Glutamic acid-based lipids, lipid nanoparticle containing glutamic acid-based lipids, and formulations thereof

Abstract

Provided is a glutamic or glutaric acid-based ionizable lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof. The compound can be used to obtain lipid nanoparticles. In some embodiments, the lipid nanoparticle can comprise (a) from about 40 to about 100 mol % of the compound of Formula (I); (b) from 0 to about 20 mol % of a neutral lipid; (c) from 0 to about 50 mol % of a helper lipid; (d) from 0 to about 5 mol % of a polymer-conjugated lipid; and (e) from 0 to about 10 mol % of a hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle. In some embodiments, the ionizable lipid compound is a glutamic acid-based ionizable lipid compound.

Description

GLUTAMIC ACID-BASED LIPIDS, LIPID NANOPARTICLE CONTAINING GLUTAMIC ACID-BASED LIPIDS, AND FORMULATIONS THEREOFFIELD OF THE DISCLOSURE

[0001] The present disclosure relates to glutamic or glutaric acid-based lipids, and their use for preparing systems for encapsulating cargos such as nucleic acid sequences, polypeptides or peptides. More particularly, the present disclosure relates to ionizable glutamic or glutaric acidbased lipid compounds useful to prepare lipid nanoparticles (LNPs). The present disclosure also relates to LNPs comprising such glutamic or glutaric acid-based lipids.

[0002] The present disclosure also relates to lipids nanoparticles (LNPs) for the delivery of cargos such as nucleic acid sequences, polypeptides or peptides and methods of use of these LNPs for the treatment of diseases, disorders and / or conditions.BACKGROUND

[0003] The development of systems, such as lipid nanoparticle delivery systems, for delivering therapeutics such as proteins, nucleic acid sequences, polypeptides or peptides to prevent or treat diseases has been increasing in the recent years. Lipid nanoparticles (LNPs) usually contain four ingredients: an ionizable lipid, a phospholipid, cholesterol and a PEGylated lipid. A major component of LNPs is the ionizable lipid. The phospholipid supports the formation of a lipid bilayer while cholesterol can stabilize the lipid bilayer. The PEGylated lipid, being amphiphilic, remains on the surface of LNPs to provide colloidal stability by steric shielding. Designing new ionizable lipids with suitable efficacy, stability and / or biodegradability to allow the preparation of LNPs is needed.

[0004] Proteins have been the standard for therapeutics but the use of nucleic acids as therapeutic modalities for a variety of diseases and therapeutic indications has gained in prominence over the past few years. Various companies have shown that nucleic acids (e.g., siRNA, mRNA, circular RNA, DNA, etc.) can be more effective when compared to protein-based therapies. There is a need for new delivery systems, such as new LNPs, for both nucleic acid and protein therapeutics.SUMMARY

[0005] The present disclosure provides new lipid compounds, more particularly lipid compounds comprising a central glutamic or glutaric acid moiety, referred to herein, as “glutamic or glutaric acid-based lipids”. In some embodiments, the lipid compounds comprise a central glutamic acid moiety and are referred to as “glutamic-based lipids”. In some embodiments, the lipid compounds comprise a central glutaric acid moiety and are referred to as “glutaric-based lipids”.

[0006] The present disclosure also provides LNPs, more particularly LNPs formulated with glutamic or glutaric acid-based lipid compounds. Particles, such as nanoparticles, comprising the compounds, constructs comprising the nanoparticles and cargos, wherein the cargo can be a small molecule, an antibody, a polynucleotide, or a polypeptide, methods of using the parti cl es / constructs, and methods of preparing the compounds, particles and constructs are also provided.

[0007] Hence, according to one aspect, there is provided a compound of Formula (I):or a pharmaceutically acceptable salt thereof, whereinA is absent or -CH2-;B is -CH2- or -CH2CH2-, wherein when A is absent then B is -CH2CH2- and when A is -CH2- then B is -CH2-;X is -NR1R2 or -O-(C=O)Ri’;Ri is H or an optionally substituted linear C1-C4 alkyl group;R2 is H, an optionally substituted linear C1-C8 alkyl group, -C(=O)R?, -S(=O)2-R?, orRr is an optionally substituted linear C1-C8 alkyl group;R20 is H or an optionally substituted linear C1-C8 alkyl group;R21 is an optionally substituted linear C1-C8 alkyl group or -(C=O)-R22, wherein R22 is an optionally substituted C1-C8 alkyl group;R3 and R4 are independently H, a linear or branched C4-C60 alkyl, a linear or branched C4-C60 alkenyl, or a linear or branched C4-C60 alkynyl group, wherein at least one of R3 or R4 is different from H;Rs and Re are independently a linear or branched C4-C60 alkyl, a linear or branched C4- C60 alkenyl, or a linear or branched C4-C60 alkynyl group;R7 is an optionally substituted linear Cl -C8 alkyl group, an optionally substituted linear C2-C8 alkenyl group, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10-membered heteroaryl;wherein when any of R3, R4, Rs and Re represents an alkenyl group, the alkenyl group independently comprises from one to six C=C bonds each independently having the E or Z configuration; wherein when any of R3, R4, Rs and Re represents an alkynyl group, the alkynyl group independently comprises from one to six C=C bonds; wherein when R7 represents an alkenyl group, the alkenyl group comprises from one to two C=C bonds each independently having the E or Z configuration; wherein when any alkyl and / or alkenyl group is substituted, this group is independently substituted with one or more -OH, -(C=O)-H, -(C=O)-CH3, -(C=O)-(CH2)pCH3, -(C=O)-(CH2)P-Ri9, -O- (C=O)-CH3, -O-(C=O)-(CH2)PCH3, -O-(C=O)-(CH2)P-Ri9, -(C=O)-OCH3, -(C=O)-O-(CH2)PCH3, -(C=O)-O-(CH2)P-Ri9, -(C=O)-(CH2)p-(C=O)-Ri9, -(C=O)-O-(CH2)PN(CH3)-(CH2)P’-Ri9, - (C=O)NH-(CH2)P-R19, -(C=S)-CH3, -0-CH3, -O-(CH2)PCH3, -NH2, -NHCH3, -N(CH3)2, - N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2)PO-(C=O)CH3)2, -N(CH3)-(CH2)P-R19, -(C=O)NH2, - N3, -NO2, -SCH3, -SCH2CH3, an optionally substituted 5 to 10-membered heterocyclyl, C5-C12 aralkyl, C5-C10 aryl or an optionally substituted 5 to 10-membered heteroaryl, with p and p’ are independently from 1 to 5; and wherein R19 is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10- membered heteroaryl; wherein when any 5 to 10-membered heteroaryl and / or 5 to 10-membered heterocyclyl is substituted, this group is independently substituted with one or more substituents selected from the group consisting of C1-C4 alkyl group, -(CH2)P”N(CH3)2and oxo (=0), with p” from 1 to 5; with the provisos that when A is absent, B is -CH2CH2-, X is -NRIR2, Ri is -CH3, and each of R3, R4, Rs, and Re is linear Cl 8 alkyl groups with one C=C bond between carbons C9 and C10 and one C=C bond between carbons C12 and C13, then R2is different from -CH3, -CH2CH2OH and - CH2CH2NH2.

[0008] In one embodiment, the compound of Formula (I) can have be of Formula (II)or a pharmaceutically acceptable salt thereof, whereinRi is H or an optionally substituted linear C1-C4 alkyl group;R.2 is H, an optionally substituted linear C1-C8 alkyl group, -C(=O)R?, -S(=O)2-R?, orR20 is H or an optionally substituted linear C1-C8 alkyl group;R21 is an optionally substituted linear C1-C8 alkyl group or -(C=O)-R22, wherein R22 is an optionally substituted C1-C8 alkyl group;R3 and R4 are independently H, a linear or branched C4-C60 alkyl, a linear or branched C4-C60 alkenyl, or a linear or branched C4-C60 alkynyl group, wherein at least one of R3 or R4 is different from H;Rs and Re are independently a linear or branched C4-C60 alkyl, a linear or branched C4-C60 alkenyl, or a linear or branched C4-C60 alkynyl group;R7 is an optionally substituted linear Cl -C8 alkyl group, an optionally substituted linear C2-C8 alkenyl group, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10-membered heteroaryl; wherein when any of R3, R4, Rs and Re represents an alkenyl group, the alkenyl group independently comprises from one to six C=C bonds each independently having the E or Z configuration; wherein when any of R3, R4, Rs and Re represents an alkynyl group, the alkynyl group independently comprises from one to six C=C bonds; wherein when R7 represents an alkenyl group, the alkenyl group comprises from one to two C=C bonds each independently having the E or Z configuration; wherein when any alkyl and / or alkenyl group is substituted, this group is independently substituted with one or more -OH, -(C=O)-H, -(C=O)-CH3, -(C=O)-(CH2)pCH3, -(C=O)-(CH2)P-Ri9, -O- (C=O)-CH3, -O-(C=O)-(CH2)PCH3, -O-(C=O)-(CH2)P-Ri9, -(C=O)-OCH3, -(C=O)-O-(CH2)PCH3, -(C=O)-O-(CH2)P-Ri9, -(C=O)-(CH2)P-(C=O)-Ri9, -(C=O)-O-(CH2)PN(CH3)-(CH2)P’-Ri9, - (C=O)NH-(CH2)P-R19, -(C=S)-CH3, -0-CH3, -O-(CH2)PCH3, -NH2, -NHCH3, -N(CH3)2, - N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2)PO-(C=O)CH3)2, -N(CH3)-(CH2)P-R19, -(C=O)NH2, -N3, -NO2, -SCH3, -SCH2CH3, an optionally substituted 5 to 10-membered heterocyclyl, C5-C12 aralkyl, C5-C10 aryl or an optionally substituted 5 to 10-membered heteroaryl, with p and p’ are independently from 1 to 5; and wherein R19 is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10- membered heteroaryl,wherein when any 5 to 10-membered heteroaryl and / or 5 to 10-membered heterocyclyl is substituted, this group is independently substituted with one or more substituents selected from the group consisting of C1-C4 alkyl group, -(CH2)P”N(CH3)2 and oxo (=0), with p” from 1 to 5.

[0009] According to another aspect, there is provided a compound selected from the group consisting of Compounds 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320a, 320b, 321a, 321b, 322, 323, 324, 325, 326, 328, 329, 330, 331,332, 333, 334, 335, 336, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 358, 359, 360 and 362 of Table 1, or a pharmaceutically acceptable salt thereof. In one embodiment, the compound can be selected from the group consisting of Compounds 301, 302, 303, 304, 305, 306, 307, 309, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320a, 320b, 321a, 321b, 322, 323, 324, 325, 326, 328, 329, 330, 331, 332, 333, 334, 335, 336, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 358, 359, 360 and 362 of Table 1, or a pharmaceutically acceptable salt thereof In one embodiment, the compound can be selected from the group consisting of Compounds 301, 302, 303, 304, 305, 306, 307, 309, 311, 312, 313, 314, 315, 316, 318, 319, 320a, 320b, 321a, 321b, 325, 326, 346, 347, 350, 351, 352, 353, 355, 358, 359, 360 and 362 of Table 1, or a pharmaceutically acceptable salt thereof. In another embodiment, the compound can be selected from the group consisting of Compounds 305, 313, 314, 315, 316, 317, 318, 320a, 320b, 321a, 321b, 323, 324, 325, 326, 328, 329, 330, 331, 332,333, 334, 335, 336, 340, 350, 351 and 360 of Table 1, or a pharmaceutically acceptable salt thereof. In yet another embodiment, the compound can be selected from the group consisting of Compounds 301, 303, 304, 305, 306, 307, 311, 312, 313, 314, 315, 319, 320a, 320b, 321a, 321b, 325, 326, 346, 347, 350, 351, 352, 353, 355, 358 and 359 of Table 1, or a pharmaceutically acceptable salt thereof. In a further embodiment, the compound can be selected from the group consisting of Compounds 305, 306, 307, 313, 314, 315, 319, 320a, 320b, 321a, 321b, 325, 326, 350, 351, 352, 353, 355, 358 and 359 of Table 1, or a pharmaceutically acceptable salt thereof.

[0010] In some embodiments, the compound of the present disclosure, i.e., the glutamic or glutaric-acid based lipid compound of the present disclosure, or the pharmaceutically acceptable salt thereof can be in the form of any enantiomers, any diastereoisomers, any cis or trans geometric isomers, or any mixtures thereof.

[0011] According to another aspect, there is provided a lipid nanoparticle comprising at least one compound of the present disclosure, i.e., the glutamic or glutaric-acid based lipid compound of the present disclosure, or the pharmaceutically acceptable salt thereof.

[0012] According to another aspect, there is provided a lipid nanoparticle comprising:(a) from about 40 to about 100 mol % of an ionizable lipid;(b) from 0 to about 20 mol % of a neutral lipid;(c) from 0 to about 50 mol % of a helper lipid;(d) from 0 to about 5 mol % of a polymer-conjugated lipid; and(e) from 0 to about 10 mol % of a hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle; and wherein the ionizable lipid is at least one compound of Formula (I):or a pharmaceutically acceptable salt thereof, whereinA is absent or -CH2-;B is -CH2- or -CH2CH2- ■ wherein when A is absent then B is -CH2CH2- and when A is -CH2- then B is -CH2-;X is -NR1R2 or -O-(C=O)Ri’;Ri is H or an optionally substituted linear C1-C4 alkyl group;R2 is H, an optionally substituted linear C1-C8 alkyl group, -C(=O)R?, -S(=O)2-R7, orRr is an optionally substituted linear C1-C8 alkyl group;R20 is H or an optionally substituted linear C1-C8 alkyl group;R21 is an optionally substituted linear C1-C8 alkyl group or -(C=O)-R22, wherein R22 is an optionally substituted C1-C8 alkyl group;R3 and R4 are independently H, a linear or branched C4-C60 alkyl, a linear or branched C4-C60 alkenyl, or a linear or branched C4-C60 alkynyl group, wherein at least one of R3 or R4 is different from H;Rs and Re are independently a linear or branched C4-C60 alkyl, a linear or branched C4- C60 alkenyl, or a linear or branched C4-C60 alkynyl group;R7 is an optionally substituted linear Cl -C8 alkyl group, an optionally substituted linear C2-C8 alkenyl group, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10-membered heteroaryl; wherein when any of R3, R4, Rs and Re represents an alkenyl group, the alkenyl group independently comprises from one to six C=C bonds each independently having the E or Z configuration;wherein when any of R3, R4, Rs and Re represents an alkynyl group, the alkynyl group independently comprises from one to six C=C bonds; wherein when R7 represents an alkenyl group, the alkenyl group comprises from one to two C=C bonds each independently having the E or Z configuration; wherein when any alkyl and / or alkenyl group is substituted, this group is independently substituted with one or more -OH, -(C=O)-H, -(C=O)-CH3, -(C=O)-(CH2)pCH3, -(C=O)-(CH2)P-Ri9, -O- (C=O)-CH3, -O-(C=O)-(CH2)PCH3, -O-(C=O)-(CH2)P-Ri9, -(C=O)-OCH3, -(C=O)-O-(CH2)PCH3, -(C=O)-O-(CH2)p-Ri9, -(C=O)-(CH2)p-(C=O)-Ri9, -(C=O)-O-(CH2)PN(CH3)-(CH2)P-RI9, - (C=O)NH-(CH2)P-R19, -(C=S)-CH3, -O-CH3, -O-(CH2)PCH3, -NH2, -NHCH3, -N(CH3)2, - N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2)PO-(C=O)CH3)2, -N(CH3)-(CH2)P-R19, -(C=O)NH2, - N3, -NO2, -SCH3, -SCH2CH3, an optionally substituted 5 to 10-membered heterocyclyl, C5-C12 aralkyl, C5-C10 aryl or an optionally substituted 5 to 10-membered heteroaryl, with p and p’ are independently from 1 to 5; and wherein R19 is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10- membered heteroaryl; wherein when any 5 to 10-membered heteroaryl and / or 5 to 10-membered heterocyclyl is substituted, this group is independently substituted with one or more substituents selected from the group consisting of C1-C4 alkyl group, -(CH2)P”N(CH3)2and oxo (=0), with p” from 1 to 5; with the provisos that when A is absent, B is -CH2CH2-, X is -NRIR2, Ri is -CH3, and each of R3, R4, Rs, and Re is linear Cl 8 alkyl groups with one C=C bond between carbons C9 and C10 and one C=C bond between carbons C12 and Ci3, then R2is different from -CH3, -CH2CH2OH and - CH2CH2NH2.

[0013] In some embodiments, the lipid nanoparticle can comprise as ionizable lipid, at least one of the compounds of Formula (I), (II), (12), (13) or (14), or the lipid(s) having a structure of Formulas (Ila), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d) or (I4e), or the lipid(s) having a structure of Formulas (I), (II), (12), (13), (11-1), (11-2), (11-3), (11-4), (11-5), (11-6), (Il-4a), (II- 4b), (Il-5a), (Il-5b), (Il-5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), or the compounds in Table 1, as defined herein.

[0014] In some embodiments, the lipid nanoparticle can comprise: (a) from about 40 to about 85 mol % of the ionizable lipid; (b) from about 1 to about 20 mol % of the neutral lipid; (c) from 0 to about 50 mol % of the helper lipid; (d) from 0 to about 5 mol % of the polymer-conjugated lipid; and (e) from 0 to about 10 mol % of the hydrophobic component.

[0015] In some embodiments, the lipid nanoparticle can comprise: (a) from about 40 to about 85 mol % of the ionizable lipid; (b) from about 1 to about 20 mol % of the neutral lipid; (c) fromabout 1 to about 50 mol % of the helper lipid; (d) from 0 to about 5 mol % of the polymer- conjugated lipid; and (e) from 0 to about 10 mol % of the hydrophobic component.

[0016] In some embodiments, the lipid nanoparticle can comprise: (a) from about 40 to about 85 mol % of the ionizable lipid; (b) from about 1 to about 20 mol % of the neutral lipid; (c) from about 1 to about 50 mol % of the helper lipid; (d) from about 1 to about 5 mol % of the polymer- conjugated lipid; and (e) from 0 to about 10 mol % of the hydrophobic component.

[0017] In some embodiments, the lipid nanoparticle can comprise: (a) from about 40 to about 85 mol % of the ionizable lipid; (b) from about 1 to about 20 mol % of the neutral lipid; (c) from about 1 to about 50 mol % of the helper lipid; (d) from about 1 to about 5 mol % of the polymer- conjugated lipid; and (e) from about 0.1 to about 10 mol % of the hydrophobic component.

[0018] In some embodiments, the lipid nanoparticle can comprise: (a) from about 40 to about 85 mol % of the ionizable lipid; (b) from about 5 to about 20 mol % of the neutral lipid; (c) from about 1 to about 50 mol % of the helper lipid; (d) from about 1 to about 4 mol % of the polymer- conjugated lipid; and (e) from about 0.1 to about 10 mol % of the hydrophobic component.

[0019] In some embodiments, the lipid nanoparticle can comprise: (a) from about 40 to about 85 mol % of the ionizable lipid; (b) from about 5 to about 10 mol % of the neutral lipid; (c) from about 1 to about 40 mol % of the helper lipid; (d) from about 1 to about 4 mol % of the polymer- conjugated lipid; and (e) from about 0 to about 5 mol % of the hydrophobic component.

[0020] In some embodiments, the lipid nanoparticle can comprise: (a) from about 40 to about 85 mol % of the ionizable lipid; (b) from about 5 to about 10 mol % of the neutral lipid; (c) from about 1 to about 40 mol % of the helper lipid; (d) from about 1 to about 4 mol % of the polymer- conjugated lipid; and (e) from about 0.1 to about 5 mol % of the hydrophobic component.

[0021] According to another aspect, there is provided a pharmaceutical composition comprising a lipid nanoparticle as defined herein, and a pharmaceutical acceptable excipient.

[0022] According to another aspect, there is provided a method for delivering a cargo to a cell comprising contacting the cell with a lipid nanoparticle as defined herein, wherein the lipid nanoparticle comprises the cargo.

[0023] According to another aspect, there is provided a use of a lipid nanoparticle as defined herein, for delivering a cargo to a cell, wherein the lipid nanoparticle comprises the cargo.

[0024] According to another aspect, there is provided a vaccine comprising a lipid nanoparticle as defined herein, wherein the lipid nanoparticle comprises at least one cargo, preferably the cargo comprises at least one of a small molecule, an antibody, a polynucleotide or a polypeptide, more preferably the cargo comprises at least one nucleic acid such as mRNA.

[0025] According to another aspect, there is provided a method of vaccinating a subject against an infectious agent comprising:(i) contacting the subject with a vaccine as defined herein, and(ii) eliciting an immune response.

[0026] According to another aspect, there is provided a use of a vaccine as defined herein for vaccinating a subject against an infectious agent.

[0027] According to another aspect, there is provided a method of treating cancer in a subject comprising administering a lipid nanoparticle as defined herein to the subject, wherein the lipid nanoparticle comprises an anti-cancer cargo or a cargo triggering an immune response against cancer cells.

[0028] According to another aspect, there is provided a lipid nanoparticle as defined herein for use in the treatment of cancer in a subject, wherein the lipid nanoparticle comprises an anti-cancer cargo or a cargo triggering an immune response against cancer cells.

[0029] According to another aspect, there is provided a use of a lipid nanoparticle as defined herein for treating cancer in a subject, wherein the lipid nanoparticle comprises an anti-cancer cargo or a cargo triggering an immune response against cancer cells.

[0030] According to another aspect, there is provided a use of a lipid nanoparticle as defined herein for the preparation of a medicament for treating cancer in a subject, wherein the lipid nanoparticle comprises an anti-cancer cargo or a cargo triggering an immune response against cancer cells.

[0031] The details of various embodiments are set forth in the description below. Other features, objects and advantages will be apparent from the description, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 shows a cryo-TEM image of LNPs-04 formulated with ionizable lipid 305 (contains spike mRNA).

[0033] Figure 2 shows a cryo-TEM image of LNPs-04 formulated with ionizable lipid 305 (contains spike mRNA).

[0034] Figure 3 shows a cryo-TEM image of LNPs-05 formulated with ionizable lipid 306 (contains spike mRNA).

[0035] Figure 4 shows a cryo-TEM image of LNPs-05 formulated with ionizable lipid 306 (contains spike mRNA).

[0036] Figure 5 shows a cryo-TEM image of LNPs-19 formulated with ionizable lipid 305(contains spike mRNA), obtained by Point-of-Care mixing.

[0037] Figure 6 shows a cryo-TEM image of LNPs-19 formulated with ionizable lipid 305(contains spike mRNA), obtained by Point-of-Care mixing.

[0038] Figure 7 shows a cryo-TEM image of LNPs-20 formulated with ionizable lipid 305 (contains spike mRNA) and squalene, obtained by Point-of-Care mixing.

[0039] Figure 8 shows a cryo-TEM image of LNPs-20 formulated with ionizable lipid 305 (contains spike mRNA) and squalene, obtained by Point-of-Care mixing.

[0040] Figure 9 is a scheme representing Point-of-Care preparation of lipid nanoparticles. A lipid solution in a prefilled syringe (A) is injected (C) into a vial containing a mRNA solution (B) followed by vigorous shaking (D) provides mRNA encapsulated in LNPs ready for dosing (E).

[0041] Figure 10 is a comparison of transfection efficiencies of LNPs-04 and PTX-COVID- 19-B in Huh-7 cells by ELISA. PTX-COVID-19-B vaccine is an mRNA-LNP vaccine comprising an mRNA encoding for the full-length spike protein of the Covid-19-B virus. PTX-COVID-19-B can be used as a positive control in experiments using the spike mRNA-LNP of the present disclosure. The formulation of PTX-COVID-19-B is described for instance in WO 2023 / 050003 (see “PTX-B”).

[0042] Figure 11 is a comparison of transfection efficiencies of LNPs-05 and PTX-COVID- 19-B in Huh-7 cells by ELISA.

[0043] Figure 12 is a comparison of transfection efficiencies of LNPs-04, LNPs-06 and PTX- COVID-19-B in Huh-7 cells by ELISA.

[0044] Figure 13 is a comparison of transfection efficiencies of LNPs-04, LNPs-19, LNPs-20 and PTX-COVID-19-B in Huh-7 cells by ELISA.

[0045] Figure 14A is a graph showing the spike protein-specific IgG response with LNPs-04 and LNPs-05 measured 14 days after the second intramuscular immunization in mice.

[0046] Figure 14B is a graph showing the Neutralizing Antibody (NAb) response against Wuhan-Hu- 1 / D614G with LNPs-04 and LNPs-05 measured 14 days after the second intramuscular immunization in mice.

[0047] Figure 15 is a graph showing the Neutralizing Antibody (NAb) response against Wuhan-Hu- 1 / D614G with LNPs-04 and LNPs-05 at different mRNA doses.

[0048] Figure 16 shows level of the antigen-specific IFNy secreting cells measured in splenocytes 14 days after the second immunization in mice with LNPs-04 and LNPs-05.

[0049] Figure 17 shows level of the antigen-specific IL-4 secreting cells measured in splenocytes 14 days after the second immunization in mice with LNPs-04 and LNPs-05.

[0050] Figure 18 shows the MC38gp tumor growth control after immunization in mice withLNPs-04-05

[0051] Figure 19 shows a comparison of transfection efficiencies of conventionally prepared LNPs-21 and Point-of-Care LNPs-22 with commercial transfection agent MessengerMAX™. (a) Flow cytometry data; (b) Percentage of transfected cells calculated from the flow cytometry data.

[0052] Figures 20A-20B show a comparison of transfection efficiencies of LNPs-35, and LNPs-38 (Figure 20A), LNPs-39, LNPs-40, LNPs-41, LNPs-43, LNPs-44, LNPs-45 and LNPs- 46 (Figure 20B) and PTX-COVID-19-B in Huh-7 cells by ELISA

[0053] Figures 21A-21B show a comparison of transfection efficiencies of LNPs-24, LNPs- 26, LNPs-42 and LNPs-49 (Figure 21A), LNPs-25, LNPs-27, LNPs-29, LNPs-41, LNPs-43 and LNPs-47 (Figure 21B), and PTX-COVID-19-B in Huh-7 cells by ELISA.

[0054] Figure 22 is a comparison of transfection efficiencies of LNPs-28 and LNPs-44 and PTX-COVID-19-B in Huh-7 cells by ELISA.

[0055] Figures 23A-23B show a comparison of transfection efficiencies of LNPs-31 and LNPs-54 (Figure 23 A), LNPs-30 and LNPs-48 (Figure 23B) and PTX-COVID-19-B in Huh-7 cells by ELISA.

[0056] Figures 24A-24D show the transfection efficiencies of LNPs-21 in different PBMC cell subsets. Figure 24A shows the transfection efficiency of LNPs-21 in PBMCs as assessed by flow cytometry and gated on the different cell subsets, as measured by tdTomato expression. Figure 24B shows the transfection efficiency of LNPs-21 in purified T cells from PBMCs from a donor, in either naive T cells or 24 hours following activation with anti-CD3 / anti-CD28 antibodies or PMA / ionomycin, as compared to MessengerMAX™, as measured by tdTomato expression. Figure 24C shows a schematic for the transfection efficiency experiment in immature / mature monocyte-derived dendritic cells (MDDCs) and unpolarized / polarized monocyte-derived macrophages (MDM). Figure 24D shows the results from the experiment of Figure 24A for the transfection efficiency of LNPs-21 and viability of transfected cells, as compared to MessengerMax, as measured by tdTomato expression.

[0057] Figures 25A-25C show the effect of LNPs on immune activation. Figures 25A-25B show PBMCs from two healthy human donors (Donor 1 and Donor 2) transfected with lOOng / well of tdTomato mRNA encapsulated in LNPs-21 or MessengerMax (Thermofisher). Supernatant from transfected cells were collected 24 hours after transfection and quantified with LEGENDplex™ assay (Biolegend). Figure 25C shows LNPs-35 and LNPs-23 encapsuling Spike mRNA added to a TLR-4 reporter cell line. 16 hours later, luciferase was measured to indicate the activation of TLR-4. Cells treated with MPLA (Monophosphoryl lipid A) or formulation buffer were used as positive and negative controls.

[0058] Figures 26A-26C show the in vivo immunogenicity of LNPs-35 and LNPs-23. Female C57BL / 6 mice were vaccinated twice at week 0 and week 2 with 5 pg COVID-19 spike mRNA encapsulated in LNPs-35 or LNPs-23. 10 days after the 2ndvaccination, serum from vaccinated mice were collected for detection of neutralizing antibodies (nAb) against Wuhan (D614G) pseudovirus (Figure 26A). Splenocytes from vaccinated mice were used for cytokine production (IFN-y and IL-4) by ELISPOT (Figures 26B-26C). Numbers on top of the column are geometric mean titers of nAb or mean of spot forming units (SFU) per million input cells.

[0059] Figures 27A-27H show the results of the in vivo induction of cytolytic T cells and immunogenicity following immunization with LNPs-04-05 and LNPs-06-02 pulsed with gp33 or adenovirus peptides (AV) (as shown in Sebzda, E. et al., J Exp Med (1996) 183 (3): 1093-1104). Figure 27A shows a schematic of the in vivo cytolytic lymphocyte assay that was performed on Day 8 post immunization. Splenocyte target cells were isolated from naive mice and stained with Cell Trace Violet (CTV) and pulsed for 1 hour with either gp33 or AV peptides. Approximately 3 x 106target cells were then reinjected i.v. into vaccinated mice and splenocytes were collected 4 hours p.i. to measure specific lysis normalized to the formulation buffer. Figure 27B shows the results of the experiment of Figure 27 A. Figures 27C-27D show the percentages of peripheral gp33+ and gp34+ CD8+ T cells in blood samples taken on Day 7 via saphenous vein. RBCs were lysed with ACK lysis buffer and stained with PE labelled tetramers to gp33 or gp34. Figures 27E- 27H show the serum cytokine / chemokine levels in blood samples taken 6 hours post immunization of the LNPs, assayed by Legendplex.

[0060] Figures 28A-28K show the results of a study for the evaluation of mRNA-LNP vaccine (LNPs-58) protectiveness with a tumor rechallenge setup using a murine, transplantable, EGFRvIII-expressing GBM model. Figure 28A shows a schematic of the immunization and assessment schedule. Briefly, syngeneic and sex-matched transplantable GBM cells (IxlO5) were transplanted into the right stratum of 6-8-week-old female recipient mice on Day 1 of the experiment. Thereafter, mice were either vaccinated with the indicated EGFRvIII LNPs at Days 10, 14, 18, and 25 or left untreated. On Day 43, all vaccinated (surviving) animals were rechallenged with another tumor dose injected into the contralateral brain side. To ensure the validity of the outcomes, a new untreated control cohort was also included, providing a robust comparison. Figure 28B shows the survival of the mice treated with EGFRvIII-encoding mRNA encapsulated in LNPs, as compared to untreated and luciferase controls. Figure 28C shows body weight measurements of mice throughout the study to monitor potential adverse effects of the LNPs. Figure 28D shows the results of the quantification of total anti-EGFRvIII IgGs in mouse plasma throughout the study (FB = formulation buffer injected mice; tumor control = tumortransplanted FB-treated). Figure 28E shows the results of the Detection of splenic cytotoxic T lymphocytes (CTLs) that specifically recognize the EGFRvIII mutation using an EGFRvIII dextramer regent coupled to the chromophore PE. Figure 28F shows the results of detection of splenic cytotoxic T lymphocytes (CTLs) that react to stimulation using EGFRvIII peptides by expressing the activation-induced marker (AIM) CD69 (as determined by flow cytometry). Spleen-derived cells were incubated overnight with EGFRvIII-derived peptides to test CTL reactivity to these peptides. Figures 28G-28I show the results of the assessment of changes in CTL cell surface marker expressions by flow cytometry at d21 of the study. Figure 28 J shows the results of the assessment of changes in gamma-delta T cell frequencies through staining for the TCRyS cell surface marker by flow cytometry at d21. Figure 28K shows the results of the assessment of changes in T helper (Th) cell surface marker expressions by flow cytometry at d21 of the study.DETAILED DESCRIPTIONI. Introduction

[0061] The following description sets forth exemplary compounds, compositions, methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.

[0062] The delivery of a therapeutic agent to a subject is important for its therapeutic effects and usually it can be impeded by limited ability of the compound to reach targeted cells and tissues. Improvement of such therapeutic agents to enter the targeted cells of tissues by a variety of means of delivery is crucial. Nucleic acid therapy has emerged as the dominant method of treating various diseases and therapeutic indications given the versatility, lower immune response and higher potency as compared to traditional therapies. For example, nucleic acid therapy includes the use of small interfering (siRNA) to reduce the translation of messenger RNA (mRNA), mRNA as a way to produce a target of interest, circular RNA (oRNA) which can provide continuous production of a polypeptide or peptide or can be a sponge to compete with other RNA molecules, and viral vectors to provide a continuous production of a target of interest. However, some nucleic acids are unstable and easily degraded so they need to be formulated to prevent the degradation and to aid in the intracellular delivery of the nucleic acids.

[0063] The present disclosure relates to novel glutamic or glutaric acid-based lipid compounds and compositions comprising the same, more particularly nanoparticles based on these glutamic or glutaric acid-based lipid compounds, capable of encapsulating a cargo such as a biologically active and therapeutic agent.

[0064] The present disclosure also relates to novel lipid nanoparticle compositions that may have improved stability, and / or increased efficacy such as increased immunogenicity when it is used in vaccines, and / or facilitate the intracellular delivery of biologically active and therapeutic agents. In some embodiments, the lipid nanoparticle compositions may have low or reduced toxicity. The present disclosure relates also to pharmaceutical compositions that comprise such lipid compositions, and that are useful to deliver therapeutically effective amounts of biologically active agents into the cells of patients.

[0065] Examples of biologically active agents include but are not limited to: (1) proteins including immunoglobin proteins, (2) polynucleotides such as genomic DNA, cDNA, or mRNA, (3) antisense polynucleotides, and (4) low molecular weight compounds, whether synthetic or naturally occurring, such as the peptide hormones and antibiotics.

[0066] “Lipid” means an organic compound that comprises an ester of fatty acid and is characterized by being insoluble in water, but soluble in many organic solvents. Lipids are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0067] “Lipid particle” or “lipid nanoparticle (i.e., “LNP”) means a lipid formulation that can be used to deliver a cargo, such as a therapeutic nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, and the like). In preferred embodiments, the lipid particle can be used to encapsulate a nucleic acid. In preferred embodiments, the lipid nanoparticle can be formed from an ionizable lipid, a neutral lipid (e.g., a phospholipid), a polymer-conjugated lipid that can prevent aggregation of the nanoparticle (e.g., a PEG-lipid), and optionally a helper lipid (e.g., cholesterol). In some embodiments, a therapeutic nucleic acid (e.g., mRNA) may be encapsulated in the lipid portion of the nanoparticle, thereby protecting it from enzymatic degradation. In another preferred embodiment, the lipid nanoparticle can comprise another component such as a hydrophobic component to improve LNP internalization, immune activation and / or antibody production.

[0068] Lipid nanoparticles generally comprise cholesterol (aids in stability and promotes membrane fusion), a phospholipid (which provides structure to the LNP bilayer and also may aid in endosomal escape), a polyethylene glycol (PEG) derivative (which reduces LNP aggregation and “shields” the LNP from non-specific endocytosis by immune cells), and an ionizable lipid (complexes negatively charged RNA and enhances endosomal escape), which form the LNP- forming composition.

[0069] Lipid nanoparticles typically can have a particle size, e.g., expressed as a mean diameter, ranging from 30 nm to 200 nm, from 40 nm to 180 nm, from 50 nm to 150 nm, from 60nm to 130 nm, from 60 nm to 120 nm, from 60 nm to 110 nm, from 60 nm to 100 nm, from 60 nm to 90 nm, from 70 nm to 110 nm, from 70 nm to 100 nm, from 80 nm to 100 nm, from 90 nm to 100 nm, from 70 to 90 nm, from 80 nm to 90 nm, or from 70 nm to 80 nm. In some embodiments, the particle size can be about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm or 200 nm and are substantially non-toxic. In addition, nucleic acids, when present in the lipid nanoparticles of the present disclosure, are resistant in aqueous solution to degradation with a nuclease.II. LipidsIonizable lipids

[0070] The present disclosure relates to compounds that are ionizable lipids, more particularly ionizable glutamic or glutaric acid-based lipids. The ionizable lipids may be cationic lipids.

[0071] In some embodiments, compounds of the present disclosure are based on a glutamic or glutaric acid core, such as the alpha-glutamic acid core, beta-glutamic acid core, alpha-ketoglutaric acid core, or beta-ketoglutaric acid core:

[0072] In some embodiments, compounds of the present disclosure are based on the glutamic acid core, such as the alpha-glutamic acid core or beta-glutamic acid core.

[0073] In some embodiments, compounds of the present disclosure comprise a glutamic acid core. Such glutamic acid core comprises two ester moieties and one primary, secondary or tertiary amino group (e g., -O-CO-CH2CH2CH(NRIR2)-CO-O- or -O-CO-CH2CH(NRIR2)CH2-CO-O-). In some embodiments, compounds of the present disclosure comprise a glutaric acid core. Such glutaric acid core comprises three ester moieties (e.g., -O-CO-CH2CH2CH(OCORI)-CO-O- or - O-CO-CH2CH(OCORI)CH2-CO-O-). Ester bonds present the particularity of being biodegradable.

[0074] In some embodiments, the ionizable lipid compound of the present disclosure can be a compound of Formula (I):or a pharmaceutically acceptable salt thereof, whereinA is absent or -CH2-;B is -CH2- or -CH2CH2- ■ wherein when A is absent then B is -CH2CH2- and when A is -CH2- then B is -CH2-;X is -NR1R2 or -O-(C=O)Rr;Ri is H or an optionally substituted linear C1-C4 alkyl group;R2 is H, an optionally substituted linear C1-C8 alkyl group, -C(=O)R?, -S(=O)2-R7, orRr is an optionally substituted linear C1-C8 alkyl group;R20 is H or an optionally substituted linear C1-C8 alkyl group;R21 is an optionally substituted linear C1-C8 alkyl group or -(C=O)-R22, wherein R22 is an optionally substituted C1-C8 alkyl group;R3 and R4 are independently H, a linear or branched C4-C60 alkyl, a linear or branched C4-C60 alkenyl, or a linear or branched C4-C60 alkynyl group, wherein at least one of R3 or R4 is different from H;Rs and Re are independently a linear or branched C4-C60 alkyl, a linear or branched C4- C60 alkenyl, or a linear or branched C4-C60 alkynyl group;R7 is an optionally substituted linear Cl -C8 alkyl group, an optionally substituted linear C2-C8 alkenyl group, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10-membered heteroaryl; wherein when any of R3, R4, Rs and Re represents an alkenyl group, the alkenyl group independently comprises from one to six C=C bonds each independently having the E or Z configuration; wherein when any of R3, R4, Rs and Re represents an alkynyl group, the alkynyl group independently comprises from one to six C=C bonds; wherein when R7 represents an alkenyl group, the alkenyl group comprises from one to two C=C bonds each independently having the E or Z configuration; wherein when any alkyl and / or alkenyl group is substituted, this group is independently substituted with one or more -OH, -(C=O)-H, -(C=O)-CH3, -(C=O)-(CH2)pCH3, -(C=O)-(CH2)P-Ri9, -O- (C=O)-CH3, -O-(C=O)-(CH2)PCH3, -O-(C=O)-(CH2)P-Ri9, -(C=O)-OCH3, -(C=O)-O-(CH2)PCH3, -(C=O)-O-(CH2)P-Ri9, -(C=O)-(CH2)P-(C=O)-Ri9, -(C=O)-O-(CH2)PN(CH3)-(CH2)P’-Ri9, -(C=O)NH-(CH2)P-Ri9, -(C=S)-CH3, -O-CH3, -O-(CH2)pCH3, -NH2, -NHCH3, -N(CH3)2, - N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2)PO-(C=O)CH3)2, -N(CH3)-(CH2)P-RI9, -(C=O)NH2, - N3, -N02, -SCH3, -SCH2CH3, an optionally substituted 5 to 10-membered heterocyclyl, C5-C12 aralkyl, C5-C10 aryl or an optionally substituted 5 to 10-membered heteroaryl, with p and p’ are independently from 1 to 5; and wherein R19 is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10- membered heteroaryl; wherein when any 5 to 10-membered heteroaryl and / or 5 to 10-membered heterocyclyl is substituted, this group is independently substituted with one or more substituents selected from the group consisting of C1-C4 alkyl group, -(CH2)P”N(CH3)2and oxo (=0), with p” from 1 to 5; with the provisos that when A is absent, B is -CH2CH2-, X is -NRIR2, Ri is -CH3, and each of R3, R4, Rs, and Re is linear Cl 8 alkyl groups with one C=C bond between carbons C9 and C10 and one C=C bond between carbons C12 and Ci3, then R2is different from -CH3, -CH2CH2OH and - CH2CH2NH2.

[0075] In some embodiments, when any group is said to be "substituted with “one or more substituents”, this group can be substituted for instance with 1 to 6 substituents, preferably with 1 to 4 substituents, more preferably with 1 to 3 substituents. In some embodiments, when any group is said to be substituted with one or more substituents, this group can be substituted with 1 or 2 substituents. In some embodiments, when any group is said to be substituted with one or more substituents, this group can be substituted with one substituent. In some embodiments, when any group is said to be substituted with one or more substituents, this group can be substituted with 2 substituents. In some embodiments, when any group is said to be substituted with one or more substituents, this group can be substituted with three substituents.

[0076] In some embodiments, the ionizable lipid compound of the present disclosure can be a compound of Formula (II):pharmaceutically acceptable salt thereof, whereinRi is H or an optionally substituted linear C1-C4 alkyl group;R2is H, an optionally substituted linear C1-C8 alkyl group, or -C(=O)R?, -S(=O)2-R?, orR.20 is H or an optionally substituted linear C1-C8 alkyl group;R21 is an optionally substituted linear C1-C8 alkyl group or -(C=O)-R.22, wherein R22 is an optionally substituted C1-C8 alkyl group;R3 and R4 are independently H, a linear or branched C4-C60 alkyl, a linear or branched C4- C60 alkenyl, or a linear or branched C4-C60 alkynyl group; wherein at least one of R3 or R4 is different from H;Rs and Re are independently a linear or branched C4-C60 alkyl, a linear or branched C4-C60 alkenyl, or a linear or branched C4-C60 alkynyl group;R7 is an optionally substituted linear C1-C8 alkyl group, an optionally substituted linear C2- C8 alkenyl group, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10-membered heteroaryl; wherein when any of R3, R4, Rs and Re represents an alkenyl group, the alkenyl group independently comprises from one to six C=C bonds each independently having the E or Z configuration; wherein when any of R3, R4, Rs and Re represents an alkynyl group, the alkynyl group independently comprises from one to six C=C bonds; wherein when R7 represents an alkenyl group, the alkenyl group comprises from one to two C=C bonds each independently having the E or Z configuration; wherein when any alkyl and / or alkenyl group is substituted, this group is independently substituted with one or more -OH, -(C=O)-H, -(C=O)-CH3, -(C=O)-(CH2)pCH3, -(C=O)-(CH2)P-Ri9, -O- (C=O)-CH3, -O-(C=O)-(CH2)PCH3, -O-(C=O)-(CH2)P-Ri9, -(C=O)-OCH3, -(C=O)-O-(CH2)PCH3, -(C=O)-O-(CH2)P-Ri9, -(C=O)-(CH2)P-(C=O)-Ri9, -(C=O)-O-(CH2)PN(CH3)-(CH2)P’-Ri9, - (C=O)NH-(CH2)P-R19, -(C=S)-CH3, -0-CH3, -O-(CH2)PCH3, -NH2, -NHCH3, -N(CH3)2, - N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2)PO-(C=O)CH3)2, -N(CH3)-(CH2)P-R19, -(C=O)NH2, -N3, -NO2, -SCH3, -SCH2CH3, an optionally substituted 5 to 10-membered heterocyclyl, C5-C12 aralkyl, C5-C10 aryl or an optionally substituted 5 to 10-membered heteroaryl, with p and p’ are independently from 1 to 5; and wherein R19 is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10- membered heteroaryl; wherein when any 5 to 10-membered heteroaryl and / or 5 to 10-membered heterocyclyl is substituted, this group is independently substituted with one or more substituents selected from the group consisting of C1-C4 alkyl group, -(CH2)P”N(CH3)2 and oxo (=0), with p” from 1 to 5.

[0077] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, Ri is H or an optionally substituted linear C1-C4 alkyl group. In some embodiments, Ri is H or an optionally substituted linear Cl -C3 alkyl group. In some embodiments,Ri is H or an optionally substituted linear C1-C2 alkyl group. In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, Ri is H or an optionally substituted linear Cl alkyl group. In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, Ri is H or CH3. In some embodiments, Ri is H.

[0078] In some embodiments, in the compound of Formula (I), or the pharmaceutically acceptable salt thereof, Rr is an optionally substituted linear C1-C6 alkyl group. In some embodiments, Rr is an optionally substituted linear C1-C4 alkyl group. In some embodiments, Rr is an optionally substituted linear C1-C3 alkyl group. In some embodiments, r is an optionally substituted linear C2-C4 alkyl group. In some embodiments, Rr is an optionally substituted linear C3-C4 alkyl group.

[0079] In some embodiments, in the compound of Formula (I), or the pharmaceutically acceptable salt thereof, Rr is substituted with any substituent that can be present on alkyl group, as defined herein. In some embodiments, Rr can be substituted with -NH2, -NHCH3, -N(CH3)2, or -N(CH2CH3)2. In some embodiments, Rr can be substituted with -NHCH3, -N(CH3)2, or - N(CH2CH3)2. In some embodiments, Rr can be substituted with -N(CH3)2.

[0080] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R2is H, an optionally substituted linear C1-C4 alkyl group, -C(=O)R7, -

[0082] In some embodiments, R2is H, an optionally substituted linear C3-C4 alkyl group, -C(=O)R7, -S(=O)2-R7, or

[0083] In some embodiments, R2 is H, an optionally substituted linear Cl alkyl group, -C(=O)R7, -S(=O)2-R7, or

[0084] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, Ri is H and R2 is H.

[0085] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, Ri is H and R2 is an optionally substituted linear C1-C8 alkyl group, -

[0086] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, Ri is H and R2 is an optionally substituted linear C1-C4 alkyl group, -

[0087] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, Ri is an optionally substituted linear C1-C4 alkyl group and R2 is an optionally substituted linear C1-C4 alkyl group, or -C(=O)R7.

[0088] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R7is an optionally substituted linear C1-C8 alkyl group, an optionally substituted linear C2-C8 alkenyl group, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10-membered heteroaryl.

[0089] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R7is an optionally substituted linear C1-C8 alkyl group, an optionally substituted linear C2-C8 alkenyl group, an optionally substituted 5 to 6-membered heterocyclyl or an optionally substituted 5 to 6-membered heteroaryl.

[0090] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R7is an optionally substituted linear C1-C4 alkyl group, an optionally substituted linear C2-C4 alkenyl group, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10-membered heteroaryl.

[0091] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R7is an optionally substituted linear C1-C4 alkyl group, an optionallysubstituted linear C2-C4 alkenyl group, an optionally substituted 5 to 6-membered heterocyclyl or an optionally substituted 5 to 6-membered heteroaryl.

[0092] In some embodiments, when R? represents an alkenyl group, the alkenyl group can comprise from one to two C=C bonds each independently having the E or Z configuration.

[0093] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R20 is H or an optionally substituted linear C1-C8 alkyl group.

[0094] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R21 is an optionally substituted linear C1-C8 alkyl group or -(C=O)-R22, wherein R22 is an optionally substituted C1-C8 alkyl group.

[0095] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R20 is H or an optionally substituted linear C1-C4 alkyl group.

[0096] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R21 is an optionally substituted linear C1-C4 alkyl group or -(C=O)-R22, wherein R22 is an optionally substituted C1-C4 alkyl group.

[0097] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, when any alkyl and / or alkenyl group is substituted (e.g., in Ri, Rr. R2, R7, R20, R21 and / or R22), this group can independently be substituted with one or more -OH, -(C=O)- H, -(C=O)-CH3, -(C=O)-(CH2)PCH3, -(C=O)-(CH2)P-RI9, -O-(C=O)-CH3, -O-(C=O)-(CH2)PCH3, -O-(C=O)-(CH2)P-Ri9, -(C=O)-OCH3, -(C=O)-O-(CH2)PCH3, -(C=O)-O-(CH2)P-Ri9, -(C=O)- (CH2)P-(C=O)-Ri9, -(C=O)-O-(CH2)PN(CH3)-(CH2)P’-Ri9, -(C=O)NH-(CH2)P-Ri9, -(C=S)-CH3, - O-CH3, -O-(CH2)PCH3, -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2)PO- (C=O)CH3)2, -N(CH3)-(CH2)P-R19, -(C=O)NH2, -N3, -NO2, -SCH3, -SCH2CH3, an optionally substituted 5 to 10-membered heterocyclyl, C5-C12 aralkyl, C5-C10 aryl or an optionally substituted 5 to 10-membered heteroaryl, with p and p’ are independently from 1 to 5; and wherein R19 is independently -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, an optionally substituted 5 to 10- membered heterocyclyl or an optionally substituted 5 to 10-membered heteroaryl. In some embodiments, p and p’ are independently from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, or both 1.

[0098] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, when any alkyl and / or alkenyl group is substituted (e.g., in Ri, Rr, R2, R7, R20, R21 and / or R22), this group can independently be substituted with one or more -OH, -(C=O)- H, -(C=O)-CH3, -(C=O)-(CH2)PCH3, -(C=O)-(CH2)P-R19, -O-(C=O)-CH3, -O-(C=O)-(CH2)PCH3, -O-(C=O)-(CH2)P-R19, -(C=O)-OCH3, -(C=O)-O-(CH2)PCH3, -(C=O)-O-(CH2)P-R19, -(C=O)- (CH2)P-(C=O)-R19, -(C=O)-O-(CH2)PN(CH3)-(CH2)P’-R19, -(C=O)NH-(CH2)P-R19, -O-CH3, -O- (CH2)PCH3, -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2)PO- (C=O)CH3)2, -N(CH3)-(CH2)P-Ri9, -(C=O)NH2, an optionally substituted 5 to 10-memberedheterocyclyl, C5-C12 aralkyl, C5-C10 aryl or an optionally substituted 5 to 10-membered heteroaryl, wherein p and p’ are independently from 1 to 5; and wherein Rig is independently - NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10-membered heteroaryl. In some embodiments, p and p’ are independently 1 or 3. In some embodiments, p and p’ are independently 1 or 2.

[0099] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, when any alkyl and / or alkenyl group is substituted (e.g., in Ri, Rr. R2, R7, R20, R21 and / or R22), this group can independently be substituted with one or more -OH, -(C=O)- (CH2)P-Ri9, -O-(C=O)-(CH2)P-RI9, -(C=O)-O-(CH2)P-RI9, -(C=O)-(CH2)P-(C=O)-Ri9, -(C=O)-O- (CH2)pN(CH3)-(CH2)p’-N(CH3)2, -(C=O)NH-(CH2)P-Ri9, -NH2, -NHCH3, -N(CH3)2, - N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2)PO-(C=O)CH3)2, -N(CH3)-(CH2)PN(CH3)2„ an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10- membered heteroaryl, wherein p and p’ are independently from 1 to 5; and wherein R19 is independently -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, an optionally substituted 5 to 6- membered heterocyclyl or optionally substituted 5 to 10-membered heteroaryl. In some embodiments, p and p’ are independently from 1 to 3. In some embodiments, p and p’ are independently 1 or 2.

[0100] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, when any alkyl and / or alkenyl group is substituted (e.g., in Ri, Rr, R2, R7, R20, R21 and / or R22), this group can independently be substituted with one or more -OH, -(C=O)- (CH2)P-Ri9, -O-(C=O)-(CH2)P-RI9, -(C=O)-O-(CH2)P-RI9, -(C=O)-(CH2)P-(C=O)-Ri9, -(C=O)-O- (CH2)pN(CH3)-(CH2)p’-N(CH3)2, -(C=O)NH-(CH2)P-Ri9, -NH2, -NHCH3, -N(CH3)2, - N(CH2CH3)2, -N((CH2)pO-(C=O)CH3)2, -N(CH3)-(CH2)pN(CH3)2, a 5 to 10-membered heterocyclyl optionally substituted with one C1-C4 alkyl group or 5 to 10-membered heteroaryl optionally substituted with one C1-C4 alkyl group, wherein p and p’ are independently from 1 to 35; and wherein R19 is independently -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, or 5 to 10- membered heteroaryl optionally substituted with one C1-C4 alkyl group. In some embodiments, p and p’ are independently from 1 to 3. In some embodiments, p and p’ are independently 1 or 2.

[0101] In some embodiments, in the compound of Formula (I) or (11), or the pharmaceutically acceptable salt thereof, when any alkyl and / or alkenyl group is substituted (e.g., in Ri, Rr, R2, R7, R20, R21 and / or R22), this group can independently be substituted with one or more -OH, -(C=O)- (CH2)P-Ri9, -O-(C=O)-(CH2)P-RI9, -(C=O)-O-(CH2)P-RI9, -(C=O)-(CH2)P-(C=O)-Ri9, -(C=O)-O- (CH2)pN(CH3)-(CH2)p’-N(CH3)2, -(C=O)NH-(CH2)P-Ri9, -N(CH3)2, -N((CH2)PO-(C=O)CH3)2, - N(CH3)-(CH2)PN(CH3)2, a 5 to 10-membered heterocyclyl optionally substituted with one C1-C2 alkyl group or 5 to 10-membered heteroaryl optionally substituted with one C1-C2 alkyl group,with p and p’ are independently from 1 to 5; and wherein R19 is -N(CH3)2, a 5 to 6-membered heterocyclyl optionally substituted with one C1-C2 alkyl group, or a 5 to 6-membered heteroaryl optionally substituted with one C1-C2 alkyl group.

[0102] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, when any 5 to 10-membered heteroaryl and / or 5 to 10-membered heterocyclyl is substituted (e.g, in R7 and R19), this group can independently be substituted with one or more substituents selected from the group consisting of C1-C4 alkyl group, - (CH2)p’ N(CH3)2 and oxo (=0), with p” from 1 to 5. In some embodiments, p” is from 1 to 3. In some embodiments, p” is 1 or 2.

[0103] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, when any 5 to 10-membered heteroaryl and / or 5 to 10-membered heterocyclyl is substituted (e.g, in R7 and R19), this group can independently be substituted with one or more substituents selected from the group consisting of C 1 -C2 alkyl group, -(CH2)2N(CH3)2 and oxo (=0).

[0104] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R20 is H or a linear C 1 -C3 alkyl group optionally substituted with -N(CH3)2.

[0105] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R21 is a linear C1-C4 alkyl group optionally substituted with -N(CH3)2 or 5 to 10-membered heterocyclyl optionally substituted with a linear C1-C3 alkyl group, or is - (C=O)-R22, wherein R22 is C1-C3 alkyl group optionally substituted with -N(CH3)2.

[0106] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R21 is a linear C1-C4 alkyl group optionally substituted with -N(CH3)2 or 5 to 10-membered heterocyclyl optionally substituted with a C1-C2 alkyl group, or is -(C=O)-R22, wherein R22 is a C1-C3 alkyl group optionally substituted with -N(CH3)2

[0107] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, any 5 to 10-membered heteroaryl can independently represent furanyl, benzofuranyl, benzimidazolyl, quinolinyl, isoquinolinyl, thiophenyl, pyrrolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazolyl, pyrazolyl, pyrazinyl, pyridinyl, pyridazinyl or pyrimidinyl. As noted above, these groups can be substituted themselves.

[0108] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, the 5 to 10-membered heterocyclyl can independently represent a lactam, morpholinyl, piperazinyl or piperidinyl. As noted above, these groups can be substituted themselves.

[0109] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, the group -NR1R2 has one of the following structures:

[0110] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, the group[oni] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R3 and R4 are independently H, a linear or branched C4-C60 alkyl, a linear or branched C4-C60 alkenyl, or a linear or branched C4-C60 alkynyl group, wherein at least one of R3 or R4 is different from H. In some embodiments, R3 and R4 are independently H, a linear or branched C4-C30 alkyl, a linear or branched C4-C30 alkenyl, or a linear or branched C4-C30 alkynyl group, wherein at least one of R3 or R4 is different from H. In some embodiments, R3 and R4 are independently H, a linear or branched C6-C25 alkyl, or a linear or branched C6-C25 alkenyl, wherein at least one of R3 or R4 is different from H.

[0112] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, Rs and Re are independently a linear or branched C4-C30 alkyl, a linear or branched C4-C30 alkenyl, or a linear or branched C4-C30 alkynyl group. In some embodiments, Rs and Re are independently a linear or branched C6-C25 alkyl, or a linear or branched C6-C25 alkenyl group.

[0113] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R3, R4, Rs and Re are independently a linear or branched C5-C20 alkyl, a linear or branched C5-C20 alkenyl, or a linear or branched C5-C20 alkynyl group. In some embodiments, in the compound of Formula (I), or the pharmaceutically acceptable salt thereof, R3, R4, Rs and Re are independently a linear or branched C6-C20 alkyl, a linear or branched C6-C20 alkenyl, or a linear or branched C6-C20 alkynyl group. In some embodiments, in the compound of Formula (I), or the pharmaceutically acceptable salt thereof, R3, R4, Rs and Re are independently a linear or branched C7-C20 alkyl, a linear or branched C7-C20 alkenyl, or a linear or branched C7-C20 alkynyl group. In some embodiments, in the compound of Formula (I), or the pharmaceutically acceptable salt thereof, R3, R4, Rs and Re are independently a linear or branched C8-C20 alkyl, a linear or branched C8-C20 alkenyl, or a linear or branched C8-C20 alkynyl group.

[0114] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R3, R4, Rs and Re are independently a linear or branched C5-C20 alkyl or a linear or branched C5-C20 alkenyl. In some embodiments, in the compound of Formula (I), or the pharmaceutically acceptable salt thereof, R3, R4, Rs and Re are independently a linear or branched C6-C20 alkyl, or a linear or branched C6-C20 alkenyl. In some embodiments, in the compound of Formula (I), or the pharmaceutically acceptable salt thereof, R3, R4, Rs and Re are independently a linear or branched C7-C20 alkyl, or a linear or branched C7-C20 alkenyl. In some embodiments, in the compound of Formula (I), or the pharmaceutically acceptable salt thereof, R3, R4, Rs and Re are independently a linear or branched C8-C20 alkyl, or a linear or branched C8- C20 alkenyl.

[0115] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, when any of R3, R4, Rs and Re is alkenyl, the alkenyl group independently comprises one to six C=C bonds, or one to five C=C bonds, or one to four C=C bonds, or one to three C=C bonds, or one or two C=C bonds.

[0116] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, when any of R3, R4, Rs and Re is alkynyl, the alkynyl group independently comprises one to six c=C bonds, or one to five c=C bonds, or one to four c=C bonds , or one to three c=c bonds, one or two c=c bonds.

[0117] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, when any of R3, R4, Rs and Re is C8-C20 alkenyl, the alkenyl group independently comprises one or two C=C bonds, and when any of R3, R4, Rs and Re is C8-C20 alkynyl, the alkynyl group independently comprises one or two c=C bonds.

[0118] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R3 and R4 are linear C6-C16 alkyl groups. In some embodiments, R3 and R4 are branched C8-C12 alkyl groups.

[0119] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R3 and R4 are linear C8-C20 alkenyl groups, and the alkenyl groups each comprise one C=C bond.

[0120] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R3 and R4 are linear C8-C16 alkenyl groups, and the alkenyl groups each comprise one C=C bond.

[0121] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R3 is H and R4 is a linear C6-C25 alkenyl group, wherein the alkenyl group comprise one C=C bond.

[0122] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R4 is H and R3 is a linear C6-C25 alkenyl group, wherein the alkenyl group comprise one C=C bond.

[0123] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, Rs and Re are independently linear C8-C20 alkyl groups.

[0124] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, Rs and Re are independently linear C10-C18 alkyl groups.

[0125] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, Rs and Re are linear C6-C16 alkyl groups. In some embodiments, Rs and Re are branched C8-C12 alkyl groups.

[0126] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, Rs and Re are linear C8-C20 alkenyl groups, wherein the alkenyl groups each comprise one C=C bond.

[0127] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, Rs and Re are linear C15-C20 alkenyl groups, wherein the alkenyl groups each comprise two C=C bonds.

[0128] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, Rs and Re are branched C22-C25 alkenyl groups, wherein the alkenyl groups each comprise one C=C bond.

[0129] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, Rs and Re are branched C13-C17 alkenyl groups, wherein the alkenyl groups each comprise three C=C bonds.

[0130] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, any of R3, R4, Rs and Re can be C6-C25 alkenyl, and the alkenyl group independently comprises one, two or three C=C bonds.

[0131] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R3, R4, Rs and Re independently have one of the following structures, considering that one of R3 and R4 is different than H:

[0132] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R3 and R4 are both different from H and are identical.

[0133] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, Rs and Re are identical.

[0134] In some embodiments, in the compound of Formula (I) or (II), or the pharmaceutically acceptable salt thereof, R3 and R4 are both different from H and R3, R4, Rs and Re are identical.

[0135] In some embodiments, the ionizable lipid compound of the present disclosure can be a compound of Formula (12) or (13):(12) (13) or a pharmaceutically acceptable salt thereof, wherein Ri, R2, R3, R4, Rs, Re and Rr are as defined herein for formula (I) and / or (II).

[0136] In some embodiments, the ionizable lipid compound of the present disclosure can be the compound of formula (12), or the pharmaceutically acceptable salt thereof, wherein Ri is H or C1-C2 alkyl, and R2 is -C(=O)R?; and wherein R3, R4, Rs, Re and R7 are as defined herein for formula (I) and / or (II).

[0137] In some embodiments, the ionizable lipid compound of the present disclosure can be the compound of formula (13), or the pharmaceutically acceptable salt thereof, wherein Rr is a linear C1-C4 alkyl group substituted with -(C=O)-(CH2)P-Ri9, -O-(C=O)-(CH2)P-Ri9, -(C=O)-O- (CH2)P-Ri9, -(C=O)-(CH2)P-(C=O)-Ri9, -(C=O)-O-(CH2)PN(CH3)-(CH2)P’-N(CH3)2, -(C=O)NH- (CH2)P-R19, -N(CH3)2, -N((CH2)PO-(C=O)CH3)2, -N(CH3)-(CH2)PN(CH3)2, an optionally substituted 5 to 6-membered heterocyclyl or an optionally substituted 5 to 6-membered heteroaryl, with p and p’ are independently from 1 to 5; and wherein R^is -N(CH3)2, an optionally substituted 5 to 6-membered heterocyclyl or an optionally substituted 5 to 6-membered heteroaryl; wherein when the 5 to 6-membered heteroaryl and / or 5 to 6-membered heterocyclyl is substituted, this group is independently substituted with one substituent selected from the group consisting of Cl- C2 alkyl group; and wherein R3, R4, Rs and Re are as defined herein for formula (I) and / or (II).

[0138] In some embodiments, the ionizable lipid compound of the present disclosure can be a compound of Formula (11 -1), (11-2), (11-3), (11-4), (11-5) or (11-6):or a pharmaceutically acceptable salt thereof, wherein Ri is H or a linear C1-C4 alkyl; wherein Rio and Rn are independently -H, -(C=O)-CH3, -(C=O)-(CH2)PCH3, -(C=O)-(CH2)P-Ri9, or -(C=O)-(CH2)P-(C=O)-RI9; and wherein R3, R4, Rs, Re, R7, R19, R20 and R21 are as defined herein for Formula (I) and / or (II) and p is from 1 to 5.

[0139] In some embodiments, in the Formulas (11-1), (11-2), (11-3), (11-4), (11-5) or (11-6), p can independently be from 1 to 5, or from 1 to 4, or from 1 to 3, or from 1 to 2, or p can be 1, 2, 3, 4, or 5.

[0140] In some embodiments, the ionizable lipid compound can be a compound of formula (Il-4a):or a pharmaceutically acceptable salt thereof, wherein Ri is H or a linear C1-C4 alkyl; and wherein R3, R4, Rs and Re are as defined herein for formula (I) and / or (II).

[0141] In some embodiments, the ionizable lipid compound can be a compound of formula (Il - 4a), or pharmaceutically acceptable salt thereof, wherein Ri is H or -CH3.

[0142] In some embodiments, the ionizable lipid compound can be a compound of formula (Il - 4a), or pharmaceutically acceptable salt thereof, wherein Ri is -CH3.

[0143] In some embodiments, the ionizable lipid compound can be a compound of formula (Il -4b):or a pharmaceutically acceptable salt thereof, wherein Ri is H or a linear C1-C4 alkyl; wherein R12 and R13 are independently an optionally substituted linear Cl -C4 alkyl; wherein when the linear alkyl group is substituted, this group is independently substituted with -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, or 5-6 membered heteroaryl; and wherein R3, R4, Rs and Re are as defined herein for formula (I) and / or (II).

[0144] In some embodiments, the ionizable lipid compound can be a compound of formula (Il - 4b), or pharmaceutically acceptable salt thereof, wherein Ri is H or -CH3.

[0145] In some embodiments, the ionizable lipid compound can be a compound of formula (Il - 4b), or pharmaceutically acceptable salt thereof, wherein Ri is -CH3.

[0146] In some embodiments, the ionizable lipid compound can be a compound of formula (Il - 4b), or pharmaceutically acceptable salt thereof, wherein R12 and R13 are independently linear Cl- C4 alkyl substituted with -NH2, -NHCH3, -N(CH3)2 or -N(CH2CH3)2.

[0147] In some embodiments, the ionizable lipid compound can be a compound of formula (Il - 4b), or pharmaceutically acceptable salt thereof, wherein R12 and R13 are independently linear Cl- C4 alkyl substituted with -N(CH3)2.

[0148] In some embodiments, the ionizable lipid compound can be a compound of formula (Il -5a):or a pharmaceutically acceptable salt thereof, wherein Ri is H or a linear C1-C4 alkyl; wherein q is from 1 to 5; wherein R14 and R’14 independently represent -H, -CH3, -CH2CH3, or -(CH2)PO-(C=O)CH3 with p is from 1 to 5, or R14 and R’14 are connected to form a 6-membered heterocyclyl optionally substituted with -CH3 or -CH2CH3; and wherein R3, R4, Rs and Re are as defined herein for formula (I) and / or (II) for formula (I) and / or (II).

[0149] In some embodiments, the ionizable lipid compound can be a compound of formula (Il - 5a), or pharmaceutically acceptable salt thereof, wherein Ri is H or -CH3.

[0150] In some embodiments, the ionizable lipid compound can be a compound of formula (Il - 5a), or pharmaceutically acceptable salt thereof, wherein Ri is H.

[0151] In some embodiments, the ionizable lipid compound can be a compound of formula (Il - 5a), or pharmaceutically acceptable salt thereof, wherein R14 and R’14 independently represent -H,-CH3, -CH2CH3, or -(CH2)PO-(C=O)CH3 with p is from 1 to 3, or R14 and R’14 are connected to form a 6-membered heterocyclyl optionally substituted with -CH3 or -CH2CH3.

[0152] In some embodiments, the ionizable lipid compound can be a compound of formula (Il -5b):or a pharmaceutically acceptable salt thereof, wherein Xi is -NH- or -O-; wherein R15 is a linear Cl -C4 alkyl, -(CH2)P-Ri9, or-(CH2)PN(CH3)-((CH2)P’-N(CH3)2) with p and p’ are independently from 1 to 5, and R3, R4, Rs, Re and R19 are as defined herein for formula (I) and / or (II).

[0153] In some embodiments, the ionizable lipid compound can be a compound of formula (Il - 5b), or pharmaceutically acceptable salt thereof, wherein R15 is -(CH2)2N(CH3)-((CH2)2-N(CH3)2) or -(CH2)P-Ri9 with p is from 1 to 3.

[0154] In some embodiments, the ionizable lipid compound can be a compound of formula (Il - 5b), or pharmaceutically acceptable salt thereof, wherein Ris is-(CH2)2N(CH3)-((CH2)2-N(CH3)2), -(CH2)P-N(CH3)2, or -(CH2)P-R23 with p is from 1 to 3 and R23 is a 5 to 6-membered heteroaryl or 5 to 6-membered heterocyclyl, optionally substituted with C1-C2 alkyl.

[0155] In some embodiments, the ionizable lipid compound can be a compound of formula (Il - 5b), or pharmaceutically acceptable salt thereof, wherein Ris is-(CH2)2N(CH3)-((CH2)2-N(CH3)2), -(CH2)P-N(CH3)2 or -(CH2)P-R23 with p is from 1 to 3 and R23 is furanyl, thiophenyl, pyrrolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazolyl, pyrazolyl, pyrazinyl, pyridinyl, pyridazinyl, pyrimidinyl, piperidinyl or piperazinyl, wherein R23 is optionally substituted with Cl- C2 alkyl.

[0156] In some embodiments, the ionizable lipid compound can be a compound of formula (Il - 5b), or pharmaceutically acceptable salt thereof, wherein R15 is -(CH2)2N(CH3)-((CH2)2-N(CH3)2), -(CH2)P-N(CH3)2or-(CH2)P-R23 with p is from 1 to 3 and R23 is imidazolyl or piperidinyl, wherein R23 is optionally substituted with a C1-C2 alkyl.

[0157] In some embodiments, the ionizable lipid compound can be a compound of formula (Il -5c):or a pharmaceutically acceptable salt thereof, wherein Rig is a 5 to 6-membered heteroaryl or a 5 to 6-membered heterocyclyl, optionally substituted with a linear C1-C4 alkyl, an oxo (=0) and / or a -(CH2)P-N(CH3)2 and wherein p is from 1 to 3, and wherein R3, R4, Rs and Re are as defined herein for formula (I) and / or (II).

[0158] In some embodiments, the ionizable lipid compound can be a compound of formula (Il - 5c) or pharmaceutically acceptable salt thereof, wherein Rie is furanyl, thiophenyl, pyrrolyl, pyrrolydinyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazolyl, pyrazolyl, pyrazinyl, pyridinyl, pyridazinyl, piperazinyl, piperidinyl or pyrimidinyl optionally substituted with a linear C1-C4 alkyl, an oxo (=0) and / or a -(CH2)p-N(CH3)2 with p from 1 to 3.

[0159] In some embodiments, the ionizable lipid compound can be a compound of formula (Il - 5c) or pharmaceutically acceptable salt thereof, wherein Rig is imidazolyl or pyrrolidinyl optionally substituted with a C1-C2 alkyl, an oxo (=0) and / or a -(CH2)p-N(CH3)2 with p from 1 to 3.

[0160] In some embodiments, the ionizable lipid compound can be a compound of formula (Il -5d):or a pharmaceutically acceptable salt thereof, wherein R17 is a 5 to 6-membered heteroaryl optionally substituted with a linear C1-C4 alkyl, or a -(CH2)P-N(CH3)2 and wherein p is from 1 to 3; wherein the C=C bonds present in Formula (Il -5d) independently have the E or Z configuration; and wherein R3, R4, Rs and Re are as defined herein for formula (I) and / or (II).

[0161] In some embodiments, the ionizable lipid compound can be a compound of formula (Il - 5d) or pharmaceutically acceptable salt thereof, wherein R17 is furanyl, thiophenyl, pyrrolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazolyl, pyrazolyl, pyrazinyl, pyridinyl, pyridazinyl or pyrimidinyl, optionally substituted with a C1-C2 alkyl.

[0162] In some embodiments, the ionizable lipid compound can be a compound of formula (Il - 5d) or pharmaceutically acceptable salt thereof, wherein R17 is imidazolyl.

[0163] In some embodiments, the ionizable lipid compound can be a compound of formula (Il - 6a), (Il -6b) or (Il -6c):or a pharmaceutically acceptable salt thereof, whereinR31 is a linear C1-C4 alkyl group optionally substituted with -N(CHs)2;R32 is -N(CHS)2 or a 5 to 6-membered heterocyclyl optionally substituted with a linear C1-C4 alkyl group, and z is from 1 to 4; and R3, R4, Rs and Re are as defined herein for formula (I) and / or (II).

[0164] In some embodiments, the ionizable lipid compound can be a compound of formula (Il - 6a), or pharmaceutically acceptable salt thereof, wherein R31 is -(CH2)3-N(CH3)2.

[0165] In some embodiments, the ionizable lipid compound can be a compound of formula (Il - 6b) or (Il -6c), or pharmaceutically acceptable salt thereof, wherein R32 is -N(CH3)2, and z is from 2 to 4.

[0166] In some embodiments, the ionizable lipid compound can be a compound of formula (Il - 6b) or (Il -6c), or pharmaceutically acceptable salt thereof, wherein R32 is piperazinyl or piperidinyl substituted with a C1-C2 alkyl group, and z is 3.

[0167] In some embodiments, in the Formulas (I), (II), (12), (Il -1), (11-2), (11-3), (11-4), (II- 4a), (Il-4b), (11-5), (Il-5a), (II-5b), (II-5c), (Il-5d), (11-6), (Il-6a), (Il-6b) or (Il-6c), R3and R4 can be identical. In some embodiments, in the Formulas (I), (II), (12), (11-1), (11-2), (11-3), (11-4),(Il-4a), (Il-4b), (11-5), (Il-5a), (II-5b), (II-5c), (II-5d), (11-6), (Il-6a), (Il-6b) or (Il-6c), R5and Re can be identical. In some embodiments, in the Formulas (I), (II), (12), (Il -1), (11-2), (11-3), (II- 4), (Il-4a), (Il-4b), (11-5), (Il-5a), (Il-5b), (Il-5c), (Il-5d), (11-6), (Il-6a), (Il-6b) or (Il-6c), R3and Rj are both identical, and Rs and Re are both identical.

[0168] In some embodiments, the ionizable lipid compound can be a compound of Formula (Ila):or a pharmaceutically acceptable salt thereof, wherein m is a number from 6 to 18 and n is a number from 2 to 14; and wherein the C=C bonds present in the Formula (Ila) independently have the E or Z configuration.

[0169] In some embodiments, the ionizable lipid compound can be a compound of Formula (I2a):(I2a), or a pharmaceutically acceptable salt thereof, wherein m is a number from 6 to 18 and n is a number from 2 to 14; and wherein the C=C bonds present in the Formula (I2a) independently have the E or Z configuration.

[0170] In some embodiments, the ionizable lipid compound can be a compound of Formula (I2b):or a pharmaceutically acceptable salt thereof, wherein m is a number from 6 to 18; and n is a number from 2 to 14; and wherein the C=C bonds present in the Formula (I2b) independently have the E or Z configuration.

[0171] In some embodiments, the ionizable lipid compound can be a compound of Formula (I3a):or a pharmaceutically acceptable salt thereof, wherein m is a number from 6 to 18; n is a number from 2 to 14; and Ri is H or a linear C1-C4 alkyl; and wherein the C=C bonds present in the Formula (I3a) independently have the E or Z configuration.

[0172] In some embodiments, in the Formula (I3a), Ri is H or C1-C3 alkyl, or Ri is H or C1-C2 alkyl, or Ri is H or -CH3, more preferably Ri is -CH3.

[0173] In some embodiments, the ionizable lipid compound can be a compound of Formula (I3b):or a pharmaceutically acceptable salt thereof, wherein m is a number from 6 to 18; n is a number from 2 to 14; Ri is H or a linear C1-C4 alkyl; and R12 and R13 are independently an optionally substituted linear C1-C4 alkyl; wherein when the linear alkyl group is substituted, this group is independently substituted with -NH2, -NHCH3, -N(CH3)2, -N CFECFE , or 5-6 membered heteroaryl; and wherein the C=C bonds present in the Formula (I3b) independently have the E or Z configuration.

[0174] In some embodiments, in the Formula (I3b), Ri is H or C1-C3 alkyl, or Ri is H or C1-C2 alkyl, or Ri is H or -CH3, more preferably Ri is -CH3.

[0175] In some embodiments, in the Formula (I3b), R12 and R13 are independently C1-C3 alkyl, or R12 and R13 are independently selected from Cl alkyl, C2 alkyl and C3 alkyl. In some embodiments, R12 and R13 can be both C3 alkyl.

[0176] In some embodiments, in the Formula (I3b), R12 and R13 are independently linear Cl- C4 alkyl substituted with -NH2, -NHCH3, -N(CH3)2 or -N(CH2CH3)2, more preferably substituted with -N(CH3)2.

[0177] In some embodiments, the ionizable lipid compound can be a compound of Formula (I4a):or a pharmaceutically acceptable salt thereof, wherein m is a number from 6 to 18; n is a number from 2 to 14; and q is a number from 1 to 4; wherein R14 and R’14 independently represent -H, -CH3, -CH2CH3, or -(CH2)PO-(C=O)CH3with p is from 1 to 5; and wherein the C=C bonds present in the Formula (I4a) independently have the E or Z configuration.

[0178] In some embodiments, in the Formula (I4a), p is from 1 to 5, or from 1 to 4, or from 1 to 3, or from 1 to 2, or p can be 1, 2, 3, 4 or 5. In some embodiments, in the Formula (I4a), q is from 1 to 4, or from 1 to 3, or from 1 to 2, or q can be 1, 2, 3 or 4. In some embodiments, in the Formula (I4a), p is from 1 to 3.

[0179] In some embodiments, the ionizable lipid compound can be a compound of Formula (I4b):or a pharmaceutically acceptable salt thereof, wherein m is a number from 6 to 18; n is a number from 2 to 14; and wherein Ri5is -CH3, -(CH2)pCH3, -(CH2)PRi9, or-(CH2)pN(CH3)-((CH2)P’- N(CH3)2) with p and p’ are independently from 1 to 5, and R19 is as defined herein for Formula (I); and wherein the C=C bonds present in the Formula (I4b) independently have the E or Z configuration.

[0180] In some embodiments, in the Formula (I4b), p and p’ can independently be from 1 to 4, or from 1 to 3, or from 1 to 2, or p and p’ can independently be 1, 2, 3, 4 or 5. In some embodiments, in the Formula (I4b), p and p’ can independently be from 1 to 3.

[0181] In some embodiments, in the Formula (I4b), R15 is preferably -(CH2)2N(CH3)-((CH2)2- N(CH3)2).

[0182] In some embodiments, the ionizable lipid compound can be a compound of Formula (I4c):or a pharmaceutically acceptable salt thereof, wherein m is a number from 6 to 18; n is a number from 2 to 14; and Ri6 is a 5-6 membered heteroaryl; and wherein the C=C bonds present in the Formula (I4c) independently have the E or Z configuration.

[0183] In some embodiments, in the Formula (I4c), Ri6 can be furanyl, thiophenyl, pyrrolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazolyl, pyrazolyl, pyrazinyl, pyridinyl, pyridazinyl or pyrimidinyl. In some embodiments, in the Formula (I4c), Ri6 is imidazolyl.

[0184] In some embodiments, the ionizable lipid compound can be a compound of Formula (I4d):or a pharmaceutically acceptable salt thereof, wherein m is a number from 6 to 18; n is a number from 2 to 14; and Rr? is a 5-6 membered heteroaryl; and wherein the C=C bonds present in the Formula (I4d) independently have the E or Z configuration.

[0185] In some embodiments, in the Formula (I4d), Rn can be furanyl, thiophenyl, pyrrolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazolyl, pyrazolyl, pyrazinyl, pyridinyl, pyridazinyl or pyrimidinyl. In some embodiments, in the Formula (I4d), Rn is imidazolyl.

[0186] In some embodiments, the ionizable lipid compound can be a compound of Formula (I4e):or a pharmaceutically acceptable salt thereof, wherein m is a number from 6 to 18; n is a number from 2 to 14; and Ris is -(CH2)P-Ri9 with p is from 1 to 5, and R19 is as defined herein for Formula (I); and wherein the C=C bonds present in the Formula (I4e) independently have the E or Z configuration.

[0187] In some embodiments, in the Formula (I4e), Ris can be -(CH2)P-Ri9 with p is from 1 to 5, or from 1 to 4, or from 1 to 3, or from 1 to 2, or p is 1, 2, 3, 4 or 5; and R19 is as defined herein for Formula (I).

[0188] In some embodiments, in the Formula (I4e), Ris is -(CH2)P-Ri9 with p is from 1 to 3, and R19 is as defined herein for Formula (I). In some embodiments, in the Formula (I4e), Ris is -(CH2)s- (5-6 membered heteroaryl) wherein 5-6 membered heteroaryl is for example furanyl, thiophenyl, pyrrolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazolyl, pyrazolyl, pyrazinyl, pyridinyl, pyridazinyl or pyrimidinyl. In some embodiments, in the Formula (I4e), Ris is -(CH2)3-imidazole.

[0189] In some embodiments, in the Formulas (Ila), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d) or (I4e), m can independently be an integer from 6 to 18, or m can be an integer from 6 to 17, or from 6 to 16, or from 6 to 15, or from 6 to 14, or from 6 to 13, or from 6 to 12, or from 6 to11, or from 6 to 10, or from 6 to 9, or from 6 to 8, or from 6 to 7, or m can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18. In some embodiments, m can be 12.

[0190] In some embodiments, in the Formulas (Ila), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d) or (I4e), n can independently be an integer from 2 to 14, or n can be an integer from 2 to 13, or from 2 to 12, or from 2 to 11, or from 2 to 10, or from 2 to 9, or from 2 to 8, or from 2 to 7, or from 2 to 6, or from 2 to 5, or from 2 to 4, or from 2 to 3, or n can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13 or 14. In some embodiments, n can be from 2 to 8, or from 2 to 7, or from 2 to 6, or from 2 to 5, or from 2 to 4, or from 2 to 3. In some embodiments, n can be from 3 to 8, or from 3 to 7, or from 3 to 6, or from 3 to 5, or from 3 to 4. In some embodiments, n can be 3, 4, or 5. In some embodiments, n can be 3 or 5.

[0191] In some embodiments, the ionizable lipid compounds of the present disclosure, without being limited to, can be selected from the group consisting of Compounds 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320a, 320b, 321a, 321b, 322, 323, 324, 325, 326, 328, 329, 330, 331, 332, 333, 334, 335, 336, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 358, 359, 360 and 362, of Table 1, or a pharmaceutically acceptable salt thereof.

[0192] In some embodiments, the ionizable lipid compounds of the present disclosure, without being limited to, can be selected from the group consisting of Compounds 301, 302, 303, 304, 305, 306, 307, 309, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320a, 320b, 321a, 321b, 322, 323, 324, 325, 326, 328, 329, 330, 331, 332, 333, 334, 335, 336, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 358, 359, 360 and 362, of Table 1, or a pharmaceutically acceptable salt thereof.

[0193] In some embodiments, the ionizable lipid compounds of the present disclosure, without being limited to, can be selected from the group consisting of Compounds 301, 302, 303, 304, 305, 306, 307, 309, 311, 312, 313, 314, 315, 316, 318, 319, 320a, 320b, 321a, 321b, 325, 326, 346, 347, 350, 351, 352, 353, 355, 358, 359, 360 and 362, of Table 1, or a pharmaceutically acceptable salt thereof.

[0194] In some embodiments, the ionizable lipid compounds of the present disclosure, without being limited to, can be selected from the group consisting of Compounds 305, 313, 314, 315, 316, 317, 318, 320a, 320b, 321a, 321b, 323, 324, 325, 326, 328, 329, 330, 331, 332, 333, 334, 335, 336, 340, 350, 351 and 360, of Table 1, or a pharmaceutically acceptable salt thereof.

[0195] In some embodiments, the ionizable lipid compounds of the present disclosure, without being limited to, can be selected from the group consisting of Compounds 346, 347, 353, 354, 355 and 359, of Table 1, or a pharmaceutically acceptable salt thereof.

[0196] In some embodiments, the ionizable lipid compounds of the present disclosure, without being limited to, can be selected from the group consisting of Compounds 301, 303, 304, 305, 306, 307, 311, 312, 313, 314, 315, 319, 320a, 320b, 321a, 321b, 325, 326, 346, 347, 350, 351, 352, 353, 355, 358 and 359, of Table 1, or a pharmaceutically acceptable salt thereof.

[0197] In some embodiments, the ionizable lipid compounds of the present disclosure, without being limited to, can be selected from the group consisting of Compounds 305, 306, 307, 313, 314, 315, 319, 320a, 320b, 321a, 321b, 325, 326, 346, 350, 351, 352, 353, 355, 358 and 359, of Table 1, or a pharmaceutically acceptable salt thereof.

[0198] In some embodiments, the ionizable lipid compounds can be in the form of any enantiomer and / or any diastereoisomer thereof, or any mixture thereof.

[0199] Table 1: Non-Limiting Examples of Ionizable Lipid Compounds

[0200] The cis (Z) or trans (E) stereochemistry as drawn in the chemical structures of Table 1was randomly attributed. In other words, each of the C=C bonds in the chemical structures of Table 1 can independently have the cis or trans configuration. In some embodiments, the compounds of Table 1 can be in the form of mixtures of cis or trans geometric isomers.

[0201] In some embodiments, the ionizable lipid compounds of Table 1 can be in the form of any enantiomers, any diastereoisomers, any cis or trans geometric isomers, or any mixtures thereof.

[0202] The term “compound”, as used herein, is meant to embrace all stereoisomers, geometric isomers, tautomers, and isotopes of a depicted or described structure associated with the compound. When referring to compound features or substituents, the terms “optional” or “optionally” refer to a feature or substituent that may or may not occur. For example, “optionally substituted alkyl” encompasses both “alkyl” and “substituted alkyl” as defined below. It will be understood by those skilled in the art, with respect to any group containing one or more substituents, that such groups are not intended to introduce any substitution or substitution patterns that are sterically impractical, synthetically non-feasible and / or inherently unstable.

[0203] The compounds herein described may have asymmetric centers, geometric centers (e.g., double bond), or both. All chiral, diastereomeric, racemic forms and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. Compounds of the present disclosure containing an asymmetrically substituted atom may be isolated in optically active or racemic forms. It is well known in the art how to prepare optically active forms, such as by resolution of racemic forms, by synthesis from optically active starting materials, or through use of chiral auxiliaries. Geometric isomers of olefins, C=N double bonds, or other types of double bonds may be present in the compounds described herein, and all such stable isomers are included in the present disclosure. Specifically, cis and trans geometric isomers of the compounds of the present disclosure may also exist and may be isolated as a mixture of isomers or as separated isomeric forms.

[0204] Compounds described herein also embrace tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Examples of prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and 4H- 1 ,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H- pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

[0205] Compounds described herein also embrace all the isotopes of the atoms occurring in the intermediate or final compounds. “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. Thus, by way of example, each individual hydrogen atom present in formula (200) may be present as a1H,2H (deuterium) or3H (tritium) atom, preferablyJH or2H. Similarly, by way of example, each individual carbon atom present in formula (200) may be present as a12C,13C or14C atom, preferably12C.

[0206] The compounds or structures and salts of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.Neutral lipids

[0207] In some embodiments, the lipid nanoparticle can also include at least one neutral lipid. In some embodiments, the neutral lipids may be phospholipids, or derivatives thereof.

[0208] Examples of phospholipids suitable for use in the present disclosure include, but are not limited to: dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), l,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1 -myristoyl-2-palmitoyl phosphatidylcholine (MPPC),1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), l-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), l,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), l,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphophatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof.

[0209] In some embodiments, the preferred phospholipids are distearoylphosphatidylcholine (DSPC) and dioleoylphosphatidylethanolamine (DOPE).

[0210] In other embodiments, the preferred phospholipids are DSPC, DOPC, DMPC and PE.Helper lipids

[0211] In some embodiments, the lipid nanoparticle can also include at least one helper lipid.

[0212] “Helper lipids” are lipids that enhance transfection, such as transfection of the lipid nanoparticle including the payloads and cargos. The mechanism by which the helper lipid enhances transfection may include enhancing particle stability and / or enhancing membranefusogenicity. Helper lipids include steroids and alkyl resorcinols. Helper lipids suitable for use in the present disclosure include, but are not limited to, cholesterol, 5 -heptadecylresorcinol, cholesterol hemisuccinate, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'- hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof.

[0213] In some embodiments, the preferred helper lipid is cholesterol.Polymer-conjugated lipids

[0214] In some embodiments, the lipid nanoparticle can further include at least one polymer- conjugated lipid.

[0215] In some embodiments, the polymer-conjugated lipid comprises a polymer conjugated to at least one lipid. In some embodiments, the polymer-conjugated lipid can comprise at least one component that reduces aggregation of particles, at least one component that decreases clearing of the lipid nanoparticle from circulation in a subject, at least component that increases the lipid nanoparticle’s ability to traverse mucus layers, at least one component that decreases a subjects immune response to administration of the lipid nanoparticle, at least one component that modifies membrane fluidity of the lipid nanoparticle, at least one component that contributes to the stability of the lipid nanoparticle, or any combination thereof. In some embodiments, the lipid nanoparticle may be essentially devoid of polymer-conjugated lipid. In some embodiments, the lipid nanoparticle may contain no amount of polymer-conjugated lipid.

[0216] In some embodiments, the polymer present in the polymer-conjugated lipid may comprise at least one polyethylene glycol (PEG), at least one polypropylene glycol (PPG), poly(2-oxazoline) (POZ), at least one polyamide (ATTA), at least one cationic polymer, or any combination thereof.

[0217] In some embodiments, the lipid conjugated to the polymer may be selected from, but is not limited to, at least one of the ionizable, neutral, or helper lipids listed previously.

[0218] In some embodiments, the polymer conjugated to at least one lipid is PEG and the polymer-conjugated lipid can be referred to as “PEG-lipid”. In some embodiments, the at least one PEG-lipid may be selected from, but is not limited to at least one of Siglec-IL-PEG-DSPE, R)- 2,3-bis(octadecyloxy)propyl-l-(methoxypoly(ethyleneglycol)2000)propylcarbamate, PEG-S- DSG, PEG-S-DMG, PEG-PE, PEG-PAA, PEG-OH DSPE Cl 8, PEG-DSPE, PEG-DSG, PEG- DPG, PEG-DOMG, PEG-DMPE Na, PEG-DMPE, PEG-DMG2000, PEG-DMG C14, PEG-DMG 2000, PEG-DMG, PEG-DMA, PEG-Ceramide Cl 6, PEG-C-DOMG, PEG-c-DMOG, PEG-c- DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k-DMG, PEG2k-Cll, PEG2000- PE, PEG2000P, PEG2000-DSPE, PEG2000-DOMG, PEG2000-DMG, PEG2000-C-DMA, PEG2000, PEG200, PEG(2k)-DMG, PEG DSPE C18, PEG DMPE C14, PEG DLPE C12, PEG Click DMG C14, PEG Click Cl 2, PEG Click CIO, N(Carbonyl-methoxypolyethylengly col-2000)-l,2-distearoyl-sn-glycero3-phosphoethanolamine, Myrj52, mPEG-PLA, MPEG-DSPE, mPEG3000-DMPE, MPEG-2000-DSPE, MPEG2000-DSPE, mPEG2000-DPPE, mPEG2000- DMPE, mPEG2000-DMG, mDPPE-PEG2000, l,2-distearoyl-sn-glycero-3- phosphoethanolamine-PEG2000, HPEG-2K-LIPD, Folate PEG-DSPE, DSPE-PEGMA 500, DSPE-PEGMA, DSPE-PEG6000, DSPE-PEG5000, DSPE-PEG2K-NAG, DSPE-PEG2k, DSPE- PEG2000maleimide, DSPE-PEG2000, DSPE-PEG, DSG-PEGMA, DSG-PEG5000, DPPE-PEG- 2K, DPPE-PEG, DPPE-mPEG2000, DPPE-mPEG, DPG-PEGMA, DGPE-PEG2000, DMPE- PEGMA, DMPE-PEG2000, DMPE-Peg, DMPE-mPEG2000, DMG-PEGMA, DMG-PEG2000, DMG-PEG, distearoyl-glycerol-polyethyleneglycol, C18PEG750, CI8PEG5000, CI8PEG3000, CI8PEG2000, CI6PEG2000, CI4PEG2000, C18-PEG5000, C18PEG, C16PEG, C16 mPEG (polyethylene glycol) 2000 Ceramide, C14-PEG-DSPE200, C14-PEG2000, C14PEG2000, C14- PEG 2000, C14-PEG, C14PEG, 14:0-PEG2KPE, l,2-distearoyl-sn-glycero-3- phosphoethanolamine-PEG2000, (R)-2,3-bis(octadecyloxy)propyl-l-(methoxypoly(ethyleneglycol)2000)propylcarbamate, (PEG)-C-DOMG, PEG-C-DMA, and DSPE-PEG-X.

[0219] In some embodiments, the preferred polymer-conjugated lipids are polyethylene glycol (PEG), polypropylene glycol (PPG), poly(2-oxazoline) (POZ), polyamide (ATTA), cationic polymer, polysarcosine (Psar), polyglutamic acid (PGA) and l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol (PEG-DMG).

[0220] In some embodiments, the preferred polymer-conjugated lipids are PEG-lipids selected from PEG-DMG or PEG-DSG.

[0221] In some embodiments, the preferred PEG-lipid is PEG2k-DMG.

[0222] In some embodiments, the average molecular weight of the polymer moiety (e.g., PEG) of the polymer-conjugated lipid may be between 500 and 20,000 daltons. In some embodiments, the molecular weight of the polymer may be about 500 to 20,000, 1,000 to 20,000, 1,500 to 20,000, 2,000 to 20,000, 2,500 to 20,000, 3,000 to 20,000, 3,500 to 20,000, 4,000 to 20,000, 4,500 to 20,000, 5,000 to 20,000, 5,500 to 20,000, 6,000 to 20,000, 6,500 to 20,000, 7,000 to 20,000, 7,500 to 20,000, 8,000 to 20,000, 8,500 to 20,000, 9,000 to 20,000, 9,500 to 20,000, 10,000 to 20,000,10.500 to 20,000, 11,000 to 20,000, 11,500 to 20,000, 12,000 to 20,000, 12,500 to 20,000, 13,000 to 20,000, 13,500 to 20,000, 14,000 to 20,000, 14,500 to 20,000, 15,000 to 20,000, 15,500 to 20,000, 16,000 to 20,000, 16,500 to 20,000, 17,000 to 20,000, 17,500 to 20,000, 18,000 to 20,000,18.500 to 20,000, 19,000 to 20,000, 19,500 to 20,000, 500 to 19,500, 1,000 to 19,500, 1,500 to 19,500, 2,000 to 19,500, 2,500 to 19,500, 3,000 to 19,500, 3,500 to 19,500, 4,000 to 19,500, 4,500 to 19,500, 5,000 to 19,500, 5,500 to 19,500, 6,000 to 19,500, 6,500 to 19,500, 7,000 to 19,500,7.500 to 19,500, 8,000 to 19,500, 8,500 to 19,500, 9,000 to 19,500, 9,500 to 19,500, 10,000 to9,500, 10,500 to 19,500, 11,000 to 19,500, 11,500 to 19,500, 12,000 to 19,500, 12,500 to 19,500,3,000 to 19,500, 13,500 to 19,500, 14,000 to 19,500, 14,500 to 19,500, 15,000 to 19,500, 15,500 19,500, 16,000 to 19,500, 16,500 to 19,500, 17,000 to 19,500, 17,500 to 19,500, 18,000 to9.500, 18,500 to 19,500, 19,000 to 19,500, 1,500 to 19,000, 2,000 to 19,000, 2,500 to 19,000,,000 to 19,000, 3,500 to 19,000, 4,000 to 19,000, 4,500 to 19,000, 5,000 to 19,000, 5,500 to9,000, 6,000 to 19,000, 6,500 to 19,000, 7,000 to 19,000, 7,500 to 19,000, 8,000 to 19,000, 8,500 19,000, 9,000 to 19,000, 9,500 to 19,000, 10,000 to 19,000, 10,500 to 19,000, 11,000 to 19,000,1.500 to 19,000, 12,000 to 19,000, 12,500 to 19,000, 13,000 to 19,000, 13,500 to 19,000, 14,000 19,000, 14,500 to 19,000, 15,000 to 19,000, 15,500 to 19,000, 16,000 to 19,000, 16,500 to9,000, 17,000 to 19,000, 17,500 to 19,000, 18,000 to 19,000, 18,500 to 19,000, 1,500 to 18,500,,000 to 18,500, 2,500 to 18,500, 3,000 to 18,500, 3,500 to 18,500, 4,000 to 18,500, 4,500 to8.500, 5,000 to 18,500, 5,500 to 18,500, 6,000 to 18,500, 6,500 to 18,500, 7,000 to 18,500, 7,500 18,500, 8,000 to 18,500, 8,500 to 18,500, 9,000 to 18,500, 9,500 to 18,500, 10,000 to 18,500,0.500 to 18,500, 11,000 to 18,500, 11,500 to 18,500, 12,000 to 18,500, 12,500 to 18,500, 13,000 18,500, 13,500 to 18,500, 14,000 to 18,500, 14,500 to 18,500, 15,000 to 18,500, 15,500 to8.500, 16,000 to 18,500, 16,500 to 18,500, 17,000 to 18,500, 17,500 to 18,500, 18,000 to 18,500,.500 to 18,000, 2,000 to 18,000, 2,500 to 18,000, 3,000 to 18,000, 3,500 to 18,000, 4,000 to 8,000, 4,500 to 18,000, 5,000 to 18,000, 5,500 to 18,000, 6,000 to 18,000, 6,500 to 18,000, 7,000 18,000, 7,500 to 18,000, 8,000 to 18,000, 8,500 to 18,000, 9,000 to 18,000, 9,500 to 18,000,0,000 to 18,000, 10,500 to 18,000, 11,000 to 18,000, 11,500 to 18,000, 12,000 to 18,000, 12,500 18,000, 13,000 to 18,000, 13,500 to 18,000, 14,000 to 18,000, 14,500 to 18,000, 15,000 to8,000, 15,500 to 18,000, 16,000 to 18,000, 16,500 to 18,000, 17,000 to 18,000, 17,500 to 18,000,.500 to 17,500, 2,000 to 17,500, 2,500 to 17,500, 3,000 to 17,500, 3,500 to 17,500, 4,000 to7.500, 4,500 to 17,500, 5,000 to 17,500, 5,500 to 17,500, 6,000 to 17,500, 6,500 to 17,500, 7,000 17,500, 7,500 to 17,500, 8,000 to 17,500, 8,500 to 17,500, 9,000 to 17,500, 9,500 to 17,500,0,000 to 17,500, 10,500 to 17,500, 11,000 to 17,500, 11,500 to 17,500, 12,000 to 17,500, 12,500 17,500, 13,000 to 17,500, 13,500 to 17,500, 14,000 to 17,500, 14,500 to 17,500, 15,000 to7,500, 15,500 to 17,500, 16,000 to 17,500, 16,500 to 17,500, 17,000 to 17,500, 1,500 to 17,000,,000 to 17,000, 2,500 to 17,000, 3,000 to 17,000, 3,500 to 17,000, 4,000 to 17,000, 4,500 to7,000, 5,000 to 17,000, 5,500 to 17,000, 6,000 to 17,000, 6,500 to 17,000, 7,000 to 17,000, 7,500 17,000, 8,000 to 17,000, 8,500 to 17,000, 9,000 to 17,000, 9,500 to 17,000, 10,000 to 17,000,0.500 to 17,000, 11,000 to 17,000, 11,500 to 17,000, 12,000 to 17,000, 12,500 to 17,000, 13,000 17,000, 13,500 to 17,000, 14,000 to 17,000, 14,500 to 17,000, 15,000 to 17,000, 15,500 to7,000, 16,000 to 17,000, 16,500 to 17,000, 1,500 to 16,500, 2,000 to 16,500, 2,500 to 16,500,,000 to 16,500, 3,500 to 16,500, 4,000 to 16,500, 4,500 to 16,500, 5,000 to 16,500, 5,500 to6.500, 6,000 to 16,500, 6,500 to 16,500, 7,000 to 16,500, 7,500 to 16,500, 8,000 to 16,500, 8,500 16,500, 9,000 to 16,500, 9,500 to 16,500, 10,000 to 16,500, 10,500 to 16,500, 11,000 to 16,500,1.500 to 16,500, 12,000 to 16,500, 12,500 to 16,500, 13,000 to 16,500, 13,500 to 16,500, 14,000 16,500, 14,500 to 16,500, 15,000 to 16,500, 15,500 to 16,500, 16,000 to 16,500, 1,500 to 16,000,,000 to 16,000, 2,500 to 16,000, 3,000 to 16,000, 3,500 to 16,000, 4,000 to 16,000, 4,500 to6,000, 5,000 to 16,000, 5,500 to 16,000, 6,000 to 16,000, 6,500 to 16,000, 7,000 to 16,000, 7,500 16,000, 8,000 to 16,000, 8,500 to 16,000, 9,000 to 16,000, 9,500 to 16,000, 10,000 to 16,000,0.500 to 16,000, 11,000 to 16,000, 11,500 to 16,000, 12,000 to 16,000, 12,500 to 16,000, 13,000 16,000, 13,500 to 16,000, 14,000 to 16,000, 14,500 to 16,000, 15,000 to 16,000, 15,500 to6,000, 1,500 to 15,500, 2,000 to 15,500, 2,500 to 15,500, 3,000 to 15,500, 3,500 to 15,500, 4,000 15,500, 4,500 to 15,500, 5,000 to 15,500, 5,500 to 15,500, 6,000 to 15,500, 6,500 to 15,500,,000 to 15,500, 7,500 to 15,500, 8,000 to 15,500, 8,500 to 15,500, 9,000 to 15,500, 9,500 to5.500, 10,000 to 15,500, 10,500 to 15,500, 11,000 to 15,500, 11,500 to 15,500, 12,000 to 15,500,2.500 to 15,500, 13,000 to 15,500, 13,500 to 15,500, 14,000 to 15,500, 14,500 to 15,500, 15,000 15,500, 1,500 to 15,000, 2,000 to 15,000, 2,500 to 15,000, 3,000 to 15,000, 3,500 to 15,000,,000 to 15,000, 4,500 to 15,000, 5,000 to 15,000, 5,500 to 15,000, 6,000 to 15,000, 6,500 to5,000, 7,000 to 15,000, 7,500 to 15,000, 8,000 to 15,000, 8,500 to 15,000, 9,000 to 15,000, 9,500 15,000, 10,000 to 15,000, 10,500 to 15,000, 11,000 to 15,000, 11,500 to 15,000, 12,000 to5,000, 12,500 to 15,000, 13,000 to 15,000, 13,500 to 15,000, 14,000 to 15,000, 14,500 to 15,000,.500 to 14,500, 2,000 to 14,500, 2,500 to 14,500, 3,000 to 14,500, 3,500 to 14,500, 4,000 to4.500, 4,500 to 14,500, 5,000 to 14,500, 5,500 to 14,500, 6,000 to 14,500, 6,500 to 14,500,,000 to 14,500, 7,500 to 14,500, 8,000 to 14,500, 8,500 to 14,500, 9,000 to 14,500, 9,500 to4,500, 10,000 to 14,500, 10,500 to 14,500, 11,000 to 14,500, 11,500 to 14,500, 12,000 to4.500, 12,500 to 14,500, 13,000 to 14,500, 13,500 to 14,500, 14,000 to 14,500, 1,500 to 14,000,,000 to 14,000, 2,500 to 14,000, 3,000 to 14,000, 3,500 to 14,000, 4,000 to 14,000, 4,500 to4,000, 5,000 to 14,000, 5,500 to 14,000, 6,000 to 14,000, 6,500 to 14,000, 7,000 to 14,000,.500 to 14,000, 8,000 to 14,000, 8,500 to 14,000, 9,000 to 14,000, 9,500 to 14,000, 10,000 to4,000, 10,500 to 14,000, 11,000 to 14,000, 11,500 to 14,000, 12,000 to 14,000, 12,500 to4,000, 13,000 to 14,000, 13,500 to 14,000, 1,500 to 13,500, 2,000 to 13,500, 2,500 to 13,500,,000 to 13,500, 3,500 to 13,500, 4,000 to 13,500, 4,500 to 13,500, 5,000 to 13,500, 5,500 to3.500, 6,000 to 13,500, 6,500 to 13,500, 7,000 to 13,500, 7,500 to 13,500, 8,000 to 13,500,.500 to 13,500, 9,000 to 13,500, 9,500 to 13,500, 10,000 to 13,500, 10,500 to 13,500, 11,000 13,500, 11,500 to 13,500, 12,000 to 13,500, 12,500 to 13,500, 13,000 to 13,500, 1,500 to3,000, 2,000 to 13,000, 2,500 to 13,000, 3,000 to 13,000, 3,500 to 13,000, 4,000 to 13,000,.500 to 13,000, 5,000 to 13,000, 5,500 to 13,000, 6,000 to 13,000, 6,500 to 13,000, 7,000 to3,000, 7,500 to 13,000, 8,000 to 13,000, 8,500 to 13,000, 9,000 to 13,000, 9,500 to 13,000,0,000 to 13,000, 10,500 to 13,000, 11,000 to 13,000, 11,500 to 13,000, 12,000 to 13,000, 12,500 13,000, 1,500 to 12,500, 2,000 to 12,500, 2,500 to 12,500, 3,000 to 12,500, 3,500 to 12,500,,000 to 12,500, 4,500 to 12,500, 5,000 to 12,500, 5,500 to 12,500, 6,000 to 12,500, 6,500 to2.500, 7,000 to 12,500, 7,500 to 12,500, 8,000 to 12,500, 8,500 to 12,500, 9,000 to 12,500,,500 to 12,500, 10,000 to 12,500, 10,500 to 12,500, 11,000 to 12,500, 11,500 to 12,500, 12,000 12,500, 1,500 to 12,000, 2,000 to 12,000, 2,500 to 12,000, 3,000 to 12,000, 3,500 to 12,000,,000 to 12,000, 4,500 to 12,000, 5,000 to 12,000, 5,500 to 12,000, 6,000 to 12,000, 6,500 to2,000, 7,000 to 12,000, 7,500 to 12,000, 8,000 to 12,000, 8,500 to 12,000, 9,000 to 12,000,.500 to 12,000, 10,000 to 12,000, 10,500 to 12,000, 11,000 to 12,000, 11,500 to 12,000, 1,500 11,500, 2,000 to 11,500, 2,500 to 11,500, 3,000 to 11,500, 3,500 to 11,500, 4,000 to 11,500,.500 to 11,500, 5,000 to 11,500, 5,500 to 11,500, 6,000 to 11,500, 6,500 to 11,500, 7,000 to1.500, 7,500 to 11,500, 8,000 to 11,500, 8,500 to 11,500, 9,000 to 11,500, 9,500 to 11,500,0,000 to 11,500, 10,500 to 11,500, 11,000 to 11,500, 1,500 to 11,000, 2,000 to 11,000, 2,500 11,000, 3,000 to 11,000, 3,500 to 11,000, 4,000 to 11,000, 4,500 to 11,000, 5,000 to 11,000,.500 to 11,000, 6,000 to 11,000, 6,500 to 11,000, 7,000 to 11,000, 7,500 to 11,000, 8,000 to1,000, 8,500 to 11,000, 9,000 to 11,000, 9,500 to 11,000, 10,000 to 11,000, 10,500 to 11,000,.500 to 10,500, 2,000 to 10,500, 2,500 to 10,500, 3,000 to 10,500, 3,500 to 10,500, 4,000 to0.500, 4,500 to 10,500, 5,000 to 10,500, 5,500 to 10,500, 6,000 to 10,500, 6,500 to 10,500,,000 to 10,500, 7,500 to 10,500, 8,000 to 10,500, 8,500 to 10,500, 9,000 to 10,500, 9,500 to0,500, 10,000 to 10,500, 1,500 to 10,000, 2,000 to 10,000, 2,500 to 10,000, 3,000 to 10,000,.500 to 10,000, 4,000 to 10,000, 4,500 to 10,000, 5,000 to 10,000, 5,500 to 10,000, 6,000 to0,000, 6,500 to 10,000, 7,000 to 10,000, 7,500 to 10,000, 8,000 to 10,000, 8,500 to 10,000,,000 to 10,000, 9,500 to 10,000, 1,500 to 9,500, 2,000 to 9,500, 2,500 to 9,500, 3,000 to 9,500,.500 to 9,500, 4,000 to 9,500, 4,500 to 9,500, 5,000 to 9,500, 5,500 to 9,500, 6,000 to 9,500,.500 to 9,500, 7,000 to 9,500, 7,500 to 9,500, 8,000 to 9,500, 8,500 to 9,500, 9,000 to 9,500,.500 to 9,000, 2,000 to 9,000, 2,500 to 9,000, 3,000 to 9,000, 3,500 to 9,000, 4,000 to 9,000,.500 to 9,000, 5,000 to 9,000, 5,500 to 9,000, 6,000 to 9,000, 6,500 to 9,000, 7,000 to 9,000,.500 to 9,000, 8,000 to 9,000, 8,500 to 9,000, 1,500 to 8,500, 2,000 to 8,500, 2,500 to 8,500,,000 to 8,500, 3,500 to 8,500, 4,000 to 8,500, 4,500 to 8,500, 5,000 to 8,500, 5,500 to 8,500,,000 to 8,500, 6,500 to 8,500, 7,000 to 8,500, 7,500 to 8,500, 8,000 to 8,500, 1,500 to 8,000,,000 to 8,000, 2,500 to 8,000, 3,000 to 8,000, 3,500 to 8,000, 4,000 to 8,000, 4,500 to 8,000,,000 to 8,000, 5,500 to 8,000, 6,000 to 8,000, 6,500 to 8,000, 7,000 to 8,000, 7,500 to 8,000,.500 to 7,500, 2,000 to 7,500, 2,500 to 7,500, 3,000 to 7,500, 3,500 to 7,500, 4,000 to 7,500,.500 to 7,500, 5,000 to 7,500, 5,500 to 7,500, 6,000 to 7,500, 6,500 to 7,500, 7,000 to 7,500,1.500 to 7,000, 2,000 to 7,000, 2,500 to 7,000, 3,000 to 7,000, 3,500 to 7,000, 4,000 to 7,000,4.500 to 7,000, 5,000 to 7,000, 5,500 to 7,000, 6,000 to 7,000, 6,500 to 7,000, 1,500 to 6,500,2,000 to 6,500, 2,500 to 6,500, 3,000 to 6,500, 3,500 to 6,500, 4,000 to 6,500, 4,500 to 6,500,5,000 to 6,500, 5,500 to 6,500, 6,000 to 6,500, 1,500 to 6,000, 2,000 to 6,000, 2,500 to 6,000,3,000 to 6,000, 3,500 to 6,000, 4,000 to 6,000, 4,500 to 6,000, 5,000 to 6,000, 5,500 to 6,000,1.500 to 5,500, 2,000 to 5,500, 2,500 to 5,500, 3,000 to 5,500, 3,500 to 5,500, 4,000 to 5,500,4.500 to 5,500, 5,000 to 5,500, 1,500 to 5,000, 2,000 to 5,000, 2,500 to 5,000, 3,000 to 5,000,3.500 to 5,000, 4,000 to 5,000, 4,500 to 5,000, 1,500 to 4,500, 2,000 to 4,500, 2,500 to 4,500,3,000 to 4,500, 3,500 to 4,500, 4,000 to 4,500, 1,500 to 4,000, 2,000 to 4,000, 2,500 to 4,000,3,000 to 4,000, 3,500 to 4,000, 1,500 to 3,500, 2,000 to 3,500, 2,500 to 3,500, 3,000 to 3,500,1.500 to 3,000, 2,000 to 3,000, 2,500 to 3,000, 1,500 to 2,500, 2,000 to 2,500, and 1,500 to 2,000 daltons.Hydrophobic components

[0223] In some embodiments, the lipid nanoparticles can also include another type of lipids, referred to as “hydrophobic components” in the present disclosure. The hydrophobic component is defined as a component, which, during the lipid nanoparticle formation, is incorporated in the lipid bilayer due to its hydrophobic nature. In some embodiments the hydrophobic component may be selected from the group consisting of cardiolipin, squalene, vitamin A and derivatives thereof, P-carotene, withaferin A and a-tocopherol. In some embodiments the hydrophobic component may be selected from the group consisting of cardiolipin, squalene, vitamin A, retinol, P-carotene, withaferin A and a-tocopherol.III. Lipid nanoparticlesGeneralities

[0224] In general, lipid nanoparticles can be characterized as small solid or semi-solid particles possessing an exterior lipid layer with a hydrophilic exterior surface that is exposed to the nonlipid nanoparticle environment, an interior space which may aqueous (vesicle like) or non-aqueous (micelle like), and at least one hydrophobic inter-membrane space. Lipid nanoparticle membranes may be lamellar or non-lamellar and may be comprised of 1, 2, 3, 4, 5 or more layers. In some embodiments, lipid nanoparticles may comprise a cargo or a payload into their interior space, into the inter membrane space, onto their exterior surface, or any combination thereof.

[0225] The present disclosure also provides lipid nanoparticle comprising a cargo or payload. As used herein, the term “cargo” or “payload” can refer to one or more molecules or structures encompassed in a lipid nanoparticle for delivery to or into a cell or tissue. Non-limiting examplesof cargo can include a nucleic acid, a polypeptide, peptide, protein, a liposome, a label, a tag, a small chemical molecule, a large biological molecule, and any combinations or fragments thereof. In the originator constructs and benchmark constructs, the region of the construct which comprises or encodes the cargo or payload is referred to as the “cargo region” or the “payload region”.

[0226] In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid of the present disclosure discussed in Section II, such as the ionizable lipid of Formulas (I), (II), (12), (13), (11-1), (11-2), (11-3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (Il-5c), (Il-5d), (Il-6a), (Il -6b) or (Il-6c), or more particularly, the compounds in Table 1. Not willing to be bound to any theory, the diester bonds of the lipids provide biodegradability, such ester bonds are stable at physiological pH, but can be enzymatically hydrolyzed within tissues and cells. The length of the R3, R.4, RS and R6groups in Formulas (I), (II), (12), (13), (11-1), (11-2), (11-3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (Il-5c), (Il-5d), (Il -6a), (Il-6b) or (Il-6c), can also be adjusted to reach the desired zeta potential, particle size or membrane rigidity.

[0227] In some embodiments, the lipid nanoparticle may comprise any lipid described in the disclosure. In some embodiments, the lipid may be any cationic lipid described in the disclosure. In some embodiments, the lipid nanoparticle may comprise neutral lipids. In some embodiments, the neutral lipid may be a phospholipid, or a derivative thereof. In some embodiments, the lipid may be any phospholipid described in the disclosure. In some embodiments, the lipid may be any cholesterol derivative described in the disclosure. In some embodiments, a polymer (e.g., PEG) may be conjugated to at least one lipid. In some embodiments, a PEG-lipid may be used in the lipid nanoparticle and can be any PEG-lipid conjugate described in the disclosure.

[0228] In some embodiments, the lipid nanoparticles can be characterized by their shape. In some embodiments, the lipid nanoparticles can comprise lipid nanoparticles which are substantially spherical. In some embodiments, the lipid nanoparticles can comprise lipid nanoparticles which can be substantially rod-shaped (i.e., cylindrical). In some embodiments, the lipid nanoparticles can comprise lipid nanoparticles which can be substantially disk shaped. In some embodiments, the lipid nanoparticles can comprise a mixture of lipid nanoparticles of such different shapes.

[0229] In some embodiments, the term “nanoparticle” as used herein refers to any particle ranging in size from 10-1000 nm. The nanoparticle may be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255,260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350,355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445,450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540,545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635,640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730,735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825,830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920,925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 nm.

[0230] In some embodiments, a population of lipid nanoparticles, such as those resulting from the same formulation, may be characterized by measuring the uniformity of size, shape, or mass of the particles in the population, uniformity may be expressed in some embodiments as the poly dispersity index (PI) of the population. In some embodiments uniformity may be expressed in some embodiments as the disparity (D) of the population. The terms “poly dispersity index” and “disparity” are understood herein to be equivalent and may be used interchangeably. In some embodiments, a population of lipid nanoparticles resulting from a given formulation can have a PI of between about 0.1 and 1. In some embodiments, a population of lipid nanoparticles resulting from a giving formulation can have a PI of less than about 1, less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0. 1. In some embodiments, a population of lipid nanoparticles resulting from a given formulation can have a PI of between about 0.1 to 1, 0.1 to 0.8, 0.1 to 0.6, 0.1 to 0.4, 0.1 to 0.2, 0.2 to 1, 0.2 to 0.8, 0.2 to 0.6, 0.2 to 0.4, 0.4 to 1, 0.4 to 0.8, 0.4 to 0.6, 0.6 to 1, 0.6 to 0.8, and 0.8 to 1. In some embodiments, the lipid nanoparticle may have PI ranging between about 0.01 to 0.3, 0.02-0.3, 0.03-0.3, 0.04-0.3, 0.05-0.3, 0.06-0.3, 0.07- 0.3, 0.08-0.3, 0.09-0.3, 0.1-0.3, 0.11-0.3, 0.12-0.3, 0.13-0.3, 0.14-0.3, 0.15-0.3, 0.16-0.3, 0.17-0.3, 0.18-0.3, 0.19-0.3, 0.2-0.3, 0.21-0.3, 0.22-0.3, 0.23-0.3, 0.24-0.3, 0.25-0.3, 0.26-0.3, 0.27-0.3, 0.28-0.3, 0.29-0.3, 0.01 to 0.25, 0.02-0.25, 0.03-0.25, 0.04-0.25, 0.05-0.25, 0.06-0.25, 0.07-0.25, 0.08-0.25, 0.09-0.25, 0.1-0.25, 0.11-0.25, 0.12-0.25, 0.13-0.25, 0.14-0.25, 0.15-0.25, 0.16-0.25, 0.17-0.25, 0.18-0.25, 0.19-0.25, 0.2-0.25, 0.21-0.25, 0.22-0.25, 0.23-0.25, 0.24-0.25, 0.01 to 0.2, 0.02-0.2, 0.03-0.2, 0.04-0.2, 0.05-0.2, 0.06-0.2, 0.07-0.2, 0.08-0.2, 0.09-0.2, 0. 1-0.2, 0.11-0.2, 0.12-0.2, 0.13-0.2, 0.14-0.2, 0.15-0.2, 0.16-0.2, 0.17-0.2, 0.18-0.2, 0.19-0.2, 0.01 to 0.15, 0.02- 0.15, 0.03-0.15, 0.04-0.15, 0.05-0.15, 0.06-0.15, 0.07-0.15, 0.08-0.15, 0.09-0.15, 0.1-0.15, 0.11- 0.15, 0.12-0.15, 0.13-0.15, 0.14-0.15, 0.01 to 0.1, 0.02-0.1, 0.03-0.1, 0.04-0.1, 0.05-0.1, 0.06-0.1, 0.07-0.1, 0.08-0.1, 0.09-0.1, 0.01 to 0.05, 0.02-0.0.5, 0.03-0.0.5, or 0.04-0.05.

[0231] In some embodiments, the total mole percentage of the lipid(s) in the LNP is between about 10% to about 95%, such as between about 10% to about 20%, between about 21% to about 30%, between about 31% to about 40%, between about 41% to about 50%, between about 51% to about 60%, between about 61% to about 70%, between about 71% to about 80%, between about 81% to about 90%, or between about 91% to about 95%.Formulations

[0232] In some embodiments, the lipids of the present disclosure, including at least the ionizable lipids disclosed herein, may be incorporated into lipid nanoparticles (LNPs). In some embodiments a lipid nanoparticle may be comprised of at least one ionizable lipid, at least one neutral lipid, at least one helper lipid, at least one polymer-conjugated lipid, or any combination thereof, wherein the neutral lipid, helper lipid, polymer-conjugated lipid are as defined herein. In some embodiments, the LNP may be comprised of at least one ionizable lipid, at least one neutral lipid, and at least one helper lipid. In some embodiments, the LNP may be comprised of at least one ionizable lipid, at least one neutral lipid, and at least one polymer-conjugated lipid. In some embodiments, the LNP may be comprised of at least one neutral lipid, at least one helper lipid, and at least one polymer-conjugated lipid. In some embodiments, the LNP may be comprised of at least one ionizable lipid and at least one neutral lipid. In some embodiments, the LNP may be comprised of at least one ionizable lipid and at least one helper lipid. In some embodiments, the LNP may be comprised of at least one ionizable lipid and at least one polymer-conjugated lipid. In some embodiments, the LNP may be comprised of at least one neutral lipid and at least one helper lipid. In some embodiments, the LNP may be comprised of at least one neutral lipid and at least one polymer-conjugated lipid. In some embodiments, the LNP may be comprised of at least one helper lipid and at least one polymer-conjugated lipid. In some embodiments, the LNP may be comprised of at least one ionizable lipid. In some embodiments, the LNP may be comprised of at least one neutral lipid. In some embodiments, a LNP may be comprised of a helper lipid. In some embodiments, the LNP may be comprised of a polymer-conjugated lipid. In some embodiments a lipid nanoparticle may be comprised of at least one ionizable lipid, at least one neutral lipid, at least one helper lipid, at least one polymer-conjugated lipid; and at least one hydrophobic component, wherein the ionizable lipid, neutral lipid, helper lipid, polymer- conjugated lipid and hydrophobic component are as defined herein.

[0233] In some embodiments, the lipid nanoparticle can further comprise an adjuvant, a cell targeting component, a fat-soluble vitamin, an immunomodulating substance or a component that promotes absorption of drugs.

[0234] In some embodiments, the adjuvant can be squalene, the cell targeting component can be cardiolipin, the fat-soluble vitamin can be vitamin A or E, the immunomodulating substance can be withaferin and the component that promote absorption of drugs can be caffeine.

[0235] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13) or (14) or the lipid(s) having a structure of Formulas (Ila), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d) or (I4e), or the lipid(s) having a structure of Formulas (I), (II), (12), (13), (11-1), (11-2), (II-3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (Il-5c), (Il-5d), (Il-6a), (Il-6b) or (II- 6c), or the compounds in Table 1, in the lipid nanoparticle, can be from 0.1 to 100 mol%. In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13) or (14), or the lipid(s) having a structure of Formulas (Ila), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d) or (I4e), or the lipid(s) having a structure of Formulas (I), (II), (12), (13), (11-1), (11-2), (11-3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (Il-5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), or the compounds in Table 1, in the lipid nanoparticle, is between 10%-95%, such as between about 10% to about 20%, between about 21% to about 30%, between about 31% to about 40%, between about 41% to about 50%, between about 51% to about 60%, between about 61% to about 70%, between about 71% to about 80%, between about 81% to about 90%, or between about 91% to about 95%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of about 40 to 100 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of about 20 to 60 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of about 50 to 85 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of less than about 20 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of more than about 50 mol% or about 70 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of about 95 mol% or less. In some embodiments, the lipid nanoparticle comprises an ionizable lipid in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of more than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%.

[0236] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13) or (14), or the lipid(s) having a structure of Formulas (Ila), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d) or (I4e), or the lipid(s) having a structure of Formulas (I), (II), (12), (13), (11-1), (11-2), (II- 3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (Il-5c), (Il-5d), (Il-6a), (Il-6b) or (II- 6c), or the compounds in Table 1, in the lipid nanoparticle, is from about 30% to 100%, from about 40% to 100%, from about 30% to about 90%, from about 30% to about 85%, from about 30% to about 80%, from about 30% to about 75%, from about 30% to about 70%, from about 30% to about 65%, or from about 30% to about 60%.

[0237] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13) or (14),or the lipid(s) having a structure of Formulas (Ila), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d) or (I4e), or the lipid(s) having a structure of Formulas (I), (II), (12), (13), (11 - 1), (11-2), (11-3), (II- 4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (Il-5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), or the compounds in Table 1, in the lipid nanoparticle, is about 40% to about 90%, from about 40% to about 85%, from about 40% to about 80%, from about 40% to about 75%, from about 40% to about 70%, from about 40% to about 65%, or from about 40% to about 60%.

[0238] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13) or (14), or the lipid(s) having a structure of Formulas (Ila), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d) or (I4e), or the lipid(s) having a structure of Formulas (I), (II), (12), (13), (11 - 1), (11-2), (11-3), (II- 4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (Il-5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), or the compounds in Table 1, in the lipid nanoparticle, is about 50% to about 90%, from about 50% to about 85%, from about 50% to about 80%, from about 50% to about 75%, from about 50% to about 70%, from about 50% to about 65%, or from about 50% to about 60%.

[0239] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13) or (14), or the lipid(s) having a structure of Formulas (Ila), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d) or (I4e), or the lipid(s) having a structure of Formulas (I), (II), (12), (13), (11 - 1), (11-2), (11-3), (II- 4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (Il-5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), or the compounds in Table 1, in the lipid nanoparticle, is about 55% to about 90%, from about 55% to about 85%, from about 55% to about 80%, from about 55% to about 75%, from about 55% to about 70%, from about 55% to about 65%, or from about 55% to about 60%.

[0240] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13) or (14), or the lipid(s) having a structure of Formulas (Ila), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d) or (I4e), or the lipid(s) having a structure of Formulas (I), (II), (12), (13), (11-1), (11-2), (II- 3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (Il-5c), (Il-5d), (Il-6a), (Il-6b) or (II- 6c), or the compounds in Table 1, in the lipid nanoparticle, is about 60% to about 90%, from about 60% to about 85%, from about 60% to about 80%, from about 60% to about 75%, from about 60% to about 70%, or from about 60% to about 65%.

[0241] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13) or (14), or the lipid(s) having a structure of Formulas (Ila), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d) or (I4e), or the lipid(s) having a structure of Formulas (I), (II), (12), (13), (11-1), (11-2), (II- 3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (Il-5c), (Il-5d), (Il-6a), (Il-6b) or (II-6c), or the compounds in Table 1, in the lipid nanoparticle, is about 65% to about 90%, from about 65% to about 85%, from about 65% to about 80%, from about 65% to about 75%, or from about 65% to about 70%.

[0242] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13) or (14), or the lipid(s) having a structure of Formulas (Ila), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d) or (I4e), or the lipid(s) having a structure of Formulas (I), (II), (12), (13), (11 - 1), (11-2), (11-3), (II- 4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (Il-5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), or the compounds in Table 1, in the lipid nanoparticle, is about 70% to about 90%, from about 70% to about 85%, from about 70% to about 80%, or from about 70% to about 75%.

[0243] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13) or (14), or the lipid(s) having a structure of Formulas (Ila), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d) or (I4e), or the lipid(s) having a structure of Formulas (I), (II), (12), (13), (11-1), (11-2), (II- 3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (Il-5c), (Il-5d), (Il-6a), (Il-6b) or (II- 6c), or the compounds in Table 1, in the lipid nanoparticle, is about 75% to about 90%, from about 75% to about 85%, or from about 75% to about 80%.

[0244] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13) or (14), or the lipid(s) having a structure of Formulas (Ila), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d) or (I4e), or the lipid(s) having a structure of Formulas (I), (II), (12), (13), (11-1), (11-2), (II-3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (Il-5c), (Il-5d), (Il-6a), (Il-6b) or (II- 6c), or the compounds in Table 1, in the lipid nanoparticle, is about 80% to about 90%, or from about 80% to about 85%.

[0245] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13) or (14), or the lipid(s) having a structure of Formulas (Ila), (I2a), (I2b), (I3a), (I3b), (I4a), (I4b), (I4c), (I4d) or (I4e), or the lipid(s) having a structure of Formulas (I), (II), (12), (13), (11 - 1), (11-2), (11-3), (II-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (Il-5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), or the compounds in Table 1, in the lipid nanoparticle, is about 85% to about 90%.

[0246] In some embodiments, the ionizable lipid mol % of the lipid nanoparticle can be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol %.

[0247] In some embodiments, transfer vehicle variability between lots can be less than 15%, less than 10% or less than 5%.

[0248] In some embodiments, when the lipid nanoparticle comprises at least one neutral lipid,the neutral lipid can be present in the lipid nanoparticle in an amount of about 0. 1 to 100 mol%. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of at most 35 mol%, or at most 30 mol%, or at most 25 mol%, or at most 20 mol%, or from about 0. 1 to about 35 mol %, or from about 5 to about 35 mol%, of from about 0.1 to about 20 mol %. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of about 5 to 25 mol%. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of less than about 5 mol%. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of more than about 25 mol% or about 35 mol%. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of about 95 mol% or less. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiment, the lipid nanoparticle comprises at least one neutral lipid in an amount of at most about 10 mol%.

[0249] In some embodiments, the total mole percentage of the neutral lipid in the lipid nanoparticle is about 5% to about 20%, from about 5% to about 18%, from about 5% to about 16%, from about 5% to about 14%, from about 5% to about 12%, from about 5% to about 10%, or from about 5% to about 8%.

[0250] In some embodiments, the total mole percentage of the neutral lipid in the lipid nanoparticle is about 7% to about 20%, from about 7% to about 18%, from about 7% to about 16%, from about 7% to about 14%, from about 7% to about 12%, from about 7% to about 10%, or from about 7% to about 8%.

[0251] In some embodiments, the total mole percentage of the neutral lipid in the lipid nanoparticle is about 9% to about 20%, from about 9% to about 18%, from about 9% to about 16%, from about 9% to about 14%, from about 9% to about 12%, or from about 9% to about 10%.

[0252] In some embodiments, the total mole percentage of the neutral lipid in the lipid nanoparticle is about 11% to about 20%, from about 11% to about 18%, from about 11% to about 16%, from about 11% to about 14%, or from about 11% to about 12%.

[0253] In some embodiments, the total mole percentage of the neutral lipid in the lipid nanoparticle is about 13% to about 20%, from about 13% to about 18%, from about 13% to about 16%, or from about 13% to about 14%.

[0254] In some embodiments, the total mole percentage of the neutral lipid in the lipid nanoparticle is about 15% to about 20%, or from about 17% to about 18%.

[0255] In some embodiments, the neutral lipid mol % of the lipid nanoparticle can be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol %.

[0256] In some embodiments, when the lipid nanoparticle comprises at least one helper lipid, thehelper lipid can be present in the lipid nanoparticle in an amount of about 0.1 to 100 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of at most about 50 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of about 20 to 45 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of about 25 to 55 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of less than about 20 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of more than about 45 mol% or about 55 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of about 95 mol% or less. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5 and 1 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of more than or equal to about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%.

[0257] In some embodiments, the helper lipid mol % of the lipid nanoparticle can be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol %.

[0258] In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 1% to about 50%, from about 1% to about 45%, from about 1% to about 40%, from about 1% to about 35%, from about 1% to about 30%, from about 1% to about 25%, from about 1% to about 20%, from about 1% to about 15%, from about 1% to about 10%, or from about 1% to about 5%.

[0259] In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 5% to about 50%, from about 5% to about 45%, from about 5% to about 40%, from about 5% to about 35%, from about 5% to about 30%, from about 5% to about 25%, from about 5% to about 20%, from about 5% to about 15%, or from about 5% to about 10%.

[0260] In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 10% to about 50%, from about 10% to about 45%, from about 10% to about 40%, from about 10% to about 35%, from about 10% to about 30%, from about 10% to about 25%, from about 10% to about 20%, or from about 10% to about 15%.

[0261] In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 15% to about 50%, from about 15% to about 45%, from about 15% to about 40%, from about 15% to about 35%, from about 15% to about 30%, from about 15% to about 25%, or from about 15% to about 20%.

[0262] In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 20% to about 50%, from about 20% to about 45%, from about 20% to about 40%, from about 20% to about 35%, from about 20% to about 30%, or from about 20% to about 25%.

[0263] In some embodiments, the total mole percentage of the helper lipid in the lipidnanoparticle is about 25% to about 50%, from about 25% to about 45%, from about 25% to about 40%, from about 25% to about 35%, or from about 25% to about 30%.

[0264] In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 30% to about 50%, from about 30% to about 45%, from about 30% to about 40%, or from about 30% to about 35%.

[0265] In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 35% to about 50%, from about 35% to about 45%, or from about 35% to about 40%. In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 40% to about 50%, or from about 40% to about 45%. In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 45% to about 50%.

[0266] In some embodiments, when the lipid nanoparticle comprises at least one polymer- conjugated lipid, the polymer-conjugated lipid can be present in the lipid nanoparticle in an amount of about 0.1 to 100 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of about 95 mol% or less. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of at most 15 mol%, or from about 0.5 to 15 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of about 15 to 40 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of less than about 0.1 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of at most about 5 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of more than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%.

[0267] In some embodiments, the polymer-conjugated lipid mol % of the lipid nanoparticle can be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol %.

[0268] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 0.1% to about 10%, from about 0.1% to about 9%, from about 0.1% to about 8%, from about 0.1% to about 7%, from about 0.1% to about 6%, from about 0.1% to about 5%, or from about 0.1% to about 4%, from about 0.1% to about 3%, from about 0.1% to about 2%, or from about 0. 1% to about 1%.

[0269] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 0.5% to about 10%, from about 0.5% to about 9%, from about 0.5% to about 8%, from about 0.5% to about 7%, from about 0.5% to about 6%, from about 0.5% to about 5%, or from about 0.5% to about 4%, from about 0.5% to about 3%, from about 0.5% to about2%, or from about 0.5% to about 1%.

[0270] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 1% to about 10%, from about 1% to about 9%, from about 1% to about 8%, from about 1% to about 7%, from about 1% to about 6%, from about 1% to about 5%, from about 1% to about 4%, from about 1% to about 3%, or from about 1% to about 2%.

[0271] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 2% to about 10%, from about 2% to about 9%, from about 2% to about 8%, from about 2% to about 7%, from about 2% to about 6%, from about 2% to about 5%, from about 2% to about 4%, or from about 2% to about 3%.

[0272] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 3% to about 10%, from about 3% to about 9%, from about 3% to about 8%, from about 3% to about 7%, from about 3% to about 6%, from about 3% to about 5%, or from about 3% to about 4%.

[0273] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 4% to about 10%, from about 4% to about 9%, from about 4% to about 8%, from about 4% to about 7%, from about 4% to about 6%, or from about 4% to about 5%.

[0274] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 5% to about 10%, from about 5% to about 9%, from about 5% to about 8%, from about 5% to about 7%, or from about 5% to about 6%.

[0275] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 6% to about 10%, from about 6% to about 9%, from about 6% to about 8%, or from about 6% to about 7%. In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 7% to about 10%, from about 7% to about 9%, or from about 7% to about 8%. In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 8% to about 10%, or from about 8% to about 9%. In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 8% to about 10%, or from about 8% to about 9%. In some embodiments, the total mole percentage of the PEG-lipid in the lipid nanoparticle is about 9% to about 10%.

[0276] In some embodiments, the lipid nanoparticle can comprise a hydrophobic component. In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle at most about 20%, or at most about 15%, or at most about 10%, or from about 1% to about 20%, from about 1% to about 15%, from about 1% to about 10%, or from about 1% to about 5%. In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 3% to about 20%, from about 3% to about 15%, from about 3% to about 10%, or from about 3% to about 5%. In some embodiments, the total mole percentage of thehydrophobic component in the lipid nanoparticle is at most about 5 %.

[0277] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 5% to about 20%, from about 5% to about 15%, or from about 5% to about 10%.

[0278] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 7% to about 20%, from about 7% to about 15%, or from about 7% to about 10%.

[0279] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 9% to about 20%, from about 9% to about 15%, or from about 9% to about 10%.

[0280] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 11% to about 20%, or from about 11% to about 15%.

[0281] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 13% to about 20%, or from about 13% to about 15%.

[0282] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about or from about 15% to about 20%.

[0283] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about or from about 17% to about 20%.

[0284] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about or from about 19% to about 20%.

[0285] In some embodiments, the lipid nanoparticle is comprised of about 30-85 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 1- 50 mol% of at least one helper lipid (e.g., cholesterol), and about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0286] In some embodiments, the at least one ionizable lipid is at least one compound having a structure of Formula (I), (II), (Ila), (12), (I2a), (I2b), (13), (I3a), (I3b), (14), (I4a), (I4b), (I4c), (I4d) or (I4e), or a structure of Formula (I), (II), (12), (13), (11-1), (11-2), (11-3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (Il-5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof.

[0287] In some embodiments, the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 30- 40 mol% of at least one helper lipid (e.g., cholesterol), and about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0288] In some embodiments, the lipid nanoparticle is comprised of about 35-45 mol% of at least one ionizable lipid, about 25-35 mol% of at least one neutral lipid (e.g., a phospholipid), about 20-30 mol% of at least one helper lipid (e.g., cholesterol), and about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0289] In some embodiments, the lipid nanoparticle is comprised of about 45-65 mol% of at least one ionizable lipid, about 5-10 mol% of at least one neutral lipid (e.g., a phospholipid), about 25- 40 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0290] In some embodiments, the lipid nanoparticle is comprised of about 40-60 mol% of at least one ionizable lipid, about 5-15 mol% of at least one neutral lipid (e.g., a phospholipid), about 35- 45 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-3 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0291] In some embodiments, the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 15- 50 mol% of at least one helper lipid (e.g., cholesterol), and about 0.01-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0292] In some embodiments, the lipid nanoparticle is comprised of about 10-75 mol% of at least one ionizable lipid, about 0.5-50 mol% of at least one neutral lipid (e.g., a phospholipid), about 5- 60 mol% of at least one helper lipid (e.g., cholesterol), and about 0.1-20 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0293] In some embodiments, the lipid nanoparticle is comprised of about 50-65 mol% of at least one ionizable lipid, about 3-15 mol% of at least one neutral lipid (e.g., a phospholipid), about 30- 40 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-2 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0294] In some embodiments, the lipid nanoparticle is comprised of about 50-85 mol% of at least one ionizable lipid, about 3-15 mol% of at least one neutral lipid (e.g., a phospholipid), about 1- 40 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-2 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0295] In some embodiments, the lipid nanoparticle is comprised of about 25-75 mol% of at least one ionizable lipid, about 0.1-15 mol% of at least one neutral lipid (e.g., a phospholipid), about 5- 50 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-20 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0296] In some embodiments, the lipid nanoparticle is comprised of about 50-65 mol% of at least one ionizable lipid, about 5-10 mol% of at least one neutral lipid (e.g., a phospholipid), about 25- 35 mol% of at least one helper lipid (e.g., cholesterol), and about 5-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0297] In some embodiments, the lipid nanoparticle is comprised of about 20-60 mol% of at leastone ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 25- 55 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-15 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0298] In some embodiments, the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 18.5-48.5 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer- conjugated lipid (e.g., a PEG-lipid) and about 0-20 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, -carotene, withaferin A and / or a-tocopherol).

[0299] In some embodiments, the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 18.5-48.5 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer- conjugated lipid (e.g., a PEG-lipid) and about 0-15 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, -carotene, withaferin A and / or a-tocopherol).

[0300] In some embodiments, the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 18.5-48.5 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer- conjugated lipid (e.g., a PEG-lipid) and about 0-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, -carotene, withaferin A and / or a-tocopherol).

[0301] In some embodiments, the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 18.5-48.5 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer- conjugated lipid (e.g., a PEG-lipid) and about 4-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, -carotene, withaferin A and / or a-tocopherol).

[0302] In some embodiments, the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 30- 45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer- conjugated lipid (e.g., a PEG-lipid) and about 0-20 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, -carotene, withaferin A and / or a-tocopherol).

[0303] In some embodiments, the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 30- 45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer- conjugated lipid (e.g., a PEG-lipid) and about 0-15 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, -carotene, withaferin A and / or a-tocopherol).

[0304] In some embodiments, the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer- conjugated lipid (e.g., a PEG-lipid) and about 0-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, -carotene, withaferin A and / or a-tocopherol).

[0305] In some embodiments, the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about SO- 45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer- conjugated (e.g., a PEG-lipid) and about 4-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, -carotene, withaferin A and / or a-tocopherol).

[0306] In some embodiments, the lipid nanoparticle is comprised of about 50-85 mol% of at least one ionizable lipid, about 5-20 mol% of at least one neutral lipid (e.g., a phospholipid), about 1- 40 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer- conjugated (e.g., a PEG-lipid) and about 0-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, -carotene, withaferin A and / or a-tocopherol).

[0307] In some embodiments, the lipid nanoparticle is comprised of about 50-85 mol% of at least one ionizable lipid, about 5-20 mol% of at least one neutral lipid (e.g., a phospholipid), about 1- 40 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer- conjugated (e.g., a PEG-lipid) and about 0.1-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, -carotene, withaferin A and / or a-tocopherol).

[0308] In some embodiments, the lipid nanoparticle is comprised of about 50-85 mol% of at least one ionizable lipid, about 5-20 mol% of at least one neutral lipid (e.g., a phospholipid), about 1- 40 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer- conjugated (e.g., a PEG-lipid) and about 0.1-5 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, -carotene, withaferin A and / or a-tocopherol).

[0309] In some embodiments, the lipid nanoparticle is comprised of about 50-85 mol% of at least one ionizable lipid which is at least one compound having a structure of Formula (I), (II), (Ila), (12), (I2a), (I2b), (13), (I3a), (I3b), (14), (I4a), (I4b), (I4c), (Eld) or (I4e), or a structure of Formula (I), (II), (12), (13), (11-1), (11-2), (11-3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (II- 5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof, about 5-20 mol% of at least one neutral (e.g., a phospholipid), about 1-40 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 0-15 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, -carotene, withaferin A and / or a-tocopherol).

[0310] In some embodiments, the lipid nanoparticle is comprised of about 50-85 mol% of at least one ionizable lipid which is at least one compound having a structure of Formula (I), (II), (Ila),(12), (I2a), (I2b), (13), (I3a), (I3b), (14), (I4a), (I4b), (I4c), (I4d) or (I4e), or a structure of Formula (I), (II), (12), (13), (11-1), (11-2), (11-3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (II- 5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof, about 5-20 mol% of at least one neutral (e.g., a phospholipid), about 1-40 mol% of at least one helper lipid (e.g., cholesterol), about 1-5 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 1-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, -carotene, withaferin A and / or a-tocopherol).

[0311] In some embodiments, the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid which is at least one compound having a structure of Formula (I), (II), (Ila), (12), (I2a), (I2b), (13), (I3a), (I3b), (14), (I4a), (I4b), (I4c), (I4d) or (I4e), or a structure of Formula (I), (II), (12), (13), (11-1), (11-2), (11-3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (II- 5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof, about 5-25 mol% of at least one neutral (e.g., a phospholipid), about 30-45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 4-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, -carotene, withaferin A and / or a-tocopherol).

[0312] In some embodiments, the lipid nanoparticle is comprised of about 40-60 mol% of at least one ionizable lipid which is at least one compound having a structure of Formula (I), (II), (Ila), (12), (I2a), (I2b), (13), (I3a), (I3b), (14), (I4a), (I4b), (I4c), (I4d) or (I4e), or a structure of Formula (I), (II), (12), (13), (11-1), (11-2), (11-3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (II- 5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof, about 5-10 mol% of at least one phospholipid or phospholipid derivative described herein, about 30-45 mol% of at least one cholesterol or cholesterol derivative described herein, about 1-4 mol% of at least one PEG-lipid described herein and about 0-5 mol% of squalene, cardiolipin, withaferin A, vitamin A, retinol, -carotene, and / or a-tocopherol.

[0313] In some embodiments, the lipid nanoparticle is comprised of about 40-60 mol% of at least one ionizable lipid which is at least one compound having a structure of Formula (I), (II), (Ila), (12), (I2a), (I2b), (13), (I3a), (I3b), (14), (I4a), (I4b), (I4c), (I4d) or (I4e), or a structure of Formula (I), (II), (12), (13), (11-1), (11-2), (11-3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (II- 5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof, about 5-10 mol% of DSPC, about 30-45 mol% of cholesterol, about 1-4 mol% of PEG-DMG and about 0-5 mol% of squalene, cardiolipin,vitamin A, retinol, [3-carotene, withaferin A and / or a-tocopherol.

[0314] In some embodiments, the lipid nanoparticle is comprised of about 50-85 mol% of at least one ionizable lipid selected from Table 1 herein, about 5-20 mol% of at least one neutral (e.g., a phospholipid), about 1-40 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 0-15 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, [3-carotene, withaferin A and / or a-tocopherol).

[0315] In some embodiments, the lipid nanoparticle is comprised of about 50-85 mol% of at least one ionizable lipid selected from Table 1 herein, about 5-20 mol% of at least one neutral (e.g., a phospholipid), about 1-40 mol% of at least one helper lipid (e.g., cholesterol), about 1-5 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 1-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, [3-carotene, withaferin A and / or a-tocopherol).

[0316] In some embodiments, the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid selected from Table 1 herein, about 5-25 mol% of at least one neutral (e.g., a phospholipid), about 30-45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 4-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, [3-carotene, withaferin A and / or a-tocopherol).

[0317] In some embodiments, the lipid nanoparticle is comprised of about 40-60 mol% of at least one ionizable lipid selected from Table 1 herein, about 5-10 mol% of at least one phospholipid or phospholipid derivative described herein, about 30-45 mol% of at least one cholesterol or cholesterol derivative described herein, about 1-4 mol% of at least one PEG-lipid described herein and about 0-5 mol% of squalene, cardiolipin, withaferin A, vitamin A, retinol, [3-carotene and / or a-tocopherol.

[0318] In some embodiments, the lipid nanoparticle is comprised of about 40-60 mol% of at least one ionizable lipid selected from Table 1 herein, about 5-10 mol% of DSPC, about 30-45 mol% of cholesterol, about 1-4 mol% of PEG-DMG and about 0-5 mol% of squalene, cardiolipin, vitamin A, retinol, [3-carotene, withaferin A and / or a-tocopherol.

[0319] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 100 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (Ila), (12), (I2a), (I2b), (13), (I3a), (I3b), (14), (I4a), (I4b), (I4c), (I4d) or (I4e), or a structure of Formula (I), (II), (12), (13), (11-1), (11-2), (11-3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (II- 5b), (Il-5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from 0 to about 10 mol % of theneutral lipid; from 0 to about 50 mol % of the helper lipid; from 0 to about 5 mol % of the polymer- conjugated lipid; and from 0 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0320] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 99 mol % ofthe ionizable lipid which is at least one compound having a structure of Formula (I), (II), (Ila), (12), (I2a), (I2b), (13), (I3a), (I3b), (14), (I4a), (I4b), (I4c), (I4d) or (I4e), or a structure of Formula (I), (II), (12), (13), (11-1), (11-2), (11-3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (II- 5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), as defined herein, oratleast one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 1 to about 10 mol % of the neutral lipid; from 0 to about 50 mol % of the helper lipid; from 0 to about 5 mol % of the polymer-conjugated lipid; and from 0 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0321] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 60 mol % ofthe ionizable lipid which is at least one compound having a structure of Formula (I), (II), (Ila), (12), (I2a), (I2b), (13), (I3a), (I3b), (14), (I4a), (I4b), (I4c), (I4d) or (I4e), or a structure of Formula (I), (II), (12), (13), (11-1), (11-2), (11-3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (II- 5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), as defined herein, oratleast one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 1 to about 10 mol % of the neutral lipid; from about 1 to about 50 mol % of the helper lipid; from 0 to about 5 mol % of the polymer- conjugated lipid; and from 0 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0322] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 85 mol % ofthe ionizable lipid which is at least one compound having a structure of Formula (I), (II), (Ila), (12), (I2a), (I2b), (13), (I3a), (I3b), (14), (I4a), (I4b), (I4c), (I4d) or (I4e), or a structure of Formula (I), (II), (12), (13), (11-1), (11-2), (11-3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (II- 5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), as defined herein, oratleast one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 1 to about 10 mol % of the neutral lipid; from about 1 to about 50 mol % of the helper lipid; from 0 to about 5 mol % of the polymer- conjugated lipid; and from 0 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0323] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 85 mol % ofthe ionizable lipid which is at least one compound having a structure of Formula (I), (II), (Ila), (12), (I2a), (I2b), (13), (I3a), (I3b), (14), (I4a), (I4b), (I4c), (I4d) or (I4e), or a structure of Formula (I), (II), (12), (13), (11-1), (11-2), (11-3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (II- 5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), as defined herein, oratleast one compound in Table 1 herein,or a pharmaceutically acceptable salt thereof; from about 1 to about 10 mol % of the neutral lipid; from about 1 to about 50 mol % of the helper lipid; from about 1 to about 5 mol % of the polymer- conjugated lipid; and from 0 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0324] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 85 mol % ofthe ionizable lipid which is at least one compound having a structure of Formula (I), (II), (Ila), (12), (I2a), (I2b), (13), (I3a), (I3b), (14), (I4a), (I4b), (I4c), (I4d) or (I4e), or a structure of Formula (I), (II), (12), (13), (11-1), (11-2), (11-3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (II- 5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), as defined herein, or atleast one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 1 to about 10 mol % of the neutral lipid; from about 1 to about 50 mol % of the helper lipid; from about 1 to about 5 mol % of the polymer- conjugated lipid; and from about 0.1 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0325] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 85 mol % ofthe ionizable lipid which is at least one compound having a structure of Formula (I), (II), (Ila), (12), (I2a), (I2b), (13), (I3a), (I3b), (14), (I4a), (I4b), (I4c), (I4d) or (I4e), or a structure of Formula (I), (II), (12), (13), (11-1), (11-2), (11-3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (II- 5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), as defined herein, or atleast one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 5 to about 10 mol % of the neutral lipid; from about 1 to about 45 mol % of the helper lipid; from about 1 to about 4 mol % of the polymer- conjugated lipid; and from about 0.1 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0326] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 85 mol % ofthe ionizable lipid which is at least one compound having a structure of Formula (I), (II), (Ila), (12), (I2a), (I2b), (13), (I3a), (I3b), (14), (I4a), (I4b), (I4c), (I4d) or (I4e), or a structure of Formula (I), (II), (12), (13), (11-1), (11-2), (11-3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (Il-5b), (II- 5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), as defined herein, or atleast one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 5 to about 10 mol % of the neutral lipid; from about 1 to about 40 mol % of the helper lipid; from about 1 to about 4 mol % of the polymer- conjugated lipid; and from 0.1 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0327] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 85 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (Ila), (12), (I2a), (I2b), (13), (I3a), (I3b), (14), (I4a), (I4b), (I4c), (I4d) or (I4e), or a structure of Formula (I), (II), (12), (13), (11-1), (11-2), (11-3), (11-4), (11-5), (11-6), (Il-4a), (Il-4b), (Il-5a), (II-5b), (Il-5c), (Il-5d), (Il-6a), (Il-6b) or (Il-6c), as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 5 to about 10 mol % of a phospholipid as the neutral lipid; from about 1 to about 40 mol % of a sterol as the helper lipid; from about 1 to about 4 mol % of aPEG-lipid as the polymer-conjugated lipid; and from 1 to about 5 mol % of squalene, cardiolipin, a-tocopherol, withaferin A, vitamin A, or a combination thereof as the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.Encapsulation

[0328] In some embodiments, the lipid nanoparticle may fully or partially encapsulate a cargo or payload. In some embodiments, essentially 0% of the cargo present in the final formulation is exposed to the environment outside of the lipid nanoparticle (i.e., the cargo is fully encapsulated. In some embodiments, the cargo is associated with the lipid nanoparticle but is at least partially exposed to the environment outside of the lipid nanoparticle. In some embodiments, the lipid nanoparticle may be characterized by the % of the cargo not exposed to the environment outside of the lipid nanoparticle, e.g., the encapsulation efficiency. For the sake of clarity, an encapsulation efficiency of about 100% refers to a lipid nanoparticle formulation where essentially all the cargo is fully encapsulated by the lipid nanoparticle, while an encapsulation rate of about 0% refers to a lipid nanoparticle where essential none of the cargo is encapsulated in the lipid nanoparticle, such as with a lipid nanoparticle where the cargo is bound to the external surface of the lipid nanoparticle. On some embodiments, a lipid nanoparticle may have an encapsulation efficiency of less than about 100%, less than about 95%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15% less than about 10%, or less than 5%. In some embodiments, a lipid nanoparticle may have an encapsulation efficiency of between about 90 to 100%, 80 to 100%, 70 to 100%, 60 to 100%, 50 to 100%, 40 to 100%, 30 to 100%, 20 to 100%, 10 to 100%, 80 to 90%, 70 to 90%, 60 to 90%, 50 to 90%, 40 to 90%, 30 to 90%, 20 to 90%, 10 to 90%, 70 to 80%, 60 to 80%, 50 to 80%, 40 to 80%, 30 to 80%, 20 to 80%, 10 to 80%, 60 to 70%, 50 to 70%, 40 to 70%, 30 to 70%, 20 to 70%, 10 to 70%, 40 to 50%, 30 to 50%, 20 to 50%, 10 to 50%, 30 to 40%, 20 to 40%, 10 to 40%, 20 to 30%, 10 to 30%, and 10 to 20%.

[0329] In some embodiments, the weight ratio of the lipid nanoparticle (including all the lipids) and the cargo or payload is between about 100: 1 to about 1: 1, such as between about 100: 1 to about 90: 1, between about 89: 1 to about 80: 1, between about 79: 1 to about 70: 1, between about 69: 1 to about 60: 1, between about 59: 1 to about 50: 1, between about 49: 1 to about 40: 1, betweenabout 39:1 to about 30:1, between about 29:1 to about 20:1, between about 19:1 to about 10:1, and between about 9: 1 to about 1:1.

[0330] In some embodiments, the lipid nanoparticle further comprises an originator construct or a benchmark construct with at least one cargo or payload. The cargo or payload can be a small molecule, an antibody, a polynucleotide or a polypeptide. The cargo or payload can comprise at least one nucleic acid, such as mRNA. The cargo or payload may be any DNA, RNA or polypeptide described herein.

[0331] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is a coding RNA.

[0332] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is a non-coding RNA.

[0333] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is an oRNA.

[0334] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is an mRNA.

[0335] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is a personalized vaccine mRNA. Non-limiting example includes 3-GP.

[0336] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is a Covid- 19 vaccine mRNA. Nonlimiting example coronavirus spike protein.

[0337] In some embodiments, the at least one RNA compound is comprised of a functional RNA where the RNA results in at least one change in a cell, tissue, organ and / or organism. Said changes in state may include, but are not limited to, altering the expression level of a polypeptide, altering the translation level of a nucleic acid, altering the expression level of a nucleic acid, altering the amount of a polypeptide present in a cell, tissue, organ and / or organism, changing a genetic sequence of a cell, tissue, organ and / or organism, adding nucleic acids to a target genome, subtracting nucleic acids from a target genome, altering physiological activity in a cell, tissue, organ and / or organism or any combination thereof.

[0338] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is DNA.

[0339] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads which are DNA. The DNA may be the same DNA or different DNA. As a non-limiting example, the DNA are the same. As a non-limiting example, the DNA are different. As a non-limiting example, the DNA are different but encode thesame payload or cargo. As a non-limiting example, the DNA are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).

[0340] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with three cargos or payloads which are DNA. The DNA may be the same DNA or different DNA. As a non-limiting example, the DNA are the same. As a non-limiting example, the DNA are different. As a non-limiting example, two DNA are the same and one is different. As a non-limiting example, the first DNA is different from the second and third DNA. As anon-limiting example, the first DNA, second DNA and third DNA are all different. As anon- limiting example, the first DNA is different from the second and third DNA, but they all encode the same payload or cargo. As a non-limiting example, the first DNA is different from the second and third DNA but the second and third DNA encode the same payload or cargo.

[0341] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is a polypeptide.

[0342] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads which are polypeptides. The polypeptides may be the same polypeptide or different polypeptides. As a non-limiting example, the polypeptides are the same. As a non-limiting example, the polypeptides are different. As a non-limiting example, the polypeptides are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).

[0343] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with three cargos or payloads which are polypeptides. The polypeptides may be the same polypeptide or different polypeptides. As a non-limiting example, the polypeptides are the same. As a non-limiting example, the polypeptides are different. As a non-limiting example, two polypeptides are the same and one is different. As a non-limiting example, the first polypeptide is different from the second and third polypeptides. As a non-limiting example, the first polypeptide, second polypeptide and third polypeptide are all different. As a non-limiting example, the first polypeptide is different from the second and third polypeptides, but they all encode the same payload or cargo. As a non-limiting example, the first polypeptide is different from the second and third polypeptides but the second and third polypeptides encode the same pay load or cargo.

[0344] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is a peptide.

[0345] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads which are peptides. The peptides may be the same peptide or different peptides. As a non-limiting example, the peptides are the same. As a non-limiting example, the peptides are different. As a non-limiting example, the peptides are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).

[0346] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with three cargos or pay loads which are peptides. The peptides may be the same peptide or different peptides. As a non-limiting example, the peptides are the same. As a non-limiting example, the peptides are different. As a non-limiting example, two peptides are the same and one is different. As anon-limiting example, the first peptide is different from the second and third peptides. As a non-limiting example, the first peptide, second peptide and third peptide are all different. As a non-limiting example, the first peptide is different from the second and third peptides, but they all encode the same payload or cargo. As a non-limiting example, the first peptide is different from the second and third peptides but the second and third peptide encode the same payload or cargo.

[0347] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is RNA.

[0348] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads which are RNAs. The RNAs may be the same RNA or different RNAs. As a non-limiting example, the RNAs are the same. As a non-limiting example, the RNAs are different. As a non-limiting example, the RNAs are different but encode the same payload or cargo. As a non-limiting example, the RNAs are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).

[0349] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with three cargos or pay loads which are RNAs. The RNAs may be the same RNA or different RNAs. As a non-limiting example, the RNAs are the same. As a non-limiting example, the RNAs are different. As a non-limiting example, two RNAs are the same and one is different. As a non-limiting example, the first RNA is different from the second and third RNAs. As a non-limiting example, the first RNA, second RNA and third RNA are all different. As a nonlimiting example, the first RNA is different from the second and third RNAs, but they all encode the same payload or cargo. As a non-limiting example, the first RNA is different from the secondand third RNA but the second and third RNAs encode the same payload or cargo.

[0350] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads where one is RNA, and one is DNA. The RNA and DNA may encode the same peptide or polypeptide or may encode different peptides or polypeptides. As a non-limiting example, the RNA and DNA may encode the same peptide or polypeptide. As a non-limiting example, the RNA and DNA may encode different peptides or polypeptides. As a non-limiting example, the RNA and DNA are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).

[0351] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads where one is RNA, and one is a peptide. The RNA may encode the same peptide as the peptide cargo / payload the RNA may encode a different peptide. As a non-limiting example, the RNA encodes the same peptide. As a non-limiting example, the RNA encodes a different peptide. As a non-limiting example, the RNA and peptide are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).

[0352] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads where one is RNA, and one is a polypeptide. The RNA may encode the same polypeptide as the polypeptide cargo / payload the RNA may encode a different polypeptide. As a non-limiting example, the RNA encodes the same polypeptide. As a non-limiting example, the RNA encodes a different polypeptide. As a nonlimiting example, the RNA and polypeptide are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).

[0353] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads where one is DNA, and one is a peptide. The DNA may encode the same peptide as the peptide cargo / payload the DNA may encode a different peptide. As a non-limiting example, the DNA encodes the same peptide. As a non-limiting example, the DNA encodes a different peptide. As a non-limiting example, the DNA and peptide are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).

[0354] In some embodiments, the lipid nanoparticle comprises an originator construct or abenchmark construct with two cargos or payloads where one is DNA, and one is a polypeptide. The DNA may encode the same polypeptide as the polypeptide cargo / payload the DNA may encode a different polypeptide. As a non-limiting example, the DNA encodes the same polypeptide. As a non-limiting example, the DNA encodes a different polypeptide. As a nonlimiting example, the DNA and polypeptide are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).Methods of preparation

[0355] The lipid nanoparticles described herein may be formed using techniques known in the art. As a non-limiting example, an organic solution containing the lipids is mixed together with an acidic aqueous solution containing the originator construct or benchmark construct in a microfluidic channel resulting in the formation of targeting system (lipid nanoparticle and the benchmark construct). Another technique can involve turbulent mixing in a T-junction, where the flow rate of the solution of the originator construct or benchmark construct and the lipid solution are substantially the same.

[0356] In some embodiments, a lipid nanoparticle formulation may be prepared by the methods described in International Publication No. W02008103276, the content of which is herein incorporated by reference in its entirety. In some embodiments, lipid nanoparticle formulations may be as described in International Publication No. W02019131770, the content of which is herein incorporated by reference in its entirety.

[0357] In some embodiments, a lipid nanoparticle formulation may be prepared by the methods described in International Publication No. WO2020237227, the content of which is herein incorporated by reference in its entirety.

[0358] In some embodiments, a lipid nanoparticle formulation may be prepared using the methodologies and devices described in US20240181406A1, the content of which is herein incorporated by reference in its entirety.

[0359] In further embodiments, a lipid nanoparticle formulation may be prepared using the FDmiX mixing systems developed by FDX Fluid Dynamix GmbH (See https : / / www.fdx, de / en / fdmix / . the content of which is herein incorporated by reference in its entirety).

[0360] In some embodiments, a lipid nanoparticle formulation may be prepared by a so-called “Point-of-Care” mixing method as described herein (see Examples). It consists in preparing separately a solution of a cargo, such as mRNA for instance, that can be frozen or lyophilized for storage, and a solution of lipid. Mixing the lipid solution and the mRNA solution followed byvigorous shaking provides mRNA encapsulated in LNPs ready for using / dosing. Point-of-Care mixing can produce mRNA-LNPs with comparable quality and efficacy to mRNA-LNPs obtained with conventional methods (conventional mRNA-LNPs). Manufacturing of conventional mRNA- LNPs is a sensitive process and usually involves specialized equipment and analytical techniques. The mRNA-LNPs are sensitive to minor changes in the manufacturing process. Long-term storage of mRNA-LNPs requires cryo-storage facilities. Still long-term stability of mRNA-LNPs may not be ensured. On the other hand, in Point-of-Care mixing, the LNPs are not manufactured ahead of time of administration. The mRNA and lipid solution can be manufactured, filled and released using conventional small-molecule pharmaceutical manufacturing units. Both the mRNA and lipid solution are stable under frozen conditions.IV. Cargo and Payloads

[0361] The present disclosure also provides compositions or constructs comprising the lipid nanoparticles of the present disclosure, wherein the lipid nanoparticles may comprise, encode or be conjugated to a cargo or payload to produce the constructs. In some embodiments, the cargo or payload is or encodes a biologically active molecule such as, but not limited to a therapeutic protein. As used herein, the term “biologically active” refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active. In some embodiments, the cargo or payload is or encodes one or more prophylactically- or therapeutically-active proteins, polypeptides, or other factors. As a nonlimiting example, the cargo or payload may be or encode an agent that enhances tumor killing activity such as, but not limited to, TRAIL or tumor necrosis factor (TNF), in a cancer. As another non-limiting example, the cargo or payload may be or encode an agent suitable for the treatment of conditions such as muscular dystrophy (e.g., cargo or payload is or encodes Dystrophin), cardiovascular disease (e.g., cargo or payload is or encodes SERCA2a, GATA4, Tbx5, Mef2C, Hand2, Myocd, etc.), neurodegenerative disease (e.g., cargo or payload is or encodes NGF, BDNF, GDNF, NT-3, etc.), chronic pain (e.g., cargo or payload is or encodes GlyRal), an enkephalin, or a glutamate decarboxylase (e.g., cargo or payload is or encodes GAD65, GAD67, or another isoform), lung disease (e.g., cargo or payload is or encodes CFTR), hemophilia (e.g., cargo or payload is or encodes Factor VIII or Factor IX), neoplasia (e.g., cargo or payload is or encodes PTEN, ATM, ATR, EGFR, ERBB2, ERBB3, ERBB4, Notchl, Notch2, Notch3, Notch4, AKT, AKT2, AKT3, HIF, HI Fla, HIF3a, Met, HRG, Bcl2, PPARalpha, PPAR gamma, WT1 (Wilms Tumor), FGF Receptor Family members (5 members: 1, 2, 3, 4, 5), CDKN2a, APC, RB (retinoblastoma), MEN1, VHL, BRCA1, BRCA2, AR (Androgen Receptor), TSG101, IGF, IGFReceptor, Igfl (4 variants), Igf2 (3 variants), Igfl Receptor, Igf2 Receptor, Bax, Bcl2, caspases family (9 members: 1, 2, 3, 4, 6, 7, 8, 9, 12), Kras, Ape), age-related macular degeneration (e.g., cargo or payload is or encodes Aber, Ccl2, Cc2, cp (ceruloplasmin), Timp3, cathepsin D, Vldlr), schizophrenia (e.g. Neuregulin (Nrgl), Erb4 (receptor for Neuregulin), Complexin-1 (Cplxl), Tphl Tryptophan hydroxylase, Tph2 Tryptophan hydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HIT (Slc6a4), COMT, DRD (Drdla), SLC6A3, DAOA, DTNBPI, Dao (Daol)), trinucleotide repeat disorders (e.g., HTT (Huntington's Dx), SBMA / SMAXI / AR (Kennedy's Dx), FXN / X25 (Friedrich's Ataxia), ATX3 (Machado-Joseph's Dx), ATXNI and ATXN2 (spinocerebellar ataxias), DMPK (myotonic dystrophy), Atrophin-1 and Atnl (DRPL A Dx), CBP (Creb-BP-global instability), VLDLR (Alzheimer's), Atxn7, AtxnlO), fragile X syndrome (e.g., cargo or payload is or encodes FMR2, FXRI, FXR2, mGLUR5), secretase related disorders (e.g., cargo or payload is or encodes APH-1 (alpha and beta), Presenilin (Psenl), nicastrin (Ncstn), PEN-2), ALS (e.g., cargo or payload is or encodes SOD1, ALS2, STEX, FUS, TARD BP, VEGF (VEGF-a, VEGF-b, VEGF-c)), autism (e.g., cargo or payload is or encodes Mecp2, BZRAP1, MDGA2, Sema5A, Neurexin 1), Alzheimer's disease (e.g., cargo or payload is or encodes El, CHIP, UCH, UBB, Tau, LRP, PICALM, Clusterin, PSI, SORL1, CR1, Vldlr, Ubal, Uba3, CHIP28 (Aqpl, Aquaporin 1), Uchll, Uchl3, APP), inflammation (e.g., cargo or payload is or encodes IL-10, IL-1 (IL-Ia, IL-Ib), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-171), 11-23, Cx3crl, ptpn22, TNFa, NOD2 / CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cll), Parkinson's Disease (e.g., x-Synuclein, DJ-1, LRRK2, Parkin, PINK1), blood and coagulation disorders, such as, e.g., anemia, bare lymphocyte syndrome, bleeding disorders, hemophagocytic lymphohistiocytosis disorders, hemophilia A, hemophilia B, hemorrhagic disorders, leukocyte deficiencies and disorders, sickle cell anemia, and thalassemia (e.g., cargo or payload is or encodes CRAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3, UMPH1, PSNI, RHAG, RH50A, NRAMP2, SPTB, ALAS2, ANH1, ASB, ABCB7, ABC7, ASAT, TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11 , MHC2TA, C2TA, RFX5, RFXAP, RFX5, TBXA2R, P2RX1, P2X1, HF1, CFH, HUS, MCFD2, FANCA, FAC A, FA1, FA, FA A, FAAP95, FAAP90, FLJ34064, FANCB, FANCC, FACC, BRCA2, FANCDI, FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF, XRCC9, FANCG, BRI PI, BACH1, FANCJ, PHF9, FANCL, FANCM, KIAA1596, PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3, HLH3, FHL3, F8, FSC, PI, ATT, F5, ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3, EIF2B5, LVWM, CACH, CLE, EIF2B4, HBB, HBA2, HBB, HBD, LCRB, HBA1), B-cell non-Hodgkin lymphoma or leukemia (e.g., cargo or payload is or encodes BCL7A, BCL7, ALI, TCL5, SCL, TAL2, FLT3, NBS1, NBS, ZNFN1AI, 1KI, LYF1, H0XD4, H0X4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AFIO, ARHGEF12, LARG, KIAA0382, CALM, CLTH, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT, LPP, NPMI,NUP214, D9S46E, CAN, CAIN, RUNXI, CBFA2, AML1, WHSC1LI, NSD3, FLT3, AF1Q, NPMI, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF1Q, CALM, CLTH, ARL11, ARLTS1, P2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF, WSS, NFNS, PTPNII, PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA, GATA1, GF1, ERYF1, NFE1, ABLI, NQ01, DIA4, NM0R1, NUP214, D9S46E, CAN, CAIN), inflammation and immune related diseases and disorders (e.g., cargo or payload is or encodes KIR3DL1, NKAT3, NKB1, AMB11, K1R3DS1, IFNG, CXCL12, TNFRSF6, APT1, FAS, CD95, ALPS1A, IL2RG, SCIDX1, SCIDX, IMD4, CCL5, SCYA5, D17S136E, TCP228, IL10, CSIF, CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5), CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSFS, CD40LG, HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI), inflammation (e.g., cargo or payload is or encodes IL-10, IL-1 (IL-IA, IL-IB), IL-13, IL- 17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-171), 11-23, Cx3crl, ptpn22, TNFa, NOD2 / CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cII), JAK3, JAKL, DCLREIC, ARTEMIS, SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDXI, SCIDX, IMD4), metabolic, liver, kidney and protein diseases and disorders (e.g., cargo or pay load is or encodes TTR, PALB, APOA1, APP, AAA, CVAP, ADI, GSN, FGA, LYZ, TTR, PALB, KRT18, KRT8, CIRH1A, NAIC, TEX292, KIAA1988, CFTR, ABCC7, CF, MRP7, SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM, TCF1, HNF1A, M0DY3, SCOD1, SCO1, CTNNB1, PDGFRL, PDGRL, PRLTS, AX1NI, AXIN, CTNNB1, TP53, P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5, UMOD, HNFJ, FJHN, MCKD2, ADMCKD2, PAH, PKU1, QDPR, DHPR, PTS, FCYT, PKHD1, ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63), muscular / skeletal diseases and disorders (e.g., cargo or payload is or encodes DMD, BMD, MYF6, LMNA, LMN1, EMD2, FPLD, CMDIA, HGPS, LGMDIB, LMNA, LMNI, EMD2, FPLD, CMDIA, FSHMD1A, FSHD1A, FKRP, MDC1C, LGMD2I, LAMA2, LAMM, LARGE, KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B, SGCG, LGMD2C, DMDA1, SCG3, SGCA, ADL, DAG2, LGMD2D, DMDA2, SGCB, LGMD2E, SGCD, SGD, LGMD2F, CMD1L, TCAP, LGMD2G, CMD1N, TRIM32, HT2A, LGMD2H, FKRP, MDCIC, LGMD21, TTN, CMD1G, TMD, LGMD2J, P0MT1, CAV3, LGMD1C, SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1, LRP5, BMND1, LRP7, LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTMI, GL, TCIRG1, TIRC7, OC116, OPTB1, VAPB, VAPC, ALS8, SMN1, SMA1, SMA2, SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2, CATF1, SMARD1), neurological and neuronal diseases and disorders (e.g., cargo or payload is or encodes SOD1, ALS2, STEX, FUS, TARDBP, VEGF (VEGF-a, VEGF-b, VEGF-c), APP, AAA, CVAP, ADI, APOE, AD2, PSEN2, AD4, STM2, APBB2, FE65LI, NOS3, PLAU, URK, ACE, DCPI, ACEI,MPO, PAC1PI, PAXIPIL, PTIP, A2M, BLMH, BMH, PSEN1, AD3, Mecp2, BZRAP1, MDGA2, Sema5A, Neurexin 1, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4, KIAA1260, AUTSX2, FMR2, FXR1, FXR2, mGLUR5, HD, IT15, PRNP, PRIP, JPH3, JP3, HDL2, TBP, SCA17, NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP, SCA17, SNCA, NACP, PARK1, PARKA, DJI, PARK7, LRRK2, PARK8, PINK1, PARK6, UCHL1, PARK5, SNCA, NACP, PARK!, PARKA, PRKN, PARK2, PDJ, DBH, NDUFV2, MECP2, RTT, PPMX, MRX16, MRX79, CDKL5, STK9, MECP2, RTT, PPMX, MRX16,MRX79, x-Synuclein, DJ-1, Neuregulin-1 (Nrgl), Erb4 (receptor for Neuregulin), Complexin-1 (Cplxl), Tphl Tryptophan hydroxylase, Tph2, Tryptophan hydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), CONT, DRD (Drdla), SLC6A , DAO A, DTNBP1, Dao (Daol), APH-l(alpha and beta), Presenilin (Psenl), Nicastrin, (Ncstn), PEN-2, Nosl, Parpl, Natl, Nat2, HTT, SBMA / SMAX1 / AR, FXN / X25, ATX3, TXN, ATXN2, DMPK, Atrophin-1, Atal, CBP, VLDLR, Atxn7, and AtxnlO), and ocular diseases and disorders (e.g., Aber, Ccl2, Cc2, cp (ceruloplasmin), Timp3, cathepsin-D, Vldlr, Ccr2, CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, CRY Al, PAX6, AN2, MGDA, CRYB Al, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP 19, CRYGD, CRYG4, BFSP2, CP49, CP47, HSF4, CTM, HSF4, CTM, MIP, AQPO, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA, CRYAI, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1, APOA1, TGFBI, CSD2, CDGG1, CSD, BIGH3, CDG2, TACSTD2, TROP2, M1SI, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, CFD, KERA, CNA2, MYOC, TIGR, GLCIA, JO AG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1BI, GLC3A, OPA1, NTG, NPG, CYP1BI, GLC3A, CRB1, RP12, CRX, CORD2, CRD, RPGRIPI, LCA6, CORD9, RPE65, RP20, AIPL1, LCA4, GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3, ELOVL4, ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, and VMD2).

[0362] In some embodiments, the cargo or payload is or encodes a factor that can affect the differentiation of a cell. As a non-limiting example, the expression of one or more of Oct4, Klf4, Sox2, c-Myc, L-Myc, dominant-negative p53, Nanog, Glisl, Lin28, TFIID, mir-302 / 367, or other miRNAs can cause the cell to become an induced pluripotent stem (iPS) cell.

[0363] In some embodiments, the cargo or payload is or encodes a factor for transdifferentiating cells. Non-limiting examples of factors include: one or more of GATA4, Tbx5, Mef2C, Myocd, Hand2, SRF, Mespl, SMARCD3 for cardiomyocytes; Ascii, Nurrl, LmxlA, Bm2, Myth, NeuroDl, FoxA2 for neural cells; and Hnf4a, Foxal, Foxa2 or Foxa3 for hepatic cells.Polypeptides, Proteins and Peptides

[0364] The lipid nanoparticles of the present disclosure may comprise, encode or be conjugated to a cargo or payload which is a polypeptide, protein or peptide. As used herein, the term“polypeptide” generally refers to polymers of amino acids linked by peptide bonds and embraces “protein and “peptides.” Polypeptides for the present disclosure include all polypeptides, proteins and / or peptides known in the art. Non-limiting categories of polypeptides include antigens, antibodies, antibody fragments, cytokines, peptides, hormones, enzymes, oxidants, antioxidants, synthetic polypeptides, and chimeric polypeptides.

[0365] As used herein, the term “peptide” generally refers to shorter polypeptides of about 50 amino acids or less. Peptides with only two amino acids may be referred to as “dipeptides.” Peptides with only three amino acids may be referred to as “tripeptides.” Polypeptides generally refer to polypeptides with from about 4 to about 50 amino acids. Peptides may be obtained via any method known to those skilled in the art. In some embodiments, peptides may be expressed in culture. In some embodiments, peptides may be obtained via chemical synthesis (e.g., solid phase peptide synthesis).

[0366] In some embodiments, the lipid nanoparticles of the present disclosure may comprise, encode or be conjugated to a cargo or payload which is a simple protein which upon hydrolysis yields the amino acids and occasionally small carbohydrate compounds. Non-limiting examples of simple proteins include albumins, albuminoids, globulins, glutelins, histones and protamines.

[0367] In some embodiments, the lipid nanoparticles of the present disclosure may comprise, encode or be conjugated to a cargo or payload which is a conjugated protein which may be a simple protein associated with a non-protein. Non-limiting examples of conjugated proteins include, glycoproteins, hemoglobins, lecithoproteins, nucleoproteins, and phosphoproteins.

[0368] In some embodiments, the lipid nanoparticles of the present disclosure may comprise, encode or be conjugated to a cargo or payload which is a derived protein which is a protein that is derived from a simple or conjugated protein by chemical or physical means. Non-limiting examples of derived proteins include denatured proteins and peptides.

[0369] In some embodiments, the polypeptide, protein or peptide may be unmodified.

[0370] In some embodiments, the polypeptide, protein or peptide may be modified. Types of modifications include, but are not limited to, Phosphorylation, Glycosylation, Acetylation, Ubiquitylation / Sumoylation, Methylation, Palmitoylation, Quinone, Amidation, Myristoylation, Pyrrolidone carboxylic acid, Hydroxylation, Phosphopantetheine, Prenylation, GPI anchoring, Oxidation, ADP-ribosylation, Sulfation, S-nitrosylation, Citrullination, Nitration, Gammacarboxyglutamic acid, Formylation, Hypusine, Topaquinone (TPQ), Bromination, Lysine topaquinone (LTQ), Tryptophan tryptophylquinone (TTQ), Iodination, and Cysteine tryptophylquinone (CTQ). In some aspects, the polypeptide, protein or peptide may be modified by a post-transcriptional modification which can affect its structure, subcellular localization, and / or function.

[0371] In some embodiments, the polypeptide, protein or peptide may be modified using phosphorylation. Phosphorylation, or the addition of a phosphate group to serine, threonine, or tyrosine residues, is one of most common forms of protein modification. Protein phosphorylation plays an important role in fine tuning the signal in the intracellular signaling cascades.

[0372] In some embodiments, the polypeptide, protein or peptide may be modified using ubiquitination which is the covalent attachment of ubiquitin to target proteins. Ubiquitination- mediated protein turnover has been shown to play a role in driving the cell cycle as well as in protein-degradation-independent intracellular signaling pathways.

[0373] In some embodiments, the polypeptide, protein or peptide may be modified using acetylation and methylation which can play a role in regulating gene expression. As a non-limiting example, the acetylation and methylation could mediate the formation of chromatin domains (e.g., euchromatin and heterochromatin) which could have an impact on mediating gene silencing.

[0374] In some embodiments, the polypeptide, protein or peptide may be modified using glycosylation. Glycosylation is the attachment of one of a large number of glycan groups and is a modification that occurs in about half of all proteins and plays a role in biological processes including, but not limited to, embryonic development, cell division, and regulation of protein structure. The two main types of protein glycosylation are N-glycosylation and O-glycosylation. For N-glycosylation the glycan is attached to an asparagine and for O-glycosylation the glycan is attached to a serine or threonine.

[0375] In some embodiments, the polypeptide, protein or peptide may be modified using Sumoylation. Sumoylation is the addition of SUMOs (small ubiquitin-like modifiers) to proteins and is a post-translational modification similar to ubiquitination.Antibodies

[0376] As used herein, the term "antibody" is referred to in the broadest sense and specifically covers various embodiments including, but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies formed from at least two intact antibodies), and antibody fragments (e.g., diabodies) so long as they exhibit a desired biological activity (e.g., “functional”). Antibodies are primarily amino acid based molecules which are monomeric or multimeric polypeptides which comprise at least one amino acid region derived from a known or parental antibody sequence and at least one amino acid region derived from a non-antibody sequence. The antibodies may comprise one or more modifications (including, but not limited to the addition of sugar moieties, fluorescent moieties, chemical tags, etc.). For the purposes herein, an "antibody" may comprise a heavy and light variable domain as well as an Fc region.

[0377] The cargo or payload may comprise or may encode polypeptides that form one or more functional antibodies.

[0378] In some embodiments, the cargo or payload may comprise or may encode polypeptides that form or function as any antibody including, but not limited to, antibodies that are known in the art and / or antibodies that are commercially available which may be therapeutic, diagnostic, or for research purposes. Additionally, the cargo or payload may comprise or may encode fragments of such antibodies or antibodies such as, but not limited to, variable domains or complementarity determining regions (CDRs).

[0379] As used herein, the term "native antibody" refers to a usually heterotetrameric glycoprotein of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Genes encoding antibody heavy and light chains are known and segments making up each have been well characterized and described (Matsuda, F. et al., 1998. The Journal of Experimental Medicine. 188(11); 2151-62 and Li, A. et al., 2004. Blood. 103(12: 4602-9, the content of each of which are herein incorporated by reference in their entirety). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. As used herein, the term "light chain" refers to a component of an antibody from any vertebrate species assigned to one of two clearly distinct types, called kappa and lambda based on amino acid sequences of constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2.

[0380] As used herein, the term "variable domain" refers to specific antibody domains found on both the antibody heavy and light chains that differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. Variable domains comprise hypervariable regions. As used herein, the term "hypervariable region" refers to a region within a variable domain comprising amino acid residues responsible for antigen binding. The amino acids present within the hypervariable regions determine the structure of the complementarity determining regions (CDRs) that become part of the antigen-binding site of the antibody. As used herein, the term “CDR” refers to a region of an antibody comprising a structure that is complimentary to its target antigen or epitope. Other portions of the variable domain, not interacting with the antigen, are referred to as framework (FW) regions. The antigen-binding site(also known as the antigen combining site or paratope) comprises the amino acid residues necessary to interact with a particular antigen. The exact residues making up the antigen-binding site are typically elucidated by co-crystallography with bound antigen, however computational assessments can also be used based on comparisons with other antibodies (Strohl, W.R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA. 2012. Ch. 3, p47-54, the contents of which are herein incorporated by reference in their entirety). Determining residues making up CDRs may include the use of numbering schemes including, but not limited to, those taught by Kabat [Wu, T.T. et al., 1970, JEM, 132(2):211-50 and Johnson, G. et al., 2000, Nucleic Acids Res. 28(1): 214-8, the contents of each of which are herein incorporated by reference in their entirety], Chothia [Chothia and Lesk, J. Mol. Biol. 196, 901 (1987), Chothia et al., Nature 342, 877 (1989) and Al-Lazikani, B. et al., 1997, J. Mol. Biol. 273(4):927-48, the contents of each of which are herein incorporated by reference in their entirety], Lefranc (Lefranc, M.P. et al., 2005, Immunome Res. 1:3) and Honegger (Honegger, A. and Pluckthun, A. 2001. J. Mol. Biol. 309(3):657-70, the contents of which are herein incorporated by reference in their entirety).

[0381] VH and VL domains each have three CDRs. VL CDRs are referred to herein as CDR-L1, CDR-L2 and CDR-L3, in order of occurrence when moving from N- to C- terminus along the variable domain polypeptide. VH CDRs are referred to herein as CDR-H1, CDR-H2, and CDR- H3, in order of occurrence when moving from N- to C-terminus along the variable domain polypeptide. Each of CDRs have favored canonical structures with the exception of the CDR-H3, which comprises amino acid sequences that may be highly variable in sequence and length between antibodies resulting in a variety of three-dimensional structures in antigen-binding domains. In some cases, CDR-H3s may be analyzed among a panel of related antibodies to assess antibody diversity.

[0382] Various methods of determining CDR sequences are known in the art and may be applied to known antibody sequences. The system described by Kabat, also referred to as “numbered according to Kabat,” “Kabat numbering,” “Kabat definitions,” and “Kabat labeling,” provides an unambiguous residue numbering system applicable to any variable domain of an antibody, and provides precise residue boundaries defining the three CDRs of each chain. (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987) and (1991), the contents of which are incorporated by reference in their entirety). Kabat CDRs and comprise about residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain, and 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain. Chothia and coworkers found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. (Chothia et al. (1987) J. Mol. Biol. 196: 901-917; and Chothia et al. (1989) Nature 342: 877-883, the contents of each of which is herein incorporated by reference in itsentirety). These CDRs can be referred to as “Chothia CDRs,” “Chothia numbering,” or “numbered according to Chothia,” and comprise about residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain, and 26-32 (CDR1), 52-56 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain. Mol. Biol. 196:901-917 (1987). The system described by MacCallum, also referred to as “numbered according to MacCallum,” or “MacCallum numbering” comprises about residues 30-36 (CDR1), 46-55 (CDR2) and 89-96 (CDR3) in the light chain variable domain, and 30-35 (CDR1), 47-58 (CDR2) and 93-101 (CDR3) in the heavy chain variable domain. (MacCallum et al. ((1996) J. Mol. Biol. 262(5): 732-745), the contents of which is herein incorporated by reference in its entirety). The system described by AbM, also referred to as “numbering according to AbM,” or “AbM numbering” comprises about residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain, and 26-35 (CDR1), 50-58 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain. The IMGT (INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM) numbering of variable regions can also be used, which is the numbering of the residues in an immunoglobulin variable heavy or light chain according to the methods of the IIMGT (Lefranc, M.-P., "The IMGT unique numbering for immunoglobulins, T cell Receptors and Ig-like domains", The Immunologist, 7, 132-136 (1999), and is herein incorporated by reference in its entirety by reference). As used herein, "IMGT sequence numbering" or “numbered according to IMTG,” refers to numbering of the sequence encoding a variable region according to the IMGT. For the heavy chain variable domain, when numbered according to IMGT, the hypervariable region ranges from amino acid positions 27 to 38 for CDR1, amino acid positions 56 to 65 for CDR2, and amino acid positions 105 to 117 for CDR3. For the light chain variable domain, when numbered according to IMGT, the hypervariable region ranges from amino acid positions 27 to 38 for CDR1, amino acid positions 56 to 65 for CDR2, and amino acid positions 105 to 117 for CDR3.

[0383] In some embodiments, the cargo or payload may comprise or may encode antibodies which have been produced using methods known in the art such as, but are not limited to immunization and display technologies (e.g., phage display, yeast display, and ribosomal display), hybridoma technology, heavy and light chain variable region cDNA sequences selected from hybridomas or from other sources,

[0384] In some embodiments, the cargo or payload may comprise or may encode antibodies which were developed using any naturally occurring or synthetic antigen. As used herein, an “antigen” is an entity which induces or evokes an immune response in an organism. An immune response is characterized by the reaction of the cells, tissues and / or organs of an organism to the presence of a foreign entity. Such an immune response typically leads to the production by the organism of one or more antibodies against the foreign entity, e.g., antigen or a portion of theantigen. As used herein, “antigens” also refer to binding partners for specific antibodies or binding agents in a display library.

[0385] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous cells (or clones), i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variants that may arise during production of the monoclonal antibodies, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen

[0386] The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. The monoclonal antibodies herein include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies.

[0387] As used herein, the term "humanized antibody" refers to a chimeric antibody comprising a minimal portion from one or more non-human (e.g., murine) antibody source(s) with the remainder derived from one or more human immunoglobulin sources. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable region from an antibody of the recipient are replaced by residues from the hypervariable region from an antibody of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and / or capacity.

[0388] In some embodiments, the cargo or payload may comprise or may encode antibody mimetics. As used herein, the term “antibody mimetic” refers to any molecule which mimics the function or effect of an antibody, and which binds specifically and with high affinity to their molecular targets. In some embodiments, antibody mimetics may be monobodies, designed to incorporate the fibronectin type III domain (Fn3) as a protein scaffold. In some embodiments, antibody mimetics may be those known in the art including, but are not limited to affibody molecules, affilins, affitins, anticalins, avimers, Centyrins, DARPINSTM, fynomers, Kunitz domains, and domain peptides. In other embodiments, antibody mimetics may include one or more non-peptide regions.Antibody Fragments and Variants

[0389] In some embodiments, the cargo or payload may comprise or may encode antibodyfragments which comprise antigen binding regions from full-length antibodies. Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site. Also produced is a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen. Compounds and / or compositions of the present disclosure may comprise one or more of these fragments.

[0390] In some embodiments, the Fc region may be a modified Fc region wherein the Fc region may have a single amino acid substitution as compared to the corresponding sequence for the wildtype Fc region, wherein the single amino acid substitution yields an Fc region with preferred properties to those of the wild-type Fc region. Non-limiting examples of Fc properties that may be altered by the single amino acid substitution include bind properties or response to pH conditions

[0391] As used herein, the term “Fv” refers to an antibody fragment comprising the minimum fragment on an antibody needed to form a complete antigen binding site. These regions consist of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. Fv fragments can be generated by proteolytic cleavage, but are largely unstable. Recombinant methods are known in the art for generating stable Fv fragments, typically through insertion of a flexible linker between the light chain variable domain and the heavy chain variable domain to form a single chain Fv (scFv) or through the introduction of a disulfide bridge between heavy and light chain variable domains.

[0392] As used herein, the term "single chain Fv" or "scFv" refers to a fusion protein of VH and VL antibody domains, wherein these domains are linked together into a single polypeptide chain by a flexible peptide linker. In some embodiments, the Fv polypeptide linker enables the scFv to form the desired structure for antigen binding. In some embodiments, scFvs are utilized in conjunction with phage display, yeast display or other display methods where they may be expressed in association with a surface member (e.g., phage coat protein) and used in the identification of high affinity peptides for a given antigen.

[0393] As used herein, the term “antibody variant” refers to a modified antibody (in relation to a native or starting antibody) or a biomolecule resembling a native or starting antibody in structure and / or function (e.g., an antibody mimetic). Antibody variants may be altered in their amino acid sequence, composition, or structure as compared to a native antibody. Antibody variants may include, but are not limited to, antibodies with altered isotypes (e.g., IgA, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM), humanized variants, optimized variants, multispecific antibody variants (e.g.,bispecific variants), and antibody fragments.Multispecific antibodies

[0394] In some embodiments, the cargo or payload may be or may encode antibodies that bind more than one epitope. As used herein, the terms “multibody” or “multispecific antibody” refer to an antibody wherein two or more variable regions bind to different epitopes. The epitopes may be on the same or different targets. In certain embodiments, a multispecific antibody is a "bispecific antibody," which recognizes two different epitopes on the same or different antigens.

[0395] In some embodiments, multi-specific antibodies may be prepared by the methods used by BIOATLA® and described in International Patent publication WO201109726, the contents of which are herein incorporated by reference in their entirety. First a library of homologous, naturally occurring antibodies is generated by any method known in the art (i.e., mammalian cell surface display), then screened by FACSAria or another screening method, for multi-specific antibodies that specifically bind to two or more target antigens. In some embodiments, the identified multi-specific antibodies are further evolved by any method known in the art, to produce a set of modified multi-specific antibodies. These modified multi-specific antibodies are screened for binding to the target antigens. In some embodiments, the multi-specific antibody may be further optimized by screening the evolved modified multi-specific antibodies for optimized or desired characteristics.

[0396] In some embodiments, multi-specific antibodies may be prepared by the methods used by BIOATLA® and described in Unites States Publication No. US20150252119, the contents of which are herein incorporated by reference in their entirety. In one approach, the variable domains of two parent antibodies, wherein the parent antibodies are monoclonal antibodies are evolved using any method known in the art in a manner that allows a single light chain to functionally complement heavy chains of two different parent antibodies. Another approach requires evolving the heavy chain of a single parent antibody to recognize a second target antigen. A third approach involves evolving the light chain of a parent antibody so as to recognize a second target antigen. Methods for polypeptide evolution are described in International Publication WO2012009026, the contents of which are herein incorporated by reference in their entirety, and include as non-limiting examples, Comprehensive Positional Evolution (CPE), Combinatorial Protein Synthesis (CPS), Comprehensive Positional Insertion (CPI), Comprehensive Positional Deletion (CPD), or any combination thereof. The Fc region of the multi-specific antibodies described in United States Publication No. US20150252119 may be created using a knob-in-hole approach, or any other method that allows the Fc domain to form heterodimers. The resultant multi-specific antibodies may be further evolved for improved characteristics or properties such as binding affinity for the target antigen.Bispecific antibodies

[0397] In some embodiments, the cargo or payload may be or may encode bispecific antibodies. As used herein, the term “bispecific antibody” refers to an antibody capable of binding two different antigens. Such antibodies typically comprise regions from at least two different antibodies. Such antibodies typically comprise antigen-binding regions from at least two different antibodies. For example, a bispecific monoclonal antibody (BsMAb, BsAb) is an artificial protein composed of fragments of two different monoclonal antibodies, thus allowing the BsAb to bind to two different types of antigen.

[0398] In some cases, the cargo or payload may be or may encode bispecific antibodies comprising antigen-binding regions from two different anti-tau antibodies. For example, such bispecific antibodies may comprise binding regions from two different antibodies

[0399] Bispecific antibody frameworks may include any of those described in Riethmuller, G., 2012. Cancer Immunity. 12: 12-18; Marvin, J.S. et al., 2005. Acta Pharmacologica Sinica. 26(6):649-58; and Schaefer, W. et al., 2011. PNAS. 108(27): 11187-92, the contents of each of which are herein incorporated by reference in their entirety.

[0400] New generations of BsMAb, called “trifunctional bispecific” antibodies, have been developed. These consist of two heavy and two light chains, one each from two different antibodies, where the two Fab regions (the arms) are directed against two antigens, and the Fc region (the foot) comprises the two heavy chains and forms the third binding site.

[0401] Of the two paratopes that form the tops of the variable domains of a bispecific antibody, one can be directed against a target antigen and the other against a T-lymphocyte antigen like CD3. In the case of trifunctional antibodies, the Fc region may additionally bind to a cell that expresses Fc receptors, like a macrophage, a natural killer (NK) cell or a dendritic cell. In sum, the targeted cell is connected to one or two cells of the immune system, which subsequently destroy it.

[0402] Other types of bispecific antibodies have been designed to overcome certain problems, such as short half-life, immunogenicity and side-effects caused by cytokine liberation. They include chemically linked Fabs, consisting only of the Fab regions, and various types of bivalent and trivalent single-chain variable fragments (scFvs), fusion proteins mimicking the variable domains of two antibodies. The furthest developed of these newer formats are the bi-specific T-cell engagers (BiTEs) and mAb2's, antibodies engineered to contain an Fcab antigen-binding fragment instead of the Fc constant region.

[0403] Using molecular genetics, two scFvs can be engineered in tandem into a single polypeptide, separated by a linker domain, called a “tandem scFv” (tascFv). TascFvs have been found to be poorly soluble and require refolding when produced in bacteria, or they may be manufactured in mammalian cell culture systems, which avoids refolding requirements but may result in poor yields. Constructionof a tascFv with genes for two different scFvs yields a “bispecific single-chain variable fragments” (bis-scFvs). Only two tascFvs have been developed clinically by commercial firms; both are bispecific agents in active early phase development by Micromet for oncologic indications and are described as “Bispecific T-cell Engagers (BiTE)”. Blinatumomab is an anti-CD19 / anti-CD3 bispecific tascFv that potentiates T-cell responses to B-cell non-Hodgkin lymphoma in Phase 2. MT110 is an anti-EP-CAM / anti-CD3 bispecific tascFv that potentiates T-cell responses to solid tumors in Phase 1. Bispecific, tetravalent “TandAbs” are also being researched by Affimed.

[0404] In some embodiments, the cargo or payload may be or may encode antibodies comprising a single antigen-binding domain. These molecules are extremely small, with molecular weights approximately one-tenth of those observed for full-sized mAbs. Further antibodies may include “nanobodies” derived from the antigen-binding variable heavy chain regions (VHHs) of heavy chain antibodies found in camels and llamas, which lack light chains.

[0405] Disclosed and claimed in PCT Publication WO2014144573 (the contents of which are herein incorporated by reference in its entirety) to Memorial Sloan-Kettering Cancer Center are multimerization technologies for making dimeric multispecific binding agents (e.g., fusion proteins comprising antibody components) with improved properties over multispecific binding agents without the capability of dimerization.

[0406] In some cases, the cargo or payload may be or may encode tetravalent bispecific antibodies (TetBiAbs as disclosed and claimed in PCT Publication WO2014144357, the contents of which are herein incorporated in its entirety). TetBiAbs feature a second pair of Fab fragments with a second antigen specificity attached to the C-terminus of an antibody, thus providing a molecule that is bivalent for each of the two antigen specificities. The tetravalent antibody is produced by genetic engineering methods, by linking an antibody heavy chain covalently to a Fab light chain, which associates with its cognate, co-expressed Fab heavy chain.

[0407] In some aspects, the cargo or payload may be or may encode biosynthetic antibodies as described in U.S. Patent No. 5,091,513 (the contents of which are herein incorporated by reference in their entirety). Such antibody may include one or more sequences of amino acids constituting a region which behaves as a biosynthetic antibody binding site (BABS). The sites comprise 1) non- covalently associated or disulfide bonded synthetic VH and VL dimers, 2) VH-VL or VL-VH single chains wherein the VH and VL are attached by a polypeptide linker, or 3) individuals VH or VL domains. The binding domains comprise linked CDR and FR regions, which may be derived from separate immunoglobulins. The biosynthetic antibodies may also include other polypeptide sequences which function, e.g., as an enzyme, toxin, binding site, or site of attachment to an immobilization media or radioactive atom. Methods are disclosed for producing the biosynthetic antibodies, for designing BABS having any specificity that can be elicited by in vivo generation ofantibody, and for producing analogs thereof.

[0408] In some embodiments, the cargo or payload may be or may encode antibodies with antibody acceptor frameworks taught in U.S. Patent No. 8,399,625. Such antibody acceptor frameworks may be particularly well suited accepting CDRs from an antibody of interest. In some cases, CDRs from anti-tau antibodies known in the art or developed according to the methods presented herein may be used.Miniaturized Antibody

[0409] In some embodiments, the cargo or payload may be or may encode a “miniaturized” antibody. Among the best examples of mAb miniaturization are the small modular immunopharmaceuticals (SMIPs) from Trubion Pharmaceuticals. These molecules, which can be monovalent or bivalent, are recombinant single-chain molecules containing one VL, one VH antigen-binding domain, and one or two constant “effector” domains, all connected by linker domains. Presumably, such a molecule might offer the advantages of increased tissue or tumor penetration claimed by fragments while retaining the immune effector functions conferred by constant domains. At least three “miniaturized” SMIPs have entered clinical development. TRU- 015, an anti-CD20 SMIP developed in collaboration with Wyeth, is the most advanced project, having progressed to Phase 2 for rheumatoid arthritis (RA). Earlier attempts in systemic lupus erythrematosus (SLE) and B cell lymphomas were ultimately discontinued. Trubion and Facet Biotechnology are collaborating in the development of TRU-016, an anti-CD37 SMIP, for the treatment of CLL and other lymphoid neoplasias, a project that has reached Phase 2. Wyeth has licensed the anti-CD20 SMIP SBI-087 for the treatment of autoimmune diseases, including RA, SLE, and possibly multiple sclerosis, although these projects remain in the earliest stages of clinical testing.Diabodies

[0410] In some embodiments, the cargo or payload may be or may encode diabodies. As used herein, the term "diabody" refers to a small antibody fragment with two antigen-binding sites. Diabodies comprise a heavy chain variable domain VH connected to a light chain variable domain VL in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0411] Diabodies are functional bispecific single-chain antibodies (bscAb). These bivalent antigen-binding molecules are composed of non-covalent dimers of scFvs, and can be produced in mammalian cells using recombinant methods. (See, e.g., Mack et al, Proc. Natl. Acad. Sci., 92: 7021-7025, 1995). Few diabodies have entered clinical development. An iodine-123-labeled diabody version of the anti-CEA chimeric antibody cT84.66 has been evaluated for pre-surgicalimmunoscintigraphic detection of colorectal cancer in a study sponsored by the Beckman Research Institute of the City of Hope (Clinicaltrials.gov NCT00647153).Unibody

[0412] In some embodiments, the cargo or payload may be or may encode a “unibody,” in which the hinge region has been removed from IgG4 molecules. While IgG4 molecules are unstable and can exchange light-heavy chain heterodimers with one another, deletion of the hinge region prevents heavy chain-heavy chain pairing entirely, leaving highly specific monovalent light / heavy heterodimers, while retaining the Fc region to ensure stability and half-life in vivo. This configuration may minimize the risk of immune activation or oncogenic growth, as IgG4 interacts poorly with FcRs and monovalent unibodies fail to promote intracellular signaling complex formation. These contentions are, however, largely supported by laboratory, rather than clinical, evidence. Other antibodies may be “miniaturized” antibodies, which are compacted 100 kDa antibodies.Intrabodies

[0413] In some embodiments, the cargo or payload may be or may encode intrabodies. The term “intrabody” refers to a form of antibody that is not secreted from a cell in which it is produced, but instead targets one or more intracellular proteins. Intrabodies may be used to affect a multitude of cellular processes including, but not limited to intracellular trafficking, transcription, translation, metabolic processes, proliferative signaling, and cell division. In some embodiments, methods of the present disclosure may include intrabody -based therapies. In some such embodiments, variable domain sequences and / or CDR sequences disclosed herein may be incorporated into one or more constructs for intrabody-based therapy. For example, intrabodies may target one or more glycated intracellular proteins or may modulate the interaction between one or more glycated intracellular proteins and an alternative protein.

[0414] More than two decades ago, intracellular antibodies against intracellular targets were first described (Biocca, Neuberger and Cattaneo EMBO J. 9: 101-108, 1990, the contents of which are herein incorporated by reference in their entirety). The intracellular expression of intrabodies in different compartments of mammalian cells allows blocking or modulation of the function of endogenous molecules (Biocca, et al., EMBO J. 9: 101-108, 1990; Colby et al., Proc. Natl. Acad. Sci. U.S.A. 101: 17616-21, 2004, the contents of which are herein incorporated by reference in their entirety). Intrabodies can alter protein folding, protein-protein, protein-DNA, protein-RNA interactions and protein modification. They can induce a phenotypic knockout and work as neutralizing agents by direct binding to the target antigen, by diverting its intracellular trafficking or by inhibiting its association with binding partners. They have been largely employed as researchtools and are emerging as therapeutic molecules for the treatment of human diseases such as viral pathologies, cancer and misfolding diseases. The fast-growing bio-market of recombinant antibodies provides intrabodies with enhanced binding specificity, stability, and solubility, together with lower immunogenicity, for their use in therapy.

[0415] In some embodiments, intrabodies have advantages over interfering RNA (iRNA); for example, iRNA has been shown to exert multiple non-specific effects, whereas intrabodies have been shown to have high specificity and affinity to target antigens. Furthermore, as proteins, intrabodies possess a much longer active half-life than iRNA. Thus, when the active half-life of the intracellular target molecule is long, gene silencing through iRNA may be slow to yield an effect, whereas the effects of intrabody expression can be almost instantaneous. Lastly, it is possible to design intrabodies to block certain binding interactions of a particular target molecule, while sparing others.

[0416] Intrabodies are often single chain variable fragments (scFvs) expressed from a recombinant nucleic acid molecule and engineered to be retained intracellularly (e.g., retained in the cytoplasm, endoplasmic reticulum, or periplasm). Intrabodies may be used, for example, to ablate the function of a protein to which the intrabody binds. The expression of intrabodies may also be regulated through the use of inducible promoters in the nucleic acid expression vector comprising the intrabody. Intrabodies may be produced using methods known in the art, such as those disclosed and reviewed in: Marasco et al., 1993 Proc. Natl. Acad. Sci. USA, 90: 7889-7893; Chen et al., 1994, Hum. Gene Then 5:595-601; Chen et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 5932-5936; Maciejewski et al., 1995, Nature Med., 1: 667-673; Marasco, 1995, Immunotech, 1: 1-19; Mhashilkar, et al., 1995, EMBO J. 14: 1542-51; Chen et al., 1996, Hum. Gene Therap., 7: 1515-1525; Marasco, Gene Ther. 4: 11-15, 1997; Rondon and Marasco, 1997, Annu. Rev. Microbiol. 51:257-283; Cohen, et al., 1998, Oncogene 17:2445-56; Probaet al., 1998, J. Mol. Biol. 275:245-253; Cohen et al., 1998, Oncogene 17:2445-2456; Hassanzadeh, et al., 1998, FEBS Lett. 437:81-6; Richardson et al., 1998, Gene Ther. 5:635-44; Ohage and Steipe, 1999, J. Mol. Biol. 291:1119-1128; Ohage et al., 1999, J. Mol. Biol. 291: 1129-1134; Wirtz and Steipe, 1999, Protein Sci. 8:2245-2250; Zhu et al., 1999, J. Immunol. Methods 231:207-222; Arafat et al., 2000, Cancer Gene Ther. 7:1250-6; der Maur et al., 2002, J. Biol. Chem. 277:45075-85; Mhashilkar et al., 2002, Gene Ther. 9:307-19; and Wheeler et al., 2003, FASEB J. 17: 1733-5; and references cited therein). In particular, a CCR5 intrabody has been produced by Steinberger et al., 2000, Proc. Natl. Acad. Sci. USA 97:805-810). See generally Marasco, WA, 1998, "Intrabodies: Basic Research and Clinical Gene Therapy Applications" Springer: New York; and for a review of scFvs, see Pluckthun in “The Pharmacology of Monoclonal Antibodies,” 1994, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315; the contents of each of which are eachincorporated by reference in their entireties.

[0417] Sequences from donor antibodies may be used to develop intrabodies. Intrabodies are often recombinantly expressed as single domain fragments such as isolated VH and VL domains or as a single chain variable fragment (scFv) antibody within the cell. For example, intrabodies are often expressed as a single polypeptide to form a single chain antibody comprising the variable domains of the heavy and light chains joined by a flexible linker polypeptide. Intrabodies typically lack disulfide bonds and are capable of modulating the expression or activity of target genes through their specific binding activity. Single chain antibodies can also be expressed as a single chain variable region fragment joined to the light chain constant region.

[0418] As is known in the art, an intrabody can be engineered into recombinant polynucleotide vectors to encode sub-cellular trafficking signals at its N or C terminus to allow expression at high concentrations in the sub-cellular compartments where a target protein is located. For example, intrabodies targeted to the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and, optionally, a C-terminal ER retention signal. Intrabodies intended to exert activity in the nucleus are engineered to include a nuclear localization signal. Lipid moi eties are joined to intrabodies in order to tether the intrabody to the cytosolic side of the plasma membrane. Intrabodies can also be targeted to exert function in the cytosol. For example, cytosolic intrabodies are used to sequester factors within the cytosol, thereby preventing them from being transported to their natural cellular destination.

[0419] There are certain technical challenges with intrabody expression. In particular, protein conformational folding and structural stability of the newly-synthesized intrabody within the cell is affected by reducing conditions of the intracellular environment.

[0420] Intrabodies may be promising therapeutic agents for the treatment of misfolding diseases, including Tauopathies, prion diseases, Alzheimer's, Parkinson's, and Huntington's, because of their virtually infinite ability to specifically recognize the different conformations of a protein, including pathological isoforms, and because they can be targeted to the potential sites of aggregation (both intra- and extracellular sites). These molecules can work as neutralizing agents against amyloidogenic proteins by preventing their aggregation, and / or as molecular shunters of intracellular traffic by rerouting the protein from its potential aggregation site.Maxibodies

[0421] In some embodiments, the cargo or payload may be or may encode a maxibody (bivalent scFV fused to the amino terminus of the Fc (CH2-CH3 domains) of IgG.Chimeric Antigen Receptors (CARs)

[0422] In some embodiments, the cargo or payload may be or may encode a chimeric antigen receptors (CARs) which when transduced into immune cells (e.g., T cells and NK cells), can re-direct the immune cells against the target (e.g., a tumor cell) which expresses a molecule recognized by the extracellular target moiety of the CAR.

[0423] As used herein, the term “chimeric antigen receptor (CAR)” refers to a synthetic receptor that mimics TCR on the surface of T cells. In general, a CAR is composed of an extracellular targeting domain, a transmembrane domain / region and an intracellular signaling / activation domain. In a standard CAR receptor, the components: the extracellular targeting domain, transmembrane domain and intracellular signaling / activation domain, are linearly constructed as a single fusion protein. The extracellular region comprises atargeting domain / moiety (e.g., a scFv) that recognizes a specific tumor antigen or other tumor cell-surface molecules. The intracellular region may contain a signaling domain of TCR complex (e.g., the signal region of CD3Q, and / or one or more costimulatory signaling domains, such as those from CD28, 4-1BB (CD137) and OX- 40 (CD 134). For example, a “first-generation CAR” only has the CD3^ signaling domain, whereas in an effort to augment T-cell persistence and proliferation, costimulatory intracellular domains are added, giving rise to second generation CARs having a CD3^signal domain plus one costimulatory signaling domain, and third generation CARs having CD3^ signal domain plus two or more costimulatory signaling domains. A CAR, when expressed by a T cell, endows the T cell with antigen specificity determined by the extracellular targeting moiety of the CAR. In some aspects, one or more elements such as homing and suicide genes can be added to develop a more competent and safer architecture of CAR (so called the fourth generation CAR).

[0424] In some embodiments, the extracellular targeting domain is j oined through the hinge (also called space domain or spacer) and transmembrane regions to an intracellular signaling domain. The hinge connects the extracellular targeting domain to the transmembrane domain which transverses the cell membrane and connects to the intracellular signaling domain. The hinge may need to be varied to optimize the potency of CAR transformed cells toward cancer cells due to the size of the target protein where the targeting moiety binds, and the size and affinity of the targeting domain itself. Upon recognition and binding of the targeting moiety to the target cell, the intracellular signaling domain leads to an activation signal to the CAR T cell, which is further amplified by the “second signal” from one or more intracellular costimulatory domains. The CAR T cell, once activated, can destroy the target cell.

[0425] In some embodiments, the CAR may be split into two parts, each part is linked a dimerizing domain, such that an input that triggers the dimerization promotes assembly of the intact functional receptor. Wu and Lim reported a split CAR in which the extracellular CD 19 binding domain and the intracellular signaling element are separated and linked to the FKBP domain and the FRB* (T2089L mutant of FKBP-rapamycin binding) domain that heterodimerize in the presence of the rapamycin analog AP21967. The split receptor is assembled in the presenceof AP21967 and together with the specific antigen binding, activates T cells (Wu et al., Science, 2015, 625(6258): aab4077, the contents of which are herein incorporated by reference in its entirety).

[0426] In some embodiments, the CAR may be designed as an inducible CAR which has an incorporation of aTet-On inducible system to a CD 19 CAR construct. The CD19 CAR is activated only in the presence of doxycycline (Dox). Sakemura reported that Tet-CD19CAR T cells in the presence of Dox were equivalently cytotoxic against CD 19+ cell lines and had equivalent cytokine production and proliferation upon CD 19 stimulation, compared with conventional CD19CAR T cells (Sakemura et al., Cancer Immuno. Res., 2016, Jun 21, Epub; the contents of which is herein incorporated by reference in its entirety). The dual systems provide more flexibility to tum-on and off of the CAR expression in transduced T cells.

[0427] In some embodiments, the cargo or payload may be or may encode a first generation CAR, or a second generation CAR, or a third generation CAR, or a fourth generation CAR. In some embodiments, the cargo or payload may be or may encode a full CAR construct composed of the extracellular domain, the hinge and transmembrane domain and the intracellular signaling region. In other embodiments, the cargo or payload may be or may encode a component of the full CAR construct including an extracellular targeting moiety, a hinge region, a transmembrane domain, an intracellular signaling domain, one or more co-stimulatory domain, and other additional elements that improve CAR architecture and functionality including but not limited to a leader sequence, a homing element and a safety switch, or the combination of such components.

[0428] In some embodiments, the cargo or payload may be or may encode a tunable CARs. The reversible on-off switch mechanism allows management of acute toxicity caused by excessive CAR- T cell expansion. The ligand conferred regulation of the CAR may be effective in offsetting tumor escape induced by antigen loss, avoiding functional exhaustion caused by tonic signaling due to chronic antigen exposure and improving the persistence of CAR expressing cells in vivo. The tunable CAR may be utilized to down regulate CAR expression to limit on target on tissue toxicity caused by tumor lysis syndrome. Down regulating the expression of the CARs following anti-tumor efficacy may prevent (1) On target off tumor toxicity caused by antigen expression in normal tissue. (2) antigen independent activation in vivo.Extracellular targeting domain / moiety

[0429] In some embodiments, the extracellular target moiety of a CAR may be any agent that recognizes and binds to a given target molecule, for example, a neoantigen on tumor cells, with high specificity and affinity. The target moiety may be an antibody and variants thereof that specifically binds to a target molecule on tumor cells, or a peptide aptamer selected from a random sequence pool based on its ability to bind to the target molecule on tumor cells, or a variant or fragment thereofthat can bind to the target molecule on tumor cells, or an antigen recognition domain from native T- cell receptor (TCR) (e.g. CD4 extracellular domain to recognize HIV infected cells), or exotic recognition components such as a linked cytokine that leads to recognition of target cells bearing the cytokine receptor, or a natural ligand of a receptor.

[0430] In some embodiments, the targeting domain of a CAR may be a Ig NAR, a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a F(ab)'3 fragment, Fv, a single chain variable fragment (scFv), a bis-scFv, a (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), a unibody, a nanobody, or an antigen binding region derived from an antibody that specifically recognizes a target molecule, for example a tumor specific antigen (TSA). In one embodiment, the targeting moiety is a scFv antibody. The scFv domain, when it is expressed on the surface of a CAR T cell and subsequently binds to a target protein on a cancer cell, is able to maintain the CAR T cell in proximity to the cancer cell and to trigger the activation of the T cell. A scFv can be generated using routine recombinant DNA technology techniques and is discussed in the present disclosure.

[0431] In some embodiments, the targeting moiety of a CAR construct may be an aptamer such as a peptide aptamer that specifically binds to a target molecule of interest. The peptide aptamer may be selected from a random sequence pool based on its ability to bind to the target molecule of interest.

[0432] In some embodiments, the targeting moiety of a CAR construct may be a natural ligand of the target molecule, or a variant and / or fragment thereof capable of binding the target molecule. In some aspects, the targeting moiety of a CAR may be a receptor of the target molecule, for example, a full length human CD27, as a CD70 receptor, may be fused in frame to the signaling domain of CD3 forming a CD27 chimeric receptor as an immunotherapeutic agent for CD70- positive malignancies.

[0433] In some embodiments, the targeting moiety of a CAR may recognize a tumor specific antigen (TSA), for example a cancer neoantigen which is restrictedly expressed on tumor cells.

[0434] As non-limiting examples, the CAR of the present disclosure may comprise the extracellular targeting domain capable of binding to a tumor specific antigen selected from 5T4, 707-AP, A33, AFP (a-fetoprotein), AKAP-4 ( A kinase anchor protein 4), ALK, a5pi-integrin, androgen receptor, annexin II, alpha- actinin-4, ART-4, Bl, B7H3, B7H4, BAGE (B melanoma antigen), BCMA, BCR-ABL fusion protein, beta-catenin, BKT-antigen, BTAA, CA-I (carbonic anhydrase I), CA50 (cancer antigen 50), CA125, CA15-3, CA195, CA242, calretinin, CAIX (carbonic anhydrase), CAMEL (cytotoxic T-lymphocyte recognized antigen on melanoma), CAM43, CAP-1, Caspase-8 / m, CD4, CD5, CD7, CD19, CD20, CD22, CD23, CD25, CD27 / m, CD28, CD30, CD33, CD34, CD36, CD38, CD40 / CD154, CD41, CD44v6, CD44v7 / 8, CD45,CD49f, CD56, CD68\KP1, CD74, CD79a / CD79b, CD103, CD123, CD133, CD138,CD171, cdc27 / m, CDK4 (cyclin dependent kinase 4), CDKN2A, CDS, CEA (carcinoembryonic antigen), CEACAM5, CEACAM6, chromogranin, c-Met, c-Myc, coa-1, CSAp, CT7, CT10, cyclophilin B, cyclin Bl, cytoplasmic tyrosine kinases, cytokeratin, DAM- 10, DAM-6, dek-can fusion protein, desmin, DEPDC1 (DEP domain containing 1), E2A-PRL, EBN A, EGF-R (epidermal growth factor receptor), EGP-1 (epithelial glycoprotein -1) (TROP-2), EGP-2, EGP-40, EGFR (epidermal growth factor receptor), EGFRvIII, EF-2, ELF2M, EMMPRIN, EpCAM (epithelial cell adhesion molecule), EphA2, Epstein Barr virus antigens, Erb (ErbBl; ErbB3; ErbB4), ETA (epithelial tumor antigen), ETV6-AML1 fusion protein, FAP (fibroblast activation protein), FBP (folate-binding protein), FGF-5, folate receptor, FOS related antigen 1, fucosyl GM1, G250, GAGE (GAGE-1; GAGE-2), galectin, GD2 (ganglioside), GD3, GFAP (glial fibrillary acidic protein), GM2 (oncofetal antigen-immunogenic- 1; OFA-I-1), GnT-V, GplOO, H4- RET, HAGE (helicase antigen), HER-2 / neu, HIFs (hypoxia inducible factors), HIF-1, HIF-2, HLA-A2, HLA-A*0201-R170I, HLA-A1 1, HMWMAA, Hom / Mel-40, HSP70-2M (Heat shock protein 70), HST-2, HTgp-175, hTERT (or hTRT), human papillomavirus-E6 / human papillomavirus-E7 and E6, iCE (immune-capture EIA), IGF-1R, IGH-IGK, IL-2R, IL-5, ILK (integrin-linked kinase), IMP3 (insulin-like growth factor II mRNA-binding protein 3), IRF4 (interferon regulatory factor 4), KDR (kinase insert domain receptor), KIAA0205, KRAB-zinc finger protein (KID)-3; KID31, KSA (17-1A), K-ras, LAGE, LCK, LDLR / FUT (LDLR- fucosyltransferaseAS fusion protein), LeY (Lewis Y), MAD-CT-1, MAGE (tyrosinase, melanoma-associated antigen) (MAGE-1; MAGE-3), melan-A tumor antigen (MART), MART- 2 / Ski, MC1R (melanocortin 1 receptor), MDM2, mesothelin, MPHOSPH1, MSA(muscle-specific actin), mTOR (mammalian targets of rapamycin), MUC-1, MUC-2, MUM-1 (melanoma associated antigen (mutated) 1), MUM-2, MUM-3, Myosin / m, MYL-RAR, NA88-A, N- acetylglucosaminyltransferase, neo-PAP, NF-KB (nuclear factor-kappa B), neurofilament, NSE (neuron- specific enolase), Notch receptors, NuMa, N-Ras, NY-BR-1, NY- CO-1, NY-ESO-1, Oncostatin M, OS-9, OY-TES1, p53 mutants, p!90 minor bcr-abl, pl5(58), pl85erbB2, pl80erbB- 3, PAGE (prostate associated gene), PAP (prostatic acid phosphatase), PAX3, PAX5, PDGFR (platelet derived growth factor receptor), cytochrome P450 involved in piperidine and pyrrolidine utilization (PIPA), Pml-RAR alpha fusion protein, PR-3 (proteinase 3), PSA (prostate specific antigen), PSM, PSMA (Prostate stem cell antigen), PRAME (preferentially expressed antigen of melanoma), PTPRK, RAGE (renal tumor antigen), Raf (A-Raf, B-Raf and C-Raf), Ras, receptor tyrosine kinases, RCAS1, RGSS, ROR1 (receptor tyrosine kinase-like orphan receptor 1), RU1, RU2, SAGE, SART-1, SART-3, SCP-1, SDCCAG16, SP-17 (sperm protein 17), src-family, SSX (synovial sarcoma X breakpoint)-!, SSX-2(HOM-MEL-40), SSX-3, SSX-4, SSX-5, STAT-3, STAT-5, STAT-6, STEAD, STn, survivin, syk-ZAP70, TA-90 (Mac-2 binding protein\cyclophilinC-associated protein), TAAL6, TACSTD1 (tumor associated calcium signal transducer 1), TACSTD2, TAG-72-4, TAGE, TARP (T cell receptor gamma alternate reading frame protein), TEL / AML1 fusion protein, TEM1, TEM8 (endosialin or CD248), TGF , TIE2, TLP, TMPRSS2 ETS fusion gene, TNF-receptor (TNF-a receptor, TNF- receptor; or TNF-y receptor), transferrin receptor, TPS, TRP-1 (tyrosine related protein 1), TRP-2, TRP-2 / INT2, TSP-180, VEGF receptor, WNT, WT-1 (Wilm’s tumor antigen) and XAGE.

[0435] In some embodiments, the cargo or payload may be or may encode a CAR which comprises a universal immune receptor which has a targeting moiety capable of binding to a labelled antigen.

[0436] In some embodiments, the cargo or payload may be or may encode a CAR which comprises a targeting moiety capable of binding to a pathogen antigen.

[0437] In some embodiments, the cargo or payload may be or may encode a CAR which comprises a targeting moiety capable of binding to non-protein molecules such as tumor- associated glycolipids and carbohydrates.

[0438] In some embodiments, the cargo or payload may be or may encode a CAR which comprises a targeting moiety capable of binding to a component within the tumor microenvironment including proteins expressed in various tumor stroma cells including tumor associated macrophages (TAMs), immature monocytes, immature dendritic cells, immunosuppressive CD4+CD25+ regulatory T cells (Treg) and MDSCs.

[0439] In some embodiments, the cargo or payload may be or may encode a CAR which comprises a targeting moiety capable of binding to a cell surface adhesion molecule, a surface molecule of an inflammatory cell that appears in an autoimmune disease, or a TCR causing autoimmunity. As non-limiting examples, the targeting moiety of the present disclosure may be a scFv antibody that recognizes a tumor specific antigen (TSA), for example scFvs of antibodies SS, SSI and HN1 that specifically recognize and bind to human mesothelin, scFv of antibody of GD2, a CD 19 antigen binding domain, aNKG2D ligand binding domain, human anti-mesothelin scFvs, an anti-CSl binding agent, an anti-BCMA binding domain, anti-CD19 scFv antibody, GFR alpha 4 antigen binding fragments, anti-CLL-1 (C-type lectin-like molecule 1) binding domains, CD33 binding domains, a GPC3 (glypican-3) binding domain, a GFR alpha4 (Glycosylphosphatidylinositol (GPI)-linked GDNF family a -receptor 4 cell-surface receptor) binding domain, CD123 binding domains, an anti-RORl antibody or fragments thereof, scFvs specific to GPC-3, scFv for CSPG4, and scFv for folate receptor alpha.Intracellular signaling domains

[0440] The intracellular domain of a CAR fusion polypeptide, after binding to its target molecule, transmits a signal to the immune effector cell, activating at least one of the normal effectorfunctions of immune effector cells, including cytolytic activity (e.g., cytokine secretion) or helper activity. Therefore, the intracellular domain comprises an “intracellular signaling domain" of a T cell receptor (TCR).

[0441] In some aspects, the entire intracellular signaling domain can be employed. In other aspects, a truncated portion of the intracellular signaling domain may be used in place of the intact chain as long as it transduces the effector function signal.

[0442] In some embodiments, the intracellular signaling domain may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of IT AM containing cytoplasmic signaling sequences include those derived from TCR CD3zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In one example, the intracellular signaling domain is a CD3 zeta (CD3Q signaling domain.

[0443] In some embodiments, the intracellular region further comprises one or more costimulatory signaling domains which provide additional signals to the immune effector cells. These costimulatory signaling domains, in combination with the signaling domain can further improve expansion, activation, memory, persistence, and tumor-eradicating efficiency of CAR engineered immune cells (e.g., CAR T cells). In some cases, the costimulatory signaling region contains 1, 2, 3, or 4 cytoplasmic domains of one or more intracellular signaling and / or costimulatory molecules. The costimulatory signaling domain may be the intracellular / cytoplasmic domain of a costimulatory molecule, including but not limited to CD2, CD7, CD27, CD28, 4-1BB (CD137), 0X40 (CD134), CD30, CD40, ICOS (CD278), GITR (glucocorticoid-induced tumor necrosis factor receptor), LFA-1 (lymphocyte function- associated antigen- 1), LIGHT, NKG2C, B7-H3. In one example, the costimulatory signaling domain is derived from the cytoplasmic domain of CD28. In another example, the costimulatory signaling domain is derived from the cytoplasmic domain of 4-1BB (CD137). In another example, the costimulatory signaling domain may be an intracellular domain of GITR as taught in U.S. Pat. NO.: 9, 175, 308; the contents of which are incorporated herein by reference in its entirety.

[0444] In some embodiments, the intracellular region may comprise a functional signaling domain from a protein selected from the group consisting of an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation protein (SLAM) such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, CD2F- 10, SLAMF6, SLAMF7, an activating NK cell receptor, BTLA, a Toll ligand receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CDlla / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, IL-15Ra, ITGA4, VLA1, CD49a, ITGA4, IA4,CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDl lb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, NKD2C SLP76, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, CD270 (HVEM), GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds with CD83, DAP 10, TRIM, ZAP70, Killer immunoglobulin receptors (KIRs) such as KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3, and KIR2DP1; lectin related NK cell receptors such as Ly49, Ly49A, and Ly49C.

[0445] In some embodiments, the intracellular signaling domain of the present disclosure may contain signaling domains derived from JAK-STAT. In other embodiments, the intracellular signaling domain of the present disclosure may contain signaling domains derived from DAP-12 (Death associated protein 12) (Topfer et al., Immunol., 2015, 194: 3201-3212; and Wang et al., Cancer Immunol., 2015, 3: 815-826). DAP-12 is a key signal transduction receptor in NK cells. The activating signals mediated by DAP-12 play important roles in triggering NK cell cytotoxicity responses toward certain tumor cells and virally infected cells. The cytoplasmic domain of DAP12 contains an Immunoreceptor Tyrosine-based Activation Motif (ITAM). Accordingly, a CAR containing a DAP12-derived signaling domain may be used for adoptive transfer of NK cells.Transmembrane domains

[0446] In some embodiments, the CAR may comprise a transmembrane domain. As used herein, the term “Transmembrane domain (TM)” refers broadly to an amino acid sequence of about 15 residues in length which spans the plasma membrane. The transmembrane domain may include at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid residues and spans the plasma membrane. In some embodiments, the transmembrane domain may be derived either from a natural or from a synthetic source. The transmembrane domain of a CAR may be derived from any naturally membrane-bound or transmembrane protein. For example, the transmembrane region may be derived from (i.e., comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD3 epsilon, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD33, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, or CD154.

[0447] Alternatively, the transmembrane domain of the present disclosure may be synthetic. In some aspects, the synthetic sequence may comprise predominantly hydrophobic residues such as leucine and valine.

[0448] In some embodiments, the transmembrane domain may be selected from the groupconsisting of a CD8a transmembrane domain, a CD4 transmembrane domain, a CD 28 transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, and a human IgG4 Fc region.

[0449] In some embodiments, the CAR may comprise an optional hinge region (also called spacer). A hinge sequence is a short sequence of amino acids that facilitates flexibility of the extracellular targeting domain that moves the target binding domain away from the effector cell surface to enable proper cell / cell contact, target binding and effector cell activation. The hinge sequence may be positioned between the targeting moiety and the transmembrane domain. The hinge sequence can be any suitable sequence derived or obtained from any suitable molecule. The hinge sequence may be derived from all or part of an immunoglobulin (e.g., IgGl, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that falls between the CHI and CH2 domains of an immunoglobulin, e.g., an IgG4 Fc hinge, the extracellular regions of type 1 membrane proteins such as CD8a CD4, CD28 and CD7, which may be a wild-type sequence or a derivative. Some hinge regions include an immunoglobulin CH3 domain or both a CH3 domain and a CH2 domain. In certain embodiments, the hinge region may be modified from an IgGl, IgG2, IgG3, or IgG4 that includes one or more amino acid residues, for example, 1, 2, 3, 4 or 5 residues, substituted with an amino acid residue different from that present in an unmodified hinge.

[0450] In some embodiments, the CAR may comprise one or more linkers between any of the domains of the CAR. The linker may be between 1-30 amino acids long. In this regard, the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. In other embodiments, the linker may be flexible.

[0451] In some embodiments, the components including the targeting moiety, transmembrane domain and intracellular signaling domains may be constructed in a single fusion polypeptide. The fusion polypeptide may be the payload.

[0452] In some embodiments, the cargo or payload may be or may encode a CD 19 specific CAR targeting different B cell malignancies and HER2-specific CAR targeting sarcoma, glioblastoma, and advanced Her2 -positive lung malignancy. Tandem CAR (TanCAR)

[0453] In some embodiments, the CAR may be a tandem chimeric antigen receptor (TanCAR) which is able to target two, three, four, or more tumor specific antigens. In some aspects, The CAR is a bispecific TanCAR including two targeting domains which recognize two different TSAs on tumor cells. The bispecific TanCAR may be further defined as comprising an extracellular region comprising a targeting domain (e.g., an antigen recognition domain) specific for a first tumor antigen and a targeting domain (e.g., an antigen recognition domain) specific for a second tumor antigen. In other aspects, the CAR is a multispecific TanCAR that includes three or more targeting domains configured in a tandem arrangement. The space between the targeting domains in theTanCAR may be between about 5 and about 30 amino acids in length, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 amino acids.Split CAR

[0454] In some embodiments, the CAR components including the targeting moiety, transmembrane domain and intracellular signaling domains may be split into two or more parts such that it is dependent on multiple inputs that promote assembly of the intact functional receptor. As a non-limiting example, the split CAR consists of two parts that assemble in a small molecule-dependent manner; one part of the receptor features an extracellular antigen binding domain (e.g., scFv) and the other part has the intracellular signaling domains, such as the CD3^ intracellular domain.

[0455] In other aspects, the split parts of the CAR system can be further modified to increase signal. As a non-limiting example, the second part of cytoplasmic fragment may be anchored to the plasma membrane by incorporating a transmembrane domain (e.g., CD8a transmembrane domain) to the construct. An additional extracellular domain may also be added to the second part of the CAR system, for instance an extracellular domain that mediates homo-dimerization. These modifications may increase receptor output activity, i.e., T cell activation.

[0456] In some embodiments, the two parts of the split CAR system contain heterodimerization domains that conditionally interact upon binding of a heterodimerizing small molecule. As such, the receptor components are assembled in the presence of the small molecule, to form an intact system which can then be activated by antigen engagement. Any known heterodimerizing components can be incorporated into a split CAR system. Other small molecule dependent heterodimerization domains may also be used, including, but not limited to, gibberellin-induced dimerization system (GID 1 -GAI), trimethoprim-SLF induced ecDHFR and FKBP dimerization and ABA (abscisic acid) induced dimerization of PP2C and PYL domains. The dual regulation using inducible assembly (e.g., ligand dependent dimerization) and degradation (e.g., destabilizing domain induced CAR degradation) of the split CAR system may provide more flexibility to control the activity of the CAR modified T cells.Switchable CAR

[0457] In some embodiments, the CAR may be a switchable CAR which is a controllable CARs that can be transiently switched on in response to a stimulus (e.g., a small molecule). In this CAR design, a system is directly integrated in the hinge domain that separate the scFv domain from the cell membrane domain in the CAR. Such system is possible to split or combine different key functions of a CAR such as activation and costimulation within different chains of a receptor complex, mimicking the complexity of the TCR native architecture. This integrated system can switch the scFv and antigen interaction between on / off states controlled by the absence / presenceof the stimulus.Reversible CAR

[0458] In some embodiments, the CAR may be a reversible CAR system. In this CAR architecture, a LID domain (ligand-induced degradation) is incorporated into the CAR system. The CAR can be temporarily down-regulated by adding a ligand of the LID domain.Inhibitory CAR (iCAR)

[0459] In some embodiments, the CAR may be inhibitory CARs. Inhibitory CAR (iCAR) refers to a bispecific CAR design wherein a negative signal is used to enhance the tumor specificity and limit normal tissue toxicity. This design incorporates a second CAR having a surface antigen recognition domain combined with an inhibitory signal domain to limit T cell responsiveness even with concurrent engagement of an activating receptor. This antigen recognition domain is directed towards a normal tissue specific antigen such that the T cell can be activated in the presence of first target protein, but if the second protein that binds to the iCAR is present, the T cell activation is inhibited.

[0460] As a non-limiting example, iCARs against Prostate specific membrane antigen (PMSA) based on CTLA4 and PD1 inhibitory domains demonstrated the ability to selectively limit cytokine secretion, cytotoxicity and proliferation induced by T cell activation.Chimeric switch receptor

[0461] In some embodiments, the cargo or payload may be or may encode a chimeric switch receptors which can switch a negative signal to a positive signal. As used herein, the term “chimeric switch receptor” refers to a fusion protein comprising a first extracellular domain and a second transmembrane and intracellular domain, wherein the first domain includes a negative signal region, and the second domain includes a positive intracellular signaling region. In some aspects, the fusion protein is a chimeric switch receptor that contains the extracellular domain of an inhibitory receptor on T cell fused to the transmembrane and cytoplasmic domain of a costimulatory receptor. This chimeric switch receptor may convert a T cell inhibitory signal into a T cell stimulatory signal.

[0462] As a non-limiting example, the chimeric switch receptor may comprise the extracellular domain of PD-1 fused to the transmembrane and cytoplasmic domain of CD28. In some aspects, Extracellular domains of other inhibitory receptors such as CTLA-4, LAG-3, TIM-3, KIRs and BTLA may also be fused to the transmembrane and cytoplasmic domain derived from costimulatory receptors such as CD28, 4-1BB, CD27, 0X40, CD40, GTIR and ICOS.

[0463] In some embodiments, chimeric switch receptors may include recombinant receptors comprising the extracellular cytokine-binding domain of an inhibitory cytokine receptor (e.g., IL-13 receptor a (IL-13Ral), IL-10R, and IL-4Ra) fused to an intracellular signaling domain of a stimulatory cytokine receptor such as IL-2R (IL-2Ra, IL-2R and IL-2Rgamma) and IL-7Ra. One example of such chimeric cytokine receptor is a recombinant receptor containing the cytokinebinding extracellular domain of IL-4Ra linked to the intracellular signaling domain of IL-7Ra.

[0464] In some embodiments, the chimeric switch receptor may be a chimeric TGF receptor. The chimeric TGF receptor may comprise an extracellular domain derived from a TGF receptor such as TGF receptor 1 , TGF receptor 2, TGF receptor 3, or any other TGF receptor or variant thereof; and a non- TGF receptor intracellular domain. The non-TGFf> receptor intracellular domain may be the intracellular domain or fragment thereof derived from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CD28, 4-1BB (CD137), 0X40 (CD134), CD3zeta, CD40, CD27, or a combination thereof.Activation-conditional CAR

[0465] In some embodiments, the cargo or payload may be or may encode an activationconditional chimeric antigen receptor, which is only expressed in an activated immune cell. The expression of the CAR may be coupled to activation conditional control region which refers to one or more nucleic acid sequences that induce the transcription and / or expression of a sequence e.g., a CAR under its control. Such activation conditional control regions may be promoters of genes that are upregulated during the activation of the immune effector cell e.g., IL2 promoter or NF AT binding sites.CAR targeting to tumor cells with specific proteoglycan markers

[0466] In some embodiments, the cargo or payload may be or may encode a CAR that targets specific types of cancer cells. Human cancer cells and metastasis may express unique and otherwise abnormal proteoglycans, such as polysaccharide chains (e.g., chondroitin sulfate (CS), dermatan sulfate (DS or CSB), heparan sulfate (HS) and heparin). Accordingly, the CAR may be fused with a binding moiety that recognizes cancer associated proteoglycans. In one example, a CAR may be fused with VAR2CSA polypeptide (VAR2-CAR) that binds with high affinity to a specific type of chondroitin sulfate A (CSA) attached to proteoglycans. The extracellular ScFv portion of the CAR may be substituted with VAR2CSA variants comprising at least the minimal CSA binding domain, generating CARs specific to chondroitin sulfate A (CSA) modifications. Alternatively, the CAR may be fused with a split-protein binding system to generate a spy-CAR, in which the scFv portion of the CAR is substituted with one portion of a split-protein binding system such as SpyTag and Spy-catcher and the cancer-recognition molecules (e.g., scFv and or VAR2-CSA) are attached to the CAR through the split-protein binding system.Nucleic Acids

[0467] The lipid nanoparticles of the present disclosure may comprise a payload region (which may also be referred to as a cargo region) which is a nucleic acid. The term “nucleic acid,” in its broadest sense, includes any compound and / or substance that comprise a polymer of nucleotides which may be referred to as polynucleotides. Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof.

[0468] In some embodiments, the payload region comprises nucleic acid sequences encoding more than one cargo or payload.

[0469] In some embodiments, the payload region may be or encode a coding nucleic acid sequence.

[0470] In some embodiments, the payload region may be or encode a non-coding nucleic acid sequence.

[0471] In some embodiments, the payload region may be or encode both a coding and anon-coding nucleic acid sequence.DNA

[0472] Deoxyribonucleic acid (DNA) is a molecule that carries genetic information for all living things and consists of two strands that wind around one another to form a shape known as a double helix. Each strand has a backbone made of alternating sugar (deoxyribose) and phosphate groups. Attached to each sugar is one of four bases: adenine (A), cytosine (C), guanine (G), and thymine (T). The two strands are held together by bonds between adenine and thymine or cytosine and guanine. The sequence of the bases along the backbones serves as instructions for assembling protein and RNA molecules.

[0473] In some embodiments, the payload region may be or encode a coding DNA.

[0474] In some embodiments, the payload region may be or encode a non-coding DNA.

[0475] In some embodiments, the payload region may be or encode both a coding and a noncoding DNA.

[0476] In some embodiments, the DNA may be modified. Types of modifications include, but are not limited to, methylation, acetylation, phosphorylation, ubiquitination, and sumoylation.Vectors

[0477] In some embodiments, the originator constructs and / or benchmark constructs described herein can be or be encoded by vectors such as plasmids or viral vectors. In some embodiments, the originator constructs and / or benchmark constructs are or are encoded by viral vectors. Viral vectors may be, but are not limited to, Herpesvirus (HSV) vectors, retroviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors, lentiviral vectors, and the like. In someembodiments, the viral vectors are AAV vectors. In some embodiments, the viral vectors are lentiviral vectors. In some embodiments, the viral vectors are retroviral vectors. In some embodiments, the viral vectors are adenoviral vectors.Adeno-Associated Viral (AAVs) Vectors

[0478] Viruses of the Parvoviridae family are small non-enveloped icosahedral capsid viruses characterized by a single stranded DNA genome. Parvoviridae family viruses consist of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. Due to its relatively simple structure, easily manipulated using standard molecular biology techniques, this virus family is useful as a biological tool. The genome of the virus may be modified to contain a minimum of components for the assembly of a functional recombinant virus, or viral particle, which is loaded with or engineered to express or deliver a desired payload, which may be delivered to a target cell, tissue, organ, or organism.

[0479] The Parvoviridae family comprises the Dependovirus genus which includes adeno- associated viruses (AAV) capable of replication in vertebrate hosts including, but not limited to, human, primate, bovine, canine, equine, and ovine species.

[0480] The AAV vector genome is a linear, single-stranded DNA (ssDNA) molecule approximately 5,000 nucleotides (nts) in length. The AAV vector genome can comprise a payload region and at least one inverted terminal repeat (ITR) or ITR region. ITRs traditionally flank the coding nucleotide sequences for the non-structural proteins (encoded by Rep genes) and the structural proteins (encoded by capsid genes or Cap genes). While not wishing to be bound by theory, an AAV vector genome typically comprises two ITR sequences. The AAV vector genome comprises a characteristic T-shaped hairpin structure defined by the self-complementary terminal 145 nucleotides of the 5’ and 3’ ends of the ssDNA which form an energetically stable double stranded region. The double stranded hairpin structures comprise multiple functions including, but not limited to, acting as an origin for DNA replication by functioning as primers for the endogenous DNA polymerase complex of the host viral replication cell.

[0481] In addition to the encoded heterologous payload, AAV vector genomes may comprise, in whole or in part, of any naturally occurring and / or recombinant AAV serotype nucleotide sequence or variant. AAV variants may have sequences of significant homology at the nucleic acid (genome or capsid) and amino acid levels (capsids), to produce constructs which are generally physical and functional equivalents, replicate by similar mechanisms, and assemble by similar mechanisms. Chiorini et al., J. Vir. 71: 6823-33(1997); Srivastava et al., J. Vir. 45:555-64 (1983); Chiorini et al., J. Vir. 73:1309-1319 (1999); Rutledge et al., J. Vir. 72:309-319 (1998); and Wu et al., J. Vir. 74: 8635-47 (2000), the contents of each of which are incorporated herein by reference in their entirety.

[0482] In some embodiments, the AAV vector genome comprises at least one control elementwhich provides for the replication, transcription, and translation of a coding sequence encoded therein. Not all of the control elements need always be present as long as the coding sequence is capable of being replicated, transcribed, and / or translated in an appropriate host cell. Non-limiting examples of expression control elements include sequences for transcription initiation and / or termination, promoter and / or enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation signals, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficacy (e.g., Kozak consensus sequence), sequences that enhance protein stability, and / or sequences that enhance protein processing and / or secretion.

[0483] AAV vector genomes may be produced recombinantly and may be based on adeno- associated virus (AAV) parent or reference sequences. As used herein, a “vector genome” is any molecule or moiety which transports, transduces, or otherwise acts as a carrier of a heterologous molecule such as the nucleic acids described herein.

[0484] In addition to single stranded AAV vector genomes (e.g., ssAAVs), the present disclosure also provides for self-complementary AAV (scAAVs) vector genomes. scAAV vector genomes contain DNA strands which anneal together to form double stranded DNA. By skipping second strand synthesis, scAAVs allow for rapid expression in the cell.

[0485] In some embodiments, the AAV vector genome is an scAAV.

[0486] In some embodiments, the AAV vector genome is an ssAAV.

[0487] In some embodiments, the AAV vector genome may be part of an AAV particles where the serotype of the capsid may be, but is not limited to, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-lb, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15,AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAVl-7 / rh.48, AAVl-8 / rh.49, AAV2-15 / rh.62, AAV2-3 / rh.61, AAV2-4 / rh.5O, AAV2-5 / rh.51, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3-ll / rh.53, AAV4-8 / rll.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5-3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.lO, AAV16.12 / hu.l l, AAV29.3 / bb.l, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV114.3 / hu.4O, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.1O / hu.6O, AAV161.6 / hu.61, AAV33.12 / hu.l7,AAV33.4 / hu.l5, AAV33.8 / hu.l6, AAV52 / hu.l9, AAV52.1 / hu.2O, AAV58.2 / hu.25, AAVA3.3,AAV A3.4, AAV A3.5, AAV A3.7, AAVC1, AAVC2, AAVC5, AAV-DJ, AAV-DJ8, AAVF3,AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi.l, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVLK03, AAVH-1 / hu.l, AAVH-5 / hu.3, AAVLG-10 / rh.40, AAVLG- 4 / rh.38, AAVLG-9 / hu.39, AAVN721-8 / rh.43, AAVCh.5, AAVCh.5Rl, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5Rl, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.ll, AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44Rl, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48Rl, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14 / 9, AAVhu.t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.l3R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64Rl, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhEl.l, AAVhErl.5, AAVhER1.14, AAVhErl.8, AAVhErl.16, AAVhErl.18, AAVhErl.35, AAVhErl.7, AAVhErl.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV- 8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.5O, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.ll, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24,AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UP ENN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-El, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt- 6.7, AAV CHt-6.8, AAV CHt-Pl, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-Bl, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-Hl, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-Fl, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg- F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1, AAV CLvl-1, AAV Clvl-10, AAV CLvl-2, AAV CLv-12, AAV CLvl-3, AAV CLv-13, AAV CLvl-4, AAV Clvl-7, AAV Clvl-8, AAV Clvl-9, AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-Dl, AAV CLv- D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-El, AAV CLv-Kl, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-Ml, AAV CLv-Mll, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-Rl, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp- 1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, AAVF9 / HSC9, PHP.B, PHP.A, G2B-26, G2B-13, TH1.1-32, and / or TH1.1-35 and variants thereof.Inverted Terminal Repeats (ITRs)

[0488] In some embodiments, the AAV vector genomes may comprise at least one ITR region and a payload region. In some embodiments, the vector genome has two ITRs. These two ITRs flank the payload region at the 5’ and 3’ ends. The ITRs flinch on as origins of replication comprising recognition sites for replication. ITRs comprise sequence regions which can be complementary and symmetrically arranged. ITRs incorporated into vector genomes may be comprised of naturally occurring polynucleotide sequences or recombinantly derived polynucleotide sequences.

[0489] The ITRs may be derived from the same serotype as the capsid or a derivative thereof. The ITR may be of a different serotype than the capsid. In some embodiments, the AAV particle has more than one ITR. In a non-limiting example, the AAV particle has a vector genomecomprising two ITRs. In some embodiments, the ITRs are of the same serotype as one another. In another embodiment, the ITRs are of different serotypes. Non-limiting examples include zero, one or both of the ITRs having the same serotype as the capsid. In some embodiments both ITRs of the vector genome of the AAV particle are AAV2 ITRs.

[0490] Independently, each ITR may be about 100 to about 150 nucleotides in length. An ITR may be about 100-105 nucleotides in length, 106-110 nucleotides in length, 111-115 nucleotides in length, 116-120 nucleotides in length, 121-125 nucleotides in length, 126-130 nucleotides in length, 131-135 nucleotides in length, 136-140 nucleotides in length, 141-145 nucleotides in length or 146-150 nucleotides in length. In some embodiments, the ITRs are 140-142 nucleotides in length. Non-limiting examples of ITR length are 102, 140, 141, 142, 145 nucleotides in length, and those having at least 95% identity thereto.Promoters

[0491] In some embodiments, the payload region of the vector genome comprises at least one element to enhance the transgene target specificity and expression (See e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the contents of which are herein incorporated by reference in its entirety). Nonlimiting examples of elements to enhance the transgene target specificity and expression include promoters, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (Poly A) signal sequences and upstream enhancers (USEs), CMV enhancers and introns.

[0492] In some embodiments, the promoter is efficient when it drives expression of the polypeptide(s) encoded in the pay load region of the vector genome of the AAV particle.

[0493] In some embodiments, the promoter is deemed to be efficient when it drives expression in the cell being targeted.

[0494] In some embodiments, the promoter drives expression of the pay load for a period of time in targeted tissues. Expression driven by a promoter may be for a period of 1 hour, 2, hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more than 10 years. Expression may be for 1-5 hours, 1-12 hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-6months, 3-9 months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 years, or 5-10 years.

[0495] In some embodiment, the promoter drives expression of the payload for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 years, 49 years, 50 years, 55 years, 60 years, 65 years, or more than 65 years.

[0496] Promoters may be naturally occurring or non-naturally occurring. Non-limiting examples of promoters include viral promoters, plant promoters and mammalian promoters. In some embodiments, the promoters may be human promoters. In some embodiments, the promoter may be truncated.

[0497] Promoters which drive or promote expression in most tissues include, but are not limited to, human elongation factor la-subunit (EFla), cytomegalovirus (CMV) immediate-early enhancer and / or promoter, chicken [3-actin (CBA) and its derivative CAG, f> glucuronidase (GUSB), or ubiquitin C (UBC). Tissue-specific expression elements can be used to restrict expression to certain cell types such as, but not limited to, muscle specific promoters, B cell promoters, monocyte promoters, leukocyte promoters, macrophage promoters, pancreatic acinar cell promoters, endothelial cell promoters, lung tissue promoters, astrocyte promoters, or nervous system promoters which can be used to restrict expression to neurons, astrocytes, or oligodendrocytes.

[0498] Non-limiting examples of muscle-specific promoters include mammalian muscle creatine kinase (MCK) promoter, mammalian desmin (DES) promoter, mammalian troponin I (TNNI2) promoter, and mammalian skeletal alpha-actin (ASKA) promoter (see, e.g., U.S. Patent Publication US20110212529, the contents of which are herein incorporated by reference in their entirety)

[0499] Non-limiting examples of tissue-specific expression elements for neurons include neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B-chain (PDGF- ), synapsin (Syn), methyl-CpG binding protein 2 (MeCP2), Ca2+ / calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light (NFL) or heavy (NFH), -globin minigene nf>2. preproenkephalin (PPE), enkephalin (Enk) and excitatory amino acid transporter 2 (EAAT2) promoters. Nonlimiting examples of tissue-specific expression elements for astrocytes include glial fibrillary acidic protein (GFAP) and EAAT2 promoters. A non-limiting example of a tissue-specificexpression element for oligodendrocytes includes the myelin basic protein (MBP) promoter.

[0500] In some embodiments, the promoter may be less than 1 kb. The promoter may have a length of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, or more than 800 nucleotides. The promoter may have a length between 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800, or 700-800.

[0501] In some embodiments, the promoter may be a combination of two or more components of the same or different starting or parental promoters such as, but not limited to, CMV and CBA. Each component may have a length of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600,610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790,800, or more than 800. Each component may have a length between 200-300, 200-400, 200-500,200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800 or 700-800. In some embodiments, the promoter is a combination of a 382 nucleotide CMV-enhancer sequence and a 260 nucleotide CBA-promoter sequence.

[0502] In some embodiments, the vector genome comprises a ubiquitous promoter. Non-limiting examples of ubiquitous promoters include CMV, CBA (including derivatives CAG, CBh, etc.), EF-la, PGK, UBC, GUSB (hGBp), and UCOE (promoter of HNRPA2B1-CBX3).

[0503] In some embodiments, the promoter is not cell specific.

[0504] In some embodiments, the vector genome comprises an engineered promoter.

[0505] In some embodiments, the vector genome comprises a promoter from a naturally expressed protein.Untranslated Regions (UTRs)

[0506] By definition, wild type untranslated regions (UTRs) of a gene are transcribed but not translated. Generally, the 5’ UTR starts at the transcription start site and ends at the start codon and the 3’ UTR starts immediately following the stop codon and continues until the termination signal for transcription.

[0507] Features typically found in abundantly expressed genes of specific target organs may be engineered into UTRs to enhance the stability and protein production. As a non-limiting example, a 5’ UTR from mRNA normally expressed in the liver (e.g., albumin, serum amyloid A,Apolipoprotein A / B / E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII) may be used in the vector genomes of the AAV particles to enhance expression in hepatic cell lines or liver.

[0508] While not wishing to be bound by theory, wild-type 5' untranslated regions (UTRs) include features which play roles in translation initiation. Kozak sequences, which are commonly known to be involved in the process by which the ribosome initiates translation of many genes, are usually included in 5’ UTRs. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (ATG), which is followed by another 'G1.

[0509] In some embodiments, the 5’UTR in the vector genome includes a Kozak sequence.

[0510] In some embodiments, the 5’UTR in the vector genome does not include a Kozak sequence.

[0511] While not wishing to be bound by theory, wild-type 3' UTRs are known to have stretches of Adenosines and Uridines embedded therein. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995, the contents of which are herein incorporated by reference in its entirety): Class I AREs, such as, but not limited to, c-Myc and MyoD, contain several dispersed copies of an AUUUA motif within U-rich regions. Class II AREs, such as, but not limited to, GM-CSF and TNF-a, possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Class III ARES, such as, but not limited to, c-Jun and Myogenin, are less well defined. These U rich regions do not contain an AUUUA motif. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules can lead to HuR binding and thus, stabilization of the message in vivo.

[0512] Introduction, removal or modification of 3' UTR AU rich elements (AREs) can be used to modulate the stability of polynucleotides. When engineering specific polynucleotides, e.g., payload regions of vector genomes, one or more copies of an ARE can be introduced to make polynucleotides less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein.

[0513] In some embodiments, the 3' UTR of the vector genome may include an oligo(dT) sequence for templated addition of a poly-A tail.

[0514] In some embodiments, the vector genome may include at least one miRNA seed, binding site or full sequence. microRNAs (or miRNA or miR) are 19-25 nucleotide noncoding RNAs that bind to the sites of nucleic acid targets and down-regulate gene expression either by reducing nucleicacid molecule stability or by inhibiting translation. A microRNA sequence comprises a “seed” region, i.e., a sequence in the region of positions 2-8 of the mature microRNA, which sequence has perfect Watson-Crick complementarity to the miRNA target sequence of the nucleic acid.

[0515] In some embodiments, the vector genome may be engineered to include, alter or remove at least one miRNA binding site, sequence, or seed region.

[0516] Any UTR from any gene known in the art may be incorporated into the vector genome of the AAV particle. These UTRs, or portions thereof, may be placed in the same orientation as in the gene from which they were selected, or they may be altered in orientation or location. In some embodiments, the UTR used in the vector genome of the AAV particle may be inverted, shortened, lengthened, made with one or more other 5' UTRs or 3' UTRs known in the art. As used herein, the term “altered” as it relates to a UTR, means that the UTR ha...

Claims

CLAIMS1- A compound of Formula (I):or a pharmaceutically acceptable salt thereof, whereinA is absent or -CH2-;B is -CH2- or -CH2CH2- ■ wherein when A is absent then B is -CH2CH2- and when A is -CH2- then B is -CH2-;X is -NR1R2 or -O-(C=O)Ri’;Ri is H or an optionally substituted linear C1-C4 alkyl group;R2 is H, an optionally substituted linear C1-C8 alkyl group, -C(=O)R?, -S(=O)2-R7, orRr is an optionally substituted linear C1-C8 alkyl group;R20 is H or an optionally substituted linear C1-C8 alkyl group;R21 is an optionally substituted linear C1-C8 alkyl group or -(C=O)-R22, wherein R22 is an optionally substituted C1-C8 alkyl group;R3 and R4 are independently H, a linear or branched C4-C60 alkyl, a linear or branched C4-C60 alkenyl, or a linear or branched C4-C60 alkynyl group, wherein at least one of R3 or R4 is different from H;Rs and Re are independently a linear or branched C4-C60 alkyl, a linear or branched C4- C60 alkenyl, or a linear or branched C4-C60 alkynyl group;R7 is an optionally substituted linear Cl -C8 alkyl group, an optionally substituted linear C2-C8 alkenyl group, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10-membered heteroaryl; wherein when any of R3, R4, Rs and Re represents an alkenyl group, the alkenyl group independently comprises from one to six C=C bonds each independently having the E or Z configuration; wherein when any of R3, R4, Rs and Re represents an alkynyl group, the alkynyl group independently comprises from one to six C=C bonds;wherein when R? represents an alkenyl group, the alkenyl group comprises from one to two C=C bonds each independently having the E or Z configuration; wherein when any alkyl and / or alkenyl group is substituted, this group is independently substituted with one or more -OH, -(C=O)-H, -(C=O)-CH3, -(C=O)-(CH2)pCH3, -(C=O)-(CH2)P-Ri9, -O- (C=O)-CH3, -O-(C=O)-(CH2)PCH3, -O-(C=O)-(CH2)P-Ri9, -(C=O)-OCH3, -(C=O)-O-(CH2)PCH3, -(C=O)-O-(CH2)P-Ri9, -(C=O)-(CH2)P-(C=O)-Ri9, -(C=O)-O-(CH2)PN(CH3)-(CH2)P’-Ri9, - (C=O)NH-(CH2)P-R19, -(C=S)-CH3, -O-CH3, -O-(CH2)PCH3, -NH2, -NHCH3, -N(CH3)2, - N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2)PO-(C=O)CH3)2, -N(CH3)-(CH2)P-R19, -(C=O)NH2, - N3, -NO2, -SCH3, -SCH2CH3, an optionally substituted 5 to 10-membered heterocyclyl, C5-C12 aralkyl, C5-C10 aryl or an optionally substituted 5 to 10-membered heteroaryl, with p and p’ are independently from 1 to 5; and wherein R19 is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10- membered heteroaryl; wherein when any 5 to 10-membered heteroaryl and / or 5 to 10-membered heterocyclyl is substituted, this group is independently substituted with one or more substituents selected from the group consisting of C1-C4 alkyl group, -(CH2)P”N(CH3)2 and oxo (=0), with p” from 1 to 5; with the provisos that when A is absent, B is -CH2CH2-, X is -NR1R2, Ri is -CH3, and each of R3, R4, Rs, and Re is linear Cl 8 alkyl groups with one C=C bond between carbons C9 and C10 and one C=C bond between carbons C12 and Ci3, then R2 is different from -CH3, -CH2CH2OH and - CH2CH2NH2.2- The compound of claim 1, wherein the compound of Formula (I) has a structure ofFormula (II):or a pharmaceutically acceptable salt thereof, whereinRi is H or an optionally substituted linear C1-C4 alkyl group;R.2 is H, an optionally substituted linear C1-C8 alkyl group, -C(=O)R?, -S(=O)2-R?, orR20 is H or an optionally substituted linear C1-C8 alkyl group;R21 is an optionally substituted linear C1-C8 alkyl group or -(C=O)-R22, wherein R22 is an optionally substituted C1-C8 alkyl group;R3 and R4 are independently H, a linear or branched C4-C60 alkyl, a linear or branched C4-C60 alkenyl, or a linear or branched C4-C60 alkynyl group, wherein at least one of R3 or R4 is different from H;Rs and Re are independently a linear or branched C4-C60 alkyl, a linear or branched C4-C60 alkenyl, or a linear or branched C4-C60 alkynyl group;R7 is an optionally substituted linear Cl -C8 alkyl group, an optionally substituted linear C2-C8 alkenyl group, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10-membered heteroaryl; wherein when any of R3, R4, Rs and Re represents an alkenyl group, the alkenyl group independently comprises from one to six C=C bonds each independently having the E or Z configuration; wherein when any of R3, R4, Rs and Re represents an alkynyl group, the alkynyl group independently comprises from one to six C=C bonds; wherein when R7 represents an alkenyl group, the alkenyl group comprises from one to two C=C bonds each independently having the E or Z configuration; wherein when any alkyl and / or alkenyl group is substituted, this group is independently substituted with one or more -OH, -(C=O)-H, -(C=O)-CH3, -(C=O)-(CH2)pCH3, -(C=O)-(CH2)P-Ri9, -O- (C=O)-CH3, -O-(C=O)-(CH2)PCH3, -O-(C=O)-(CH2)P-Ri9, -(C=O)-OCH3, -(C=O)-O-(CH2)PCH3, -(C=O)-O-(CH2)P-Ri9, -(C=O)-(CH2)P-(C=O)-Ri9, -(C=O)-O-(CH2)PN(CH3)-(CH2)P’-Ri9, - (C=O)NH-(CH2)P-R19, -(C=S)-CH3, -0-CH3, -O-(CH2)PCH3, -NH2, -NHCH3, -N(CH3)2, - N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2)PO-(C=O)CH3)2, -N(CH3)-(CH2)P-R19, -(C=O)NH2, -N3, -NO2, -SCH3, -SCH2CH3, an optionally substituted 5 to 10-membered heterocyclyl, C5-C12 aralkyl, C5-C10 aryl or an optionally substituted 5 to 10-membered heteroaryl, with p and p’ are independently from 1 to 5; and wherein R19 is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10- membered heteroaryl, wherein when any 5 to 10-membered heteroaryl and / or 5 to 10-membered heterocyclyl is substituted, this group is independently substituted with one or more substituents selected from the group consisting of C1-C4 alkyl group, -(CH2)P”N(CH3)2 and oxo (=0), with p” from 1 to 5.3- The compound of claim 1 or 2, or the pharmaceutically acceptable salt thereof, wherein Ri is H or an optionally substituted linear C1-C4 alkyl group;R.2 is H, an optionally substituted linear C1-C4 alkyl group, -C(=O)R?, -S(=O)2-R?, orR20 is H or an optionally substituted linear C1-C8 alkyl group;R21 is an optionally substituted linear C1-C8 alkyl group or -(C=O)-R22, wherein R22 is an optionally substituted C1-C8 alkyl group;R3 and R4 are independently H, a linear or branched C4-C30 alkyl, a linear or branched C4-C30 alkenyl, or a linear or branched C4-C30 alkynyl group, wherein at least one of R3 or R4 is different from H;Rs and Re are independently a linear or branched C4-C30 alkyl, a linear or branched C4-C30 alkenyl, or a linear or branched C4-C30 alkynyl group;R7 is an optionally substituted linear C1-C8 alkyl group, an optionally substituted linear C2-C8 alkenyl group, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10-membered heteroaryl; wherein when any of R3, R4, Rs and Re represents an alkenyl group, the alkenyl group independently comprises from one to six C=C bonds each independently having the E or Z configuration; wherein when any of R3, R4, Rs and Re represents an alkynyl group, the alkynyl group independently comprises from one to six C=C bonds; wherein when R7 represents an alkenyl group, the alkenyl group comprises from one to two C=C bonds each independently having the E or Z configuration; wherein when any alkyl and / or alkenyl group is substituted, this group is independently substituted with one or more -OH, -(C=O)-H, -(C=O)-CH3, -(C=O)-(CH2)pCH3, -(C=O)-(CH2)P-Ri9, -O- (C=O)-CH3, -O-(C=O)-(CH2)PCH3, -O-(C=O)-(CH2)P-Ri9, -(C=O)-OCH3, -(C=O)-O-(CH2)PCH3, -(C=O)-O-(CH2)P-Ri9, -(C=O)-(CH2)P-(C=O)-Ri9, -(C=O)-O-(CH2)PN(CH3)-(CH2)P’-Ri9, - (C=O)NH-(CH2)P-R19, -(C=S)-CH3, -0-CH3, -O-(CH2)PCH3, -NH2, -NHCH3, -N(CH3)2, - N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2)PO-(C=O)CH3)2, -N(CH3)-(CH2)P-R19, -(C=O)NH2, -N3, -NO2, -SCH3, -SCH2CH3, an optionally substituted 5 to 10-membered heterocyclyl, C5-C12 aralkyl, C5-C10 aryl or an optionally substituted 5 to 10-membered heteroaryl, with p and p’ are independently from 1 to 5; and wherein R19 is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10- membered heteroaryl,wherein when any 5 to 10-membered heteroaryl and / or 5 to 10-membered heterocyclyl is substituted, this group is independently substituted with one or more substituents selected from the group consisting of C1-C4 alkyl group, -(CH2)P”N(CH3)2 and oxo (C=O), with p” from 1 to 5.4- The compound of any one of claims 1 to 3, or the pharmaceutically acceptable salt thereof, whereinRi is H or an optionally substituted linear C1-C4 alkyl group;R.2 is H, an optionally substituted linear C1-C4 alkyl group, -C(=O)R?, -S(=O)2-R?, orR20 is H or an optionally substituted linear C1-C8 alkyl group;R21 is an optionally substituted linear C1-C8 alkyl group or -(C=O)-R22, wherein R22 is an optionally substituted C1-C8 alkyl group;R3 and R4 are independently H, a linear or branched C4-C30 alkyl, a linear or branched C4- C30 alkenyl, or a linear or branched C4-C30 alkynyl group, wherein at least one of R3 or R4 is different from H;Rs and Re are independently a linear or branched C4-C30 alkyl, a linear or branched C4-C30 alkenyl, or a linear or branched C4-C30 alkynyl group;R7 is an optionally substituted linear C1-C8 alkyl group, an optionally substituted linear C2- C8 alkenyl group, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10-membered heteroaryl; wherein when any of R3, R4, Rs and Re represents an alkenyl group, the alkenyl group independently comprises from one to six C=C bonds each independently having the E or Z configuration; wherein when any of R3, R4, Rs and Re represents an alkynyl group, the alkynyl group independently comprises from one to six C=C bonds; wherein when R7 represents an alkenyl group, the alkenyl group comprises from one to two C=C bonds each independently having the E or Z configuration; wherein when any alkyl and / or alkenyl group is substituted, this group is independently substituted with one or more -OH, -(C=O)-H, -(C=O)-CH3, -(C=O)-(CH2)pCH3, -(C=O)-(CH2)P-Ri9, -O- (C=O)-CH3, -O-(C=O)-(CH2)PCH3, -O-(C=O)-(CH2)P-Ri9, -(C=O)-OCH3, -(C=O)-O-(CH2)PCH3, -(C=O)-O-(CH2)P-Ri9, -(C=O)-(CH2)P-(C=O)-Ri9, -(C=O)-O-(CH2)PN(CH3)-(CH2)P’-Ri9, - (C=O)NH-(CH2)P-R19, -0-CH3, -O-(CH2)PCH3, -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2)PO-(C=O)CH3)2, -N(CH3)-(CH2)p-Ri9, -(C=O)NH2, an optionally substituted 5 to 10-membered heterocyclyl, C5-C12 aralkyl, C5-C10 aryl or an optionally substituted 5 to 10-membered heteroaryl, with p and p’ are independently from 1 to 5; and wherein R19 is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10-membered heteroaryl, wherein when any 5 to 10-membered heteroaryl and / or 5 to 10-membered heterocyclyl is substituted, this group is independently substituted with one or more substituents selected from the group consisting of C1-C4 alkyl group, -(CH2)P”N(CH3)2and oxo (=0), with p” from 1 to 5.5- The compound of any one of claims 1 to 4, or the pharmaceutically acceptable salt thereof, whereinRi is H or an optionally substituted linear C1-C4 alkyl group;R2is H, an optionally substituted linear C1-C4 alkyl group, -C(=O)R?, -S(=O)2-R7, orR2O is H or an optionally substituted linear C1-C4 alkyl group;R2I is an optionally substituted linear C1-C4 alkyl group or -(C=O)-R22, wherein R22is an optionally substituted C1-C4 alkyl group;R3and R4 are independently H, a linear or branched C6-C25 alkyl, or a linear or branched C6- C25 alkenyl group, wherein at least one of R3or R4 is different from H;Rs and Re are independently a linear or branched C6-C25 alkyl, or a linear or branched C6- C25 alkenyl group;R7 is an optionally substituted linear C1-C4 alkyl group, an optionally substituted linear C2- C4 alkenyl group, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10-membered heteroaryl; wherein when any of R3, R4, Rs and Re represents an alkenyl group, the alkenyl group independently comprises from one to four C=C bonds each independently having the E or Z configuration; wherein when R7 represents an alkenyl group, the alkenyl group comprises from one to two C=C bonds each independently having the E or Z configuration; wherein when any alkyl and / or alkenyl group is substituted, this group is independently substituted with one or more -OH, -(C=O)-(CH2)P-RI9, -O-(C=O)-(CH2)P-RI9, -(C=O)-O-(CH2)P-RI9, -(C=O)- (CH2)P-(C=O)-Ri9, -(C=O)-O-(CH2)PN(CH3)-(CH2)P’-N(CH3)2, -(C=O)NH-(CH2)P-Ri9, -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2)pO-(C=O)CH3)2, -N(CH3)- (CH2)PN(CH3)2, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10-membered heteroaryl, with p and p’ are independently from 1 to 5; and wherein R19 is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10-membered heteroaryl, wherein when any 5 to 10-membered heteroaryl and / or 5 to 10-membered heterocyclyl is substituted, this group is independently substituted with one or more substituents selected from the group consisting of C1-C4 alkyl group, -(CH2)P”N(CH3)2 and oxo (=0), with p” from 1 to 5.6- The compound of any one of claims 1 to 5, or the pharmaceutically acceptable salt thereof, whereinRi is H or an optionally substituted C1-C2 alkyl group;R2is H, an optionally substituted linear C1-C4 alkyl group, -C(=O)R?, -S(=O)2-R7, orR20 is H or a linear C1-C3 alkyl group optionally substituted with -N(CH3)2;R21 is a linear C1-C4 alkyl group optionally substituted with -N(CH3)2 or 5 to 10-membered heterocyclyl optionally substituted with a linear Cl -C3 alkyl group, or is -(C=O)-R22, wherein R22 is C1-C3 alkyl group optionally substituted with -N(CH3)2;R3and R4 are independently H, a linear or branched C6-C25 alkyl, or a linear or branched C6- C25 alkenyl group, wherein at least one of R3or R4 is different from H;Rs and Re are independently a linear or branched C6-C25 alkyl or a linear or branched C6-C25 alkenyl group;R7 is an optionally substituted linear C1-C4 alkyl group, an optionally substituted linear C2- C4 alkenyl group, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10-membered heteroaryl, wherein when the 5 to 10-membered heteroaryl and / or 5 to 10-membered heterocyclyl is substituted, this group is independently substituted with one or more substituents selected from the group consisting of C1-C2 alkyl group, - (CH2)p’’N(CH3)2and oxo (=0), with p” from 1 to 3; wherein when any of R3, R4, Rs and Re represents an alkenyl group, the alkenyl group independently comprises from one to four C=C bonds each independently having the E or Z configuration;wherein when R7 represents an alkenyl group, the alkenyl group comprises from one to two C=C bonds each independently having the E or Z configuration; wherein when any alkyl or alkenyl group in Ri, R2 and / or R7 is substituted, this group is independently substituted with one or more -OH, -(C=O)-(CH2)P-Ri9, -O-(C=O)-(CH2)P-Ri9, - (C=O)-O-(CH2)P-Ri9, -(C=O)-(CH2)P-(C=O)-Ri9, -(C=O)-O-(CH2)PN(CH3)-(CH2)P’-N(CH3)2, - (C=O)NH-(CH2)P-R19, -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, -N((CH2)PO-(C=O)CH3)2, - N(CH3)-(CH2)PN(CH3)2, a 5 to 10-membered heterocyclyl optionally substituted with one C1-C4 alkyl group or a 5 to 10-membered heteroaryl optionally substituted with one C1-C4 alkyl group, with p and p’ are independently from 1 to 5; and wherein R19 is -NH2, -NHCH3, -N(CH3)2, - N(CH2CH3)2, a 5 to 10-membered heterocyclyl optionally substituted with one C 1 -C4 alkyl group, or a 5 to 10-membered heteroaryl optionally substituted with one C1-C4 alkyl group.7- The compound of any one of claims 1 to 6, or the pharmaceutically acceptable salt thereof, whereinRi is H or an optionally substituted C1-C2 alkyl group;R2 is H, an optionally substituted linear C1-C4 alkyl group, -C(=0)R?, -S(=0)2-R?, orR.20 is H or a linear C1-C3 alkyl group optionally substituted with -NfCHsh:R21 is a linear C1-C4 alkyl group optionally substituted with -NfCHsh or 5 to 10-membered heterocyclyl optionally substituted with a C1-C2 alkyl group, or is -(C=O)-R.22, wherein R22 is a C1-C3 alkyl group optionally substituted with -NfCHsh:R3 and R4 are independently H, a linear or branched C6-C25 alkyl, or a linear or branched C6- C25 alkenyl group, wherein at least one of R3 or R4 is different from H;Rs and Re are independently a linear or branched C6-C25 alkyl or a linear or branched C6-C25 alkenyl group;R7 is an optionally substituted linear C1-C4 alkyl group, an optionally substituted linear C2- C4 alkenyl group, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10-membered heteroaryl, wherein when the 5 to 10-membered heteroaryl and / or 5 to 10-membered heterocyclyl is substituted, this group is independently substituted with one or more substituents selected from the group consisting of C1-C2 alkyl group, - (CH2)2N(CH3)2and oxo (=0);wherein when any of R3, R4, Rs and Re represents an alkenyl group, the alkenyl group independently comprises from one to four C=C bonds each independently having the E or Z configuration; wherein when R7 represents an alkenyl group, the alkenyl group comprises from one to two C=C bonds each independently having the E or Z configuration; wherein when any alkyl or alkenyl group in Ri, R2 and / or R7 is substituted, this group is independently substituted with one or more -OH, -(C=O)-(CH2)P-Ri9, -O-(C=O)-(CH2)P-Ri9, - (C=O)-O-(CH2)P-Ri9, -(C=O)-(CH2)P-(C=O)-Ri9, -(C=O)-O-(CH2)PN(CH3)-(CH2)P’-N(CH3)2, - (C=O)NH-(CH2)P-R19, -N(CH3)2, -N((CH2)PO-(C=O)CH3)2, -N(CH3)-(CH2)PN(CH3)2, a 5 to 10- membered heterocyclyl optionally substituted with one C1-C2 alkyl group or a 5 to 10-membered heteroaryl optionally substituted with one C1-C2 alkyl group, with p and p’ are independently from 1 to 5; and wherein R^is -N(CH3)2, a 5 to 10-membered heterocyclyl optionally substituted with one C1-C2 alkyl group, or a 5 to 10-membered heteroaryl optionally substituted with one Cl- C2 alkyl group.8- The compound of any one of claims 1 to 7, or the pharmaceutically acceptable salt thereof, wherein the 5 to 10-membered heteroaryl independently represents furanyl, benzofuranyl, benzimidazolyl, quinolinyl, isoquinolinyl, thiophenyl, pyrrolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazolyl, pyrazolyl, pyrazinyl, pyridinyl, pyridazinyl or pyrimidinyl.9- The compound of any one of claims 1 to 7, or the pharmaceutically acceptable salt thereof, wherein the 5 to 10-membered heterocyclyl independently represents a lactam, morpholinyl, piperazinyl or piperidinyl.10- The compound of any one of claims 1 to 9, or the pharmaceutically acceptable salt thereof, wherein Ri is H or CH3, preferably H.11- The compound of any one of claims 1 to 10, or the pharmaceutically acceptable salt thereof, wherein R2 is H, an optionally substituted linear C3-C4 alkyl group, -C(=O)R?, -S(=O)2-R?, or12- The compound of any one of claims 1 to 11, or the pharmaceutically acceptable salt thereof, wherein -NR1R2 has one of the following structures:13- The compound of any one of claims 1 to 12, or the pharmaceutically acceptable salt thereof, wherein -14- The compound of any one of claims 1 to 13, or the pharmaceutically acceptable salt thereof, wherein any of R3, R4, Rs and Re is C6-C25 alkenyl, and the alkenyl group independently comprises one, two or three C=C bonds.15- The compound of any one of claims 1 to 13, or the pharmaceutically acceptable salt thereof, wherein R3 and R4 are linear C6-C16 alkyl groups.16- The compound of any one of claims 1 to 13, or the pharmaceutically acceptable salt thereof, wherein R3 and R4 are branched C8-C12 alkyl groups.17- The compound of any one of claims 1 to 13, or the pharmaceutically acceptable salt thereof, wherein R3 and R4 are linear C8-C20 alkenyl groups, wherein the alkenyl groups each comprise one C=C bond.18- The compound of any one of claims 1 to 13, or the pharmaceutically acceptable salt thereof, wherein R3 is H and R4 is a linear C6-C25 alkenyl group, wherein the alkenyl group comprise one C=C bond.19- The compound of any one of claims 1 to 13, or the pharmaceutically acceptable salt thereof, wherein R4 is H and R3 is a linear C6-C25 alkenyl group, wherein the alkenyl group comprise one C=C bond.20- The compound of any one of claims 1 to 13 and 15 to 19, or the pharmaceutically acceptable salt thereof, wherein Rs and Re are linear C6-C16 alkyl groups.21- The compound of any one of claims 1 to 13 and 15 to 19, or the pharmaceutically acceptable salt thereof, wherein Rs and Re are branched C8-C12 alkyl groups.22- The compound of any one of claims 1 to 13 and 15 to 19, or the pharmaceutically acceptable salt thereof, wherein Rs and Re are linear C8-C20 alkenyl groups, wherein the alkenyl groups each comprise one C=C bond.23- The compound of any one of claims 1 to 13 and 15 to 19, or the pharmaceutically acceptable salt thereof, wherein Rs and Re are linear C15-C20 alkenyl groups, wherein the alkenyl groups each comprise two C=C bonds.24- The compound of any one of claims 1 to 13 and 15 to 19, or the pharmaceutically acceptable salt thereof, wherein Rs and Re are branched C22-C25 alkenyl groups, wherein the alkenyl groups each comprise one C=C bond.25- The compound of any one of claims 1 to 13 and 15 to 19, or the pharmaceutically acceptable salt thereof, wherein Rs and Re are branched C13-C17 alkenyl groups, wherein the alkenyl groups each comprise three C=C bonds.26- The compound of any one of claims 1 to 13, or the pharmaceutically acceptable salt thereof, wherein R3, R4, Rs and Re independently have one of the following structures:27- The compound of any one of claims 1 to 26, or the pharmaceutically acceptable salt thereof, wherein R3 and R4 are both different from H and are identical.28- The compound of any one of claims 1 to 27, or the pharmaceutically acceptable salt thereof, wherein Rs and Re are identical.29- The compound of any one of claims 1 to 28, or the pharmaceutically acceptable salt thereof, wherein R3 and R4 are both different from H and R3, R4, Rs and Re are identical.30- The compound of claim 1, wherein the compound of Formula (I) has a structure of Formula (12) or (13):or a pharmaceutically acceptable salt thereof, wherein Ri, R2, R3, R4, Rs, Re and Rr are as defined in any one of claims 1 to 29.31- The compound of claim 30, wherein the compound has a structure of formula (12), or a pharmaceutically acceptable salt thereof, wherein Ri is H or C1-C2 alkyl, and R2is -C(=O)R?; and wherein R3, R4, Rs, Re and R7 are as defined in any one of claims 1 to 29.32- The compound of claim 30, wherein the compound has a structure of formula (13), or a pharmaceutically acceptable salt thereof, wherein Rr is a linear C1-C4 alkyl group substituted with -(C=O)-(CH2)P-Ri9, -O-(C=O)-(CH2)P-RI9, -(C=O)-O-(CH2)P-RI9, -(C=O)-(CH2)P-(C=O)- R19, -(C=O)-O-(CH2)PN(CH3)-(CH2)P-N(CH3)2, -(C=O)NH-(CH2)P-Ri9, -N(CH3)2, -N((CH2)PO- (C=O)CH3)2, -N(CH3)-(CH2)PN(CH3)2, an optionally substituted 5 to 6-membered heterocyclyl or an optionally substituted 5 to 6-membered heteroaryl, with p and p’ are independently from 1 to 5; and wherein R19 is -N(CH3)2, an optionally substituted 5 to 6-membered heterocyclyl or an optionally substituted 5 to 6-membered heteroaryl; wherein when the 5 to 6-membered heteroaryl and / or 5 to 6-membered heterocyclyl is substituted, this group is independently substituted with one substituent selected from the group consisting of C1-C2 alkyl group; and wherein R3, R4, Rs and Re are as defined in any one of claims 1 to 29.33- The compound of claim 1 or 2, wherein the compound has a structure of Formula (11 - 1), (II- 2), (11-3), (11-4), (11-5) or (11-6):or a pharmaceutically acceptable salt thereof, wherein Ri is H or a linear C1-C4 alkyl; wherein Rio and Rn are independently -H, -(C=O)-CH3, -(C=O)-(CH2)PCH3, -(C=O)-(CH2)P-Ri9, or -(C=O)-(CH2)P-(C=O)-RI9; and wherein R3, R4, Rs, Re, R7, R19, R20 and R21 are as defined in any one of claims 1 to 29 and p is from 1 to 5.34- The compound of claim 33, wherein the compound has a structure of formula (Il-4a):or a pharmaceutically acceptable salt thereof, wherein Ri is H or a linear C1-C4 alkyl, preferably Ri is H or -CH3, more preferably Ri is - CH3; and wherein R3, R4, Rs and Re are as defined in any one of claims 1 to 29.35- The compound of claim 33, wherein the compound has a structure of formula (Il -4b):or a pharmaceutically acceptable salt thereof, wherein Ri is H or a linear C1-C4 alkyl, preferably Ri is H or -CH3, more preferably Ri is - CH3; wherein R12 and R13 are independently an optionally substituted linear C1-C4 alkyl; wherein when the linear alkyl group is substituted, this group is independently substituted with -NH2, - NHCH3, -N(CH3)2, -N(CH2CH3)2, or 5-6 membered heteroaryl, preferably R12 and R13 are independently linear C1-C4 alkyl substituted with -NH2, -NHCH3, -N(CH3)2 or -N(CH2CH3)2, more preferably substituted with -N(CH3)2; and wherein R3, R4, Rs and Re are as defined in any one of claims 1 to 29.36- The compound of claim 33, wherein the compound has a structure of formula (Il-5a):or a pharmaceutically acceptable salt thereof, wherein Ri is H or a linear C1-C4 alkyl, preferably Ri is H or -CH3, more preferably Ri is H; wherein q is from 1 to 5; wherein R14 and R’14 independently represent -H, -CH3, -CH2CH3, or -(CH2)PO-(C=O)CH3 with p is from 1 to 5, preferably p is from 1 to 3, or R14 and R’14 are connected to form a 6- membered heterocyclyl optionally substituted with -CH3 or -CH2CH3; and wherein R3, R4, Rs and Re are as defined in any one of claims 1 to 29.37- The compound of claim 33, wherein the compound has a structure of formula (Il -5b):or a pharmaceutically acceptable salt thereof, wherein Xi is -NH- or -O-; wherein R15 is a linear C1-C4 alkyl, -(CH2)P-Ri9, or -(CH2)PN(CH3)-((CH2)P’-N(CH3)2) with p and p’ are independently from 1 to 5, and R19 is as defined in any one of claims 1 to 9; preferably R15 is -(CH2)2N(CH3)-((CH2)2-N(CH3)2) or -(CH2)P-Ri9 with p is from 1 to 3; more preferably Ris is -(CH2)2N(CH3)-((CH2)2-N(CH3)2), -(CH2)P-N(CH3)2, or -(CH2)P-R23 with p is from 1 to 3 and R23 is a 5 to 6-membered heteroaryl or 5 to 6-membered heterocyclyl, optionally substituted with C1-C2 alkyl; even more preferably R15 is -(CH2)2N(CH3)-((CH2)2-N(CH3)2), -(CH2)P-N(CH3)2 or -(CH2)P- R23 with p is from 1 to 3 and R23 is furanyl, thiophenyl, pyrrolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazolyl, pyrazolyl, pyrazinyl, pyridinyl, pyridazinyl, pyrimidinyl, piperidinyl or piperazinyl, wherein R23 is optionally substituted with C1-C2 alkyl; most preferably Ris is -(CH2)2N(CH3)-((CH2)2-N(CH3)2), -(CH2)P-N(CH3)2 or -(CH2)P-R23 with p is from 1 to 3 and R23 is imidazolyl or piperidinyl, wherein R23 is optionally substituted with a C1-C2 alkyl; and wherein R3, R4, Rs and Re are as defined in any one of claims 1 to 29.38- The compound of claim 33, wherein the compound has a structure of formula (Il-5c):or a pharmaceutically acceptable salt thereof, whereinRi6 is a 5 to 6-membered heteroaryl or a 5 to 6-membered heterocyclyl, optionally substituted with a linear C1-C4 alkyl, an oxo (=0) and / or a -(CH2)P-N(CH3)2 and wherein p is from 1 to 3, preferably Rie is furanyl, thiophenyl, pyrrolyl, pyrrolydinyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazolyl, pyrazolyl, pyrazinyl, pyridinyl, pyridazinyl, piperazinyl, piperidinyl or pyrimidinyl optionally substituted with a linear C1-C4 alkyl, an oxo (=0) and / or a -(CH2)p-N(CH3)2 with p from 1 to 3, more preferably Ri6 is imidazolyl or pyrrolidinyl optionally substituted with a C1-C2 alkyl, an oxo (=0) and / or a -(CH2)p-N(CH3)2 with p from 1 to 3; and wherein R3, R4, Rs and Re are as defined in any one of claims 1 to 29.39- The compound of claim 33, wherein the compound has a structure of formula (Il -5d):or a pharmaceutically acceptable salt thereof, whereinR17 is a 5 to 6-membered heteroaryl optionally substituted with a linear C1-C4 alkyl, or a - (CH2)P-N(CH3)2 and wherein p is from 1 to 3, preferably R17 is furanyl, thiophenyl, pyrrolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazolyl, pyrazolyl, pyrazinyl, pyridinyl, pyridazinyl or pyrimidinyl, optionally substituted with a C1-C2 alkyl, more preferably R17 is imidazolyl; wherein the C=C bonds present in Formula (Il-5d) independently have the E or Z configuration; and wherein R3, R4, Rs and Re are as defined in any one of claims 1 to 29.40- The compound of claim 33, wherein the compound has a structure of Formula (Il-6a), (Il -6b) or (Il -6c):or a pharmaceutically acceptable salt thereof, whereinR31 is a linear C1-C4 alkyl group optionally substituted with -N(CH3)2; preferably R31 is - (CH2)3-N(CH3)2;R-32 is -N(CH3)2or a 5 to 6-membered heterocyclyl optionally substituted with a linear C1-C4 alkyl group, and z is from 1 to 4; preferably2is -N(CH3)2, and z is from 2 to 4; more preferably R.32 is piperazinyl or piperidinyl substituted with a C1-C2 alkyl group, and z is 3; and R3, R4, Rs and Re are as defined in any one of claims 1 to 29.41- The compound of any one of claims 30 to 40 or a pharmaceutically acceptable salt thereof, wherein R3 and R4 are both identical, and Rs and Re are both identical.42- A compound selected from the group consisting of Compounds 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320a, 320b, 321a, 321b, 322, 323, 324, 325, 326, 328, 329, 330, 331, 332, 333, 334, 335, 336, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 358, 359, 360 and 362 of Table 1, or a pharmaceutically acceptable salt thereof.43- The compound of claim 42, or a pharmaceutical acceptable salt thereof, selected from the group consisting of 301, 302, 303, 304, 305, 306, 307, 309, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320a, 320b, 321a, 321b, 322, 323, 324, 325, 326, 328, 329, 330, 331, 332, 333, 334,335, 336, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355,358, 359, 360 and 362 of Table 1.44- The compound of claim 42, or a pharmaceutical acceptable salt thereof, selected from the group consisting of 301, 302, 303, 304, 305, 306, 307, 309, 311, 312, 313, 314, 315, 316, 318, 319, 320a, 320b, 321a, 321b, 325, 326, 346, 347, 350, 351, 352, 353, 355, 358, 359, 360 and 362 of Table 1.45- The compound of claim 42, or a pharmaceutical acceptable salt thereof, selected from the group consisting of 305, 313, 314, 315, 316, 317, 318, 320a, 320b, 321a, 321b, 323, 324, 325, 326, 328, 329, 330, 331, 332, 333, 334, 335, 336, 340, 350, 351 and 360 of Table 1.46- The compound of claim 42, or a pharmaceutical acceptable salt thereof, selected from the group consisting of 301, 303, 304, 305, 306, 307, 311, 312, 313, 314, 315, 319, 320a, 320b, 321a, 321b, 325, 326, 346, 347, 350, 351, 352, 353, 355, 358 and 359 of Table 147- The compound of claim 42 or 46, or a pharmaceutical acceptable salt thereof, selected from the group consisting of 305, 306, 307, 313, 314, 315, 319, 320a, 320b, 321a, 321b, 325, 326, 346, 350, 351, 352, 353, 355, 358 and 359 of Table 1.48- The compound of any one of claims 1 to 47, wherein the compound or the pharmaceutically acceptable salt thereof is in the form of any enantiomers, any diastereoisomers, any cis or trans geometric isomers, or any mixtures thereof.49- A lipid nanoparticle comprising at least one compound of any one of claims 1 to 48 or the pharmaceutically acceptable salt thereof.50- A lipid nanoparticle comprising:(a) from about 40 to about 100 mol % of an ionizable lipid;(b) from 0 to about 20 mol % of a neutral lipid;(c) from 0 to about 50 mol % of a helper lipid;(d) from 0 to about 5 mol % of a polymer-conjugated lipid; and(e) from 0 to about 10 mol % of a hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle; and wherein the ionizable lipid is at least one compound of Formula (I):or a pharmaceutically acceptable salt thereof, whereinA is absent or -CH2-;B is -CH2- or -CH2CH2- ■ wherein when A is absent then B is -CH2CH2- and when A is -CH2- then B is -CH2-;X is -NR1R2 or -O-(C=O)Rr;Ri is H or an optionally substituted linear C1-C4 alkyl group;R2 is H, an optionally substituted linear C1-C8 alkyl group, -C(=O)R?, -S(=O)2-R7, orRr is an optionally substituted linear C1-C8 alkyl group;R20 is H or an optionally substituted linear C1-C8 alkyl group;R21 is an optionally substituted linear C1-C8 alkyl group or -(C=O)-R22, wherein R22 is an optionally substituted C1-C8 alkyl group;R3 and R4 are independently H, a linear or branched C4-C60 alkyl, a linear or branched C4-C60 alkenyl, or a linear or branched C4-C60 alkynyl group, wherein at least one of R3 or R4 is different from H;Rs and Re are independently a linear or branched C4-C60 alkyl, a linear or branched C4- C60 alkenyl, or a linear or branched C4-C60 alkynyl group;R7 is an optionally substituted linear Cl -C8 alkyl group, an optionally substituted linear C2-C8 alkenyl group, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10-membered heteroaryl; wherein when any of R3, R4, Rs and Re represents an alkenyl group, the alkenyl group independently comprises from one to six C=C bonds each independently having the E or Z configuration; wherein when any of R3, R4, Rs and Re represents an alkynyl group, the alkynyl group independently comprises from one to six C=C bonds; wherein when R7 represents an alkenyl group, the alkenyl group comprises from one to two C=C bonds each independently having the E or Z configuration; wherein when any alkyl and / or alkenyl group is substituted, this group is independently substituted with one or more -OH, -(C=0)-H, -(C=O)-CH3, -(C=O)-(CH2)pCH3, -(C=O)-(CH2)P-Ri9, -O- (C=O)-CH3, -O-(C=O)-(CH2)PCH3, -O-(C=O)-(CH2)P-Ri9, -(C=O)-OCH3, -(C=O)-O-(CH2)PCH3, -(C=O)-O-(CH2)P-Ri9, -(C=O)-(CH2)P-(C=O)-Ri9, -(C=O)-O-(CH2)PN(CH3)-(CH2)P’-Ri9, -(C=O)NH-(CH2)P-Ri9, -(C=S)-CH3, -O-CH3, -O-(CH2)pCH3, -NH2, -NHCH3, -N(CH3)2, - N(CH2CH3)2, -N(CH2CH2OH)2, -N((CH2)PO-(C=O)CH3)2, -N(CH3)-(CH2)P-RI9, -(C=O)NH2, - N3, -N02, -SCH3, -SCH2CH3, an optionally substituted 5 to 10-membered heterocyclyl, C5-C12 aralkyl, C5-C10 aryl or an optionally substituted 5 to 10-membered heteroaryl, with p and p’ are independently from 1 to 5; and wherein R19 is -NH2, -NHCH3, -N(CH3)2, -N(CH2CH3)2, an optionally substituted 5 to 10-membered heterocyclyl or an optionally substituted 5 to 10- membered heteroaryl; wherein when any 5 to 10-membered heteroaryl and / or 5 to 10-membered heterocyclyl is substituted, this group is independently substituted with one or more substituents selected from the group consisting of C1-C4 alkyl group, -(CH2)P”N(CH3)2and oxo (=0), with p” from 1 to 5; with the provisos that when A is absent, B is -CH2CH2-, X is -NRIR2, Ri is -CH3, and each of R3, R4, Rs, and Re is linear Cl 8 alkyl groups with one C=C bond between carbons C9 and C10 and one C=C bond between carbons C12 and Ci3, then R2is different from -CH3, -CH2CH2OH and - CH2CH2NH2.51 - The lipid nanoparticle according to claim 50, wherein the compound or the pharmaceutically acceptable salt thereof, is as defined in any one of claims 1 to 48.52- The lipid nanoparticle according to claim 50 or 51, comprising:(a) from about 40 to about 85 mol % of the ionizable lipid;(b) from about 1 to about 20 mol % of the neutral lipid;(c) from 0 to about 50 mol % of the helper lipid;(d) from 0 to about 5 mol % of the polymer-conjugated lipid; and(e) from 0 to about 10 mol % of the hydrophobic component.53- The lipid nanoparticle according to claim 50 or 51, comprising:(a) from about 40 to about 85 mol % of the ionizable lipid;(b) from about 1 to about 20 mol % of the neutral lipid;(c) from about 1 to about 50 mol % of the helper lipid;(d) from 0 to about 5 mol % of the polymer-conjugated lipid; and(e) from 0 to about 10 mol % of the hydrophobic component.54- The lipid nanoparticle according to claim 50 or 51, comprising:(a) from about 40 to about 85 mol % of the ionizable lipid;(b) from about 1 to about 20 mol % of the neutral lipid;(c) from about 1 to about 50 mol % of the helper lipid;(d) from about 1 to about 5 mol % of the polymer-conjugated lipid; and(e) from 0 to about 10 mol % of the hydrophobic component.55- The lipid nanoparticle according to claim 50 or 51, comprising:(a) from about 40 to about 85 mol % of the ionizable lipid;(b) from about 1 to about 20 mol % of the neutral lipid;(c) from about 1 to about 50 mol % of the helper lipid;(d) from about 1 to about 5 mol % of the polymer-conjugated lipid; and(e) from about 0.1 to about 10 mol % of the hydrophobic component.56- The lipid nanoparticle according to claim 50 or 51, comprising:(a) from about 40 to about 85 mol % of the ionizable lipid;(b) from about 5 to about 20 mol % of the neutral lipid;(c) from about 1 to about 50 mol % of the helper lipid;(d) from about 1 to about 4 mol % of the polymer-conjugated lipid; and(e) from about 0.1 to about 10 mol % of the hydrophobic component.57- The lipid nanoparticle according to claim 50 or 51, comprising:(a) from about 40 to about 85 mol % of the ionizable lipid;(b) from about 5 to about 10 mol % of the neutral lipid;(c) from about 1 to about 40 mol % of the helper lipid;(d) from about 1 to about 4 mol % of the polymer-conjugated lipid; and(e) from about 0 to about 5 mol % of the hydrophobic component.58- The lipid nanoparticle according to claim 50 or 51, comprising:(a) from about 40 to about 85 mol % of the ionizable lipid;(b) from about 5 to about 10 mol % of the neutral lipid;(c) from about 1 to about 40 mol % of the helper lipid;(d) from about 1 to about 4 mol % of the polymer-conjugated lipid; and(e) from about 0.1 to about 5 mol % of the hydrophobic component.59- The lipid nanoparticle according to any one of claims 50 to 58, wherein the neutral lipid comprises at least one phospholipid.60- The lipid nanoparticle according to any one of claims 50 to 59, wherein the neutral lipid comprises at least one phospholipid selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine(DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl- sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), 1 -myristoyl-2 -palmitoyl phosphatidylcholine (MPPC), l-palmitoyl-2 -myristoyl phosphatidylcholine (PMPC), l-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), l,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl- 2-palmitoyl phosphatidylcholine (SPPC), l,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphophatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof.61- The lipid nanoparticle according to any one of claims 50 to 60, wherein the neutral lipid comprises the phospholipid DSPC, DOPC, DMPC, PE or a combination thereof.62- The lipid nanoparticle according to any one of claims 50 to 61, wherein the helper lipid comprises a sterol, an alkyl resorcinol or a combination thereof.63- The lipid nanoparticle according to any one of claims 50 to 62, wherein the helper lipid comprises cholesterol, 5-heptadecylresorcinol and cholesterol hemisuccinate, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'- hydroxybutyl ether, or any combination thereof.64- The lipid nanoparticle according to any one of claims 50 to 63, wherein the helper lipid comprises cholesterol, 5-heptadecylresorcinol, cholesterol hemi succinate, or any combination thereof.65- The lipid nanoparticle according to any one of claims 50 to 64, wherein the helper lipid comprises at least cholesterol.66- The lipid nanoparticle according to any one of claims 50 to 65, wherein the polymer- conjugated lipid comprises polyethylene glycol (PEG), polypropylene glycol (PPG), poly(2- oxazoline) (POZ), polyamide (ATTA), cationic polymer, polysarcosine (Psar), polyglutamic acid (PGA), l,2-dimyristoyl-rac-glycero-3-methoxypoly ethylene glycol (PEG-DMG), or any combination thereof.67- The lipid nanoparticle according to any one of claims 50 to 66, wherein the polymer- conjugated lipid comprises at least one PEG-lipid.68- The lipid nanoparticle according to any one of claims 50 to 67, wherein the polymer- conjugated lipid comprises at least one PEG2k-DMG.69- The lipid nanoparticle according to any one of claims 50 to 68, wherein the hydrophobic component comprises squalene, cardiolipin, a-tocopherol, withaferin A, vitamin A, retinol, f>- carotene, or any combination thereof.70- The lipid nanoparticle according to claim 50 or 51, comprising:(a) from about 40 to about 80 mol% of the ionizable lipid;(b) from about 5 to about 15 mol% of a phospholipid as the neutral lipid;(c) from about 1 to about 40 mol% of a sterol as the helper lipid;(d) from about 1 to about 4 mol% of a PEG-lipid as the polymer-conjugated lipid; and(e) from about 0 to about 5 mol% of squalene, cardiolipin, a-tocopherol, withaferin A, vitamin A, retinol, P-carotene, or a combination thereof as the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.71- The lipid nanoparticle according to claim 70, wherein the phospholipid is DSPC.72- The lipid nanoparticle according to claims 70 or 71, wherein the sterol is cholesterol.73- The lipid nanoparticle according to any one of claims 70 to 72, wherein the PEG-lipid is PEG2k-DMG.74- The lipid nanoparticle according to any one of claims 50 to 73, wherein the hydrophobic component comprises squalene, cardiolipin, a-tocopherol, or a combination thereof.75- The lipid nanoparticle according to any one of claims 49 to 74, further comprising at least one cargo.76- The lipid nanoparticle according to claim 75, wherein the cargo comprises at least one of a small molecule, an antibody, a polynucleotide or a polypeptide.77- The lipid nanoparticle according to claim 75, wherein the cargo comprises at least one nucleic acid, such as mRNA.78- A pharmaceutical composition comprising the lipid nanoparticle according to any one of claims 49 to 77, and a pharmaceutical acceptable excipient.79- The pharmaceutical composition according to claim 78, which is formulated for intramuscular administration.80- A method for delivering a cargo to a cell comprising contacting the cell with the lipid nanoparticle according to any one of claims 49 to 77, wherein the lipid nanoparticle comprises the cargo.81- Use of the lipid nanoparticle according to any one of claims 49 to 77, for delivering a cargo to a cell, wherein the lipid nanoparticle comprises the cargo.82- A vaccine comprising the lipid nanoparticle according to any one of claims 49 to 74, wherein the lipid nanoparticle comprises at least one cargo, preferably the cargo comprises at least one of a small molecule, an antibody, a polynucleotide or a polypeptide, more preferably the cargo comprises at least one nucleic acid such as mRNA.83- A method of vaccinating a subject against an infectious agent comprising:(i) contacting the subject with the vaccine according to claim 82, and(ii) eliciting an immune response.84- The method according to claim 83, wherein the infectious agent is a virus, a bacterium, or a parasite.85- The method according to claim 83, wherein the infectious agent is Campylobacter jejuni, Clostridium difficile, entamoeba histolytica, enterotoxin B, Norwalk virus or norovirus, Helicobacter pylori, rotavirus, Candida yeast, coronavirus including SARS-CoV, SARS-CoV-2, MERS-CoV, Enterovirus 71, Epstein-Barr virus, Gram-Negative Bacteria including Bordetella, Gram-Positive Bacteria including Clostridium Tetani, Francisella Tularensis, Streptococcus bacteria, Staphylococcus bacteria, Salmonella bacteria, Hepatitis, Human Cytomegalovirus, Human Immunodeficiency Virus, Human Papilloma Virus, Influenza, John Cunningham Virus, Mycobacterium, Poxviruses, Pseudomonas Aeruginosa, Respiratory Syncytial Virus, Rubella virus, Varicella zoster virus, Zika virus, Chikungunya virus, Dengue virus, Rabies virus, Trypanosoma cruzi disease, Chagas disease, Ebola virus, Plasmodium falciparum, Marburg virus, Japanese encephalitis virus, St. Louis encephalitis virus, West Nile Virus, Yellow Fever virus, Bacillus anthracis, Botulinum toxin, Ricin, Nipah, Shiga toxin and / or Shiga-like toxin.86- Use of the vaccine of claim 83 for vaccinating a subject against an infectious agent.87- The use of claim 86, wherein the infectious agent is a virus, a bacterium, or a parasite.88- The use of claim 86, wherein the infectious agent is Campylobacter jejuni, Clostridium difficile, entamoeba histolytica, enterotoxin B, Norwalk virus or norovirus, Helicobacter pylori, rotavirus, Candida yeast, coronavirus including SARS-CoV, SARS-CoV-2, MERS-CoV, Enterovirus 71, Epstein-Barr virus, Gram-Negative Bacteria including Bordetella, Gram-Positive Bacteria including Clostridium Tetani, Francisella Tularensis, Streptococcus bacteria, Staphylococcus bacteria, Salmonella bacteria, Hepatitis, Human Cytomegalovirus, Human Immunodeficiency Virus, Human Papilloma Virus, Influenza, John Cunningham Virus, Mycobacterium, Poxviruses, Pseudomonas Aeruginosa, Respiratory Syncytial Virus, Rubella virus, Varicella zoster virus, Zika virus, Chikungunya virus, Dengue virus, Rabies virus, Trypanosoma cruzi disease, Chagas disease, Ebola virus, Plasmodium falciparum, Marburg virus, Japanese encephalitis virus, St. Louis encephalitis virus, West Nile Virus, Yellow Fever virus, Bacillus anthracis, Botulinum toxin, Ricin, Nipah, Shiga toxin and / or Shiga-like toxin.89- A method of treating cancer in a subject comprising administering the lipid nanoparticle according to any one of claims 49 to 74 to the subject, wherein the lipid nanoparticle comprises an anti-cancer cargo or a cargo triggering an immune response against cancer cells.90- The method according to claim 89, wherein the cancer is lung cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, colorectal cancer, bladder cancer, prostate cancer, cervical cancer, renal cancer, leukemia, central nervous system cancers, myeloma, or melanoma.91 - A lipid nanoparticle according to any one of claims 49 to 74 for use in the treatment of cancer in a subject, wherein the lipid nanoparticle comprises an anti-cancer cargo or a cargo triggering an immune response against cancer cells.92- The lipid nanoparticle for use according to claim 91 , wherein the cancer is lung cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, colorectal cancer, bladder cancer, prostate cancer, cervical cancer, renal cancer, leukemia, central nervous system cancers, myeloma, or melanoma.93- Use of the lipid nanoparticle according to any one of claims 46 to 74 for treating cancer in a subject, wherein the lipid nanoparticle comprises an anti-cancer cargo or a cargo triggering an immune response against cancer cells.94- The use according to claim 93, wherein the cancer is lung cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, colorectal cancer, bladder cancer, prostate cancer, cervical cancer, renal cancer, leukemia, central nervous system cancers, myeloma, or melanoma.95- Use of the lipid nanoparticle according to any one of claims 49 to 74 for the preparation of a medicament for treating cancer in a subject, wherein the lipid nanoparticle comprises an anticancer cargo or a cargo triggering an immune response against cancer cells.96- The use according to claim 95, wherein the cancer is lung cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, colorectal cancer, bladder cancer, prostate cancer, cervical cancer, renal cancer, leukemia, central nervous system cancers, myeloma, or melanoma.97- The method of claim 89 or 90, the lipid nanoparticle for use of claim 91 or 92, or the use of any one of claims 93 to 96, wherein the cargo comprises at least one of a small molecule, an antibody, a polynucleotide or a polypeptide.98- The method of claim 89 or 90, the lipid nanoparticle for use of claim 91 or 92 or the use of any one of claims 93 to 96, wherein the cargo comprises at least one nucleic acid, such as mRNA.

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