 mol%, or about 30 mol%, or about 35 mol%, or about 40 mol%. In some embodiments, the helper lipid (e.g., DSPC, DOPE, ceramide, etc.) comprises about 10% mol to about 20 mol % of the total lipids present in the LNP, and such LNPs comprise about 10% mol to about 20 mol % of the total lipids present in the LNP, and exhibit overall improved tolerability (e.g., as demonstrated by weight loss profiles and reduced cytokine responses in subjects) compared to LNPs comprising less than 10% of the same helper lipid.

[0694] D. Lipid-anchored polymers

[0695] In certain embodiments, the LNP provided by the present disclosure comprises at least one type of lipid-anchored polymer, i.e., a first lipid-anchored polymer. As used herein, the term "lipid-anchored polymer" refers to a molecule comprising a lipid portion covalently attached to a polymer (optionally via a linker). Without wishing to be bound by a particular theory, it is believed that lipid-anchored polymers can inhibit the aggregation of LNP and provide steric stability. In certain embodiments, the LNP provided by the present disclosure comprises two lipid-anchored polymers, i.e., a first lipid-anchored polymer and a second lipid-anchored polymer.

[0696] Lipid moiety in lipid-anchored polymers

[0697] More specifically, in one embodiment, a lipid-anchored polymer, e.g., a first lipid-anchored polymer according to the present disclosure, comprises:

[0698] (i) polymers;

[0699] (ii) a lipid portion comprising at least one hydrophobic tail (which may be linear or branched); and

[0700] (iii) optionally a linker connecting the polymer to the lipid moiety;

[0701] wherein the at least one hydrophobic tail (which may be linear or branched) comprises 16 to 22 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, 18, 19, 20, 21 or 22 carbon atoms in a single aliphatic chain backbone. In one embodiment, the lipid-anchored polymer (e.g., a first lipid-anchored polymer) comprises a lipid portion comprising a single or two hydrophobic tails, wherein each of the single or two hydrophobic tails comprises 16 to 22 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, 18, 19, 20, 21 or 22 carbon atoms in a single aliphatic chain backbone. In one embodiment, each of the single or two hydrophobic tails comprises 18 to 22 carbon atoms in a single aliphatic chain backbone. In another embodiment, each of the single or two hydrophobic tails comprises 18 to 20 carbon atoms in a single aliphatic chain backbone. In a specific embodiment, the single or dual hydrophobic tails each comprise 18 carbon atoms in a single aliphatic chain backbone. In another embodiment, the single or dual hydrophobic tails each comprise at least 18 carbon atoms in a single aliphatic chain backbone.

[0702] 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.

[0703] 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, homosapiens acid, oleic acid, elaidic acid, vaccinic acid, linoleic acid, trans-linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, and derivatives thereof.

[0704] When used herein to refer to the hydrophobic tail in a lipid-anchored polymer, the term "derivative" refers to a hydrophobic tail that has been modified compared to the original or native hydrophobic tail. In some embodiments, the derivative contains one or more of the following modifications compared to the original or native hydrophobic tail: a) the carboxylate group has been replaced by an amine, amide, ether, or carbonate group; b) one or more saturation points (e.g., double bonds) have been introduced (e.g., by dehydrogenation) into the hydrophobic tail; c) one or more saturation points (e.g., double bonds) have been removed from the hydrophobic tail (e.g., by hydrogenation); and d) the configuration of one or more double bonds (if present) has been changed, for example, from a cis configuration to a trans configuration, or from a trans configuration to a cis configuration. A derivative contains the same number of carbon atoms as its original or native hydrophobic tail.

[0705] As used herein, when referring to the hydrophobic tail in a lipid-anchored polymer, the term "single aliphatic chain backbone" refers to the main straight aliphatic chain or carbon chain, i.e., the longest continuous straight aliphatic chain or carbon chain. For example, the following alkyl chain with several branches contains 18 carbon atoms in the single aliphatic chain backbone, i.e., the longest continuous straight alkyl chain contains 18 carbon atoms. Note that one or two carbon atoms in the several branch points (all indicated by *) are not included in the carbon atom count in the single aliphatic chain backbone.

[0706]

[0707] In one embodiment, the lipid-anchored polymer or the first lipid-anchored polymer according to the present disclosure comprises:

[0708] (i) polymers;

[0709] (ii) a lipid portion comprising at least two hydrophobic tails (which may be linear or branched); and

[0710] (iii) optionally a linker connecting the polymer to the lipid moiety;

[0711] wherein the at least two hydrophobic tails (which may be linear or branched) comprise 16 to 22 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, 18, 19, 20, 21 or 22 carbon atoms in a single aliphatic chain backbone. In one embodiment, the lipid-anchored polymer or the first lipid-anchored polymer comprises a lipid portion comprising two hydrophobic tails, wherein each of the two hydrophobic tails independently comprises 16 to 22 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, 18, 19, 20, 21 or 22 carbon atoms in a single aliphatic chain backbone. In one embodiment, each of the two hydrophobic tails independently comprises 16 to 21 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, 18, 19, 20 or 21 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 16 to 20 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, 18, 19 or 20 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 16 to 19 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, 18 or 19 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 16 to 18 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17 or 18 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 16 or 18 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 16 or 20 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 18 or 20 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each comprise 16 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each comprise 17 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each comprise 18 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each comprise 19 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each comprise 20 carbon atoms in a single aliphatic chain backbone.

[0712] In one embodiment, the at least two hydrophobic tails (e.g., two) are each a fatty acid. Non-limiting examples of at least two hydrophobic tails comprising 16 to 22 carbon atoms in a single aliphatic chain backbone include octadecylamine, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, homosapiens acid, oleic acid, elaidic acid, vaccinic acid, linoleic acid, trans-linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, and derivatives thereof.

[0713] In one embodiment, the lipid-anchored polymer or the first lipid-anchored polymer according to the present disclosure comprises:

[0714] (i) polymers;

[0715] (ii) a lipid portion comprising at least two hydrophobic tails (which may be linear or branched); and

[0716] (iii) optionally a linker connecting the polymer to the lipid moiety;

[0717] wherein the at least two hydrophobic tails (which may be linear or branched) comprise 12 to 15 carbon atoms in a single aliphatic chain backbone, i.e., 12, 13, 14, or 15 carbon atoms in a single aliphatic chain backbone. In one embodiment, the lipid-anchored polymer or the first lipid-anchored polymer comprises a lipid portion comprising two hydrophobic tails, wherein each of the two hydrophobic tails independently comprises 12 to 15 carbon atoms in a single aliphatic chain backbone, i.e., 12, 13, 14, or 15 carbon atoms in a single aliphatic chain backbone. In one embodiment, each of the two hydrophobic tails independently comprises 12 to 14 carbon atoms in a single aliphatic chain backbone, i.e., 12, 13, or 14 carbon atoms in a single aliphatic chain backbone. In one embodiment, each of the two hydrophobic tails independently comprises 12 or 14 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each contain 12 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each contain 13 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each contain 14 carbon atoms in a single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each contain 15 carbon atoms in a single aliphatic chain backbone.

[0718] In one embodiment, one of the two hydrophobic tails is a fatty acid. Non-limiting examples of at least two hydrophobic tails comprising 12 to 15 carbon atoms in a single aliphatic chain backbone include lauric acid, myristic acid, myristoleic acid, and derivatives thereof.

[0719] In one embodiment, the lipid-anchored polymer or the first lipid-anchored polymer according to the present disclosure comprises:

[0720] (i) polymers;

[0721] (ii) a lipid portion comprising a single hydrophobic tail (which may be linear or branched); and optionally

[0722] (iii) a linker connecting the polymer to the lipid moiety;

[0723] wherein the at least single hydrophobic tail (which may be linear or branched) comprises from 12 to 22 carbon atoms in a single aliphatic chain backbone, i.e., comprises 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 carbon atoms in a single aliphatic chain backbone. In one embodiment, the lipid-anchored polymer or the first lipid-anchored polymer comprises a lipid portion comprising a single hydrophobic tail, wherein the single hydrophobic tail comprises from 12 to 22 carbon atoms in a single aliphatic chain backbone, i.e., comprises 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 carbon atoms in a single aliphatic chain backbone. In one embodiment, the single hydrophobic tail comprises 12, 14, 16, 18, 20 or 22 carbon atoms in a single aliphatic chain backbone. In one embodiment, the single hydrophobic tail comprises 16 to 20 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, 18, 19, or 20 carbon atoms in a single aliphatic chain backbone. In one embodiment, the single hydrophobic tail comprises 16 to 19 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, 18, or 19 carbon atoms in a single aliphatic chain backbone. In one embodiment, the single hydrophobic tail comprises 16 to 18 carbon atoms in a single aliphatic chain backbone, i.e., 16, 17, or 18 carbon atoms in a single aliphatic chain backbone. In one embodiment, the single hydrophobic tail comprises 12 carbon atoms in a single aliphatic chain backbone. In one embodiment, the single hydrophobic tail comprises 13 carbon atoms in a single aliphatic chain backbone. In one embodiment, the single hydrophobic tail comprises 14 carbon atoms in a single aliphatic chain backbone. In one embodiment, the single hydrophobic tail comprises 15 carbon atoms in a single aliphatic chain backbone. In one embodiment, the single hydrophobic tail comprises 16 carbon atoms in a single aliphatic chain backbone. In one embodiment, the single hydrophobic tail comprises 17 carbon atoms in a single aliphatic chain backbone. In one embodiment, the single hydrophobic tail comprises 18 carbon atoms in a single aliphatic chain backbone. In one embodiment, the single hydrophobic tail comprises 19 carbon atoms in a single aliphatic chain backbone. In one embodiment, the single hydrophobic tail comprises 20 carbon atoms in a single aliphatic chain backbone. In one embodiment, the single hydrophobic tail comprises 21 carbon atoms in a single aliphatic chain backbone. In one embodiment, the single hydrophobic tail comprises 22 carbon atoms in a single aliphatic chain backbone.

[0724] 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, homosapiens acid, oleic acid, elaidic acid, vaccinic acid, linoleic acid, trans-linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, and derivatives thereof.

[0725] Linkers in lipid-anchored polymers

[0726] In some embodiments, in the lipid-anchored polymers of the present disclosure, the lipid moiety is directly covalently attached to the polymer, or is optionally connected 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 phosphorylcholine linker, or any combination thereof. In some embodiments, the linker in the lipid-anchored polymer in the LNP of the present disclosure is a glycerol linker. Therefore, in some embodiments, the lipid-anchored polymer in the LNP of the present disclosure is a glycerolipid, wherein the glycerolipid comprises glycerol as a linker and one or more two lipid moieties as described above, such as distearoyl-rac-glycerol (DSG).

[0727] In some embodiments, the linker in the lipid-anchored polymer in the LNP of the present disclosure is a phosphate linker. Thus, in some embodiments, the lipid-anchored polymer in the LNP of the present disclosure is a phospholipid, wherein the phospholipid comprises a phosphate group as a linker and one or more lipid moieties as described above.

[0728] In some embodiments, the lipid-anchoring polymer in the LNPs of the present disclosure is both a glycerolipid and a phospholipid, such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

[0729] In some embodiments, the first lipid-anchoring polymer comprises 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: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), , 1,2-dioleoyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 18-1-trans PE, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE); and dioctadecylamine (DODA), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), derivatives thereof, and combinations of any of the foregoing.

[0730] As used herein, the term "derivative" when used in reference to a linker lipid moiety means a linker lipid moiety containing one or more of the following modifications: a) the phosphatidylethanolamine (PE) head group (if present) is 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, such as -OH, -OCH3, -NH2, maleimide, azide, or cyclooctyne, such as dibenzocyclooctyne (DBCO).

[0731] In one embodiment, the first lipid-anchoring polymer comprises 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 DOPE, DSPE, DSG, DODA, DPG, derivatives thereof, and combinations of any of the foregoing.

[0732] In some embodiments, the first lipid-anchoring polymer comprises a linker lipid moiety (i.e., having one or more hydrophobic tails containing 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-dimyristyloxy-propyl-3-amine, derivatives thereof, and combinations of any of the foregoing. In one embodiment, the first lipid-anchoring polymer comprises DMG.

[0733] Polymers in lipid-anchored polymers

[0734] 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.

[0735] In one embodiment, the polymer is polyethylene glycol (PEG). In another embodiment, the polymer is polyglycerol (PG).

[0736] In some embodiments, the molecular weight of the polymer in the lipid-anchored polymer is 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 or less, or about 50 Da or less. In some embodiments, the average molecular weight of the polymer in the lipid-anchored polymer is 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, for example, 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 average molecular weight of the polymer in the lipid-anchored polymer is 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 average molecular weight of the polymer in the lipid-anchored polymer is about 2000 Da. In some embodiments, the average molecular weight of the polymer in the lipid-anchored polymer is about 2000 Da. In some embodiments, the average molecular weight of the polymer in the lipid-anchored polymer is about 3200 Da to about 3500 Da. In some embodiments, the average molecular weight of the polymer in the lipid-anchored polymer is about 3300 Da. In some embodiments, the average molecular weight of the polymer in the lipid-anchored polymer is about 3350 Da. In some embodiments, the average molecular weight of the polymer in the lipid-anchored polymer is about 3400 Da. In some embodiments, the average molecular weight of the polymer in the lipid-anchored polymer is about 3500 Da.

[0737] Targeting moiety and second lipid-anchored polymer

[0738] In some embodiments, the LNP of the present disclosure further comprises one or more targeting moieties. The targeting moiety targets the LNP to be delivered to a specific cell type or tissue in the subject, for example, liver, bone marrow, spleen, blood, etc. In some embodiments, the targeting moiety is capable of binding to a specific cell type (e.g., hepatocytes, T cells, B cells, NK cells, dendritic cells, etc.). 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 antibodies.

[0739] The antibody can be a complete monoclonal antibody or a polyclonal antibody, as well as an immunologically active fragment (e.g., a 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, for example, an antibody containing the binding specificity of a murine antibody but the remainder being of human origin. Antibodies (including monoclonal antibodies 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, for example, an antibody or antibody fragment that can specifically bind to an antigen present on the surface of a cell. In one embodiment, the antibody or antibody fragment is a monoclonal antibody (mAb), a single-chain variable fragment (scFv), a heavy chain antibody (hcAb), a nanobody (Nb), a heavy chain immunoglobulin only (HClg), an immunoglobulin new antigen receptor (IgNAR), a variable domain of an immunoglobulin new antigen receptor (VNAR), a single domain antibody or a variable heavy chain antibody only (VHH). In one embodiment, the antibody target moiety is scFv. In another embodiment, the antibody targeting moiety is IgG. In 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 a T cell receptor (TCR), CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, CD19, CD21, CD28, or PD-1.

[0740] In some embodiments, the LNP of the present disclosure further comprises one or more targeting moieties capable of binding to specific liver cells (livercell) (such as liver cells (hepatocytes)). In one embodiment, the targeting moiety can bind to asialoglycoprotein receptor (ASGPR), i.e., liver cell-specific ASGPR. In one embodiment, the targeting moiety comprises N-acetylgalactosamine molecule (GalNAc) or its GalNAc derivative. As used herein, "GalNAc derivative" refers to a conjugate covalently attached to, for example, a modified GalNAc molecule or one or more GalNAc molecules (modified or unmodified) of a lipid-anchored polymer as defined herein. In one embodiment, the targeting moiety is a triantennary or trivalent GalNAc conjugate (i.e., GalNAc3), which is a ligand conjugate with three GalNAc molecules or three GalNAc derivatives. In one embodiment, the targeting moiety is a triantennary GalNAc represented by the following structural formula:

[0741]

[0742] In one embodiment, the targeting moiety is a tetraantennary GalNAc conjugate. In one embodiment, the targeting moiety is a tetraantennary or tetravalent GalNAc conjugate (ie, GalNAc4), which is a ligand having four GalNAc molecules or four GalNAc derivatives.

[0743] In one embodiment, the targeting moiety is capable of binding to a low-density lipoprotein receptor (LDLR) (e.g., a hepatocyte-specific LDLR). In one embodiment, the targeting moiety comprises apolipoprotein E (ApoE) protein, an ApoE polypeptide (or peptide), an apolipoprotein B (ApoB) protein, an ApoB polypeptide (or polypeptide), a fragment of any of the foregoing, or a derivative of any of the foregoing. In one embodiment, the ApoE polypeptide, the ApoB polypeptide, or a fragment thereof is an ApoE polypeptide, an ApoB polypeptide, or a fragment thereof as disclosed in International Patent Application Publication No. WO2022 / 261101, which is incorporated herein by reference in its entirety. In one embodiment, the ApoE protein is a modified ApoE protein, and the ApoB protein is a modified ApoB protein. In one embodiment, the ApoE protein has an amino acid sequence that has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the following amino acid sequence:

[0744] MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVR (SEQ ID NO: 1). In one embodiment, the ApoE protein comprises or consists of the amino acid sequence shown in SEQ ID NO: 1. In one embodiment, the ApoE protein has an amino acid sequence that has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the following amino acid sequence:

[0745] MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRHHHHHH (SEQ ID NO: 2). In one embodiment, the ApoE protein comprises the amino acid sequence shown in SEQ ID NO: 2. In one embodiment, the ApoE protein consists of the amino acid sequence shown in SEQ ID NO: 2. In one embodiment, the ApoE protein has an amino acid sequence that has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the following amino acid sequence:

[0746] MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVSGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVR (SEQ ID NO: 3). In one embodiment, the ApoE protein comprises or consists of the amino acid sequence shown in SEQ ID NO: 3. In one embodiment, the ApoE protein has an amino acid sequence that has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the following amino acid sequence:

[0747] MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVSGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRHHHHHHGGSSGSGC (SEQ ID NO: 4). In one embodiment, the ApoE protein comprises the amino acid sequence shown in SEQ ID NO: 4. In one embodiment, the ApoE protein consists of the amino acid sequence shown in SEQ ID NO: 4.

[0748] As used herein, the term "sequence identity" refers to the ratio of the number of identical amino acids between two compared sequences over the length of the comparison, expressed as a percentage. In some embodiments, the two compared sequences are the same length, i.e., have the same number of amino acids.

[0749] In one embodiment, the targeting moiety in the LNP of the present disclosure is an ApoE protein conjugate in an ApoB protein conjugate, which is a conjugate of one or more ApoE and / or ApoB protein molecules (native or modified) or fragments thereof covalently linked to a lipid-anchored polymer, e.g., as defined herein. In one embodiment, the targeting moiety in the LNP of the present disclosure is an ApoE polypeptide conjugate in an ApoB polypeptide conjugate, which is a conjugate of one or more ApoE and / or ApoB polypeptide molecules or fragments thereof covalently linked to a lipid-anchored polymer, e.g., as defined herein.

[0750] Thus, a key embodiment of the LNPs disclosed herein is that the LNPs comprise a second lipid-anchored polymer, and a targeting moiety as defined herein (and including GalNAc, ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide) is conjugated to the second lipid-anchored polymer. The second lipid-anchored polymer is structurally similar to the first lipid-anchored polymer as described herein in that the second lipid-anchored polymer also contains a lipid portion covalently linked to the polymer via a linker. In one embodiment, the second lipid anchoring polymer comprises a linker lipid moiety selected from the group consisting of: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-phosphatidylethanolamine (PPE), 1,2-dioleoyl-sn-phosphatidylethanolamine (DSPE), 1,2-dioleoyl-sn-phosphatidylethanolamine (PPE), 1,2-dioleoyl-sn-phosphatidylethanolamine (PPE), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-phosphatidylethanolamine (PPE ... amine (DEPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 18-1-trans PE, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE); and dioctadecylamine (DODA), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), derivatives thereof, and combinations of any one of the foregoing. In one embodiment, the second lipid-anchoring polymer comprises a linker lipid portion selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, derivatives thereof, and combinations of any one of the foregoing.

[0751] In one embodiment, the ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide, or fragment thereof is covalently linked to a lipid anchoring polymer (e.g., a second lipid anchoring polymer) or to the LNP of the present disclosure via strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry, such as by an azide-modified lipid anchoring polymer (e.g., DSG-PEG2000 azide, DSPE-PEG2000 azide, DSG-PEG3400 azide, DSPE-PEG3400 azide, DSG-PEG5000 azide, DSPE-PEG5000 azide) and dibenzocyclooctyne (DBCO)-functionalized ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide, or fragment thereof.

[0752] In some embodiments, the LNPs of the present disclosure may comprise a first lipid-anchored polymer and a second lipid-anchored polymer. For example, the LNPs of the present disclosure may comprise a first lipid-anchored polymer that does not comprise a targeting moiety and a second lipid-anchored polymer that comprises a targeting moiety, such as GalNAc. For example, the LNPs of the present disclosure may comprise DSG-PEG2000 modified to comprise an additional OCH3 group (DSG-PEG2000-OMe) as the first lipid-anchored polymer, and DSPE-PEG2000-GalNAc3 as the second lipid-anchored polymer.

[0753] In some embodiments, the LNPs of the present disclosure may comprise a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the second lipid-anchored polymer comprises a targeting moiety. In some embodiments, the second lipid-anchored polymer comprises a lipid moiety selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, and derivatives thereof. In some embodiments, the first lipid-anchored polymer is any lipid-anchored polymer as described above.

[0754] In one embodiment, the LNP of the present disclosure comprises a second lipid-anchored polymer, and a targeting moiety as defined herein (e.g., mAb, IgG, scFv, VHH, GalNAc, ApoE protein or peptide, ApoB protein or peptide) is conjugated to the second lipid-anchored polymer. The second lipid-anchored polymer is structurally similar to the first lipid-anchored polymer in that the second lipid-anchored polymer also contains a lipid portion comprising a hydrophobic fatty acid tail having a C-terminal end covalently attached to the polymer via a linker. 18 -C 22In one embodiment, the second lipid-anchoring polymer comprises a lipid linker portion (also referred to as a "linker lipid portion") selected from the group consisting of: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-di ... n-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-anchoring polymer comprises a lipid linker moiety (linker lipid moiety) selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, derivatives thereof, and combinations of any of the foregoing.

[0755] The lipid-anchored polymers of the present disclosure may also include 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 polymers of the present disclosure can be used for conjugation, for example, with 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 thiol reagent, a maleimide reagent, or a click chemistry reagent, for example, a reagent selected from the group consisting of: an alkyne reagent (such as a dibenzocyclooctyne (DBCO) reagent), a trans-cyclooctene (TCO) reagent, a tetrazine (TZ) reagent, and an azide (AZ) reagent.

[0756] In one embodiment, the antibody or fragment thereof (e.g., IgG, scFv, VHH) is covalently linked to a lipid-anchoring polymer (e.g., a second lipid-anchoring polymer) via strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry, such as via an azide-modified lipid-anchoring polymer (e.g., DSG-PEG2000 azide, DSPE-PEG2000 azide, DSG-PEG3400 azide, DSPE-PEG3400 azide, DSG-PEG5000 azide, DSPE-PEG5000 azide; DODA-PG46 azide) and dibenzocyclooctyne (DBCO)-functionalized scFv, VHH, IgG, or fragment thereof.

[0757] In an exemplary embodiment, the second lipid-anchored polymer conjugated to the targeting moiety is represented by the following structure:

[0758]

[0759] In another exemplary embodiment, the second lipid-anchored polymer conjugated to the targeting moiety is represented by the following structure:

[0760]

[0761] In one embodiment, the ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide, or fragment thereof is covalently linked to a lipid-anchoring polymer (e.g., a second lipid-anchoring polymer) via strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry, such as via an azide-modified lipid-anchoring polymer (e.g., DSG-PEG2000 azide, DSPE-PEG2000 azide, DSG-PEG3400 azide, DSPE-PEG3400 azide, DSG-PEG5000 azide, DSPE-PEG5000 azide; DODA-PG azide) and dibenzocyclooctyne (DBCO)-functionalized ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide, or fragment thereof.

[0762] In some embodiments, the LNP of the present disclosure may comprise a first lipid-anchored polymer and a second lipid-anchored polymer. For example, the LNP of the present disclosure may comprise a first lipid-anchored polymer that does not comprise a targeting moiety and a second lipid-anchored polymer that comprises a targeting moiety, such as scFv, VHH, GalNAc, ApoE protein / peptide, ApoB protein / peptide. For example, the LNP of the present disclosure may comprise DSG-PEG2000 modified to comprise an additional OCH3 group (DSG-PEG2000-OMe) as the first lipid-anchored polymer, and DSPE-PEG2000-scFv as the second lipid-anchored polymer.

[0763] In a specific embodiment, the first lipid-anchoring polymer is a polymer-conjugated lipid of the present disclosure, e.g., DODA-PG34, DODA-PG45, DODA-PG46, or DODA-PG58. For example, the LNP of the present disclosure may comprise DODA-PG45 as the first lipid-anchoring polymer and DSPE-PEG2000-scFv as the second lipid-anchoring polymer.

[0764] In some embodiments, the LNPs of the present disclosure may comprise a first lipid-anchoring polymer and a second lipid-anchoring polymer, wherein the second lipid-anchoring polymer comprises a targeting moiety. In some embodiments, the second lipid-anchoring 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-anchoring polymer is any lipid-anchoring polymer as described above.

[0765] In some embodiments, the LNPs of the present disclosure may comprise a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the second lipid-anchored polymer comprises a targeting moiety, and the lipid linker of the first lipid-anchored polymer and the second lipid-anchored polymer are the same, but their hydrophilic polymers are different.

[0766] In some embodiments, the LNPs of the present disclosure may comprise a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the second lipid-anchored polymer comprises a targeting moiety, and the lipid linker (linker lipid portion) of the first lipid-anchored polymer and the second lipid-anchored polymer are different, as shown below:

[0767] DSG-PEG (the first lipid-anchoring polymer) and DSPE-PEG (the second lipid-anchoring polymer);

[0768] DSPE-PEG (the first lipid-anchoring polymer) and DSG-PEG (the second lipid-anchoring polymer);

[0769] DODA-PG (the first lipid-anchoring polymer) and DSPE-PEG (the second lipid-anchoring polymer);

[0770] DPG-PEG (the first lipid-anchoring polymer) and DSPE-PEG (the second lipid-anchoring polymer);

[0771] DODA-PG (the first lipid-anchoring polymer) and DSG-PEG (the second lipid-anchoring polymer);

[0772] DPG-PEG (the first lipid-anchoring polymer) and DSG-PEG (the second lipid-anchoring polymer); and

[0773] DPG-PEG (the first lipid anchoring polymer) and DODA-PG (the second lipid anchoring polymer).

[0774] In some embodiments, the LNPs of the present disclosure may comprise a first lipid-anchoring polymer and a second lipid-anchoring polymer, wherein the second lipid-anchoring polymer comprises a targeting moiety, and the first lipid-anchoring polymer and the second lipid-anchoring polymer are the same lipid-anchoring polymer and are selected from one of the following combinations:

[0775] DSG-PEG (the first lipid-anchoring polymer) and DSG-PEG (the second lipid-anchoring polymer);

[0776] DSPE-PEG (the first lipid-anchored polymer) and DSPE-PEG (the second lipid-anchored polymer);

[0777] DODA-PG (the first lipid-anchored polymer) and DODA-PG (the second lipid-anchored polymer); and

[0778] DPG-PEG (the first lipid anchoring polymer) and DPG-PEG (the second lipid anchoring polymer).

[0779] In some embodiments, the targeting moiety is conjugated to a DSPE anchoring polymer. In some embodiments, the DSPE anchoring polymer is DSPE-PEG or a derivative thereof.

[0780] In some embodiments, the targeting moiety is conjugated to a DSG anchoring polymer. In some embodiments, the DSG anchoring polymer is DSG-PEG or a derivative thereof.

[0781] In some embodiments, the 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, the 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, the 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.

[0782] In some embodiments, the LNPs provided herein 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, the LNPs provided herein 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, the LNPs provided herein 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.

[0783] In some embodiments, the LNPs provided herein 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, the LNPs provided herein 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, the LNPs provided herein 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.

[0784] In some embodiments, the LNPs provided herein 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, the LNPs provided herein 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, the LNPs provided herein 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.

[0785] In some embodiments, the LNPs provided herein 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 having an average of 46 glycerol repeating units). In some embodiments, the LNPs provided herein consist essentially of: a therapeutic nucleic acid (TNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; and DODA-PG; and bis-DODA-PG46 (e.g., d18:1 / 2:0 or d14:1 / 2:0). In some embodiments, the LNPs provided herein consist of: a therapeutic nucleic acid (TNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; and DODA-PG46.

[0786] In some embodiments, the LNPs provided herein 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 having an average of 34 glycerol units). In some embodiments, the LNPs provided herein 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, the LNPs provided herein consist of: a therapeutic nucleic acid (TNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; and DODA-PG34.

[0787] In some embodiments, the LNPs provided herein 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, the LNPs provided herein 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, the LNPs provided herein 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.

[0788] In some embodiments, the LNPs provided herein 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, the LNPs provided herein 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, the LNPs provided herein 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.

[0789] In some embodiments, the 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, the 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, the 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.

[0790] In some embodiments, the 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, the 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, the 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.

[0791] In some embodiments, the LNPs provided herein 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, the LNPs provided herein 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, the LNPs provided herein consist of: a therapeutic nucleic acid (TNA); an ionizable lipid; a helper lipid (e.g., DSPC, DOPE, ceramide); cholesterol; and DSG-PEG2000-OMe.

[0792] In some embodiments, the LNPs provided by the present disclosure comprise: therapeutic nucleic acids (TNAs); ionizable lipids; helper lipids (e.g., DSPC, DOPE, ceramides); cholesterol; and DSG-PEG2000-OH. In some embodiments, the LNPs provided by the present disclosure consist essentially of: therapeutic nucleic acids (TNAs); ionizable lipids; helper lipids (e.g., DSPC, DOPE, ceramides); cholesterol; DSG-PEG2000-OH; and DSPE-PEG2000-VHH. In some embodiments, the LNPs provided by the present disclosure consist of: therapeutic nucleic acids (TNAs); ionizable lipids; helper lipids (e.g., DSPC, DOPE, ceramides); cholesterol; DSG-PEG2000-OH; and DSPE-PEG2000-VHH.

[0793] In some embodiments, the LNPs provided herein 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, the LNPs provided herein 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, the LNPs provided herein 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.

[0794] In some embodiments, the 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, the 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, the 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.

[0795] In some embodiments, the LNPs provided herein 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, the LNPs provided herein 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, the LNPs provided herein 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.

[0796] In some embodiments, the LNPs provided herein 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, the LNPs provided herein 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, the LNPs provided herein 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.

[0797] In some embodiments, the lipid-anchoring polymer (combination of the first and second lipid-anchoring polymers) comprises about 0.1 mol% to about 20 mol% of the total lipid present in the LNP. In some embodiments, the lipid-anchoring polymer comprises about 0.5 mol% to about 10 mol% present in the LNP. In some embodiments, the lipid-anchoring polymer comprises about 1 mol% to about 10 mol% present in the LNP. In some embodiments, the lipid-anchoring polymer comprises about 2 mol% to about 10 mol% present in the LNP. In some embodiments, the lipid-anchoring 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% present in the LNP. In some embodiments, the lipid-anchoring polymer comprises about 3 mol% to about 8 mol% present in the LNP. In some embodiments, the lipid-anchoring polymer comprises about 3 mol% to about 7 mol% present in the LNP. In some embodiments, the lipid-anchoring polymer comprises about 3 mol% to about 5 mol% present in the LNP. In some embodiments, the lipid-anchored polymer constitutes about 2 mol% to about 4 mol% of the LNP. In some embodiments, the lipid-anchored polymer constitutes about 2% to about 3% of the LNP. In some embodiments, the lipid-anchored polymer constitutes about 2 mol% of the LNP. In some embodiments, the lipid-anchored polymer constitutes about 2.5 mol% of the LNP. In some embodiments, the lipid-anchored polymer constitutes about 3 mol% of the LNP. In some embodiments, the lipid-anchored polymer constitutes about 3.5 mol% of the LNP. In some embodiments, the lipid-anchored polymer constitutes about 4 mol% of the LNP. In some embodiments, the lipid-anchored polymer constitutes about 5 mol% of the LNP. In some embodiments, the lipid-anchored polymer constitutes about 6 mol% of the LNP. In some embodiments, the lipid-anchored polymer constitutes about 7 mol% of the LNP. In some embodiments, the lipid-anchored polymer constitutes about 8 mol% of the LNP. In some embodiments, the lipid-anchored polymer constitutes about 9 mol% of the LNP. In some embodiments, the lipid-anchored polymer constitutes about 10 mol% of the LNP.

[0798] In some embodiments, the first lipid-anchoring polymer comprises about 0.1 mol% to about 10 mol%, or about 0.2 mol% to about 8 mol%, or about 0.2 mol% to about 7 mol%, or about 0.2 mol% to about 5 mol%, or about 0.3 mol% to about 4 mol%, or about 0.4 mol% to about 4 mol%, or about 0.5 mol% to about 5 mol%, or about 0.5 mol% to 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 mol% to about 3 mol%, or about 1 mol% to about 4 mol%. From about 1.5 mol% to about 5 mol%, or from about 1.5 mol% to about 4 mol%, or from about 1.5 mol% to about 3.5 mol%, or from about 1.5 mol% to about 3 mol%, or from about 2 mol% to about 5 mol%, or from about 2 mol% to about 4 mol%, or from about 2 mol% to about 3.5 mol%, or from about 2 mol% to about 3 mol%, or from about 2.5 mol% to about 5 mol%, or from about 2.5 mol% to about 4 mol%, or from about 2.5 mol% to about 3.5 mol%, or from about 2.5 mol% to about 3 mol%, or from about 3 mol% to about 5 mol%, or from about 3 mol% to about 4.5 mol%, or from about 3 mol% to about 4 mol%, or from about 3 mol% to about 3.5 mol%, or from about 3.5 mol% to about 5 mol%, or from about 3.5 mol% to about 4 mol%, or from about 3 mol% to about 7 mol% is present.

[0799] In some embodiments, the second lipid-anchoring polymer, if present, comprises about 0.005 mol% to about 5 mol%, or about 0.005 mol% to about 3 mol%, or about 0.005 mol% to about 2 mol%, or about 0.005 mol% to about 1 mol%, or about 0.005 mol% to about 0.5 mol%, or about 0.01 mol% to about 3 mol%, or about 0.01 mol% 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 about 0.025 mol% to about 0.25 mol%, or about 0.025 mol% to about 0.2 mol%, or about 0.025 mol% to about 0.1 mol%, or about 0.05 mol% to about 0.4 mol%, or about 0.05 mol% to about 0.3 mol%, or about 0.05 mol% to about 0.25 mol%, or about 0.05 mol% to about 0.2 mol%, or about 0.05 mol% to about 0.1 mol%. In some embodiments, the second lipid-anchoring polymer is present at about 0.5 mol%.

[0800] In some embodiments, the targeting moiety is conjugated to a DSPE anchoring polymer. In some embodiments, the DSPE anchoring polymer is DSPE-PEG or a derivative thereof.

[0801] In some embodiments, the targeting moiety is conjugated to a DSG anchoring polymer. In some embodiments, the DSG anchoring polymer is DSG-PEG or a derivative thereof.

[0802] In some embodiments, the LNPs provided herein comprise: a therapeutic nucleic acid (TNA); an ionizable lipid; a C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0); cholesterol; and DSG-PEG2000-OMe. In some embodiments, the LNPs provided herein consist essentially of: a therapeutic nucleic acid (TNA); an ionizable lipid; a C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0); cholesterol; and DSG-PEG2000-OMe. In some embodiments, the LNPs provided herein consist of: a therapeutic nucleic acid (TNA); an ionizable lipid; a C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0); cholesterol; and DSG-PEG2000-OMe.

[0803] In some embodiments, the LNPs provided by the present disclosure comprise: a therapeutic nucleic acid (TNA); an ionizable lipid; a C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0); 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; a C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0); 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; a C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0); cholesterol; and DSG-PEG2000-OH.

[0804] In some embodiments, the LNPs provided herein comprise: a therapeutic nucleic acid (TNA); an ionizable lipid; a C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0); cholesterol; and bis-DSG-PEG2000-OMe. In some embodiments, the LNPs provided herein consist essentially of: a therapeutic nucleic acid (TNA); an ionizable lipid; a C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0); cholesterol; and bis-DSG-PEG2000-OMe. In some embodiments, the LNPs provided herein consist of: a therapeutic nucleic acid (TNA); an ionizable lipid; a C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0); cholesterol; and bis-DSG-PEG2000-OMe.

[0805] In some embodiments, the LNPs provided herein comprise: a therapeutic nucleic acid (TNA); an ionizable lipid; a 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 repeating units). In some embodiments, the LNPs provided herein consist essentially of: a therapeutic nucleic acid (TNA); an ionizable lipid; a C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0); cholesterol; and DODA-PG; and bis-DODA-PG46 (e.g., d18:1 / 2:0 or d14:1 / 2:0). In some embodiments, the LNPs provided herein consist of: a therapeutic nucleic acid (TNA); an ionizable lipid; a C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0); cholesterol; and DODA-PG46.

[0806] In some embodiments, the LNPs provided herein comprise: a therapeutic nucleic acid (TNA); an ionizable lipid; a 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, the LNPs provided herein consist essentially of: a therapeutic nucleic acid (TNA); an ionizable lipid; a C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0); cholesterol; and bis-DODA-PG34. In some embodiments, the LNPs provided herein consist of: a therapeutic nucleic acid (TNA); an ionizable lipid; a C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0); cholesterol; and DODA-PG34.

[0807] In some embodiments, the LNPs provided herein comprise: a therapeutic nucleic acid (TNA); an ionizable lipid; DSPC; cholesterol; and DSG-PEG2000-OMe. In some embodiments, the LNPs provided herein consist essentially of: a therapeutic nucleic acid (TNA); an ionizable lipid; DSPC; cholesterol; and DSG-PEG2000-OMe. In some embodiments, the LNPs provided herein consist of: a therapeutic nucleic acid (TNA); an ionizable lipid; DSPC; cholesterol; and DSG-PEG2000-OMe.

[0808] In some embodiments, the LNPs provided herein comprise: a therapeutic nucleic acid (TNA); an ionizable lipid; DSPC; cholesterol; and DSG-PEG2000-OH. In some embodiments, the LNPs provided herein consist essentially of: a therapeutic nucleic acid (TNA); an ionizable lipid; DSPC; cholesterol; and DSG-PEG2000-OH. In some embodiments, the LNPs provided herein consist of: a therapeutic nucleic acid (TNA); an ionizable lipid; DSPC; cholesterol; and DSG-PEG2000-OH.

[0809] In some embodiments, the LNPs provided herein comprise: a therapeutic nucleic acid (TNA); an ionizable lipid; DOPE; cholesterol; and DSG-PEG2000-OMe. In some embodiments, the LNPs provided herein consist essentially of: a therapeutic nucleic acid (TNA); an ionizable lipid; DOPE; cholesterol; and DSG-PEG2000-OMe. In some embodiments, the LNPs provided herein consist of: a therapeutic nucleic acid (TNA); an ionizable lipid; DOPE; cholesterol; and DSG-PEG2000-OMe.

[0810] In some embodiments, the LNPs provided herein comprise: a therapeutic nucleic acid (TNA); an ionizable lipid; DSPC; cholesterol; and DSPE-PEG2000-OH. In some embodiments, the LNPs provided herein consist essentially of: a therapeutic nucleic acid (TNA); an ionizable lipid; DSPC; cholesterol; and DSPE-PEG2000-OH. In some embodiments, the LNPs provided herein consist of: a therapeutic nucleic acid (TNA); an ionizable lipid; DSPC; cholesterol; and DSPE-PEG2000-OH.

[0811] In some embodiments, the LNPs provided herein comprise: a therapeutic nucleic acid (TNA); an ionizable lipid; DOPE; cholesterol; and DSG-PEG2000-OMe. In some embodiments, the LNPs provided herein consist essentially of: a therapeutic nucleic acid (TNA); an ionizable lipid; DOPE; cholesterol; and DSG-PEG2000-OMe. In some embodiments, the LNPs provided herein consist of: a therapeutic nucleic acid (TNA); an ionizable lipid; DOPE; cholesterol; and DSG-PEG2000-OMe.

[0812] In some embodiments, the LNPs provided herein comprise: a therapeutic nucleic acid (TNA); an ionizable lipid; DOPE; cholesterol; and DSPE-PEG2000-OH. In some embodiments, the LNPs provided herein consist essentially of: a therapeutic nucleic acid (TNA); an ionizable lipid; DOPE; cholesterol; and DSPE-PEG2000-OH. In some embodiments, the LNPs provided herein consist of: a therapeutic nucleic acid (TNA); an ionizable lipid; DOPE; cholesterol; and DSPE-PEG2000-OH.

[0813] In some embodiments, the LNPs provided herein comprise: a therapeutic nucleic acid (TNA); an ionizable lipid; DSPC; cholesterol; DSG-PEG2000-OMe and DSPE-PEG2000-GalNAc3. In some embodiments, the LNPs provided herein consist essentially of: a therapeutic nucleic acid (TNA); an ionizable lipid; DSPC; cholesterol; DSG-PEG2000-OMe and DSPE-PEG2000-GalNAc3. In some embodiments, the LNPs provided herein consist of: a therapeutic nucleic acid (TNA); an ionizable lipid; DSPC; cholesterol; DSG-PEG2000-OMe and DSPE-PEG2000-GalNAc3.

[0814] In some embodiments, the LNPs provided herein comprise: a therapeutic nucleic acid (TNA); an ionizable lipid; a 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 herein consist essentially of: a therapeutic nucleic acid (TNA); an ionizable lipid; a 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 herein consist of: a therapeutic nucleic acid (TNA); an ionizable lipid; a C2 ceramide (e.g., d18:1 / 2:0 or d14:1 / 2:0); cholesterol; DSG-PEG2000-OH and DSPE-PEG2000-GalNAc3.

[0815] In some embodiments, the LNPs provided herein comprise: a therapeutic nucleic acid (TNA); an ionizable lipid; DSPC; cholesterol; bis-DSG-PEG2000 and DSPE-PEG2000. In some embodiments, the LNPs provided herein consist essentially of: a therapeutic nucleic acid (TNA); an ionizable lipid; DSPC; cholesterol; bis-DSG-PEG2000 and DSPE-PEG2000. In some embodiments, the LNPs provided herein consist of: a therapeutic nucleic acid (TNA); an ionizable lipid; DSPC; cholesterol; bis-DSG-PEG2000 and DSPE-PEG2000.

[0816] In some embodiments, the LNPs provided herein comprise: a therapeutic nucleic acid (TNA); an ionizable lipid; DSPC; cholesterol; DODA-PG46 and DSPE-PEG2000-GalNAc3. In some embodiments, the LNPs provided herein consist essentially of: a therapeutic nucleic acid (TNA); an ionizable lipid; DSPC; cholesterol; DODA-PG46 and DSPE-PEG2000-GalNAc3. In some embodiments, the LNPs provided herein consist of: a therapeutic nucleic acid (TNA); an ionizable lipid; DSPC; cholesterol; DODA-PG46 and DSPE-PEG2000-GalNAc3.

[0817] In some embodiments, the lipid-anchoring polymer (combination of the first and second lipid-anchoring polymers) comprises about 0.1 mol% to about 20 mol% of the total lipid present in the LNP. In some embodiments, the lipid-anchoring polymer comprises about 0.5 mol% to about 10 mol% present in the LNP. In some embodiments, the lipid-anchoring polymer comprises about 1 mol% to about 10 mol% present in the LNP. In some embodiments, the lipid-anchoring polymer comprises about 2 mol% to about 10 mol% present in the LNP. In some embodiments, the lipid-anchoring 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% present in the LNP. In some embodiments, the lipid-anchoring polymer comprises about 3 mol% to about 8 mol% present in the LNP. In some embodiments, the lipid-anchoring polymer comprises about 3 mol% to about 7 mol% present in the LNP. In some embodiments, the lipid-anchoring polymer comprises about 3 mol% to about 5 mol% present in the LNP. In some embodiments, the lipid-anchored polymer constitutes about 2 mol% to about 4 mol% of the LNP. In some embodiments, the lipid-anchored polymer constitutes about 2% to about 3% of the LNP. In some embodiments, the lipid-anchored polymer constitutes about 2 mol% of the LNP. In some embodiments, the lipid-anchored polymer constitutes about 2.5 mol% of the LNP. In some embodiments, the lipid-anchored polymer constitutes about 3 mol% of the LNP. In some embodiments, the lipid-anchored polymer constitutes about 3.5 mol% of the LNP. In some embodiments, the lipid-anchored polymer constitutes about 4 mol% of the LNP.

[0818] In some embodiments, the first lipid-anchoring polymer comprises about 0.1 mol% to about 10 mol%, or about 0.2 mol% to about 8 mol%, or about 0.2 mol% to about 7 mol%, or about 0.2 mol% to about 5 mol%, or about 0.3 mol% to about 4 mol%, or about 0.4 mol% to about 4 mol%, or about 0.5 mol% to about 5 mol%, or about 0.5 mol% to 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 % to 4 mol%, or about 3 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%.

[0819] In some embodiments, the second lipid-anchoring polymer, if present, comprises about 0.005 mol% to about 5 mol%, or about 0.005 mol% to about 3 mol%, or about 0.005 mol% to about 2 mol%, or about 0.005 mol% to about 1 mol%, or about 0.005 mol% to about 0.5 mol%, or about 0.01 mol% to about 3 mol%, or about 0.01 mol% 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 about 0.025 mol% to about 0.25 mol%, or about 0.025 mol% to about 0.2 mol%, or about 0.025 mol% to about 0.1 mol%, or about 0.05 mol% to about 0.4 mol%, or about 0.05 mol% to about 0.3 mol%, or about 0.05 mol% to about 0.25 mol%, or about 0.05 mol% to about 0.2 mol%, or about 0.05 mol% to about 0.1 mol% is present.

[0820] Lipid nanoparticles (LNPs) containing ceDNA are disclosed in International Patent Application No. PCT / US2018 / 050042, filed September 7, 2018, the entire contents of which are incorporated herein and contemplated for use in the methods and compositions disclosed herein.

[0821] 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 average diameter of the LNPs of the present disclosure as determined by light scattering is less than about 90 nm, e.g., less than about 80 nm or less than about 75 nm. According to some embodiments, the average diameter of the LNPs of the present disclosure as determined by light scattering is from about 50 nm to about 75 nm or from about 50 nm to about 70 nm.

[0822] The pKa of the formulated cationic lipid can be correlated with the effectiveness of the LNP in delivering nucleic acids (see Jayaraman et al., Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al., Nature Biotechnology 28, 172-176 (2010), both of which are incorporated by reference in their entirety). In one embodiment, the pKa of each cationic lipid is determined in lipid nanoparticles using an assay based on the fluorescence of 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS). LNPs can be prepared in PBS at a concentration of 0.4 mM total lipid using an online process as 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 a buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, with a pH range of 2.5 to 11. An aliquot of the TNS solution can be added to a final concentration of 1 mM and, after vortex mixing, the fluorescence intensity can be measured at room temperature in an SLM Aminco Series 2 luminescence spectrophotometer using excitation and emission wavelengths of 321 nm and 445 nm. A sigmoidal best fit analysis can be applied to the fluorescence data, and the pKa can be measured as the pH that produces half-maximal fluorescence intensity.

[0823] In one embodiment, relative activity can be determined by measuring luciferase expression in the liver 4 hours after administration by tail vein injection. Activity is compared at doses of 0.3 and 1.0 mg ceDNA / kg and expressed as ng luciferase / g liver measured 4 hours after administration.

[0824] Without limitation, the LNPs of the present disclosure include lipid formulations that can be used to deliver uncoated non-viral DNA vectors to target sites of interest (e.g., cells, tissues, organs, specific cell types, etc.). Typically, the LNPs comprise uncoated non-viral DNA vectors and cationic lipids or salts thereof.

[0825] Additional exemplary lipid anchoring 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 conjugate, for example, a (methoxypolyethylene glycol)-conjugated lipid. PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl) succinate (PEG-S-DMG)), PEG dialkoxypropyl carbamate, N-(carbonyl-methoxy) Polyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or a mixture thereof. For example, in International Patent Application Publication Nos. WO1996 / 010392, WO1998 / 051278, WO2002 / 087541, WO2005 / 026372, WO2008 / 147438, WO2009 / 086558, WO2012 / 000104, WO2017 / 117528, WO2017 / 099823, WO201 No. 5 / 199952, No. WO2017 / 004143, No. WO2015 / 095346, No. WO2012 / 000104, No. WO2012 / 000104, and No. WO2010 / 006282, U.S. Patent Application Publication Nos. US2003 / 0077829, US2005 / 0175682, US2008 / 0020058, US2011 / 0117125, US2013 / 0303587, US2018 / 0028664, U Additional exemplary PEG-lipid conjugates are described in US2015 / 0376115, US2016 / 0376224, US2016 / 0317458, US2013 / 0303587, US2013 / 0303587, and US20110123453, and U.S. Pat. Nos. 5,885,613, 6,287,591, 6,320,017, and 6,586,559, the contents of all of which are incorporated herein by reference in their entirety.

[0826] PEG-DAA Additional examples of PEGylated lipids include, for example, PEG-dilaurylpropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of the following: PEG-DMG, PEG-dilauroylglycerol, PEG-dipalmitoylglycerol, PEG-ditertiary glycerol, PEG-dilauroylglyceramide, PEG-dimyristoylglyceramide, PEG-dipalmitoylglyceramide, PEG-diglycerol, PEG-cholesterol (1-[8'-(cholest-5-en-3[β]-oxy)formamido-3',6'-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol)), PEG-DMB (3,4-tetracosylbenzyl-[ω]-methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In one embodiment, the PEG-lipid can be selected from the group consisting of PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000],

[0827]

[0828] as well as

[0829]

[0830] Yet other exemplary lipid-anchored polymers include N-(carbonyl-methoxypolyethylene glycol n)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG n , wherein N is 350, 500, 750, 1000 or 2000), N-(carbonyl-methoxypolyethylene glycol n )-1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG n , where N is 350, 500, 750, 1000 or 2000), DSPE-polyglycerol-cyclohexyl-carboxylic acid, DSPE-polyglycerol-2-methylglutaric acid-carboxylic acid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated with polyethylene glycol (DSPE-PEG-OH), polyethylene glycol-dimyristylglycerol (PEG-DMG) or polyethylene glycol-distearoylglycerol (PEG-DSG). In DMPE-PEG nIn some examples, wherein n is 350, 500, 750, 1000 or 2000, the PEG lipid is N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG 2,000). n In some examples, wherein n is 350, 500, 750, 1000 or 2000, the PEG lipid is N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3...

Claims

1. A lipid nanoparticle (LNP), comprising: Therapeutic nucleic acids (TNA); ionizable lipids; sterols; a first lipid-anchored polymer; wherein the first lipid-anchored polymer comprises: i) polymers; ii) a lipid portion comprising at least one hydrophobic tail; and iii) optionally a linker, which connects the polymer to the lipid moiety; wherein the at least one hydrophobic tail comprises 12 to 22 carbon atoms in a single aliphatic chain backbone; and Helper lipid represented by formula (I): or a salt or ester thereof, wherein: Is a single bond or a double bond; A is hydrogen, R 1 It is C1-C 17 Alkyl or C2-C 17 alkenyl; R 2 It is C1-C 22 Alkyl or C2-C 22 alkenyl; R 3 is hydrogen or C1-C2 alkyl; and R 4 is hydrogen or C1-C2 alkyl.

2. A lipid nanoparticle (LNP), comprising: Therapeutic nucleic acids (TNA); ionizable lipids; sterols; a first lipid-anchored polymer; wherein the first lipid-anchored polymer comprises: i) polymers; ii) a lipid portion comprising at least two hydrophobic tails; and iii) optionally a linker, which connects the polymer to the lipid moiety; wherein the at least two hydrophobic tails each comprise from 16 to 22 carbon atoms in a single aliphatic chain backbone; and Helper lipid represented by formula (I): or a salt or ester thereof, wherein: Is a single bond or a double bond; A is hydrogen, R 1 It is C1-C 17 Alkyl or C2-C 17 alkenyl; R 2 It is C1-C 22 Alkyl or C2-C 22 alkenyl; R 3 is hydrogen or C1-C2 alkyl; and R 4 is hydrogen or C1-C2 alkyl.

3. A lipid nanoparticle (LNP), comprising: Therapeutic nucleic acids (TNA); ionizable lipids; sterols; a first lipid-anchored polymer; wherein the first lipid-anchored polymer comprises: i) polymers; ii) a lipid portion comprising at least two hydrophobic tails; and iii) optionally a linker, which connects the polymer to the lipid moiety; wherein the at least two hydrophobic tails each comprise 12 to 15 carbon atoms in a single aliphatic chain backbone; and Helper lipid represented by formula (I): or a salt or ester thereof, wherein: Is a single bond or a double bond; A is hydrogen, R 1 It is C1-C 17 Alkyl or C2-C 17 alkenyl; R 2 It is C1-C 22 Alkyl or C2-C 22 alkenyl; R 3 is hydrogen or C1-C2 alkyl; and R 4 is hydrogen or C1-C2 alkyl.

4. A lipid nanoparticle (LNP), comprising: Therapeutic nucleic acids (TNA); ionizable lipids; sterols; a first lipid-anchored polymer; wherein the first lipid-anchored polymer comprises: i) polymers; ii) a lipid portion comprising a single hydrophobic tail; and iii) optionally a linker, which connects the polymer to the lipid moiety; wherein the single hydrophobic tail comprises 12 to 22 carbon atoms in a single aliphatic chain backbone; and Helper lipid represented by formula (I): or a salt or ester thereof, or a deuterated analog of any of the foregoing, wherein: Is a single bond or a double bond; A is hydrogen, R 1 It is C1-C 17 Alkyl or C2-C 17 alkenyl; R 2 It is C1-C 22 Alkyl or C2-C 22 alkenyl; R 3 is hydrogen or C1-C2 alkyl; and R 4 is hydrogen or C1-C2 alkyl.

5. The lipid nanoparticle (LNP) according to any one of claims 1 to 4, wherein the helper lipid is represented by formula (II): or a salt or ester thereof, or a deuterated analog of any of the foregoing.

6. The lipid nanoparticle (LNP) according to any one of claims 1 to 4, wherein the helper lipid is represented by formula (III): or a salt or ester thereof, or a deuterated analog of any of the foregoing.

7. The lipid nanoparticle (LNP) according to any one of claims 1 to 4, wherein the helper lipid is represented by formula (IV): or a salt or ester thereof, or a deuterated analog of any of the foregoing.

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

9. A lipid nanoparticle (LNP) according to 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 there is a helper lipid represented by (I), (II), (III) or (IV), or a salt or ester thereof, or a deuterated analogue of any of the foregoing.

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

11. according to any one of claims 1 to 7 lipid nanoparticles (LNP), wherein R 1 It is C1-C 10 Alkyl or C2-C 10 Alkenyl.

12. The lipid nanoparticle (LNP) according to any one of claims 1 to 11, wherein It's a double bond.

13. according to any one of claims 1 to 11 lipid nanoparticles (LNP), wherein R 1 It is a C1-C8 alkyl group or a C2-C8 alkenyl group.

14. The lipid nanoparticle (LNP) according to claim 13, wherein R 1 It is a C1-C7 alkyl group or a C2-C7 alkenyl group.

15. The lipid nanoparticle (LNP) according to claim 14, wherein R 1 It is a C1 alkyl group, a C3 alkyl group, a C5 alkyl group or a C7 alkyl group.

16. The lipid nanoparticle (LNP) according to claim 15, wherein R 1 It is a C1 alkyl group.

17. according to any one of claims 1 to 16 lipid nanoparticles (LNP), wherein R 2 It is C3-C 15 Alkyl or C3-C 15 Alkenyl.

18. The lipid nanoparticle (LNP) according to claim 17, wherein R 2 It is C9 alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 14 Alkyl or C 15 alkyl.

19. The lipid nanoparticle (LNP) according to claim 18, wherein R 2 It is C 12 Alkyl, C 13 Alkyl or C 14 alkyl.

20. The lipid nanoparticle (LNP) according to claim 19, wherein R 2 It is C 13 alkyl.

21. according to any one of claims 1 to 20 lipid nanoparticles (LNP), wherein R 3 It's hydrogen.

22. according to any one of claims 1 to 20 lipid nanoparticles (LNP), wherein R 3 It is a C1 alkyl group.

23. according to any one of claims 1 to 22 lipid nanoparticles (LNP), wherein R 4 It's hydrogen.

24. according to any one of claims 1 to 22 lipid nanoparticles (LNP), wherein R 4 It is a C1 alkyl group.

25. The lipid nanoparticle (LNP) of claim 1, wherein 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; or the helper lipid represented by formula (I) is selected from: as well as or a salt or ester thereof, or a deuterated analog of any of the foregoing.

26. The lipid nanoparticle (LNP) of claim 25, wherein the helper lipid represented by formula (I) is: or a salt or ester thereof, or a deuterated analog of any of the foregoing.

27. The lipid nanoparticle (LNP) of claim 25, wherein the lipid represented by formula (I) is: or a salt or ester thereof, or a deuterated analog of any of the foregoing.

28. A lipid nanoparticle (LNP), comprising: Therapeutic nucleic acids (TNA); ionizable lipids; sterols; a first lipid-anchored polymer; wherein the first lipid-anchored polymer comprises: i) polymers; ii) a lipid portion comprising at least two hydrophobic tails; and iii) optionally a linker, which connects the polymer to the lipid moiety; wherein the at least two hydrophobic tails each comprise from 16 to 22 carbon atoms in a single aliphatic chain backbone; and 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); The whole blood half-life of the LNP (t 1 / 2 ) is at least about 3 hours.

29. The lipid nanoparticle (LNP) according to claim 28, wherein the blood half-life (t 1 / 2 ) is from about 3 hours to about 8 hours, or from about 3 hours to about 7.5 hours, or from about 3 hours to about 7 hours, or from about 3 hours to about 6.5 hours, or from about 3 hours to about 6 hours, or from about 3 hours to about 9 hours, or from about 3 hours to about 10 hours, or from about 3 hours to about 11 hours, or from about 3 hours to about 12 hours, or from about 3 hours to about 13 hours, or from about 3 hours to about 14 hours, or from about 3 hours to about 15 hours, or from about 3 hours to about 16 hours, or from about 3 hours to about 24 hours.

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

31. according to any one of claims 28 to 30 lipid nanoparticles (LNP), wherein the blood clearance rate (CI) of the LNP is about 10mL / min / kg to about 50mL / min / kg, or about 10mL / min / kg to about 45mL / min / kg, or about 10mL / min / kg to about 40mL / min / kg.

32. The lipid nanoparticle (LNP) of claim 31, wherein the LNP has a blood clearance (CI) 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. according to any one of claims 28 to 32 lipid nanoparticles (LNP), wherein the whole blood terminal time point exposure (AUC last ) is the AUC of the reference LNP with C14-15 lipid polymer last At least 5 times.

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

35. The lipid nanoparticle (LNP) of claim 33, wherein the whole blood terminal time point exposure (AUC last ) is about 200 hours*ng / mL to about 250 hours*ng / mL, or about 200 hours*ng / mL to about 300 hours*ng / mL, or about 500 hours*ng / mL to about 700 hours*ng / mL, or about 500 hours*ng / mL to about 550 hours*ng / mL, or about 500 hours*ng / mL to about 600 hours*ng / mL, or about 550 hours*ng / mL to about 600 hours*ng / mL, or about 600 hours*ng / mL to about 700 hours*ng / mL, or about 600 hours*ng / mL to about 650 hours*ng / mL, or about 650 hours*ng / mL to about 700 hours*ng / mL.

36. The lipid nanoparticle (LNP) of claim 34 or claim 35, wherein the terminal 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 portion 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 portion comprising two hydrophobic tails.

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

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

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

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

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

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

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

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

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

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, homosapiens acid, oleic acid, elaidic acid, vaccinic acid, linoleic acid, elaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, and derivatives thereof.

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

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

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

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

53. The lipid nanoparticle (LNP) according to 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. The lipid nanoparticle (LNP) of claim 37, wherein the first lipid-anchored polymer comprises a lipid portion comprising a single hydrophobic tail.

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

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

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

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

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

61. 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, homosapiens acid, oleic acid, elaidic acid, vaccinic acid, linoleic acid, elaidic acid, α-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-anchoring polymer is a glycerolipid.

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

64. The lipid nanoparticle (LNP) of any one of claims 1 to 48 and 62 to 63, wherein the first lipid-anchored polymer comprises a linker-lipid moiety selected from the group consisting of: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 18-1-trans PE, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE); and dioctadecylamine (DODA), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), derivatives thereof, and combinations of any of the foregoing.

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

66. The lipid nanoparticle (LNP) of any one of claims 1 to 38, 49 to 53, 62 and 68, wherein the first lipid-anchoring 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-dimyristyloxy-propyl-3-amine, derivatives thereof, and combinations of any one of the foregoing.

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

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. 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 average molecular weight of the polymer is between about 1000 Da and about 5000 Da.

71. The lipid nanoparticle (LNP) of claim 70, wherein the average molecular weight of the polymer is between about 2000 Da and about 5000 Da.

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

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 β-sitosterol.

78. The lipid nanoparticle (LNP) of any one of claims 1 to 77, wherein the ionizable lipid is a lipid represented by: a) Formula (A): or a pharmaceutically acceptable salt thereof, wherein: R 1 and R 1' Each independently is an optionally substituted linear or branched C 1-3 alkylene; R 2 and R 2' Each independently is an optionally substituted linear or branched C 1-6 alkylene; R 3 and R 3' Each independently is an optionally substituted linear or branched C 1-6 alkyl; Or alternatively, when R 2 is an optionally substituted branched C 1-6 When alkylene, R 2 and R 3 Together with its central nitrogen atom, it forms a 4- to 8-membered heterocyclic group; Or alternatively, when R 2' is an optionally substituted branched C 1-6 When alkylene, R 2' and R 3' Together with its central nitrogen atom, it forms a 4- to 8-membered heterocyclic group; R 4 and R 4' Each independently is -CR a 、-C(R a )2CR a or -[C(R a )2]2CR a ; R a Each occurrence is independently H or C 1-3 alkyl; Or alternatively, R 4 Yes-C(R a )2CR a or -[C(R a )2]2CR a , and when R a It is C 1-3 When alkyl, R 3 and R 4 Together with its central nitrogen atom, it forms a 4- to 8-membered heterocyclic group; Or alternatively, R 4' Yes-C(R a )2CR a or -[C(R a )2]2CR a , and when R a It is C 1-3 When alkyl, R 3' and R 4' Together with its central nitrogen atom, it forms a 4- to 8-membered heterocyclic group; R 5 and R 5' are independently hydrogen, C 1-20 Alkylene or C 2-20 alkenylene; R 6 and R 6' Each occurrence is independently C 1-20 Alkylene, C 3-20 Cycloalkylene or C 2-20 alkenylene; and m and n are each independently an integer selected from 1, 2, 3, 4 and 5; or b) Formula (B): or a pharmaceutically acceptable salt thereof, wherein: a is an integer ranging from 1 to 20; b is an integer ranging from 2 to 10; R 1 Not present or selected from (C2-C 20 )alkenyl, -C(O)O(C2-C 20 ) alkyl and (C2-C 20 ) alkyl-substituted cyclopropyl; and R 2 Yes (C2-C 20 )alkyl; or c) Formula (C): or a pharmaceutically acceptable salt thereof, wherein: R 1 and R 1' Each independently is optionally selected from one or more R a (C1-C6)alkylene substituted with a group; R 2 and R 2' are each independently (C1-C2)alkylene; R 3 and R 3' Each independently is optionally selected from one or more R b (C1-C6) alkyl substituted with a group; Or alternatively, R 2 and R 3 and / or R 2' and R 3' Together with its central nitrogen atom, it forms a 4- to 7-membered heterocyclic group; R 4 and R 4 'Each is a (C2-C6)alkylene group interrupted by -C(O)O-; R 5 and R 5 'Each independently is (C2-C 30 )alkyl or (C2-C 30 )alkenyl, each of which is optionally interrupted by -C(O)O- or (C3-C6)cycloalkyl; and R a and R b are each halo or cyano; or d) Formula (D): or a pharmaceutically acceptable salt thereof, wherein: R' is absent and is hydrogen or C1-C6 alkyl; provided that when R' is hydrogen or C1-C6 alkyl, R', R 1 and R 2 The nitrogen atoms they are all connected to have a positive charge; R 1 and R 2 are each independently hydrogen, C1-C6 alkyl or C2-C6 alkenyl; R 3 It is C1-C 12 Alkylene or C2-C 12 alkenylene; R 4 It is C1-C 18 Unbranched alkyl, C2-C 18 Unbranched alkenyl or Where: R 4a and R 4b Each is independently C1-C 16 Unbranched alkyl or C2-C 16 Unbranched alkenyl; R 5 Not present, C1-C8 alkylene or C2-C8 alkenylene; R 6a and R 6b Each independently is C7-C 16 Alkyl or C7-C 16 alkenyl; provided that the combined R 6a and R 6b The total number of carbon atoms in is greater than 15; X 1 and X 2 Each is 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)-; wherein: R a Each occurrence is independently hydrogen or C1-C6 alkyl; and n is an integer selected from 1, 2, 3, 4, 5 and 6; e) Formula (E): or a pharmaceutically acceptable salt thereof, wherein: R' is absent and is hydrogen or C1-C3 alkyl; provided that when R' is hydrogen or C1-C3 alkyl, R', R 1 and R 2 The nitrogen atoms they are all connected to have a positive charge; R 1 and R 2 are each independently hydrogen or C1-C3 alkyl; R 3 It is C3-C 10 Alkylene or C3-C 10 alkenylene; R 4 It is C1-C 16 Unbranched alkyl, C2-C 16 Unbranched alkenyl or in: R 4a and R 4b Each is independently C1-C 16 Unbranched alkyl or C2-C 16 unbranched alkenyl; R 5 Not present, C1-C6 alkylene or C2-C6 alkenylene; R 6a and R 6b Each independently is C7-C 14 Alkyl or C7-C 14 alkenyl; X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -SS-, -C(R a )=N-, -N=C(R a )-, -C(R a )=NO-, -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 )2O-, -C(=O)(CR a 2)C(=O)O- or OC(=O)(CR a 2)C(=O)-; wherein: R a Each occurrence is independently hydrogen or C1-C6 alkyl; and n is an integer selected from 1, 2, 3, 4, 5 and 6; or f) an ionizable lipid selected from any one of the ionizable lipids in Table 1, 4, 5, 6 or 7.

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

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 lipid nanoparticle (LNP) of claim 80, wherein the second lipid-anchoring polymer comprises a linker-lipid moiety selected from the group consisting of: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-ditrans Oleoyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 18-1-trans PE, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE); and dioctadecylamine (DODA), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), derivatives thereof, and combinations of any of the foregoing.

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

83. The lipid nanoparticle (LNP) of claim 82, wherein the first lipid-anchoring polymer and the second lipid-anchoring polymer are different lipid-anchoring polymers; and wherein the linker-lipid of the first lipid-anchoring polymer and the second lipid-anchoring polymer comprises one of 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-anchoring polymer) and DSPE (the second lipid-anchoring polymer); DPG (the first lipid-anchoring polymer) and DSPE (the second lipid-anchoring 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-anchoring polymer) and DODA (the second lipid-anchoring polymer); DMG (the first lipid-anchored polymer) and DODA (the second lipid-anchored polymer); or DMG (the first lipid-anchoring polymer) and DPG (the second lipid-anchoring polymer).

84. The lipid nanoparticle (LNP) of claim 82, wherein the first lipid anchoring polymer and the second lipid anchoring polymer are the same lipid anchoring polymer; and wherein the linker-lipid of the first lipid anchoring polymer and the second lipid anchoring polymer comprises one of 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 DPG (the first lipid-anchoring polymer) and DPG (the second lipid-anchoring polymer).

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

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

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

88. The lipid nanoparticle (LNP) of claim 87, wherein the DSG anchoring 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 hepatocytes.

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

91. 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. 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, fragments thereof, and derivatives of any of the foregoing.

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 lipid nanoparticle (LNP) of any one of claims 1, 3 to 27, 37 to 39, and 49 to 95, wherein the ionizable lipid is ionizable lipid 81: 4-Decyltetadecyl 6-((4-(dimethylamino)butanoyl)oxy)tridecanoate or a pharmaceutically acceptable salt thereof.

97. The lipid nanoparticle (LNP) of any one of claims 1, 3 to 27, 37 to 39, and 49 to 95, wherein the ionizable lipid is ionizable lipid 89: 4-Octyldodecyl 6-((4-(dimethylamino)butanoyl)oxy)tridecanoate or a pharmaceutically acceptable salt thereof.

98. The lipid nanoparticle (LNP) of any one of claims 1 to 48 and 62 to 95, wherein the ionizable lipid is ionizable lipid 87: Heptadecan-9-yl 9-((4-(dimethylamino)butanoyl)oxy)hexadecanoate or a pharmaceutically acceptable salt thereof.

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

100. The lipid nanoparticle (LNP) of 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. The lipid nanoparticle (LNP) of 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 lipid present in the LNP.

103. The lipid nanoparticle (LNP) of any one of claims 1 to 102, wherein the first lipid-anchoring polymer is present in the LNP in an amount of about 2 mol% to about 5 mol% of the total lipids 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 about 0.005 mol% to about 5 mol% of the total lipids present in the LNP.

105. The lipid nanoparticle (LNP) of claim 104, wherein the second lipid-anchoring 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-anchoring 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-anchoring 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 lipids present in the LNP.

109. A lipid nanoparticle (LNP) according to any one of claims 1 to 27 and 37 to 108, wherein the helper lipid represented by formula (I), (II), (III) or (IV), or a salt or ester thereof, or a deuterated analogue 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), (II), (III) or (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), (II), (III) or (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), (II), (III) or (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), (II), (III) or (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), (II), (III) or (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 lipids present in the LNP.

116. The lipid nanoparticle (LNP) of 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: (i) does not comprise the helper lipid represented by formula (I), (II), (III) or (IV), or a salt or ester thereof, or a deuterated analog of any of the foregoing; or (ii) comprises a helper lipid and a reference lipid polymer, wherein the helper lipid is 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 the reference lipid polymer comprises at least two hydrophobic tails, each hydrophobic tail comprising 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 claim 118, wherein the LNP elicits an expression level of TNA in a target cell that is equal to or higher than the expression level of TNA in a target cell elicited by the reference LNP.

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

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

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

123. The lipid nanoparticle (LNP) according to 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, an end-blocked DNA (ceDNA), a single-stranded DNA (ssDNA), a ministring, a doggybone TM , protelomeric end-blocked DNA, dumbbell-shaped linear DNA, Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), mRNA, tRNA, rRNA, gRNA, DNA viral vectors, viral RNA vectors, non-viral vectors, and 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 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 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 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 or greater than about 4000 nt in length.

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

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

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

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

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

133. A method of producing a lipid nanoparticle (LNP) according to any one of claims 1 to 131, said method comprising combining: the therapeutic nucleic acid (TNA); the ionizable lipid; the sterol; said first lipid-anchored polymer; The helper lipid represented by formula (I), (II), (III) or (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 said second lipid-anchored polymer; and Optionally said targeting moiety.

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

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

136. The method of claim 134 or 135, wherein the genetic disorder is selected from the group consisting of sickle cell anemia; melanoma; hemophilia A (factor VIII (FVIII) deficiency); hemophilia B (factor IX (FIX) deficiency); cystic fibrosis (CFTR); familial hypercholesterolemia (LDL receptor deficiency); hepatoblastoma; Wilson's disease; phenylketonuria (PKU); congenital hepatic porphyria; inherited liver metabolic disorders, Lesch Nyhan syndrome; thalassemia; xeroderma pigmentosum, Fanconi's anemia; retinitis pigmentosa; ataxia telangiectasia; Bloom's syndrome; retinoblastoma; mucopolysaccharidoses; Niemann-Pick Disease; Fabry disease. 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 (LAMP-2 deficiency); lysosomal acid lipase (LAL) deficiency; neuronal ceroid lipofuscinosis (NCL); sphingolipidosis; galactosialidosis; amyotrophic lateral sclerosis (ALS); Parkinson's disease disease; Alzheimer's disease; Huntington's disease; spinocerebellar ataxia; spinal muscular atrophy (SMA); Friedreich's ataxia; Duchenne muscular dystrophy (DMD); Becker muscular dystrophy (BMD); dystrophic epidermolysis bullosa (DEB); ectonucleotide pyrophosphatase 1 deficiency; systemic arterial calcification of infancy (GACI); Leber congenital amaurosis;Stargardt disease; wet macular degeneration (wet AMD); ornithine transcarbamylase (OTC) deficiency; Usher syndrome; alpha-1 antitrypsin deficiency; progressive familial intrahepatic cholestasis (PFIC); and cathepsin A deficiency.

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

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

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

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

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

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

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

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

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

146. A therapeutic method for providing anti-tumor immunity in a subject, the method comprising administering to the subject an effective amount of the LNP according to any one of claims 1 to 131 or the pharmaceutical composition according to 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. The method of any one of claim 146 or claim 147, wherein after administration, 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.

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

150. A method of treating a blood disease, disorder or condition in 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.

151. The method of claim 150, wherein 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 myeloid leukemia (CML), Hodgkin lymphoma (HL), multiple myeloma, myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), adrenoleukodystrophy (ALD), Hurler syndrome, Krabbe disease (globular cell leukodystrophy or GLD), metachromatic leukodystrophy (MLD), severe aplastic anemia (SAA), severe combined immunodeficiency (SCID), sickle cell disease (SCD), thalassemia, Wiskott-Aldrich syndrome, Diamond-Blackfan syndrome, anemia), essential thrombocythaemia, Fanconi anemia, hemophagocytic lymphohistiocytosis (HLH), juvenile myelomonocytic leukemia (JMML), myelofibrosis, polycythemia vera, and combinations thereof.

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

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

154. A lipid nanoparticle (LNP), comprising: Therapeutic nucleic acids (TNA); ionizable lipids; helper lipids; sterols; A lipid-anchored polymer; wherein the lipid-anchored polymer comprises: i) polymers; ii) a lipid portion comprising at least one hydrophobic tail; and wherein said polymer is attached to said lipid moiety; wherein the at least one hydrophobic tail comprises 18 to 22 carbon atoms in a single aliphatic chain backbone; 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; wherein the lipid-anchored polymer is present in the LNP in an amount of about 2 mol % to about 7 mol % of the total lipids present in the LNP; and The average particle size of the LNP is 50-100 nm in diameter.

155. The lipid nanoparticle (LNP) of claim 154, wherein the LNP has an average particle size of 60-80 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 carbohydrate ligands.

160. The lipid nanoparticle (LNP) of claim 154, wherein the lipid-anchoring 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), comprising: Therapeutic nucleic acids (TNA); ionizable lipids; helper lipids; sterols; a first lipid-anchored polymer; wherein the first lipid-anchored polymer comprises: i) polymers; and ii) a lipid portion comprising at least one hydrophobic tail, wherein the polymer is attached to the lipid moiety; and a second lipid-anchored polymer; wherein the second lipid-anchored polymer comprises: i) polymers; ii) a reactive moiety for conjugation to a targeting moiety; and iii) a lipid portion comprising at least one hydrophobic tail, wherein said polymer is attached to said lipid moiety; wherein the first lipid-anchored polymer comprises at least one hydrophobic tail, the at least one hydrophobic tail comprising 18 to 22 carbon atoms in a single aliphatic chain backbone; wherein the second lipid-anchored polymer comprises at least one hydrophobic tail, the at least one hydrophobic tail comprising 18 to 22 carbon atoms in a single aliphatic chain backbone; 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; wherein the first lipid-anchored polymer is present in the LNP in an amount from about 2 mol % to about 7 mol % of the total lipids present in the LNP; wherein the second lipid-anchored polymer is present in the LNP in an amount from about 0.2 mol % to about 2 mol % of the total lipid present in the LNP; and The average particle size of the LNP is 50-100 nm in diameter.

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

163. A lipid nanoparticle (LNP), comprising: Therapeutic nucleic acids (TNA); ionizable lipids; helper lipids; sterols; a first lipid-anchored polymer; wherein the first lipid-anchored polymer comprises: i) polymers; and ii) a lipid portion comprising at least one hydrophobic tail, wherein the polymer is attached to the lipid moiety; and a second lipid-anchored polymer; wherein the second lipid-anchored polymer comprises: i) polymers; and ii) a lipid portion comprising at least one hydrophobic tail; and iii) optionally a reactive moiety for conjugation to a targeting moiety; or a targeting moiety, wherein said polymer is attached to said lipid moiety; wherein the first lipid-anchored polymer comprises at least one hydrophobic tail, the at least one hydrophobic tail comprising 18 to 22 carbon atoms in a single aliphatic chain backbone; wherein the second lipid-anchored polymer comprises at least one hydrophobic tail, the at least one hydrophobic tail comprising 18 to 22 carbon atoms in a single aliphatic chain backbone; 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; wherein the first lipid-anchored polymer is present in the LNP in an amount from about 2 mol % to about 7 mol % of the total lipids present in the LNP; wherein the second lipid-anchored polymer is present in the LNP in an amount from about 0.2 mol % to about 2 mol % of the total lipid present in the LNP; and The average particle size of the LNP is 50-100 nm in diameter.

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