Lipid structure and composition containing the same

Abstract

The present disclosure relates to ionizable lipids and compositions containing ionizable lipids. Ionizable lipids for delivery to mammalian cells or organs, optionally in combination with other lipid components such as helper lipids, stabilizing lipids, and structural lipids, and therapeutic agents such as nucleic acids, are described, which are composed of lipid-nanoparticle compositions.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 355,024, filed on June 23, 2022; U.S. Provisional Application No. 63 / 386,482, filed on December 7, 2022; and U.S. Provisional Application No. 63 / 464,022, filed on May 4, 2023, each of which is hereby incorporated by reference in its entirety.

[0002] The present disclosure relates to ionizable lipids and helper lipids that can be used in combination with other lipid components such as stabilizing lipids and structural lipids. The present disclosure also provides lipid - nanoparticle compositions containing such lipids for the delivery of therapeutic molecules, particularly therapeutic nucleic acids.

Background Art

[0003] Nucleic - acid - based therapies have received increasing attention in recent years because they have great potential to treat diseases by targeting their genetic blueprints in vivo. Nucleic - acid - based therapeutics can achieve long - term effects, or even curative effects, through gene inhibition, addition, replacement, or editing. However, the clinical translation of both nucleic acid agents and other therapeutic molecules depends on delivery technologies that improve stability, promote internalization, and / or increase target affinity.

[0004] Lipid - based delivery systems, including but not limited to lipid nanoparticles (LNPs), can provide an approach for stabilizing and delivering nucleic acids and other therapeutic molecules, and there remains an important need for the evolution of this technology. Further advancing design characteristics such as optimal particle size, encapsulation efficiency, robust manufacturing processes, different lipid affinities, and appropriate surface charges can provide an efficient lipid - based delivery system for nucleic acids and other therapeutic molecules.

Brief Description of the Drawings

[0005]

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Mode for Carrying Out the Invention

[0006] Summary of the Invention The following aspects and their embodiments described below are meant to be illustrative and exemplary and not limiting in scope.

[0007] In one aspect, the present disclosure relates to an ionizable lipid of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof,

Chemical formula

[0008] In another aspect, the ionizable lipid of formula (I) has one of the following structures:

Chemical formula

[0009] In one aspect, the present disclosure relates to an ionizable lipid of formula (I-A), or a pharmaceutically acceptable salt or stereoisomer thereof,

Chemical formula

[0010] In another aspect, the ionizable lipid of formula (I-A) has the following structure:

Chemical formula

[0011] In one aspect, the present disclosure relates to an ionizable lipid of formula (I-B) or a pharmaceutically acceptable salt or stereoisomer thereof, [Chemical formula] In the formula, L 1 is C1-C6 alkylene, and L 2 is C1-C8 alkylene, and L 3 is C1-C8 alkylene, and R 3 and R 4 are each independently H or C1-C3 alkyl, and R 6 is C4-C 20 alkyl, and R 7 is C4-C 20 alkyl, and R 8 is C4-C 20 alkyl, and R 10 is C4-C 20 alkyl.

[0012] In another aspect, the ionizable lipid of formula (I-B) has the following structure: [Chemical formula]

[0013] In one aspect, the present disclosure relates to an ionizable lipid of formula (I-B), or a pharmaceutically acceptable salt or stereoisomer thereof, [Chemical formula] In the formula, L 1 is C1-C6 alkylene, and L 2 is C1-C8 alkylene, and L 3 is C1-C8 alkylene, and R 3 and R 4 are each independently H, C1-C4 alkyl, -CH2-cyclopropyl, -(CH2) n OH, or R 3 and R 4form an N - heterocycle together, and R 6 is C4 - C 20 alkyl, and R 7 is C4 - C 20 alkyl, and R 8 is C4 - C 20 alkyl, and R 10 is C4 - C 20 alkyl, and n is 2, 3, or 4. In other embodiments, the ionizable lipid of formula (I - B) has one of the following structures:

Chemical formula

[0014] In one embodiment, the present disclosure relates to an ionizable lipid of formula (II), or a pharmaceutically acceptable salt or stereoisomer thereof,

Chemical formula

[0015] In other embodiments, the ionizable lipid of formula (II) has one of the following structures:

Chemical formula

[0016] In one aspect, the present disclosure relates to an ionizable lipid of formula (III), or a pharmaceutically acceptable salt or stereoisomer thereof,

Chemical formula

[0017] In other aspects, the ionizable lipid of formula (III) has one of the following structures:

Chemical formula

[0018] In one aspect, the present disclosure relates to an ionizable lipid of formula (IV), or a pharmaceutically acceptable salt or stereoisomer thereof,

Chemical formula

[0019] In other embodiments, the ionizable lipid of formula (IV) has one of the following structures:

Chemical formula

[0020] In one embodiment, the present disclosure relates to an ionizable lipid of formula (V), or a pharmaceutically acceptable salt or stereoisomer thereof,

Chemical formula

[0021] In other embodiments, the ionizable lipid of formula (V) has the following structure:

Chemical formula

[0022] In one embodiment, the present disclosure relates to an ionizable lipid of formula (VI), or a pharmaceutically acceptable salt or stereoisomer thereof,

Chemical formula

[0023] In other embodiments, the ionizable lipid of formula (VI) has one of the following structures:

Chemical formula

[0024] In one embodiment, the present disclosure relates to an ionizable lipid of formula (VII), or a pharmaceutically acceptable salt or stereoisomer thereof,

Chemical formula

[0025] In other embodiments, the ionizable lipid of formula (VII) has one of the following structures: [Chemical formula]

[0026] In one embodiment, the present disclosure relates to an ionizable lipid of formula (VIII), or a pharmaceutically acceptable salt or stereoisomer thereof, [Chemical formula] wherein L 1 is C1-C6 alkylene, L 1’ is C1-C6 alkylene, R 2 is C6-C 20 alkyl, R 2’ is C6-C 20 alkyl, R 9 is H, C1-C6 alkyl, or -(CH2) n OH, R 14 is C6-C 20 alkyl, R 14’ is C6-C 20 alkyl, R 15 is C6-C 20 alkyl, and n is 2, 3, or 4.

[0027] In other embodiments, the ionizable lipid of formula (VIII) has the following structure: [Chemical formula] In one embodiment, the present disclosure relates to an ionizable lipid of formula (IX), or a pharmaceutically acceptable salt or stereoisomer thereof, [Chemical formula] In the formula, R 1 is C6-C 20 alkenyl, and R 9 is H, C1-C6 alkyl, or -(CH2) n OH, and R 12 is C6-C 20 alkenyl, and n is 2, 3, or 4.

[0028] In another aspect, the ionizable lipid of formula (IX) has one of the following structures: [Chemical formula]

[0029] In one aspect, the present disclosure relates to an ionizable lipid of formula (X), or a pharmaceutically acceptable salt or stereoisomer thereof, [Chemical formula] In the formula, L 4 is absent or C1-C6 alkylene, and L 5 is absent or C1-C6 alkylene, and R 1 is C6-C 20 alkenyl, and R 2 is C6-C 20 alkyl, and R 2’ is C6-C 20 alkyl, and R 9 is H, C1-C6 alkyl, or -(CH2) n OH, and R 12 is C6-C 20 alkenyl, and n is 2, 3, or 4.

[0030] In other embodiments, the ionizable lipid of formula (X) has one of the following structures:

Chemical formula

[0031] In one embodiment, the present disclosure relates to an ionizable lipid of formula (XI), or a pharmaceutically acceptable salt or stereoisomer thereof,

Chemical formula

[0032] In other embodiments, the ionizable lipid of formula (XI) has the following structure:

Chemical formula

[0033] In one embodiment, the present disclosure relates to an ionizable lipid of formula (XII), or a pharmaceutically acceptable salt or stereoisomer thereof,

Chemical formula

[0034] In another aspect, the ionizable lipid of formula (XII) has the following structure: [Chemical formula]

[0035] In one aspect, the present disclosure relates to an ionizable lipid of formula (XIII), or a pharmaceutically acceptable salt or stereoisomer thereof, [Chemical formula] In the formula, L 2 is C1-C8 alkylene, and L 2’ is C1-C8 alkylene, and R 7 is C4-C 20 alkyl, and R 7’ is C4-C 20 alkyl, and R 8 is C4-C 20 alkyl, and R 8’ is C4-C 20 alkyl, and R 13 is H, C1-C6 alkyl, -(CH2)n OH, or -(CH2) q is N(CH3)2, n is 2, 3, or 4, and q is 2, 3, or 4.

[0036] In some embodiments, in formula (XIII), L 2 is C1-C8 alkylene, L 2’ is C1-C8 alkylene, R 7 is C4-C 20 alkyl, R 7’ is C4-C20 alkyl, R8 is C4-C20 alkyl, R8’ is C4-C20 alkyl, and R13 is H.

[0037] In some embodiments, in formula (XIII), L2 is C1-C8 alkylene, L2’ is C1-C8 alkylene, R7 is C4-C20 alkyl, R7’ is C4-C20 alkyl, R8 is C4-C20 alkyl, R8’ is C4-C20 alkyl, and R13 is methyl.

[0038] In some embodiments, in formula (XIII), L2 is C1-C8 alkylene, L2’ is C1-C8 alkylene, R7 is C4-C20 alkyl, R7’ is C4-C20 alkyl, R8 is C4-C20 alkyl, R8’ is C4-C20 alkyl, and R13 is -(CH2)nOH where n is 2.

[0039] In some embodiments, in formula (XIII), L2 is C1-C8 alkylene, L2’ is C1-C8 alkylene, R7 is C4-C20 alkyl, R7’ is C4-C20 alkyl, R8 is C4-C20 alkyl, R8’ is C4-C20 alkyl, and R13 is -(CH2)qN(CH3)2 where q is 3.

[0040] Exemplary examples of the ionizable lipid of formula (XIII) can include, but are not limited to: [Chemical formula]

[0041] In one embodiment, the ionizable lipid is of formula (XIV), or a pharmaceutically acceptable salt or stereoisomer thereof, [Chemical formula] wherein, L1 is C1-C6 alkylene, L2 is C1-C8 alkylene, R3 and R4 are each independently H or C1-C3 alkyl, R6 is C4-C20 alkyl, and R7 is C4-C20 alkyl.

[0042] In some embodiments, in formula (XIV), L 1 is C1-C6 alkylene, L 2 is C1-C8 alkylene, R 3 is methyl, R 4 is methyl, R 6 is C4-C 20 alkyl, and R 7 is C4-C 20 alkyl.

[0043] Exemplary examples of the ionizable lipid of formula (XIV) can include, but are not limited to: [Chemical formula]

[0044] In one embodiment, the ionizable lipid is of formula (XV), or a pharmaceutically acceptable salt or stereoisomer thereof, [Chemical formula] wherein, L2 is C1-C8 alkylene, L2’ is C1-C8 alkylene, R6 is C4-C20 alkyl, R6’ is C4-C20 alkyl, R7 is C4-C20 alkyl, R7’ is C4-C20 alkyl, R8 is C4-C20 alkyl, R8’ is C4-C20 alkyl, R10 is C4-C20 alkyl, R10’ is C4-C20 alkyl, R13 is H, C1-C6 alkyl, -(CH2)nOH, or -(CH2)qN(CH3)2, n is 2, 3 or 4, p1 is absent or 1, p2 is absent or 1, and q is 2, 3, or 4.

[0045] In some embodiments, in formula (XV), L2 is C1-C8 alkylene, L2’ is C1-C8 alkylene, R6 is C4-C20 alkyl, R6’ is C4-C20 alkyl, R7 is C4-C20 alkyl, R7’ is C4-C20 alkyl, R8 is C4-C20 alkyl, R8’ is C4-C20 alkyl, R10 is C4-C20 alkyl, R10’ is C4-C20 alkyl, R13 is H, p1 is absent, and p2 is absent.

[0046] In some embodiments, in formula (XV), L2 is C1-C8 alkylene, L2’ is C1-C8 alkylene, R6 is C4-C20 alkyl, R6’ is C4-C20 alkyl, R7 is C4-C20 alkyl, R7’ is C4-C20 alkyl, R8 is C4-C20 alkyl, R8’ is C4-C20 alkyl, R10 is C4-C20 alkyl, R10’ is C4-C20 alkyl, R13 is H, p1 is 1, and p2 is 1.

[0047] In some embodiments, in formula (XV), L2 is C1-C8 alkylene, L2’ is C1-C8 alkylene, R6 is C4-C20 alkyl, R6’ is C4-C20 alkyl, R7 is C4-C20 alkyl, R7’ is C4-C20 alkyl, R8 is C4-C20 alkyl, R8’ is C4-C20 alkyl, R10 is C4-C20 alkyl, R10’ is C4-C20 alkyl, R13 is methyl, p1 is absent, and p2 is absent.

[0048] In some embodiments, in formula (XV), L2 is C1-C8 alkylene, L2’ is C1-C8 alkylene, R6 is C4-C20 alkyl, R6’ is C4-C20 alkyl, R7 is C4-C20 alkyl, R7’ is C4-C20 alkyl, R8 is C4-C20 alkyl, R8’ is C4-C20 alkyl, R10 is C4-C20 alkyl, R10’ is C4-C20 alkyl, R13 is methyl, p1 is 1, and p2 is 1.

[0049] In some embodiments, in formula (XV), L2 is C1-C8 alkylene, L2’ is C1-C8 alkylene, R6 is C4-C20 alkyl, R6’ is C4-C20 alkyl, R7 is C4-C20 alkyl, R7’ is C4-C20 alkyl, R8 is C4-C20 alkyl, R8’ is C4-C20 alkyl, R10 is C4-C20 alkyl, R10’ is C4-C20 alkyl, R13 is -(CH2)nOH, n is 4, p1 is absent, and p2 is absent.

[0050] In some embodiments, in formula (XV), L2 is C1-C8 alkylene, L2' is C1-C8 alkylene, R6 is C4-C20 alkyl, R6' is C4-C20 alkyl, R7 is C4-C20 alkyl, R7' is C4-C20 alkyl, R8 is C4-C20 alkyl, R8' is C4-C20 alkyl, R10 is C4-C20 alkyl, R10' is C4-C20 alkyl, R13 is -(CH2)nOH, n is 4, p1 is 1, and p2 is 1.

[0051] Exemplary examples of the ionizable lipid of formula (XV) can include, but are not limited to:

Chemical formula

[0052] In one embodiment, the ionizable lipid is of formula (XVI), or a pharmaceutically acceptable salt or stereoisomer thereof,

Chemical formula

[0053] In one embodiment, the ionizable lipid is of formula (XVI-A), or a pharmaceutically acceptable salt or stereoisomer thereof:

Chemical formula

[0054] In some embodiments, in formula (XVI) and formula (XVI-A), L 2 is C1-C8 alkylene, and L 2’ is C1-C8 alkylene, and R 6 is C4-C 20 alkyl, and R 6’ is C4-C 20 alkyl, and R 7 is C4-C 20 alkyl, and R 7’ is C4-C 20 alkyl, and R 13 is H, p1 is absent or 1, and p2 is absent or 1.

[0055] In some embodiments, in formula (XVI) and formula (XVI-A), L 2 is C1-C8 alkylene, and L 2’ is C1-C8 alkylene, and R 6 is C4-C 20 alkyl, and R 6’ is C4-C 20 alkyl, and R 7 is C4-C20 is alkyl, and R 7’ is C4-C 20 is alkyl, and R 13 is methyl, p1 is absent or 1, and p2 is absent or 1. In other embodiments, in formulas (XVI) and (XVI-A), L 2 is C1-C8 alkylene, L 2’ is C1-C8 alkylene, and R 6 is C4-C 20 is alkyl, and R 6’ is C4-C 20 is alkyl, and R 7 is C4-C 20 is alkyl, and R 7’ is C4-C 20 is alkyl, and R 13 is -(CH2) n OH, n is 4, p1 is absent, and p2 is absent.

[0056] In some embodiments, in formulas (XVI) and (XVI-A), L 2 is C1-C8 alkylene, L 2’ is C1-C8 alkylene, and R 6 is C4-C 20 is alkyl, and R 6’ is C4-C 20 is alkyl, and R 7 is C4-C 20 is alkyl, and R 7’ is C4-C 20 is alkyl, and R 13 is -(CH2) n OH, n is 4, p1 is 1, and p2 is 1.

[0057] Exemplary examples of the ionizable lipids of formulas (XVI) and (XVI-A) can include, but are not limited to:

Chemical formula

[0058] In one embodiment, it is an ionizable lipid of formula (I-C), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

Chemical formula

[0059] In some embodiments, the ionizable lipid has the following structure:

Chemical formula

[0060] In one embodiment, it is an ionizable lipid of formula (XXII), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

Chemical formula

[0061] In some embodiments, the ionizable lipid has the following structure:

Chemical formula

[0062] In one aspect, the present disclosure relates to a lipid-nanoparticle composition comprising any one of the ionizable lipids of Formula (I) to Formula (XXII). Throughout the disclosure herein, "lipid-nanoparticle composition" can refer to a composition containing lipid nanoparticles or the lipid nanoparticles themselves. The lipid-nanoparticle composition can further comprise helper lipids, stabilizing lipids, structural lipids, and an active agent, and the active agent is a nucleic acid, a small molecule, a protein or peptide, or a combination thereof. In embodiments, the lipid-nanoparticle composition excludes stabilizing lipids. In embodiments, the lipid-nanoparticle composition excludes lipids conjugated to polyethylene glycol (PEG-lipids).

[0063] In some embodiments, the helper lipid in the lipid nanoparticle composition is selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-didodecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), l-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and mixtures thereof.

[0064] In some embodiments, the stabilizing lipid in the lipid-nanoparticle composition is 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG), having an average PEG molecular weight of about 2000 Daltons. In some embodiments, the stabilizing lipid is a polysarcosine-lipid conjugate. In some embodiments, the polysarcosine-lipid conjugate does not associate with RNA in the lipid-nanoparticle composition. In some embodiments, the polysarcosine-lipid conjugate does not form RNA particles in the lipid-nanoparticle composition. In some embodiments, the polysarcosine-lipid conjugate forms RNA particles without associating with RNA in the lipid-nanoparticle composition.

[0065] In some embodiments, the structural lipid in the lipid-nanoparticle composition is selected from the group consisting of cholesterol, cholesterol derivatives, fucosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof.

[0066] In some embodiments, the nucleic acid is selected from the group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), guide RNA (gRNA), plasmid DNA (pDNA), antisense oligodeoxynucleotide (ODN), RNA or DNA vaccine, and mixtures thereof.

[0067] In some embodiments, the nucleic acid or mRNA is self - amplifying RNA. In some embodiments, the nucleic acid or mRNA is polycistronic RNA. In some embodiments, the nucleic acid or mRNA is self - amplifying polycistronic RNA. In some embodiments, the nucleic acid or mRNA is circular RNA. In some embodiments, the nucleic acid or mRNA expresses a protein or peptide. In some embodiments, the protein or peptide expressed from the nucleic acid or mRNA is an antibody, a human antibody, a camelid antibody, a nanobody, a humanized antibody, a bispecific antibody, an enzyme, a genome - editing enzyme or nuclease, a growth factor, a cytokine, a chemokine, a small - molecule mimetic peptide, a transcription factor, a structural molecule, a signaling molecule, a reprogramming factor, a vaccine antigen, or a combination thereof. In some embodiments, the mRNA encodes a protein or peptide that acts intracellularly. In some embodiments, the mRNA encodes at least one reprogramming factor.

[0068] In some embodiments, the protein or peptide expressed from the nucleic acid or mRNA is at least one extracellular matrix protein. In some embodiments, the extracellular matrix protein is collagen, laminin, elastin, fibronectin, integrin, tenascin, proteoglycan, fibrin, or a combination thereof. In some embodiments, the collagen is collagen I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XXII, XXIII, XXIV, XXV, XXVI, XXVII, XXVIII, or a combination thereof. In some embodiments, the collagen is collagen VII. In some embodiments, collagen VII is used in a method of rejuvenating, treating, remodeling, or improving the skin or extracellular matrix. In some embodiments, collagen VII is used in a method of wound healing.

[0069] In some embodiments, the protein or peptide expressed from the nucleic acid or mRNA is a growth factor, cytokine, or a combination thereof. In some embodiments, the growth factor is EGF, FGF, NGF, CNTF, PDGF, VEGF, IGF, GMCSF, GCSF, TGF, erythropoietin, ephrin, GDNF, GDF9, KGF, angiopoietin, TPO, BMP, HGF, BDNF, GDF, HGH (somatotropin), neurotrophin, MSF, SGF, GDF (including GDF11), TGF (including TGF-β), or a combination thereof. In some embodiments, the cytokine is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, TNF-α, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, CXCL8 (formerly IL-18), IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, or a combination thereof.

[0070] In some embodiments, the protein or peptide expressed from the nucleic acid or mRNA is a human antibody, humanized antibody, camelid antibody, companion animal antibody, or nanobody. In some embodiments, the protein or peptide expressed from the nucleic acid or mRNA is an enzyme such as a nuclease, for example, a nuclease used in genome editing. In some embodiments, the protein or peptide expressed from the nucleic acid or mRNA acts intracellularly. In some embodiments, the protein or peptide expressed from the nucleic acid or mRNA is secreted. In some embodiments, the antibody is trastuzumab, glofitamab, miliximab, mirvetuximab, nirsevimab, tremelimumab, teclistamab, donanemab, spesolimab, lecanemab, tislelizumab, pemtumumab, sintilimab, teprotumumab, tripalimumab, omburtamab, retifanlimab, ublituximab, inolimomab, oportuzumab, narsoplimab, mosunetuzumab, tixagevimab, cilgavimab, relatlimab, tebentafusp, faricimab, stimtuzumab, sotrovimab, leguvimab, casirivimab, imdevimab, tezepelumab, chisotuzumab, amivantamab, anifrolumab, loncastuximab, bimekizumab, tralokinumab, evinacumab, sacituzumab, teprotumumab, isatuximab, eptinezumab, dostarlimab, ansuvimab, margetuximab, naxitamab, atorvastatinumab, mafutivimab, odesivimab, belantamab, tafasitamab, satralizumab, inebilizumab, enfuvirtide, crisantaspase, brodalumab, polatuzumab, lisankizumab, romosozumab, caplacizumab, labralizumab, emapalumab, semaprimab, fremtuzumab, moxetumomab, galcanezumab, ranalizumab, mogamulizumab, elotuzumab, tiludakizumab, ibalizumab, brospatumumab, durvalumab, emicizumab, benralizumab, ocrelizumab, guselkumab, inotuzumab, sarlumab, dupilumab, abemaciclib, brodalumab, atezolizumab, bezlotoxumab, orlatumumab, reslizumab, obiltoxaximab, ixekizumab, daratumumab, elotuzumab, nesvacumab, idarucizumab, allerocumab, mapatumumab, evolocumab, dinutuximab, secukinumab,It is at least one of nivolumab, blinatumomab, pembrolizumab, ramucirumab, vedolizumab, siltuximab, obinutuzumab, lirilumab, pertuzumab, brentuximab, belimumab, ipilimumab, denosumab, tocilizumab, ofatumumab, canakinumab, golimumab, ustekinumab, certolizumab, catumaxomab, eculizumab, ranibizumab, panitumumab, natalizumab, bevacizumab, cetuximab, efalizumab, omalizumab, tositumomab, ibritumomab, adalimumab, alemtuzumab, gemtuzumab, infliximab, palivizumab, basiliximab, daclizumab, rituximab, abciximab, edrecolomab, nebacumab, or muromonab, or is substantially similar thereto. In some embodiments, the protein or peptide is used in a method for treating a human or veterinary disease.

[0071] In some aspects, the small molecule is a chemotherapeutic agent, a GPCR agonist or antagonist, a transcriptional regulator, or an RNA splicing regulator. In some aspects, the small molecule acts intracellularly.

[0072] In some aspects, the protein or peptide is an antibody, a humanized antibody, a bispecific antibody, an enzyme, a genome editing enzyme or nuclease, a growth factor, a cytokine, a chemokine, a small molecule mimetic peptide, a transcription factor, a structural molecule, a signaling molecule, a reprogramming factor, a vaccine antigen, or a combination thereof. In some aspects, the protein or peptide acts intracellularly.

[0073] In some embodiments, the small molecule, protein, or peptide incorporated into the lipid nanoparticles and / or delivered by the lipid nanoparticle composition is a component of an "artificial niche" used to maintain quiescence of progenitor cells. In some embodiments, the artificial niche component is selected from the group consisting of elcatonin, MGCD-265, JNJ-7706621, forskolin, fomestatin, SB203580, SU5402, TGF-β, insulin-transferrin-selenium, and combinations thereof. These and other artificial niche components are disclosed in U.S. Patent No. 10,688,136, which is incorporated herein by reference.

[0074] In some embodiments, the disclosure relates to a pharmaceutical composition comprising a lipid-nanoparticle composition and a pharmaceutically acceptable carrier therefor.

[0075] In some embodiments, the methods and compositions provided herein are applied to cells, tissues, or organs of the nervous system, muscular system, respiratory system, cardiovascular system, skeletal system, genital system, integumentary system, lymphatic system, excretory system, immune system, endocrine system (e.g., endocrine and exocrine), or digestive system. As described herein, any type of cell can potentially be rejuvenated, including, but not limited to, epithelial cells (e.g., squamous epithelial cells, cuboidal epithelial cells, columnar epithelial cells, and pseudostratified epithelial cells), endothelial cells (e.g., venous endothelial cells, arterial endothelial cells, and lymphatic endothelial cells), and cells of connective tissue, muscle, and the nervous system. Such cells include, but are not limited to, epidermal cells, fibroblasts, chondrocytes, skeletal muscle cells, satellite cells, cardiomyocytes, smooth muscle cells, keratinocytes, basal cells, ameloblasts, exocrine cells, myoepithelial cells, osteoblasts, osteoclasts, neurons (e.g., sensory neurons, motor neurons, and interneurons), glial cells (e.g., oligodendrocytes, astrocytes, ependymal cells, microglia, Schwann cells, and satellite cells), columnar cells, adipocytes, pericytes, stellate cells, lung cells, blood and immune system cells (e.g., erythrocytes, monocytes, dendritic cells, macrophages, neutrophils, eosinophils, mast cells, T cells, B cells, natural killer cells), hormone-secreting cells, germ cells, stromal cells, lens cells, photoreceptor cells, taste receptor cells, and olfactory cells; and cells and / or tissues from the kidney, liver, pancreas, stomach, spleen, gallbladder, intestine, bladder, lung, prostate, breast, urogenital tract, pituitary cells, oral cavity, esophagus, skin, hair, nails, thyroid, parathyroid, adrenal glands, eyes, nose, or brain may be included, but are not limited thereto.

[0076] In some embodiments, the cells are selected from fibroblasts, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells. In an embodiment, the cell is a fibroblast. In an embodiment, the cell is an endothelial cell. In an embodiment, the cell is a chondrocyte. In an embodiment, the cell is a skeletal muscle stem cell. In an embodiment, the cell is a keratinocyte. In an embodiment, the cell is a mesenchymal stem cell. In an embodiment, the cell is a corneal epithelial cell.

[0077] In some embodiments, the methods and compositions of the present technology are applied to immune cells including, but not limited to, lymphocytes, granulocytes, monocytes, macrophages, microglia, or dendritic cells. In some embodiments, the lymphocytes are T cells, B cells, or natural killer (NK) cells. In some embodiments, the lymphocytes are tumor-infiltrating lymphocytes.

[0078] In some embodiments, the lymphocytes are T cells. In some embodiments, the T cells are cytotoxic T cells (CD8+), helper T cells (CD4+), suppressor or regulatory T cells (Tregs), memory T cells, natural killer T cells (NKT cells), or gamma delta T cells. In other embodiments, the helper T cells are Th1, Th2, Th17, Th9, or Tfh T cells. In some embodiments, the memory T cells are central memory T cells, effector memory T cells, tissue-resident memory T cells, or virtual memory T cells. In some embodiments, the suppressor or regulatory T cells of the present technology are FOXP3+ T cells or FOXP3− T cells. In some embodiments, the NKT cells are a subset of CD1d-restricted T cells.

[0079] In some embodiments, the granulocytes of the present technology are neutrophils, eosinophils, basophils, or mast cells.

[0080] In other embodiments, the lymphocytes of the present technology are B cells. In some embodiments, the B cells are memory B cells or plasma cells.

[0081] In other embodiments, the immune cells are monocytes, macrophages, microglia cells, or dendritic cells.

[0082] In some embodiments, the methods and compositions described herein can be used where the cell is an immune cell such as a natural immune cell or an engineered immune cell. In some embodiments, the methods and compositions described herein are used in parallel with, or consecutive to, a method of engineering a cell, including an engineered immune cell, such that the method is performed before, during, and / or after the engineering of the cell. In some embodiments, the methods and compositions described herein are used to engineer a cell, including an engineered immune cell. In some embodiments, such engineering includes engineering the cell to express a chimeric antigen receptor, such as an immune cell that expresses a chimeric antigen. In some embodiments, such chimeric antigen receptor targets CD19, CD30, CD33, CD123, FLT3, BCMA, GD2, or any other antigen suitable for immunotherapy. In some embodiments, such engineering includes engineering a cell including an immune cell to express other proteins or peptides, such as growth factors and cytokines. In some embodiments, such cytokine includes IL-15. In some embodiments, such engineering of a cell such as an immune cell is performed ex vivo, for example, in the manufacture of a cell therapy product such as an autologous or allogeneic chimeric antigen receptor (CAR)-T, CAR-NK, CAR-M, or CAR-NKT cell. In some embodiments, the CAR-NKT cells provided herein are engineered to target GD2 with a chimeric antigen receptor and to express IL-15. In such embodiments, the immune cell rejuvenation method described herein is performed ex vivo during or after the manufacture of the cell therapy product. In other embodiments, such engineering of the cell, and / or the immune cell, is performed ex vivo, for example, in the so-called "in situ" generation of CAR-engineered cells.In such embodiments, the RNA and / or mRNA encoding a CAR or a growth factor or a cytokine contained in the lipid-containing composition or lipid-nanoparticle composition of the present disclosure is injected in vivo into a subject or patient for in vivo CAR engineering of immune cells of the patient, such as, for example, T cells, NK cells, macrophages, tumor-infiltrating lymphocytes, dendritic cells, and / or NKT cells, i.e., "in situ", without the need to remove cells for ex vivo transfection. In such embodiments, the immune cell rejuvenation method described herein is also performed in vivo, and the mRNA encoding a reprogramming factor or factors is injected into the patient before, simultaneously with, or after the mRNA encoding a CAR or other cell engineering molecule. In some embodiments, the lipids and lipid-nanoparticles of the present disclosure are selected for in vivo targeted delivery to any cell, including immune cells such as T cells, NK cells, macrophages, tumor-infiltrating cells, dendritic cells, and / or NKT cells in vivo. In still other embodiments, the in vivo treatment is performed in the absence of any other in vivo cell engineering to enhance or restore the efficacy of the immune system, improve the effect of the immune system against cancer or an infection, or treat a disease associated with an immune dysfunction or dysregulation such as reducing inflammation.

[0083] In some embodiments, the rejuvenating immune cells are non-adherent cells such as non-adherent immune cells. In some embodiments, non-adherent cells, including non-adherent immune cells, are processed, transiently reprogrammed, rejuvenated, or manufactured in a manner such that the cells remain non-adherent without adhering to a tissue culture substrate or forming or giving rise to adherent cells or colonies of cells on the tissue culture substrate. In some embodiments, the reprogramming intervals and factors are selected such that the cells remain non-adherent without adhering to a tissue culture substrate or forming or giving rise to adherent cells or colonies of cells on the tissue culture substrate and such that the cells rejuvenate with retention of cell identity. Thus, in some embodiments, the technology provides lipid-containing compositions and lipid-nanoparticle compositions for delivering mRNA encoding at least one reprogramming factor for cell rejuvenation, and cells comprising any non-adherent cells and / or non-adherent immune cells (e.g., non-adherent T cells, NK cells, macrophages, tumor infiltrating cells, dendritic cells, and / or NKT cells) are reprogrammed in a manner such that the cells rejuvenate with retention of cell identity, the cells remain in suspension and are non-adherent, and they do not become adherent, form adherent colonies, or give rise to such cells.

[0084] In some embodiments, the methods described herein, which include a method of rejuvenating immune cells, reversing, preventing, or inhibiting exhaustion in immune cells, or inducing the proliferation of immune cells, comprise administering to the immune cells a lipid-containing composition or a lipid-nanoparticle composition of the present disclosure comprising mRNA encoding at least one reprogramming factor one, two, three, four, five, six, seven, eight, nine, or ten times over a period of one, two, three, four, five, six, seven, eight, nine, or ten days. For example, the mRNA can be administered once on the first or second day of a five- or six-day period, or it can be administered once on the first and third days of a five- or six-day period, or it can be administered once over a one-day period. In some embodiments, the mRNA is administered to the immune cells one, two, three, four, five, or six times over a period of one, two, three, four, five, or six days. In some embodiments, the mRNA is administered after an immune cell activation step. In some embodiments, the immune cell activation step comprises activating the immune cells for one, two, or three days. In some embodiments, the immune cell activation step comprises activating the immune cells using at least one of CD3, CD28, and IL-2. In some embodiments, the immune cells are activated with CD3 and CD28. In some embodiments, the mRNA administration period occurs immediately after the immune cell activation step. In some embodiments, the mRNA administration period occurs one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen days after the immune cell activation step. In some embodiments, administration of the mRNA encoding the reprogramming factor reverses immune cell exhaustion caused by the immune cell activation step. In some embodiments, administration of the mRNA encoding the reprogramming factor reverses immune cell exhaustion in immune cells from an elderly patient or donor. In some embodiments, the mRNA is administered during a manufacturing process to produce immune cells for transplantation, such as CAR-T, CAR-M, or CAR-NK cells.

[0085] In some embodiments, the use of the lipids or lipid-nanoparticles of the present disclosure for the delivery of mRNA results in enhanced rejuvenation, proliferation, recovery from or prevention of exhaustion, therapeutic effect, anti-pathogenic effect, anti-cancer effect, anti-immunogenic effect, or anti-inflammatory effect in cells that are treated or rejuvenated using the methods and compositions herein, as compared to using different delivery mechanisms for mRNA. In some embodiments, such enhanced rejuvenation, proliferation, recovery from or prevention of exhaustion, therapeutic effect, anti-pathogenic effect, anti-cancer effect, anti-immunogenic effect, or anti-inflammatory effect is due to lower toxicity, immunogenicity, and / or lower physiological impact on the cells as compared to different delivery mechanisms. In some embodiments, the different delivery mechanism is electroporation, and the use of the lipids or lipid-nanoparticle compositions of the present disclosure for the delivery of mRNA results in enhanced rejuvenation, proliferation, recovery from or prevention of exhaustion, therapeutic effect, anti-pathogenic effect, anti-cancer effect, anti-immunogenic effect, or anti-inflammatory effect in cells that are treated or rejuvenated, as compared to using electroporation. This improvement as compared to electroporation may be due to reduced toxicity or reduced physiological impact on the cells as compared to electroporation.

[0086] In some aspects, the lipid or lipid-nanoparticle compositions of the present disclosure are used in a method of delivering a therapeutic or diagnostic agent to the skin, comprising administering a lipid-containing composition or lipid-nanoparticle composition comprising at least one therapeutic or diagnostic agent. In some embodiments, the lipid or lipid-nanoparticle compositions of the present technology provide delivery of a therapeutic or diagnostic agent, such as a reprogramming factor, in a method that achieves transient reprogramming of cells, such as skin cells or immune cells. In some embodiments, transient reprogramming of the cells provides transient expression of a therapeutic or diagnostic agent, such as a reprogramming factor, and the agent reprograms and / or rejuvenates the cells for a period sufficient to effect rejuvenation without changing the identity of the cells, i.e., the skin or immune cells are rejuvenated while maintaining their identity as skin or immune cells and exhibit characteristics or a more youthful phenotype of skin or immune cells.

[0087] In some embodiments, the therapeutic agent is the mRNA disclosed herein. In some embodiments, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for treating or preventing a dermatological disease or condition, treating or preventing a disease or condition of the skin, or for cosmetic application on the skin, and comprises administering a lipid-containing composition or lipid-nanoparticle composition comprising at least one therapeutic or diagnostic agent. In some embodiments, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for treating or preventing a dermatological disease or condition or a disease or condition of the skin, and comprises administering a lipid-containing composition or lipid-nanoparticle composition comprising at least one therapeutic or diagnostic agent. In some embodiments, the therapeutic agent is the mRNA disclosed herein. In some embodiments, the mRNA encodes at least one reprogramming factor. In some embodiments, the lipid or lipid-nanoparticle composition of the present disclosure is used for cosmetic application on the skin, and comprises administering a lipid-containing composition or lipid-nanoparticle composition comprising at least one therapeutic or diagnostic agent. In some embodiments, the therapeutic agent is the mRNA disclosed herein. In some embodiments, the mRNA encodes at least one reprogramming factor. In some embodiments, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for rejuvenating the skin, and comprises administering a lipid-containing composition or lipid-nanoparticle composition comprising a therapeutic or diagnostic agent. In some embodiments, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for rejuvenating the skin, and comprises administering a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor. In such methods, the mRNA can be bound to the lipid or contained in the lipid-nanoparticle. In some embodiments, such methods further comprise transfecting skin cells with the lipid-containing composition or lipid-nanoparticle composition to deliver the mRNA. In some embodiments, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for rejuvenating the skin, and comprises administering a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor to achieve skin rejuvenation while maintaining cell identity.In such a method, the mRNA can be bound to a lipid or contained in a lipid-nanoparticle. In some embodiments, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method of rejuvenating the skin and comprises administering a lipid-containing composition or a lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor to skin cells, wherein the expression of at least one reprogramming factor in the skin cells results in an increase in fibroblast proliferation while maintaining skin cell identity. In some embodiments, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method of increasing skin thickness and comprises administering a lipid-containing composition or a lipid-nanoparticle composition comprising a therapeutic or diagnostic agent. In some embodiments, the therapeutic agent is mRNA. In some embodiments, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method of increasing skin thickness and comprises administering a lipid-containing composition or a lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor to skin cells, wherein the expression of at least one reprogramming factor in the skin cells results in an increase in skin thickness while maintaining skin cell identity. In some embodiments, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method of increasing skin elasticity and comprises administering a lipid-containing composition or a lipid-nanoparticle composition comprising a therapeutic or diagnostic agent. In some embodiments, the therapeutic agent is mRNA. In some embodiments, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method of increasing skin elasticity and comprises administering a lipid-containing composition or a lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor to skin cells, wherein the expression of at least one reprogramming factor in the skin cells results in an increase in skin elasticity while maintaining skin cell identity. In such a method, the mRNA can be bound to a lipid or contained in a lipid-nanoparticle.

[0088] In some aspects, the lipid or lipid-nanoparticle compositions of the present disclosure are used in methods of wound healing and include administering a lipid-containing composition or lipid-nanoparticle composition comprising a therapeutic or diagnostic agent. In some aspects, the lipid or lipid-nanoparticle compositions of the present disclosure are used in methods of wound healing and include administering a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor. In such methods, the mRNA can be bound to the lipid or contained within the lipid-nanoparticle. In some aspects, the lipid or lipid-nanoparticle compositions of the present disclosure are used in methods of wound healing, and the lipid-containing composition or lipid-nanoparticle composition delivers mRNA encoding at least one reprogramming factor to achieve wound healing while maintaining cell identity. In such methods, the mRNA can be bound to the lipid or contained within the lipid-nanoparticle. In some aspects, the lipid or lipid-nanoparticle compositions of the present disclosure are used in methods of wound healing and include administering to skin cells a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor, wherein expression of the at least one reprogramming factor in the skin cells results in an increase in fibroblast proliferation while maintaining skin cell identity. In such methods, the mRNA can be bound to the lipid or contained within the lipid-nanoparticle.

[0089] In some embodiments, the lipid or lipid-nanoparticle compositions of the disclosure are used in a method for treating or preventing a dermatological disease or condition, or a disease or condition of the skin, or for cosmetic application to the skin, and comprise administering a lipid-containing composition or a lipid-nanoparticle composition comprising a therapeutic agent to achieve reversal of at least one skin aging marker. In some embodiments, the lipid or lipid-nanoparticle compositions of the disclosure are used in a method for rejuvenating the skin, and comprise administering a lipid-containing composition or a lipid-nanoparticle composition to deliver to skin cells an mRNA encoding at least one reprogramming factor to achieve reversal of at least one skin aging marker while maintaining cell identity. In some embodiments, the lipid or lipid-nanoparticle compositions of the disclosure are used in a method for wound healing, and comprise administering a lipid-containing composition or a lipid-nanoparticle composition to deliver to skin cells an mRNA encoding at least one reprogramming factor to achieve reversal of at least one skin aging marker while maintaining cell identity. In some embodiments, reversal of at least one skin aging marker refers to producing rejuvenated cells that express at least one skin aging marker in a manner similar to the expression of that marker observed in youthful skin cells as compared to aged skin cells.

[0090] In some embodiments, the marker is mRNA or protein expression of IL6, CXCL8, CSF3, CXCL1, SERPINB2, LIF, IL11, CXCL2, IL24, PTGS2, MMP3, CCL2, TFPI2, IER3, ACKR3, PTGES, SLC16A6, TNFAIP6, PTPRN, IL1RN, IL1B, CXCL5, CXCL6, HAS1, HSD11B1, CH25H, ADGRD1, C3, RASD1, NR4A3, STC1, TCIM, SRGN, AC003092.1, LRRN3, CHI3L1, NR4A2, NAMPT, PRSS23, MMP1, SOD2, LOXL4, MMP11, ELN, CREG1, C15orf48, NFKBIZ, PID1, or any combination thereof. In some embodiments, the reversal of at least one skin aging marker is downregulation of mRNA or protein expression of IL6, CXCL8, CSF3, CXCL1, SERPINB2, LIF, IL11, CXCL2, IL24, PTGS2, MMP3, CCL2, TFPI2, IER3, ACKR3, PTGES, SLC16A6, TNFAIP6, PTPRN, IL1RN, IL1B, CXCL5, CXCL6, HAS1, HSD11B1, CH25H, ADGRD1, C3, RASD1, NR4A3, STC1, TCIM, SRGN, AC003092.1, LRRN3, CHI3L1, NR4A2, NAMPT, MMP1, SOD2, CREG1, C15orf48, NFKBIZ, PID1, or any combination thereof. In some embodiments, the reversal of at least one skin aging marker is upregulation of mRNA or protein expression of PRSS23, LOXL4, MMP11, ELN, or any combination thereof. In some embodiments, the reversal of at least one skin aging marker is upregulation of mRNA or protein expression of PRSS23. In some embodiments, the reversal of at least one skin aging marker is upregulation of mRNA or protein expression of LOXL4. In some embodiments, the reversal of at least one skin aging marker is upregulation of mRNA or protein expression of MMP11. In some embodiments, the reversal of at least one skin aging marker is upregulation of mRNA or protein expression of ELN.In some embodiments, the reversal of at least one skin aging marker is downregulation of the mRNA or protein expression of MMP3, MMP1, SOD2, or any combination thereof. In some embodiments, the reversal of at least one skin aging marker is downregulation of the mRNA or protein expression of MMP3. In some embodiments, the reversal of at least one skin aging marker is downregulation of the mRNA or protein expression of MMP1. In some embodiments, the reversal of at least one skin aging marker is downregulation of the mRNA or protein expression of SOD2. In some embodiments, the reversal of at least one skin aging marker is upregulation of the mRNA or protein expression of at least one of PRSS23, LOXL4, MMP11, or ELN; downregulation of the mRNA or protein expression of at least one of MMP3, MMP1, SOD2; or any combination thereof.

[0091] In some embodiments, the lipid or lipid-nanoparticle compositions of the present disclosure are used in methods for treating or preventing dermatological diseases or conditions, treating or preventing skin diseases or conditions, or for cosmetic applications on the skin, and comprise administering a lipid-containing composition or lipid-nanoparticle composition comprising a therapeutic agent to achieve an improvement in at least one skin quality marker. In some embodiments, the lipid or lipid-nanoparticle compositions of the present disclosure are used in methods for increasing skin thickness and comprise administering a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor to skin cells, wherein expression of at least one reprogramming factor in the skin cells results in an increase in skin thickness while maintaining skin cell identity. In some embodiments, the lipid or lipid-nanoparticle compositions of the present disclosure are used in methods for increasing skin elasticity and comprise administering a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor to skin cells, wherein expression of at least one reprogramming factor in the skin cells results in an increase in skin elasticity while maintaining skin cell identity. In some embodiments, the lipid or lipid-nanoparticle compositions of the present disclosure are used in methods for rejuvenating the skin and comprise administering to skin cells a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor to achieve an improvement in at least one skin quality marker. In some embodiments, the lipid or lipid-nanoparticle compositions of the present disclosure are used in methods for rejuvenating the skin and comprise administering to skin cells a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor to achieve an improvement in at least one skin quality marker while maintaining cell identity. In some embodiments, at least one skin quality marker comprises skin thickness. In some embodiments, the skin thickness is increased. In some embodiments, at least one skin quality marker is skin elasticity. In some embodiments, the skin elasticity is increased. In some embodiments, at least one skin quality marker is transepidermal water loss. Skin thickness, skin elasticity, and transepidermal water loss can be measured according to any method known to those of skill in the art.

[0092] In some embodiments, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method of wound healing, comprising administering a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor to skin cells to achieve an improvement in at least one skin quality marker. In some embodiments, the marker is the mRNA or protein expression of type I collagen, type III collagen, type V collagen, type VI collagen, type XI collagen, elastin, microfibril-associated protein 5, periostin, versican, connective tissue growth factor, lysyl oxidase, SPARC, secreted phosphoprotein 1, cartilage oligomeric matrix protein, MMP1, MMP3, MMP12, SOD2, or any combination thereof. In some embodiments, the improvement in at least one skin quality marker is a downregulation of the mRNA or protein expression of MMP1, MMP3, MMP12, SOD2, or any combination thereof. In some embodiments, the improvement in at least one skin quality marker is an upregulation of the mRNA or protein expression of type I collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type XI collagen, elastin, microfibril-associated protein 5, periostin, versican, connective tissue growth factor, lysyl oxidase, SPARC, secreted phosphoprotein 1, cartilage oligomeric matrix protein, or any combination thereof. In some embodiments, the improvement in at least one skin quality marker is an upregulation of the mRNA or protein expression of collagen. In some embodiments, the improvement in at least one skin quality marker is an upregulation of the mRNA or protein expression of type I collagen. In some embodiments, the improvement in at least one skin quality marker is an upregulation of the mRNA or protein expression of type III collagen. In some embodiments, the improvement in at least one skin quality marker is an upregulation of the mRNA or protein expression of type IV collagen. In some embodiments, the improvement in at least one skin quality marker is an upregulation of the mRNA or protein expression of type V collagen. In some embodiments, the improvement in at least one skin quality marker is an upregulation of the mRNA or protein expression of type VI collagen.In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of type XI collagen. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of type XI collagen. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of elastin. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of microfibril-associated protein 5. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of periostin. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of versican. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of connective tissue growth factor. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of lysyl oxidase. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of SPARC. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of secreted phosphoprotein 1. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of cartilage oligomeric matrix protein. In some embodiments, improvement of at least one skin quality marker is downregulation of the mRNA or protein expression of MMP3, MMP1, SOD2, or any combination thereof. In some embodiments, improvement of at least one skin quality marker is downregulation of the mRNA or protein expression of MMP3. In some embodiments, improvement of at least one skin quality marker is downregulation of the mRNA or protein expression of MMP1. In some embodiments, improvement of at least one skin quality marker is downregulation of the mRNA or protein expression of SOD2. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of collagen VII and elastin.In some embodiments, improvement of at least one skin quality marker is upregulation of at least one of collagen VII and elastin mRNA or protein expression; downregulation of at least one of MMP3, MMP1, SOD2 mRNA or protein expression; or any combination thereof.

[0093] In some embodiments, the lipid-nanoparticle composition of the present disclosure or the composition containing the lipid of the present disclosure is administered topically to the skin. In some embodiments, the lipid-nanoparticle composition of the present disclosure or the composition containing the lipid of the present disclosure is administered to the skin in an ointment, cream, or plaster. In some embodiments, the lipid-nanoparticle composition of the present disclosure or the composition containing the lipid of the present disclosure is administered to the skin via intradermal or subcutaneous injection. In some embodiments, the lipid-nanoparticle composition of the present disclosure or the composition containing the lipid of the present disclosure is administered to the skin via a gel. In some embodiments, the lipid-nanoparticle composition of the present disclosure or the composition containing the lipid of the present disclosure is administered in vivo, in vitro, or ex vivo. In some embodiments, the lipid-nanoparticle composition of the present disclosure or the composition containing the lipid of the present disclosure is administered in vivo. In some embodiments, the lipid-nanoparticle composition of the present disclosure or the composition containing the lipid of the present disclosure is for human or animal subjects.

[0094] In some embodiments, the lipid-nanoparticle compositions of the disclosure or the compositions containing the disclosed lipids transfect skin cells to deliver at least one therapeutic or diagnostic agent to the skin cells. In some embodiments, the therapeutic agent is a nucleic acid. In some embodiments, the therapeutic agent is mRNA. In some embodiments, the therapeutic agent is a combination of mRNA and siRNA. In some embodiments, the therapeutic agent is a combination of mRNA and miRNA. In some embodiments, the skin cells are keratinocytes, melanocytes, Langerhans cells, follicular cells, fibroblasts, endothelial cells, smooth muscle cells, Merkel cells, basal cells, squamous epithelial cells, apocrine gland cells, eccrine gland cells, sebaceous gland cells, lymphatic endothelial cells, or combinations thereof. In some embodiments, the lipid or lipid-nanoparticle composition is selected to provide selective transfection to a specific cell type or cell types. In some embodiments, the lipid or lipid-nanoparticle composition is selected to provide diffusion within the skin or within at least one layer of the skin.

[0095] In some embodiments, the dermatological disease or condition or skin disease or condition that is treated or prevented using the lipid composition or lipid-nanoparticle composition of the present disclosure is skin laxity or a chronic wound. In some embodiments, the skin laxity or chronic wound is a diabetic, ischemic, and compressive ulcer. In some embodiments, the dermatological disease or condition or skin disease or condition that is treated or prevented using the lipid composition or lipid-nanoparticle composition of the present disclosure is an inflammatory skin disease. In some embodiments, the inflammatory skin disease is psoriasis, atopic dermatitis, vitiligo, alopecia areata, or hidradenitis suppurativa. In some embodiments, the dermatological disease or condition or skin disease or condition that is treated or prevented using the lipid composition or lipid-nanoparticle composition of the present disclosure is a hair disorder. In some embodiments, the hair disorder is non-scarring or scarring alopecia, hair graying, or hirsutism. In some embodiments, the hair disorder is non-scarring alopecia that is androgenetic alopecia. In some embodiments, the scarring alopecia is lichen planopilaris. In some embodiments, the dermatological disease or condition or skin disease or condition that is treated or prevented using the lipid composition or lipid-nanoparticle composition of the present disclosure is skin cancer. In some embodiments, the skin cancer is basal cell carcinoma, squamous cell carcinoma, or actinic keratosis. In some embodiments, the dermatological disease or condition or skin disease or condition that is treated or prevented using the lipid composition or lipid-nanoparticle composition of the present disclosure is prurigo nodularis, acne, rosacea, or solar lentigines. In some embodiments, the method of treating any of the above dermatological diseases or conditions or diseases comprises administering a lipid-containing composition of the present disclosure or a lipid-nanoparticle composition of the present disclosure comprising a therapeutic agent. In some embodiments, the therapeutic agent is mRNA. In some embodiments, the therapeutic agent is an antibody, a human antibody, a humanized antibody, a nanobody, a camelid antibody, a bispecific antibody, an enzyme, a genome editing enzyme or nuclease, a growth factor, a cytokine, a chemokine, a transcription factor, a structural molecule, a signaling molecule, an mRNA encoding a reprogramming factor. In some embodiments, the therapeutic agent is an mRNA encoding a protein or peptide that acts intracellularly.In some embodiments, the therapeutic agent is an mRNA encoding at least one reprogramming factor.

[0096] In some embodiments, the lipid composition or lipid-nanoparticle composition of the present disclosure is used in a method of wound healing where the wound is a skin laxity or chronic wound. In some embodiments, the skin laxity or chronic wound is a diabetic, ischemic, and pressure ulcer. In some embodiments, the lipid composition or lipid-nanoparticle composition of the present disclosure is used in a method of wound healing where the wound is a lesion from skin cancer. In some embodiments, the skin cancer is basal cell carcinoma, squamous cell carcinoma, or actinic keratosis. In some embodiments, any of the above wound healing methods includes administering a lipid-containing composition of the present disclosure or a lipid-nanoparticle composition of the present disclosure that contains a therapeutic agent. In some embodiments, the therapeutic agent is an mRNA. In some embodiments, the therapeutic agent is an antibody, a human antibody, a humanized antibody, a nanobody, a camelid antibody, a bispecific antibody, an enzyme, a genome editing enzyme or nuclease, a growth factor, a cytokine, a chemokine, a transcription factor, a structural molecule, a signaling molecule, an mRNA encoding a reprogramming factor. In some embodiments, the therapeutic agent is an mRNA encoding a protein or peptide that acts intracellularly. In some embodiments, the therapeutic agent is an mRNA encoding at least one reprogramming factor.

[0097] In some embodiments, some of the lipid-nanoparticle compositions of the present disclosure containing lipid formula (II) have a higher transfection efficiency in skin cells compared to other lipid-nanoparticle compositions containing lipid formula (II) or other lipid-nanoparticle compositions containing other lipids of the present disclosure, and when used to transfect at least one reprogramming factor, result in a greater improvement in at least one skin quality marker. In some embodiments, the at least one skin quality marker is skin thickness, skin elasticity, transepidermal water loss, or any combination thereof.

[0098] In some embodiments, the lipid-nanoparticle composition of the present disclosure or the composition containing the disclosed lipid transfects skin cells to deliver at least one therapeutic or diagnostic agent to eye cells. In some embodiments, the therapeutic agent is a nucleic acid. In some embodiments, the therapeutic agent is mRNA. In some embodiments, the therapeutic agent is a combination of mRNA and siRNA. In some embodiments, the therapeutic agent is a combination of mRNA and miRNA. In some embodiments, the eye cells are retinal pigment epithelial cells, ganglion cells, photoreceptor cells, rod cells, cone cells, choroid cells, corneal cells, conjunctival cells, corneal epithelial cells, extraocular muscle cells, or a combination thereof. In some embodiments, the lipid or lipid-nanoparticle composition is selected to provide selective transfection into a particular cell type or cell types. In some embodiments, the lipid or lipid-nanoparticle composition is selected to provide diffusion within the eye, such as within the vitreous humor or the retina.

[0099] In some embodiments, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method of treating or preventing an eye disease or condition, or a disease or condition of the eye, and comprises administering to the eye a lipid-containing composition or a lipid-nanoparticle composition comprising a therapeutic agent.

[0100] In some embodiments, the eye diseases or conditions treated or prevented using the lipid composition or lipid-nanoparticle composition of the present disclosure, or the eye diseases or conditions treated, are age-related macular degeneration, glaucoma, cataract, dry eye, diabetic retinopathy, vision loss, myopia, presbyopia, dry macular degeneration, or exudative macular degeneration. In some embodiments, a method of treating any of the above eye diseases or conditions or diseases comprises administering a lipid-containing composition of the present disclosure or a lipid-nanoparticle composition of the present disclosure that comprises a therapeutic agent. In some embodiments, the therapeutic agent is mRNA. In some embodiments, the therapeutic agent is an antibody, a human antibody, a humanized antibody, a nanobody, a camelid antibody, a bispecific antibody, an enzyme, a genome editing enzyme or nuclease, a growth factor, a cytokine, a chemokine, a transcription factor, a structural molecule, a signaling molecule, an mRNA encoding a reprogramming factor. In some embodiments, the therapeutic agent is an mRNA encoding a protein or peptide that acts intracellularly. In some embodiments, the therapeutic agent is an mRNA encoding at least one reprogramming factor.

[0101] The methods provided herein involve using a lipid or lipid-nanoparticle composition to transfect a cell with one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors, thereby producing rejuvenated cells. The cells to be rejuvenated can be of any cell type. In embodiments, the cell is contacted with, exposed to, or transfected with the mRNA for a period not exceeding about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day. In embodiments, the cell is contacted with, exposed to, or transfected with the mRNA for a period not exceeding about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day. In embodiments, the cell is contacted with, exposed to, or transfected with the mRNA for a period not exceeding about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day. In embodiments, the cell is contacted with, exposed to, or transfected with the mRNA for a period not exceeding about 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day. In embodiments, the cell is contacted with, exposed to, or transfected with the mRNA for a period not exceeding about 5, 4, 3, 2, or 1 day, or less than 1 day. In embodiments, at least one reprogramming factor is expressed from the transfected mRNA within the cell, or the cell is exposed to at least one reprogramming factor expressed from the transfected mRNA for a period not exceeding about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day. In embodiments, at least one reprogramming factor is expressed from the transfected mRNA within the cell, or the cell is exposed to at least one reprogramming factor expressed from the transfected mRNA for a period not exceeding about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day.In embodiments, at least one reprogramming factor is expressed from transfected mRNA within the cell, or the cell is exposed to at least one reprogramming factor expressed from transfected mRNA for at least about 2 days and not more than about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days. In embodiments, at least one reprogramming factor is expressed from transfected mRNA within the cell, or the cell is exposed to at least one reprogramming factor expressed from transfected mRNA for not more than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or for less than 1 day. In embodiments, at least one reprogramming factor is expressed from transfected mRNA within the cell, or the cell is exposed to at least one reprogramming factor expressed from transfected mRNA for at least about 2 days and not more than about 10, 9, 8, 7, 6, 5, 4, 3, or 2 days. In embodiments, at least one reprogramming factor is expressed from transfected mRNA within the cell, or the cell is exposed to at least one reprogramming factor expressed from transfected mRNA for not more than about 7, 6, 5, 4, 3, 2, or 1 day, or for less than 1 day. In embodiments, at least one reprogramming factor is expressed from transfected mRNA within the cell, or the cell is exposed to at least one reprogramming factor expressed from transfected mRNA for at least about 2 days and not more than about 7, 6, 5, 4, 3, or 2 days. In embodiments, at least one reprogramming factor is expressed from transfected mRNA within the cell, or the cell is exposed to at least one reprogramming factor expressed from transfected mRNA for not more than about 5, 4, 3, 2, or 1 day, or for less than 1 day.In embodiments, at least one reprogramming factor is expressed from transfected mRNA within the cell, or the cell is exposed to at least one reprogramming factor expressed from transfected mRNA for at least about 2 days and not more than about 5, 4, 3, or 2 days. In embodiments, the rejuvenated cells have a phenotype or activity profile similar to that of young cells. The phenotype or activity profile includes one or more of a transcriptome profile, gene expression of one or more nuclear markers and / or epigenetic markers, proteolytic activity, mitochondrial integrity and function, SASP cytokine expression, and methylation landscape.

[0102] In some embodiments, the rejuvenated cells have a transcriptome profile that is further similar to the transcriptome profile of young cells. In embodiments, the transcriptome profile of the rejuvenated cells includes an increase in gene expression of one or more genes selected from RPL37, RHOA, SRSF3, EPHB4, ARHGAP18, RPL31, FKBP2, MAP1LC3B2, Elfl, Phf8, Pol2s2, Tafl, and Sin3a.

[0103] In some embodiments, the rejuvenated cells exhibit increased gene expression of one or more nuclear markers and / or epigenetic markers as compared to a reference value. In embodiments, the one or more nuclear markers and / or epigenetic markers are selected from Hpl gamma, H3K9me3, lamin support protein LAP2 alpha, and SIRTl protein. In embodiments, the rejuvenated cells have proteolytic activity similar to that of young cells. In embodiments, the proteolytic activity is measured as increased cellular autophagosome formation, increased chymotrypsin-like proteasome activity, or a combination thereof. In embodiments, the rejuvenated cells exhibit improved mitochondrial integrity and function as compared to a reference value. In embodiments, the improved mitochondrial integrity and function are measured as increased mitochondrial membrane potential, decreased reactive oxygen species (ROS), or a combination thereof.

[0104] In some embodiments, the rejuvenated cells exhibit decreased expression of one or more SASP cytokines as compared to a reference value. In embodiments, the one or more SASP cytokines include IL18, ILIA, GROA, IL22, and IL9. In embodiments, the rejuvenated cells exhibit a reversal of the methylation landscape. In embodiments, the reversal of the methylation landscape is measured by Horvath clock estimation. In some embodiments, the reference value is obtained from aged cells.

[0105] In embodiments, the cells are rejuvenated by transient reprogramming with mRNA encoding one or more cell reprogramming factors transfected into the cells using the lipids or lipid-nanoparticle compositions of the present disclosure. Transient reprogramming is achieved, in some embodiments, by transfecting the cells with non-integrating mRNA for a period not exceeding about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day. Transient reprogramming is achieved, in some embodiments, by transfecting the cells with non-integrating mRNA for a period not exceeding about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day. Transient reprogramming is achieved, in some embodiments, by transfecting the cells with non-integrating mRNA for a period not exceeding about 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day. Transient reprogramming is achieved, in some embodiments, by transfecting the cells with non-integrating mRNA for a period not exceeding about 6, 5, 4, 3, 2, or 1 day, or less than 1 day. Transient reprogramming is achieved, in some embodiments, by expressing at least one reprogramming factor from non-integrating mRNA transfected into the cells, or by exposing the cells to at least one reprogramming factor expressed from non-integrating mRNA transfected into the cells for a period not exceeding about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day. Transient reprogramming is achieved, in some embodiments, by expressing at least one reprogramming factor from non-integrating mRNA transfected into the cells, or by exposing the cells to at least one reprogramming factor expressed from non-integrating mRNA transfected into the cells for at least 2 days and not exceeding about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days.Transient reprogramming is, in some embodiments, achieved by expressing at least one reprogramming factor from non-integrating mRNA transfected intracellularly, or by exposing cells to at least one reprogramming factor expressed from non-integrating mRNA transfected intracellularly for no more than about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day. Transient reprogramming is, in some embodiments, achieved by expressing at least one reprogramming factor from non-integrating mRNA transfected intracellularly, or by exposing cells to at least one reprogramming factor expressed from non-integrating mRNA transfected intracellularly for at least 2 days and no more than about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days. Transient reprogramming is, in some embodiments, achieved by expressing at least one reprogramming factor from non-integrating mRNA transfected intracellularly, or by exposing cells to at least one reprogramming factor expressed from non-integrating mRNA transfected intracellularly for no more than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day. Transient reprogramming is, in some embodiments, achieved by expressing at least one reprogramming factor from non-integrating mRNA transfected intracellularly, or by exposing cells to at least one reprogramming factor expressed from non-integrating mRNA transfected intracellularly for at least 2 days and no more than about 10, 9, 8, 7, 6, 5, 4, 3, or 2 days. Transient reprogramming is, in some embodiments, achieved by expressing at least one reprogramming factor from non-integrating mRNA transfected intracellularly, or by exposing cells to at least one reprogramming factor expressed from non-integrating mRNA transfected intracellularly for no more than about 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day.Transient reprogramming is achieved, in some embodiments, by expressing at least one reprogramming factor from non-integrating mRNA transfected intracellularly, or by exposing the cells to at least one reprogramming factor expressed from non-integrating mRNA transfected intracellularly for at least 2 days and not more than about 7, 6, 5, 4, 3, or 2 days. Transient reprogramming is achieved, in some embodiments, by expressing at least one reprogramming factor from non-integrating mRNA transfected intracellularly, or by exposing the cells to at least one reprogramming factor expressed from non-integrating mRNA transfected intracellularly for not more than about 5, 4, 3, 2, or 1 days, or less than 1 day. Transient reprogramming is achieved, in some embodiments, by expressing at least one reprogramming factor from non-integrating mRNA transfected intracellularly, or by exposing the cells to at least one reprogramming factor expressed from non-integrating mRNA transfected intracellularly for at least 2 days and not more than about 5, 4, 3, or 2 days. In embodiments, transient reprogramming of the cells eliminates various features of aging while avoiding complete dedifferentiation of the cells into stem cells.

[0106] In embodiments of the methods and compositions provided herein, cellular age reversal, or rejuvenation, is achieved by transient overexpression of one or more mRNAs encoding cellular reprogramming factors that are delivered by the lipids or lipid-nanoparticle compositions of the present disclosure. Such cellular reprogramming factors can include transcription factors, epigenetic remodelers, or small molecules that affect mitochondrial function, proteolytic activity, heterochromatin levels, histone methylation, nuclear lamina polypeptides, cytokine secretion, or aging. In embodiments, the cellular reprogramming factors include one or more of OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG. In embodiments, the cellular reprogramming factors are OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG in different molar ratios, e.g., a:b:c:d:e:f molar ratio, where a, b, c, d, e, and f are all the same number (e.g., 1:1:1:1:1:1), some of the same number and some different numbers (e.g., 3:1:1:1:1:1, 2:1:1:1:1:1, 2:2:1:1:1:1, 2:2:2:1:1:1, 2:2:2:2:1:1, 2:2:2:2:2:1, 3:3:3:3:2:2), or all different numbers (e.g., 6:4:5:3:2:1), and a, b, c, d, e, and f can each be from 1 to 7, i.e., 1 to 7:1 to 7:1 to 7:1 to 7:1 to 7:1 to 7 (or in the case of combinations of fewer than six factors, 1 to 7:1 to 7:1 to 7:1 to 7:1 to 7, 1 to 7:1 to 7:1 to 7:1 to 7, 1 to 7:1 to 7:1 to 7, 1 to 7:1 to 7, or 1 to 7:1), and are applicable to OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG in an a:b:c:d:e:f molar ratio.

[0107] In embodiments, the methods and compositions provided herein can be applied to any type of cell, tissue, or organ in need of rejuvenation. The methods and compositions of the disclosure can be used to rejuvenate cells in culture (e.g., ex vivo or in vitro) to improve function and efficacy for use in cell therapy. The cells used for treating a patient can be autologous or allogeneic. The cells can be derived from the patient or a matched donor, or they can be obtained from a cell bank or be derived from iPS cells. For example, in autologous ex vivo therapy, cells can be obtained directly from the patient being treated, transfected with mRNA encoding the cell reprogramming factors described herein, and re-transplanted into the patient. Such cells can be obtained, for example, from a biopsy or surgical procedure performed on the patient. Alternatively, in allogeneic ex vivo therapy, cells can be obtained from a cell bank or a cell line derived from iPS cells, transfected with mRNA encoding the cell reprogramming factors as described herein, and re-transplanted into the patient. Alternatively, cells in need of rejuvenation can be directly transfected in vivo with mRNA encoding the cell reprogramming factors.

[0108] In another aspect, provided herein is a pharmaceutical composition comprising rejuvenated cells obtained by transiently reprogramming cells for rejuvenation by transfecting the cells with a lipid-containing composition or a lipid-nanoparticle composition of the disclosure comprising one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors for no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive days. In another aspect, provided herein is a pharmaceutical composition comprising rejuvenated cells obtained by transiently reprogramming cells for rejuvenation by transfecting the cells with a lipid-containing composition or a lipid-nanoparticle composition of the disclosure comprising one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors for no more than 4, 5, 6, or 7 consecutive days.

[0109] In some embodiments, the lipid-containing composition or lipid-nanoparticle composition of the present disclosure is optimized such that the reprogramming factor reduces any induced immune response against the protein / polypeptide and increases the stability of the protein / polypeptide, and changes the protein / polypeptide activity, such as increased activity compared to the wild-type reprogramming factor, and is used for the delivery of mRNA encoding a reprogramming factor that provides a more robust cellular rejuvenation.

[0110] In some embodiments, the methods of the present disclosure include administering to a cell or subject a lipid-containing composition or lipid-nanoparticle composition of the present disclosure that comprises RNA, or treating or transfecting a cell with a lipid-containing composition or lipid-nanoparticle composition of the present disclosure that comprises RNA, for consecutive days with a dosing interval not exceeding 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 days. In some embodiments, administration of a lipid-containing composition or lipid-nanoparticle composition of the present disclosure that comprises RNA is performed at least once per day during the dosing interval period. In some embodiments, administration is at a less frequent rate of less than once per day during the dosing interval, for example, once every 2 days, once every 3 days, once every 4 days, once every x days (where x is a number from 4 to 25). Thus, in such embodiments, for example, administering a lipid-containing composition or lipid-nanoparticle composition of the present disclosure that comprises RNA once every 5 days with a 5-day dosing interval means that the RNA is administered once during the interval, i.e., once during the total treatment period of 5 days, whereas administering the RNA twice a day with a 5-day dosing interval means that the RNA is administered 10 times during the interval, i.e., 10 times in 5 days. In some embodiments, the methods of the present disclosure include administering to a cell or subject a lipid-containing composition or lipid-nanoparticle composition of the present disclosure that comprises RNA, or treating or transfecting a cell with a lipid-containing composition or lipid-nanoparticle composition of the present disclosure that comprises RNA, for consecutive days with a dosing interval not exceeding 21, 18, 14, 10, 7, or 5 days.In some embodiments, the methods of the disclosure include administering to a cell or subject a lipid-containing composition or lipid-nanoparticle composition of the disclosure that comprises RNA, or treating or transfecting a cell with a lipid-containing composition or lipid-nanoparticle composition of the disclosure that comprises RNA for no more than 18 consecutive days. In some embodiments, the methods of the disclosure include administering to a cell or subject a lipid-containing composition or lipid-nanoparticle composition of the disclosure that comprises RNA, or treating or transfecting a cell with a lipid-containing composition or lipid-nanoparticle composition of the disclosure that comprises RNA for no more than 14 consecutive days. In some embodiments, the methods of the disclosure include administering to a cell or subject a lipid-containing composition or lipid-nanoparticle composition of the disclosure that comprises RNA, or treating or transfecting a cell with a lipid-containing composition or lipid-nanoparticle composition of the disclosure that comprises RNA for consecutive days with a dosing interval of no more than 10 days. In some embodiments, the methods of the disclosure include administering to a cell or subject a lipid-containing composition or lipid-nanoparticle composition of the disclosure that comprises RNA, or treating or transfecting a cell with a lipid-containing composition or lipid-nanoparticle composition of the disclosure that comprises RNA for consecutive days with a dosing interval of no more than 7 days. In some embodiments, the methods of the disclosure include administering to a cell or subject a lipid-containing composition or lipid-nanoparticle composition of the disclosure that comprises RNA, or treating or transfecting a cell with a lipid-containing composition or lipid-nanoparticle composition of the disclosure that comprises RNA for consecutive days with a dosing interval of no more than 5 days. In other embodiments, the exposing comprises interrupting the exposing and repeating the exposing after the interrupting.In some embodiments, the exposing, treating, transfecting, expressing, or administering comprises exposing, treating, transfecting, expressing, or administering immune cells for a period of about 2 to 5 consecutive days, about 5 to 7 consecutive days, about 7 to 10 consecutive days, about 10 to 12 consecutive days, about 12 to 14 consecutive days, about 14 to 17 consecutive days, about 17 to 19 consecutive days, or about 19 to 21 consecutive days, and in some embodiments further comprises interrupting the exposing and, after the interruption, repeating the exposing.

[0111] In some embodiments, the duration of the exposure is controlled by mechanisms such as self-amplifying RNAs, circular RNAs, B18R and other decoys, and / or on / off switches such as L7Ae or its family members. In some embodiments, the repeating is performed any number of times, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, or up to 20 times, or up to 30 times, or more. In in vivo applications, the repeating can continue for any period, for example, until the disease is successfully treated or cured, or throughout the life of the subject or patient. In some embodiments, the repeating is performed at any time after the interrupting, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the interrupting, up to 20 days, up to 30 days, up to 3 months, up to 6 months, or up to 1 year. One exposure period is considered one dosing interval, and for example, one continuous exposure-interrupt-repeated exposure sequence would include two dosing intervals.

[0112] In one embodiment, a compound having the structure of formula (XVII):

Chemical formula

[0113] In one embodiment, a compound having the structure of formula (XVIII): [Chemical formula] or a stereoisomer, salt, or tautomer thereof is provided, wherein G 3 , G 4 , R 27 , m 1 , and m 2 are as defined herein.

[0114] In one embodiment, a compound having the structure of formula (XIX): [Chemical formula] or a stereoisomer, salt, or tautomer thereof is provided, wherein R 29a , R 29b , R 30 , and n are as defined herein.

[0115] Also provided is a pharmaceutical composition comprising one or more of the compounds of formulas (XVII)-(XIX) described above and a therapeutic agent.

[0116] In other embodiments, provided is a method of treatment by administering to a subject in need thereof a pharmaceutical composition comprising a compound of formula (I) described above or a compound of formulas (XVII)-(XIX) to treat a disease.

[0117] In one embodiment, provided is also a lipid nanoparticle comprising one or more of the compounds of formulas (XVII)-(XIX) described above and a therapeutic agent comprising a nucleic acid.

[0118] Here, various aspects and embodiments are more fully described below. Such aspects and embodiments can take many different forms, and the exemplary ones disclosed herein should not be construed as limiting, but rather these embodiments are provided so that this disclosure will be thorough and complete and the scope thereof will be fully conveyed to those skilled in the art.

[0119] The present disclosure relates to ionizable lipids that can provide certain advantages when used in nanoparticle compositions for delivering active or therapeutic agents, such as nucleic acids, to cells.

[0120] Detailed Description The ionizable lipids of the present disclosure include an ionizable group, one or more ester groups, and one or more hydrophobic tail groups. The ionizable group is an amine containing a head group having an acid dissociation constant (pKa) greater than 7. Without wishing to be bound by any theory, it is believed that the high pKa allows the ionizable lipid to be positively charged at acidic pH (<6.0) and neutral at physiological pH (7.4). This then results in high encapsulation efficiency of nucleic acids at acidic pH. The ionizable lipids and other helper lipids also interact with the negatively charged membrane of the endosome, resulting in disruption of the membrane and thereby facilitating the release of the nucleic acid.

[0121] One or more ester groups in the ionizable lipids of the present disclosure confer biodegradability, which is an important feature for the clinical translation of the ionizable lipids. The inclusion of an ester bond to the ionizable group reduces accumulation and potential side effects, thereby leading to the degradation of the ionizable lipid into non-toxic metabolites after successful delivery of intracellular cargo such as nucleic acids, small molecules, or peptides or proteins.

[0122] One or more hydrophobic tail groups form an aliphatic carbon chain. The aliphatic carbon chain can further include unsaturated bonds such as double bonds or triple bonds. The hydrophobic tail can further include a branched tail. It is considered that the length and saturation of the tail can greatly affect the fluidity and delivery efficiency of the ionizable lipid. The molecular hypothesis is that the ionizable lipids of the present disclosure are expected to generate a conical structure with enhanced endosomal disruption ability due to the increased cross-section of the tail region. This then promotes endosomal release due to the protonation of the ionizable lipid at endosomal pH. The nanoparticle compositions of the present disclosure containing ionizable lipids are extremely useful for systemic delivery applications, and they can exhibit an extended circulation lifetime and mediate the expression of transfected genes or the silencing of target gene expression in vivo. The nanoparticle compositions of the present disclosure containing ionizable lipids are extremely useful for local delivery applications, and their properties, which are not limited to ionization constant, size, and surface charge, etc., can be adjusted for delivery and transfection to specific tissues.

[0123] The design of ionizable lipids is inspired by lipid-based natural products such as cephalin and sphingomyelin, as well as other common compounds such as glycerides, in order to target better in vivo delivery and subsequent clearance. Such lipids are also thought to be useful for higher transfection efficiency and cell selectivity.

[0124] The introduction of aromatic rings in the hydrophobic chain can introduce a more organized architecture thanks to π-stacking, resulting in better packing and stability of the LNP. The aromatic rings also provide easy handling for further structural improvements that change the conical shape by introducing more aliphatic chains and / or changing the attachment points at different positions.

[0125] The use of heterocycles in the lipid head group is intended to provide more defined conical lipids and better nucleic acid encapsulation.

[0126] I. Definitions For convenience, specific terms used in this specification, the examples, and the claims are gathered herein. Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0127] When ranges of values are provided, each intervening value between the upper and lower limits of that range, as well as any other stated value or intervening value within the stated range, is intended to be encompassed by this disclosure. For example, if a range of 1 mg to 8 mg is recited, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, and 7 mg, as well as ranges of values greater than or equal to 1 mg and less than or equal to 8 mg, are also intended to be explicitly disclosed.

[0128] As used herein, the term "Cn-m alkyl" refers to a saturated hydrocarbon group that may be straight-chain or branched. An alkyl group formally corresponds to an alkane having one C-H bond replaced by the point of attachment of the alkyl group to the rest of the compound. The term "Cn-m alkyl" refers to an alkyl group having n to m carbon atoms. Examples of alkyl groups include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, iso-butyl, sec-butyl, and higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl. In some embodiments, the alkyl group contains 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. In some embodiments, the alkyl group contains 6 to 20 carbon atoms, 6 to 18 carbon atoms, 6 to 17 carbon atoms, 6 to 15 carbon atoms, 6 to 14 carbon atoms, 6 to 13 carbon atoms, 6 to 9 carbon atoms, 6 to 8 carbon atoms, or 6 to 7 carbon atoms. In some embodiments, the alkyl group is optionally substituted with 1, 2, 3 or more halo groups. In some embodiments, the alkyl group is optionally substituted with 1, 2, 3, or more hetero groups.

[0129] As used herein, the term "Cn-m alkenyl" refers to a straight-chain or branched unsaturated hydrocarbon group corresponding to an alkyl group having one or more carbon-carbon double bonds. Formally, an alkenyl group corresponds to an alkene in which one C-H bond has been replaced by the point of attachment of the alkenyl group to the rest of the compound. The term "Cn-m alkenyl" refers to an alkenyl group having from n to m carbons. Examples of alkenyl groups include, but are not limited to, chemical groups such as ethenyl, propenyl, iso-propenyl, n-butenyl, sec-butenyl, etc. In some embodiments, the alkenyl group contains from 6 to 20 carbon atoms, from 6 to 18 carbon atoms, from 6 to 17 carbon atoms, from 6 to 15 carbon atoms, from 6 to 14 carbon atoms, from 6 to 13 carbon atoms, from 6 to 9 carbon atoms, from 6 to 8 carbon atoms, or from 6 to 7 carbon atoms. In some embodiments, the alkenyl group is optionally substituted with 1, 2, 3, or more halo groups. In some embodiments, the alkenyl group is optionally substituted with 1, 2, 3, or more hetero groups.

[0130] As used herein, the term "Cn-m alkylene" refers to a divalent alkyl linking group. Formally, an alkylene group corresponds to an alkane having two C-H bonds replaced by the points of attachment of the alkylene group to the rest of the compound. The term "Cn-m alkylene" refers to an alkylene group having from n to m carbon atoms. Examples of alkylene groups include, but are not limited to, ethan-1,2-diyl, propan-1,3-diyl, propan-1,2-diyl, butan-1,4-diyl, butan-1,3-diyl, butan-1,2-diyl, 2-methyl-propan-1,3-diyl, etc. In some embodiments, the alkylene group is optionally substituted with 1, 2, 3, or more halo groups. In some embodiments, the alkylene group is optionally substituted with 1, 2, 3, or more hetero groups.

[0131] As used herein, the terms "about" and "approximately" refer to values similar to the recited reference values. In certain embodiments, the term "about" or "approximately" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less) of the recited reference value, unless otherwise stated or otherwise apparent from the context (except where such numerical values exceed 100% of the possible values). For example, when used in the context of the amount of lipid component of a nanoparticle composition, "about" may mean + / - 10% of the recited value. For example, a nanoparticle composition comprising a lipid component having about 40% of a given lipid may contain from 30 to 50% lipid.

[0132] As used herein, the terms "disease" and "condition" may be used interchangeably or may differ in that a particular disease or condition may not have a known causative agent (and thus the etiology has not yet been elucidated), and is therefore not yet recognized as a disease but only as an undesirable state or syndrome, although to a greater or lesser extent a particular set of symptoms has been identified by a clinician.

[0133] As used herein, the term "encapsulated" can refer to lipid particles that provide a payload such as a nucleic acid (e.g., interfering RNA, plasmid or oligonucleotide DNA, or mRNA) having complete encapsulation, partial encapsulation, or both. In one embodiment, the nucleic acid is completely encapsulated in the lipid particle to form a lipid nanoparticle (LNP). The term "completely encapsulated" indicates that the nucleic acid within the lipid particle is not significantly degraded after exposure to serum or nuclease or protease assays that would significantly degrade free RNA or protein. Complete encapsulation can be determined by the Oligreen® assay. Oligreen® is a hypersensitive fluorescent nucleic acid stain for quantifying RNA in solution (available from Invitrogen Corporation; Carlsbad, California). "Completely encapsulated" also indicates that the lipid particle is stable in serum, i.e., it does not rapidly degrade into its component parts upon in vivo administration. In one embodiment, the nucleic acid is at least 50% encapsulated in the lipid. In one embodiment, the nucleic acid is at least 75% encapsulated in the lipid. In one embodiment, the nucleic acid is at least 90% encapsulated in the lipid. In one embodiment, the nucleic acid is completely encapsulated in the lipid.

[0134] As used herein, the term "halo" or "halogen" refers to F, Cl, Br, or I.

[0135] As used herein, the term "hetero" refers to a heteroatom selected from oxygen, nitrogen, and sulfur.

[0136] As used herein, the term "interfering RNA" refers to single-stranded RNA (e.g., mature miRNA, circular RNA, guide RNA) or double-stranded RNA (i.e., double-stranded RNA such as siRNA, aiRNA, or pre-miRNA), or an RNA vector that can reduce or inhibit the expression of a target gene or sequence (e.g., by mediating the degradation of mRNA complementary to the interfering RNA sequence or by inhibiting its translation) when the interfering RNA is in the same cell as the target gene or sequence. Thus, interfering RNA refers to single-stranded RNA that is complementary to a target mRNA sequence or to a double-stranded RNA formed by two complementary strands or a single self-complementary strand. The interfering RNA may have substantial or complete identity to the target gene or sequence, or may contain mismatch regions (i.e., mismatch motifs). The sequence of the interfering RNA can correspond to the full-length target gene or a partial sequence thereof.

[0137] Interfering RNAs include "small interfering RNAs" or "siRNAs", for example, interfering RNAs having a length of about 15 to 60, 15 to 50, or 15 to 40 (double-stranded) nucleotides, more typically having a length of about 15 to 30, 15 to 25, or 19 to 25 (double-stranded) nucleotides, preferably having a length of about 20 to 24, 21 to 22, or 21 to 23 (double-stranded) nucleotides (e.g., each complementary sequence of a double-stranded siRNA has a length of 15 to 60, 15 to 50, 15 to 40, 15 to 30, 15 to 25, or 19 to 25 nucleotides, preferably having a length of about 20 to 24, 21 to 22, or 21 to 23 nucleotides, and the double-stranded siRNA has a length of about 15 to 60, 15 to 50, 15 to 40, 15 to 30, 15 to 25, or 19 to 25 base pairs, preferably having a length of about 18 to 22, 19 to 20, or 19 to 21 base pairs). The siRNA duplex may include a 3' overhang of about 1 to about 4 nucleotides or about 2 to about 3, and a 5' phosphate terminus. Examples of siRNAs include double-stranded polynucleotide molecules assembled from two separate linear molecules, where one strand is the sense strand and the other is the complementary antisense strand; double-stranded polynucleotide molecules assembled from a single-stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; double-stranded polynucleotide molecules having a hairpin secondary structure with self-complementary sense and antisense regions; and circular single-stranded polynucleotide molecules having two or more loop structures and a stem with self-complementary sense and antisense regions, where the circular polynucleotide can be processed in vivo or in vitro to generate an active double-stranded siRNA molecule, but are not limited thereto.

[0138] siRNA can also be chemically synthesized. siRNA can also be generated by cleavage of longer dsRNA (e.g., dsRNA longer than about 25 nucleotides) by E. coli RNase III or Dicer. These enzymes process dsRNA into biologically active siRNA (see, for example, Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002), Calegari et al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002), Byrom et al., Ambion TechNotes, 10(l):4-6 (2003), Kawasaki et al., Nucleic Acids Res., 31:981-987 (2003), Knight et al., Science, 293:2269-2271 (2001), and Robertson et al., Biol. Chem., 243:82 (1968)). Preferably, the dsRNA is at least 50 nucleotides in length up to about 100, 200, 300, 400, or 500 nucleotides. The dsRNA can be on the order of 1000, 1500, 2000, 5000 nucleotides, or more in length. The dsRNA can encode an entire gene transcript or a partial gene transcript. In certain cases, siRNA can be encoded by a plasmid (e.g., transcribed as a sequence that automatically folds into a double-stranded hairpin loop).

[0139] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within a living organism (e.g., an animal, a plant, or a microorganism).

[0140] As used herein, the term "in vivo" refers to events that occur within a living organism (e.g., an animal, a plant, or a microorganism, or their cells or tissues).

[0141] As used herein, the term "rejuvenated cell(s)" refers to an aged cell that has been treated or transiently reprogrammed with one or more cell reprogramming factors such that the cell has a transcriptome profile of a younger cell while still retaining one or more cell identity markers. In some embodiments, the treated cell is rejuvenated and reprogrammed, and the rejuvenated cell that is being reprogrammed exhibits at least a 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, or greater than 75% increase / improvement in the expression of at least one rejuvenation marker compared to an untreated cell, and while still retaining cell identity markers, expresses markers and a transcriptome profile of a younger cell.

[0142] As used herein, the terms "subject", "individual", and "patient" are used interchangeably herein and include humans, as well as other primates including chimpanzees and other apes and monkey species, other mammals such as livestock animals including cows, sheep, pigs, goats, and horses, companion mammals such as dogs and cats, rodents such as mice, rats, rabbits, hamsters, and guinea pigs, birds including chickens, turkeys and other large birds, and game, wild, and hunted birds such as ducks, geese, etc., but are not limited thereto. In some cases, the methods of the disclosure find use in experimental animals, in veterinary applications, and in the development of animal models for diseases. The terms do not indicate a particular age. Thus, adults, juveniles, and neonatal individuals are intended to be encompassed.

[0143] As used herein, the term "transfection" refers to the uptake of exogenous DNA or RNA by a cell. A cell is "transfected" when exogenous DNA or RNA has been introduced within the cell membrane.

[0144] As used herein, the term "transfection efficiency" refers to the extent to which exogenous DNA or RNA is taken up by cells being transfected. Transfection efficiency can be measured, for example, as the percentage of cells in a sample that express the gene product of the transfected exogenous DNA or RNA. The percentage of cells in a sample that express the gene product of the transfected exogenous DNA or RNA can be measured by determining, according to methods well known to those skilled in the art, using flow cytometry, at 18 to 24 hours after transfection, the percentage of cells in a sample that express a reporter gene such as green fluorescent protein.

[0145] As used herein, the term "transfection ability" refers to the average expression level of the gene product of the transfected exogenous DNA or RNA per cell in a sample. The average expression level of the gene product of the transfected exogenous DNA or RNA per cell in a sample can be measured, according to methods well known to those skilled in the art, at 18 to 24 hours after transfection, using flow cytometry, and is determined by dividing the total fluorescence intensity of cells in a sample that express a reporter gene such as green fluorescent protein by the number of cells in the sample.

[0146] As used herein, the term "survival rate" refers to the extent to which cells continue to survive after transfection, and is measured as the percentage of cells that continue to survive in a sample subjected to transfection. The survival rate can be measured, at 18 to 24 hours after transfection, according to methods well known to those skilled in the art, using flow cytometry, as the percentage of cells in a sample that are positively stained with propidium iodide.

[0147] As used herein, the term "transient reprogramming" refers to the exposure of a cell to cell reprogramming factors for a period of time that is sufficient to rejuvenate the cell (i.e., to remove all or some of the characteristics of aging) but not so long as to cause dedifferentiation into a stem cell. Such transient reprogramming results in rejuvenated cells that retain their identity (i.e., differentiated cell type).

[0148] The term "treating" as used herein with respect to a method of treating a cell, tissue, or subject generally includes administering a compound or composition that reduces the frequency of symptoms of aging or a medical condition in a subject, or delays the onset thereof, as compared to a subject not receiving the compound or composition. This includes means for ameliorating or stabilizing a medical condition in a subject and includes reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of the medical condition.

[0149] As used herein, the term "isomer" means any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer, or diastereomer of a compound. A compound may contain one or more chiral centers and / or double bonds and thus may exist as double bond isomers (i.e., geometric E / Z isomers) or stereoisomers such as diastereomers (e.g., enantiomers (i.e., (+) or (-), or cis / trans isomers)). The present disclosure encompasses any and all isomers of the compounds described herein, in stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure), as well as enantiomeric and stereoisomeric mixtures, e.g., racemates. Means for separating enantiomeric and stereoisomeric mixtures of compounds into their component enantiomers or stereoisomers are well known.

[0150] In the present disclosure, although in some cases the structural formula of a lipid may represent a specific isomer for convenience, the present disclosure includes all isomers such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, etc., and it is understood that not all isomers may have the same level of activity. Additionally, crystal polymorphs may be present in the lipids represented by the formula. It should be noted that any crystalline form, mixture of crystalline forms, or their anhydrides or hydrates are included within the scope of the present disclosure.

[0151] As used herein, the term "lipid-based delivery system" includes, but is not limited to, liposomes, polyplexes, lipoplexes, and lipid nanoparticles for the delivery of any payload described herein, including but not limited to nucleic acids.

[0152] As used herein, the term "lipid nanoparticle" or "LNP" refers to nanoparticles formed from at least one lipid or nanoparticles containing at least one lipid. A "lipid-nanoparticle composition" refers to a composition containing lipid nanoparticles or the lipid nanoparticles themselves. In some embodiments, the lipid nanoparticles are lipid-nucleic acid particles or nucleic acid-lipid particles (e.g., stable nucleic acid-lipid particles). The LNP can be a particle formed from or containing the ionizable lipid of the present disclosure and a cargo encapsulated within the lipid. In some embodiments, the cargo is a nucleic acid.

[0153] The LNP can typically have an average diameter of about 10 nm to about 200 nm, about 15 nm to about 150 nm, about 20 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or 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, or 150 nm.

[0154] As used herein, the term "mammal" includes both humans and domesticated animals such as laboratory animals and household pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), and non-domesticated animals such as wild animals.

[0155] As used herein, the term "nanoparticle composition" refers to a composition comprising one or more lipids. Nanoparticle compositions are typically sized on the order of micrometers or less and may include lipid bilayers. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), polyplexes, and lipoplexes. For example, the nanoparticle composition can be a lipid nanoparticle having a diameter of 500 nm or less.

[0156] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, including DNA and RNA. DNA can be, for example, in the form of antisense molecules, plasmid DNA, pre-condensed DNA, PCR products, vectors (PI, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. RNA can be in the form of siRNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), self-amplifying RNA, polycistronic RNA, self-amplifying polycistronic RNA, circular RNA, and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring and have binding properties similar to those of the reference nucleic acid. Examples of such modifications or analogs include, but are not limited to, 5-methylcytidine, 5-methyluridine, 2-thiouridine, N6-methyladenosine, pseudouridine, and N1-methylpseudouridine. Examples of such analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2'-O-methyl ribonucleotide, and peptide nucleic acid (PNA). Unless otherwise specifically limited, the term includes nucleic acids containing known analogs of natural nucleotides having binding properties similar to those of the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly shown sequences.Specifically, codon degeneracy substitution can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res., 19:5081 (1991), Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985), Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together via phosphate groups. "Bases" include purines and pyrimidines, and further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that place new reactive groups (such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides).

[0157] As used herein, the term "optionally substituted" means unsubstituted or substituted. As used herein, the term "substituted" means that a hydrogen atom is removed and replaced by a substituent. It should be understood that substitution at a given atom is limited by the valence.

[0158] As used herein, the term "pharmaceutically acceptable" is used herein to refer to compounds, salts, compositions, dosage forms, etc. that are suitable for use in contact with the tissues of humans and / or other mammals within the scope of sound medical judgment, without undue toxicity, irritation, allergic reaction, or other problems or complications, commensurate with a reasonable benefit / risk ratio. In some embodiments, "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals (e.g., animals), more particularly humans.

[0159] As used herein, the term "pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizing agent, isotonic agent, solvent, or emulsifying agent that is approved by the U.S. Food and Drug Administration as being acceptable for use in humans or livestock. In some embodiments, the "pharmaceutically acceptable carrier", or a carrier, adjuvant, excipient therein, is a component of an "artificial niche" used to maintain quiescence of progenitor cells. In some embodiments, the artificial niche component is selected from the group consisting of elcatonin, MGCD-265, JNJ-7706621, forskolin, fumagillin, SB203580, SU5402, TGF-β, insulin-transferrin-selenium, and combinations thereof. These and other "artificial niche" components are disclosed in U.S. Patent No. 10,688,136 and are incorporated herein by reference. When used as an excipient, such artificial niche components can be encapsulated by lipid nanoparticles, can be present within the shell of lipid nanoparticles, or can be included in the formulation separately from the lipid nanoparticles.

[0160] As used herein, the term "pharmaceutically acceptable salt" includes both acid addition salts and base addition salts.

[0161] As used herein, the term "pharmaceutical composition" refers to a nanoparticle composition and a medium generally acceptable in the art for delivery of a biologically active compound to a mammal, such as a pharmaceutically acceptable carrier.

[0162] As used herein, the term "subject" or "patient" refers to any organism to which a composition according to the present disclosure can be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include, but are not limited to, mammals such as mice, rats, rabbits, non-human primates, and humans.

[0163] As used herein, the term "systemic delivery" refers to the delivery of a therapeutic product that can result in widespread exposure of an active agent within an organism. Some administration techniques can result in systemic delivery of a particular agent, while others cannot. Systemic delivery means that a useful, preferably therapeutic, amount of the agent is exposed to most parts of the body. Systemic delivery of the compositions of the present disclosure, for example, lipid nanoparticles, can be by any means known in the art, including, for example, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of the lipid nanoparticles is by intravenous delivery.

[0164] As used herein, the term "local delivery" refers to the delivery of a therapeutic product that can result in local exposure of an active agent within a specific site, tissue, organ, or cell type in an organism. Some ionizable lipid compositions, including some lipid nanoparticles, can result in local delivery of a specific agent, while other agents cannot. Local delivery means that a useful, preferably therapeutic, amount of the agent is exposed to a specific site, tissue, organ, or cell type in an organism. Local delivery of the compositions of the present disclosure, such as lipid nanoparticles, can be by any means known in the art, including, for example, intravenous, intraarterial, subcutaneous, intradermal, cutaneous, topical, intratissue, intraorgan, and intraperitoneal delivery. In some embodiments, local delivery of the compositions of the present disclosure, such as lipid nanoparticles, is effected by intravenous delivery, and the properties of the nanoparticles, such as size, shape, ionization constant, and surface charge, determine the specific site, tissue, organ, or cell type targeted by the LNC. In some embodiments, local delivery of the compositions of the present disclosure, such as lipid nanoparticles, results from local injection into a targeted site, tissue, or organ, and the compositions of the present disclosure, such as lipid nanoparticles, then enable transfection in that local environment. In some embodiments, local delivery of the compositions of the present disclosure, such as lipid nanoparticles, results from cutaneous, intradermal, or subcutaneous injection, and the compositions of the present disclosure, such as lipid nanoparticles, then enable transfection in that local environment. In some embodiments, local delivery of the compositions of the present disclosure, such as lipid nanoparticles, results from topical administration, and the compositions of the present disclosure, such as lipid nanoparticles, then enable transfection in that local environment.

[0165] As used herein, the term "therapeutic agent" or "preventive agent" refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or preventive effect and / or induces a desired biological and / or pharmacological effect. A therapeutic agent is also referred to as an "active substance" or "active agent".

[0166] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of the nanoparticle composition sufficient to effect treatment in a mammal, preferably a human, when administered to the mammal, preferably a human. The amount of the lipid nanoparticles of the present disclosure that constitutes a "therapeutically effective amount" will vary depending on the nature of the composition of the present disclosure, e.g., the particular lipid nanoparticles used, the condition and its severity, the method of administration, and the age of the subject being treated, but can be routinely determined by one of ordinary skill in the art in view of the knowledge of one of ordinary skill in the art and the present disclosure.

[0167] As used herein, the term "treating" or "treatment" as used herein encompasses the treatment of a target disease or condition in a subject, preferably a mammal, more preferably a human, and in particular, when the subject has a predisposition to the condition but has not yet been diagnosed as having it, preventing the occurrence of the disease or condition, inhibiting the disease or condition, i.e., preventing its onset, reducing the disease or condition, i.e., causing regression of the disease or condition, or reducing the symptoms resulting from the disease or condition, e.g., reducing pain without addressing the underlying disease or condition.

[0168] As used herein, the term "zeta potential" refers to the electrokinetic potential at the interface of the lipids in the nanoparticle composition or the electrokinetic potential at the interface of the nanoparticles, e.g., that of the lipid nanoparticles.

[0169] The compositions of the present disclosure can comprise, consist essentially of, or consist of the disclosed components.

[0170] All percentages, amounts, and ratios are based on the total weight of the composition unless otherwise specified, and all measurements are made at about 25°C.

[0171] Any such group may be claimed for any reason, without meeting all the means of the present disclosure, by excepting or excluding any individual member of any such group, including any arbitrary partial range or combination of partial ranges within the group, and reserving the right to claim according to the part or by any similar means. Further, any individual replacement element, analog, compound, ligand, structure, or group thereof, or any member of the claimed group may be excepted or excluded, and the patent claims may be made for any reason without meeting all the means of the present disclosure by reserving the right to do so.

[0172] Throughout the present disclosure, various patents, patent applications, and publications are referenced. The entire disclosures of these patents, patent applications, and publications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art as known to those of ordinary skill in the art as of the date of the present disclosure. The present disclosure shall control in the event of any conflict between the present disclosure and the cited patents, patent applications, and publications.

[0173] For convenience, certain terms used in this specification, examples, and claims are gathered herein. Unless otherwise defined, all technical and scientific terms used in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0174] II. Ionizable Lipids The ionizable lipids of the present disclosure are positively charged at acidic pH and condense nucleic acids such as RNA into lipid nanoparticles (LNPs). Ionizable lipids are neutral at physiological pH and minimize toxicity. They are protonated in acidic endosomes after cellular uptake and can interact with anionic endosomal phospholipids to form conical ion pairs that are incompatible with the bilayer. These cationic-anionic lipid pairs drive the transition from the bilayer structure to the inverted hexagonal HII phase, which promotes membrane fusion / fragmentation, endosomal escape, and cargo release into the cytoplasm (Semple, S.C. et al., Nat. Biotechnol. 2010, 28, 172 - 176).

[0175] The pKa of the ionizable lipid disclosed in this specification is from about 8.5 to about 9.5. Lipid nanoparticles containing one or more of the ionizable lipids described in this specification can have a pKa from about 6.5 to about 7.5.

[0176] This disclosure provides ionizable lipids of Formula (I) to Formula (XVI). These lipids can have a positive charge or a partial positive charge at physiological pH.

[0177] In one embodiment, the ionizable lipid is of Formula (I), or a pharmaceutically acceptable salt or stereoisomer thereof, where: [Chemical Structure] In the formula, L 1 is C1-C6 alkylene, R 1 is C6-C 20 alkenyl, R 2 is C6-C 20 alkyl, R 3 and R 4 are each independently H or C1-C3 alkyl, q1 is absent or 1, and q2 is absent or 1.

[0178] In some embodiments, in Formula (I), L1 is C1-C6 alkylene, R1 is C6-C20 alkenyl, R2 is C6-C20 alkyl, R3 is methyl, R4 is methyl, q1 is absent, and q2 is absent.

[0179] In some embodiments, in Formula (I), L1 is C1-C6 alkylene, R1 is C6-C20 alkenyl, R2 is C6-C20 alkyl, R3 is methyl, R4 is methyl, q1 is 1, and q2 is 1.

[0180] In some embodiments, in formula (I), L1 is C1-C6 alkylene, R1 is C6-C20 alkenyl, R2 is C6-C20 alkyl, R3 is methyl, R4 is methyl, q1 is absent, and q2 is 1.

[0181] In some embodiments, in formula (I), L1 is C1-C6 alkylene, R1 is C6-C20 alkenyl, R2 is C6-C20 alkyl, R3 is methyl, R4 is methyl, q1 is 1, and q2 is absent.

[0182] Exemplary examples of the ionizable lipid of formula (I) can include, but are not limited to:

Chemical formula

[0183] In one embodiment, the ionizable lipid is of formula (I-A), or a pharmaceutically acceptable salt or stereoisomer thereof:

Chemical formula

[0184] In some embodiments, in formula (I-A), L 1 is C1-C6 alkylene, L 2is C1-C8 alkylene, R 2 is C6-C 20 alkyl, R 3 is methyl, R 4 is methyl, R 7 is C4-C 20 alkyl, R 8 is C4-C 20 alkyl.

[0185] Exemplary examples of ionizable lipids of formula (I-A) can include, but are not limited to:

Chemical formula

[0186] In one embodiment, the ionizable lipid is of formula (I-B), or a pharmaceutically acceptable salt or stereoisomer thereof:

Chemical formula

[0187] In some embodiments, in formula (I-B), L 1 is C1-C6 alkylene, L 2 is C1-C8 alkylene, L 3is C1-C8 alkylene, R 3 is methyl, R 4 is methyl, R 6 is C4-C 20 alkyl, R 7 is C4-C 20 alkyl, R 8 is C4-C 20 alkyl, R 10 is C4-C 20 alkyl.

[0188] Exemplary examples of ionizable lipids of formula (I-B) can include, but are not limited to:

Chemical formula

[0189] In one embodiment, the ionizable lipid is of formula (II), or a pharmaceutically acceptable salt or stereoisomer thereof:

Chemical formula

[0190] In some embodiments, in formula (II), L 1 is C1-C6 alkylene, R 1 is C6-C 20 alkenyl, R 2 is C6-C 20 alkyl, R 3 is methyl, R4 is methyl, and R 5 is H.

[0191] In some embodiments, in formula (II), L 1 is C1-C6 alkylene, and R 1 is C6-C 20 alkenyl, and R 2 is C6-C 20 alkyl, and R 3 is methyl, and R 4 is methyl, and R 5 is methyl.

[0192] Exemplary examples of ionizable lipids of formula (II) can include, but are not limited to:

Chemical formula

[0193] In one embodiment, the ionizable lipid is of formula (III), or a pharmaceutically acceptable salt or stereoisomer thereof:

Chemical formula

[0194] In some embodiments, in formula (III), L 1 is C1-C6 alkylene, and R 1is C6-C 20 alkenyl, and R 2 is C6-C 20 alkyl, and R 2’ is C6-C 20 alkyl, and R 3 is methyl, and R 4 is methyl, and R 5 is H.

[0195] In some embodiments, in formula (III), L 1 is C1-C6 alkylene, and R 1 is C6-C 20 alkenyl, and R 2 is C6-C 20 alkyl, and R 2’ is C6-C 20 alkyl, and R 3 is methyl, and R 4 is methyl, and R 5 is methyl.

[0196] Exemplary examples of the ionizable lipid of formula (III) can include, but are not limited to:

Chemical formula

[0197] In one embodiment, the ionizable lipid is of formula (IV), or a pharmaceutically acceptable salt or stereoisomer thereof:

Chemical formula

[0198] In some embodiments, in formula (IV), L 1 is C1-C6 alkylene, L 2 is C1-C8 alkylene, R 2 is C6-C 20 alkyl, R 3 is methyl, R 4 is methyl, R 5 is H, R 7 is C4-C 20 alkyl, R 8 is C4-C 20 alkyl.

[0199] In some embodiments, in formula (IV), L 1 is C1-C6 alkylene, L 2 is C1-C8 alkylene, R 2 is C6-C 20 alkyl, R 3 is methyl, R 4 is methyl, R 5 is methyl, R 7 is C4-C 20 alkyl, R 8 is C4-C 20 alkyl.

[0200] Exemplary examples of ionizable lipids of formula (IV) can include, but are not limited to:

Chemical formula

[0201] In one embodiment, the ionizable lipid is of formula (V), or a pharmaceutically acceptable salt or stereoisomer thereof: [Chemical formula] In the formula, L 1 is C1-C6 alkylene, and R 1 is C6-C 20 alkenyl, and R 3 and R 4 are each independently H or C1-C3 alkyl, and R 12 is C6-C 20 alkenyl.

[0202] In some embodiments, in formula (V), L 1 is C1-C6 alkylene, and R 1 is C6-C 20 alkenyl, and R 3 is methyl, and R 4 is methyl, and R 12 is C6-C 20 alkenyl.

[0203] Exemplary examples of the ionizable lipid of formula (V) can include, but are not limited to: [Chemical formula]

[0204] In one embodiment, the ionizable lipid is of formula (VI), or a pharmaceutically acceptable salt or stereoisomer thereof: [Chemical formula] In the formula, L 1 is C1-C6 alkylene, and L 1’ is C1-C6 alkylene, and R 1 is C6-C 20 alkenyl, and R 1’ is C6-C 20 alkenyl, and R9 is H, C1-C6 alkyl, or -(CH2) n OH, and R 12 is C6-C 20 alkenyl, and R 12’ is C6-C 20 alkenyl, and n is 2, 3, or 4. In some embodiments, in formula (VI), L 1 is C1-C6 alkylene, and L 1’ is C1-C6 alkylene, and R 1 is C6-C 20 alkenyl, and R 1’ is C6-C 20 alkenyl, and R 9 is H, and R 12 is C6-C 20 alkenyl, and R 12’ is C6-C 20 alkenyl.

[0205] In some embodiments, in formula (VI), L 1 is C1-C6 alkylene, and L 1’ is C1-C6 alkylene, and R 1 is C6-C 20 alkenyl, and R 1’ is C6-C 20 alkenyl, and R 9 is methyl, and R 12 is C6-C 20 alkenyl, and R 12’ is C6-C 20 alkenyl.

[0206] In some embodiments, in formula (VI), L 1 is C1-C6 alkylene, and L 1’ is C1-C6 alkylene, and R 1 is C6-C 20 alkenyl, and R 1’ is C6-C 20 alkenyl, and R 9 is -(CH2) n OH, and R12 is C6-C 20 alkenyl, and R 12’ is C6-C 20 alkenyl, and n is 2, 3, or 4.

[0207] In some embodiments, in formula (VI), L 1 is C1-C6 alkylene, L 1’ is C1-C6 alkylene, and R 1 is C6-C 20 alkenyl, and R 1’ is C6-C 20 alkenyl, and R 9 is -(CH2) n OH, and R 12 is C6-C 20 alkenyl, and R 12’ is C6-C 20 alkenyl, and n is 2.

[0208] Exemplary examples of the ionizable lipid of formula (VI) can include, but are not limited to:

Chemical formula

[0209] In one embodiment, the ionizable lipid is of formula (VII), or a pharmaceutically acceptable salt or stereoisomer thereof:

Chemical formula

[0210] In some embodiments, in formula (VII), L 1 is C1-C6 alkylene, and L 1’ is C1-C6 alkylene, and R 2 is C6-C 20 alkyl, and R 2’ is C6-C 20 alkyl, and R 9 is H, and R 11 is H, and R 14 is C6-C 20 alkyl, and R 14’ is C6-C 20 alkyl, and R 15 is C6-C 20 alkyl.

[0211] In some embodiments, in formula (VII), L 1 is C1-C6 alkylene, and L 1’ is C1-C6 alkylene, and R 2 is C6-C 20 alkyl, and R 2’ is C6-C 20 alkyl, and R 9 is methyl, and R 11 is H, and R 14 is C6-C 20 alkyl, and R 14’ is C6-C 20 alkyl, and R 15 is C6-C 20 alkyl.

[0212] In some embodiments, in formula (VII), L1 is C1-C6 alkylene, L1' is C1-C6 alkylene, R2 is C6-C20 alkyl, R2' is C6-C20 alkyl, R9 is -(CH2)nOH, R11 is H, R14 is C6-C20 alkyl, R14' is C6-C20 alkyl, R15 is C6-C20 alkyl, and n is 2.

[0213] Exemplary examples of the ionizable lipid of formula (VII) can include, but are not limited to:

Chemical formula

[0214] In one embodiment, the ionizable lipid is of formula (VIII), or a pharmaceutically acceptable salt or stereoisomer thereof:

Chemical formula

[0215] In some embodiments, in formula (VIII), L 1 is C1-C6 alkylene, L 1’is C1-C6 alkylene, R 2 is C6-C 20 alkyl, R 2’ is C6-C 20 alkyl, R 9 is H, R 14 is C6-C 20 alkyl, R 14’ is C6-C 20 alkyl, R 15 is C6-C 20 is alkyl.

[0216] In some embodiments, in formula (VIII), L1 is C1-C6 alkylene, L1' is C1-C6 alkylene, R2 is C6-C20 alkyl, R2' is C6-C20 alkyl, R9 is methyl, R14 is C6-C20 alkyl, R14' is C6-C20 alkyl, and R15 is C6-C20 alkyl.

[0217] In some embodiments, in formula (VIII), L1 is C1-C6 alkylene, L1' is C1-C6 alkylene, R2 is C6-C20 alkyl, R2' is C6-C20 alkyl, R9 is -(CH2)nOH, R14 is C6-C20 alkyl, R14' is C6-C20 alkyl, R15 is C6-C20 alkyl, and n is 2.

[0218] Exemplary examples of the ionizable lipid of formula (VIII) can include, but are not limited to:

Chemical formula

[0219] In one embodiment, the ionizable lipid is of formula (IX), or a pharmaceutically acceptable salt or stereoisomer thereof:

Chemical formula

[0220] In some embodiments, in formula (IX), R1 is C6-C20 alkenyl, R9 is H, and R12 is C6-C20 alkenyl.

[0221] In some embodiments, in formula (IX), R1 is C6-C20 alkenyl, R9 is methyl, and R12 is C6-C20 alkenyl.

[0222] In some embodiments, in formula (IX), R1 is C6-C20 alkenyl, R9 is -(CH2)nOH, R12 is C6-C20 alkenyl, and n is 2.

[0223] Exemplary examples of the ionizable lipid of formula (IX) can include, but are not limited to:

Chemical formula

[0224] In one embodiment, the ionizable lipid is of formula (X), or a pharmaceutically acceptable salt or stereoisomer thereof:

Chemical formula

[0225] In some embodiments, in formula (X), L4 is absent, L5 is absent, R1 is C6-C20 alkenyl, R2 is C6-C20 alkyl, R2' is C6-C20 alkyl, R9 is H, C1-C6 alkyl, or -(CH2)nOH, R12 is C6-C20 alkenyl, and n is 2, 3, or 4.

[0226] In some embodiments, in formula (X), L4 is C1-C6 alkylene, L5 is C1-C6 alkylene, R1 is C6-C20 alkenyl, R2 is C6-C20 alkyl, R2' is C6-C20 alkyl, R9 is H, C1-C6 alkyl, or -(CH2)nOH, R12 is C6-C20 alkenyl, and n is 2, 3, or 4.

[0227] In some embodiments, in formula (X), L4 is absent, L5 is C1-C6 alkylene, R1 is C6-C20 alkenyl, R2 is C6-C20 alkyl, R2' is C6-C20 alkyl, R9 is H, C1-C6 alkyl, or -(CH2)nOH, R12 is C6-C20 alkenyl, and n is 2, 3, or 4.

[0228] In some embodiments, in formula (X), L4 is C1-C6 alkylene, L5 is absent, R1 is C6-C20 alkenyl, R2 is C6-C20 alkyl, R2' is C6-C20 alkyl, R9 is H, C1-C6 alkyl, or -(CH2)nOH, R12 is C6-C20 alkenyl, and n is 2, 3, or 4.

[0229] In some embodiments, in formula (X), L4 is absent or C1-C6 alkylene, L5 is absent or C1-C6 alkylene, R1 is C6-C20 alkenyl, R2 is C6-C20 alkyl, R2' is C6-C20 alkyl, R9 is H, and R12 is C6-C20 alkenyl.

[0230] In some embodiments, in formula (X), L 4 is absent or C1-C6 alkylene, L 5 is absent or C1-C6 alkylene, R 1 is C6-C 20 alkenyl, R 2 is C6-C 20 alkyl, R 2’ is C6-C 20 alkyl, R 9 is methyl, R 12 is C6-C 20 alkenyl.

[0231] In some embodiments, in formula (X), L 4 is absent or C1-C6 alkylene, L 5 is absent or C1-C6 alkylene, R 1 is C6-C 20 alkenyl, R 2 is C6-C 20 alkyl, R 2’ is C6-C 20 alkyl, R 9 is -(CH2) n OH, R 12 is C6-C20 It is alkenyl, and n is 2.

[0232] Exemplary examples of the ionizable lipid of formula (X) can include, but are not limited to:

Chemical formula

[0233] In one embodiment, the ionizable lipid is of formula (XI), or a pharmaceutically acceptable salt or stereoisomer thereof:

Chemical formula

[0234] In some embodiments, in formula (XI), L3 is absent, L4 is absent, R1 is C6-C20 alkenyl, R2 is C6-C20 alkyl, R2' is C6-C20 alkyl, R12 is C6-C20 alkenyl, R13 is H, C1-C6 alkyl, -(CH2)nOH, or -(CH2)qN(CH3)2, n is 2, 3 or 4, and q is 2, 3, or 4.

[0235] In some embodiments, in formula (XI), L3 is C1-C6 alkylene, L4 is C1-C6 alkylene, R1 is C6-C20 alkenyl, R2 is C6-C20 alkyl, R2' is C6-C20 alkyl, R12 is C6-C20 alkenyl, R13 is H, C1-C6 alkyl, -(CH2)nOH, or -(CH2)qN(CH3)2, n is 2, 3, or 4, and q is 2, 3, or 4.

[0236] In some embodiments, in formula (XI), L3 is C1-C6 alkylene, L4 is absent, R1 is C6-C20 alkenyl, R2 is C6-C20 alkyl, R2' is C6-C20 alkyl, R12 is C6-C20 alkenyl, R13 is H, C1-C6 alkyl, -(CH2)nOH, or -(CH2)qN(CH3)2, n is 2, 3, or 4, and q is 2, 3, or 4.

[0237] In some embodiments, in formula (XI), L3 is absent, L4 is C1-C6 alkylene, R1 is C6-C20 alkenyl, R2 is C6-C20 alkyl, R2' is C6-C20 alkyl, R12 is C6-C20 alkenyl, R13 is H, C1-C6 alkyl, -(CH2)nOH, or -(CH2)qN(CH3)2, n is 2, 3, or 4, and q is 2, 3, or 4.

[0238] In some embodiments, in formula (XI), L3 is absent or C1-C6 alkylene, L4 is absent or C1-C6 alkylene, R1 is C6-C20 alkenyl, R2 is C6-C20 alkyl, R2' is C6-C20 alkyl, R12 is C6-C20 alkenyl, and R13 is H.

[0239] In some embodiments, in formula (XI), L3 is absent or is C1-C6 alkylene, L4 is absent or is C1-C6 alkylene, R1 is C6-C20 alkenyl, R2 is C6-C20 alkyl, R 2’ is C6-C 20 alkyl, R 12 is C6-C 20 alkenyl, R 13 is methyl.

[0240] In some embodiments, in formula (XI), L3 is absent or is C1-C6 alkylene, L4 is absent or is C1-C6 alkylene, R1 is C6-C20 alkenyl, R2 is C6-C20 alkyl, R2’ is C6-C20 alkyl, R12 is C6-C20 alkenyl, R13 is -(CH2)nOH, and n is 2.

[0241] In some embodiments, in formula (XI), L3 is absent or is C1-C6 alkylene, L4 is absent or is C1-C6 alkylene, R1 is C6-C20 alkenyl, R2 is C6-C20 alkyl, R2’ is C6-C20 alkyl, R12 is C6-C20 alkenyl, R13 is -(CH2)qN(CH3)2, and q is 3.

[0242] Exemplary examples of the ionizable lipid of formula (XI) can include, but are not limited to:

Chemical formula

[0243] In one embodiment, the ionizable lipid is of formula (XII), or a pharmaceutically acceptable salt or stereoisomer thereof:

Chemical formula

[0244] In some embodiments, in formula (XII), L2 is C1-C8 alkylene, L2’ is C1-C8 alkylene, R7 is C4-C20 alkyl, R7’ is C4-C20 alkyl, R8 is C4-C20 alkyl, R8’ is C4-C20 alkyl, and R13 is H.

[0245] In some embodiments, in formula (XII), L2 is C1-C8 alkylene, L2’ is C1-C8 alkylene, R7 is C4-C20 alkyl, R7’ is C4-C20 alkyl, R8 is C4-C20 alkyl, R8’ is C4-C20 alkyl, and R13 is methyl.

[0246] In some embodiments, in formula (XII), L2 is C1-C8 alkylene, L2’ is C1-C8 alkylene, R7 is C4-C20 alkyl, R7’ is C4-C20 alkyl, R8 is C4-C20 alkyl, R8’ is C4-C20 alkyl, and R13 is -(CH2)nOH, where n is 4.

[0247] Exemplary examples of the ionizable lipid of formula (XII) can include, but are not limited to: [Chemical formula]

[0248] In one embodiment, the ionizable lipid is of formula (XIII), or a pharmaceutically acceptable salt or stereoisomer thereof: [Chemical formula] Wherein L 2 is C1-C8 alkylene, and L 2’ is C1-C8 alkylene, R 7 is C4-C 20 alkyl, R 7’ is C4-C 20 alkyl, R 8 is C4-C 20 alkyl, R 8’ is C4-C 20 alkyl, R 13 is H, C1-C6 alkyl, -(CH2) n OH, or -(CH2) q N(CH3)2, n is 2, 3, or 4, and q is 2, 3, or 4.

[0249] In some embodiments, in formula (XIII), L2 is C1-C8 alkylene, L2' is C1-C8 alkylene, R7 is C4-C20 alkyl, R7' is C4-C20 alkyl, R8 is C4-C20 alkyl, R8' is C4-C20 alkyl, and R13 is H.

[0250] In some embodiments, in formula (XIII), L 2 is C1-C8 alkylene, L 2’ is C1-C8 alkylene, R 7 is C4-C 20 alkyl, R 7’ is C4-C 20is alkyl, and R 8 is C4-C 20 is alkyl, and R 8’ is C4-C 20 is alkyl, and R 13 is methyl.

[0251] In some embodiments, in formula (XIII), L2 is C1-C8 alkylene, L2’ is C1-C8 alkylene, R7 is C4-C20 alkyl, R7’ is C4-C20 alkyl, R8 is C4-C20 alkyl, R8’ is C4-C20 alkyl, R13 is -(CH2)nOH, and n is 2.

[0252] In some embodiments, in formula (XIII), L2 is C1-C8 alkylene, L2’ is C1-C8 alkylene, R7 is C4-C20 alkyl, R7’ is C4-C20 alkyl, R8 is C4-C20 alkyl, R8’ is C4-C20 alkyl, R13 is -(CH2)qN(CH3)2, and q is 3.

[0253] Exemplary examples of the ionizable lipid of formula (XIII) can include, but are not limited to:

Chemical formula

[0254] In one embodiment, the ionizable lipid is of formula (XIV), or a pharmaceutically acceptable salt or stereoisomer thereof:

Chemical formula

[0255] In some embodiments, for formula (XIV), L 1 is C1-C6 alkylene, L 2 is C1-C8 alkylene, R 3 is methyl, R 4 is methyl, R 6 is C4-C 20 alkyl, R 7 is C4-C 20 alkyl.

[0256] Exemplary examples of ionizable lipids of formula (XIV) can include, but are not limited to:

Chemical formula

[0257] In one embodiment, the ionizable lipid is of formula (XV), or a pharmaceutically acceptable salt or stereoisomer thereof:

Chemical formula

[0258] In some embodiments, in formula (XV), L 2 is C1-C8 alkylene, L 2’ is C1-C8 alkylene, and R 6 is C4-C 20 alkyl, and R 6’ is C4-C 20 alkyl, and R 7 is C4-C 20 alkyl, and R 7’ is C4-C 20 alkyl, and R 8 is C4-C 20 alkyl, and R 8’ is C4-C 20 alkyl, and R 10 is C4-C 20 alkyl, and R 10’ is C4-C 20 alkyl, and R 13 is H, p1 is absent, and p2 is absent.

[0259] In some embodiments, in formula (XV), L 2 is C1-C8 alkylene, L 2’ is C1-C8 alkylene, and R 6 is C4-C 20 alkyl, and R 6’ is C4-C 20 alkyl, and R 7 is C4-C 20 alkyl, and R7’ is C4-C 20 alkyl, and R 8 is C4-C 20 alkyl, and R 8’ is C4-C 20 alkyl, and R 10 is C4-C 20 alkyl, and R 10’ is C4-C 20 alkyl, and R 13 is H, p1 is 1, and p2 is 1.

[0260] In some embodiments, in formula (XV), L2 is C1-C8 alkylene, L2' is C1-C8 alkylene, R6 is C4-C20 alkyl, R6' is C4-C20 alkyl, R7 is C4-C20 alkyl, R7' is C4-C20 alkyl, R8 is C4-C20 alkyl, R8' is C4-C20 alkyl, R10 is C4-C20 alkyl, R10' is C4-C20 alkyl, R13 is methyl, p1 is absent, and p2 is absent.

[0261] In some embodiments, in formula (XV), L2 is C1-C8 alkylene, L2' is C1-C8 alkylene, R6 is C4-C20 alkyl, R6' is C4-C20 alkyl, R7 is C4-C20 alkyl, R7' is C4-C20 alkyl, R8 is C4-C20 alkyl, R8' is C4-C20 alkyl, R10 is C4-C20 alkyl, R10' is C4-C20 alkyl, R13 is methyl, p1 is 1, and p2 is 1.

[0262] In some embodiments, in formula (XV), L2 is C1-C8 alkylene, L2’ is C1-C8 alkylene, R6 is C4-C20 alkyl, R6’ is C4-C20 alkyl, R7 is C4-C20 alkyl, R7’ is C4-C20 alkyl, R8 is C4-C20 alkyl, R8’ is C4-C20 alkyl, R10 is C4-C20 alkyl, R10’ is C4-C20 alkyl, R13 is -(CH2)nOH, n is 4, p1 is absent, and p2 is absent.

[0263] In some embodiments, in formula (XV), L2 is C1-C8 alkylene, L2’ is C1-C8 alkylene, R6 is C4-C20 alkyl, R6’ is C4-C20 alkyl, R7 is C4-C20 alkyl, R7’ is C4-C20 alkyl, R8 is C4-C20 alkyl, R8’ is C4-C20 alkyl, R10 is C4-C20 alkyl, R10’ is C4-C20 alkyl, R13 is -(CH2)nOH, n is 4, p1 is 1, and p2 is 1.

[0264] Exemplary examples of ionizable lipids of formula (XV) can include, but are not limited to:

Chemical formula

[0265] In one embodiment, the ionizable lipid is of formula (XVI), or a pharmaceutically acceptable salt or stereoisomer thereof:

Chemical formula

[0266] In one embodiment, the ionizable lipid is of formula (XVI-A), or a pharmaceutically acceptable salt or stereoisomer thereof:

Chemical formula

[0267] In some embodiments, in Formula (XVI) and Formula (XVI-A), L2 is C1-C8 alkylene, L2’ is C1-C8 alkylene, R6 is C4-C20 alkyl, R6’ is C4-C20 alkyl, R7 is C4-C20 alkyl, R7’ is C4-C20 alkyl, R13 is H, p1 is absent or 1, and p2 is absent or 1.

[0268] In some embodiments, in Formula (XVI) and Formula (XVI-A), L2 is C1-C8 alkylene, L2’ is C1-C8 alkylene, R6 is C4-C20 alkyl, R6’ is C4-C20 alkyl, R7 is C4-C20 alkyl, R7’ is C4-C20 alkyl, R13 is methyl, p1 is absent or 1, and p2 is absent or 1. In Formula (XVI) and Formula (XVI-A), L2 is C1-C8 alkylene, L2’ is C1-C8 alkylene, R6 is C4-C20 alkyl, R6’ is C4-C20 alkyl, R7 is C4-C20 alkyl, R7’ is C4-C20 alkyl, R13 is -(CH2)nOH, n is 4, p1 is absent, and p2 is absent.

[0269] In some embodiments, in Formula (XVI) and Formula (XVI-A), L2 is C1-C8 alkylene, L2’ is C1-C8 alkylene, R6 is C4-C20 alkyl, R6’ is C4-C20 alkyl, R7 is C4-C20 alkyl, R7’ is C4-C20 alkyl, R13 is -(CH2)nOH, n is 4, p1 is 1, and p2 is 1.

[0270] Exemplary examples of the ionizable lipids of Formula (XVI) and Formula (XVI-A) can include, but are not limited to:

Chemical formula

[0271] In one embodiment, the compound has the structure of formula (XVII) below, or a stereoisomer, salt, or tautomer thereof:

Chemical formula

[0272] In one embodiment, G 1 and G 2 are each independently -OC(=O)-, -NR 25 C(=O)-, or -CH=CH-. In some embodiments, G 1 and G 2 are each -OC(=O)-. In some embodiments, one of G 1 or G 2 is -NR 25 C(=O)-, and G 1 or G 2The other of them is -CH=CH-.

[0273] In one embodiment, m 2 is 0. In some embodiments, m 2 is 1.

[0274] In one embodiment, the compound has one of the following structures of formula (XVIIA)-(XVIIB):

Chemical formula

Chemical formula

Chemical formula

[0275] In one embodiment, R24 is C1-C8 heteroalkyl. In some embodiments, R24 is C3-C7 heteroalkyl. In some embodiments, R24 is C3 heteroalkyl. In some embodiments, R24 is C4 heteroalkyl. In some embodiments, R24 is C5 heteroalkyl. In some embodiments, R24 is C6 heteroalkyl. In some embodiments, R24 is C7 heteroalkyl. In some embodiments, R24 is C4 alkylamine. In some embodiments, R24 is C5 alkylamine. In some embodiments, R24 is C6 alkylamine. In some embodiments, R24 is C7 alkylamine. In some specific embodiments, R24 is

Chemical formula

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0276] In some embodiments, R 24 's C1-C8 heteroalkyl is further substituted with cycloalkyl. In some specific embodiments, R 24 is

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0277] In one embodiment, R 25 is H or C1-C4 alkyl. In some embodiments, R 25 is H. In some embodiments, R 25is C1-C4 alkyl. In some embodiments, R 25 is C1 alkyl. In some embodiments, R 25 is C2 alkyl. In some embodiments, R 25 is C3 alkyl. In some embodiments, R 25 is C4 alkyl. In some specific embodiments, R 25 is -CH3. In some other specific embodiments, R 25 is -CH2CH3.

[0278] In one embodiment, R 21 and R 22 are each independently C1-C6 alkyl, straight-chain C 10 -C 20 alkyl, straight-chain C 10 -C 20 alkenyl, or branched C 10 -C 35 alkenyl, and the C1-C6 alkyl is substituted with -OC(=O)R 26 In some embodiments, R 21 and R 22 are each independently C2-C5 alkyl substituted with -OC(=O)R 26 , straight-chain C 12 -C 18 alkyl, straight-chain C 12 -C 18 alkenyl, or branched C 14 -C 32 alkenyl. In some embodiments, one of R 21 or R 22 is straight-chain C 12 -C 18 alkyl, and the other of R 21 or R 22 is straight-chain C 12 -C 18 alkenyl. In some embodiments, one of R 21 or R 22 is C2-C5 alkyl substituted with -OC(=O)R 26 , and the other of R 21 or R 22Of which the other is a linear C 12 -C 18 alkyl. In some embodiments, R 21 or R 22 Of which one is a linear C 12 -C 18 alkyl and the other of R 21 or R 22 is a branched C 14 -C 32 alkenyl. In some embodiments, R 21 and R 22 are each C2-C5 alkyl substituted with -OC(=O)R 26 . In some embodiments, R 21 and R 22 are each C2 alkyl substituted with -OC(=O)R 26 . In some embodiments, R 21 and R 22 are each C3 alkyl substituted with -OC(=O)R 26 . In some embodiments, R 21 and R 22 are each C4 alkyl substituted with -OC(=O)R 26 . In some embodiments, R 21 and R 22 are each C5 alkyl substituted with -OC(=O)R 26 . In some embodiments, R 21 and R 22 are each linear C 12 -C 18 alkenyl. In some embodiments, R 21 and R 22 are each linear C 12 alkenyl. In some embodiments, R 21 and R 22 are each linear C 13 alkenyl. In some embodiments, R 21 and R 22 are each linear C 14 alkenyl. In some embodiments, R 21 and R 22 are each linear C 15It is alkenyl. In some embodiments, R 21 and R 22 are each linear C 16 alkenyl. In some embodiments, R 21 and R 22 are each linear C 17 alkenyl. In some embodiments, R 21 and R 22 are each linear C 18 alkenyl.

[0279] In one embodiment, R 26 is branched C 10 -C 30 alkyl. In some embodiments, R 26 is branched C 10 -C 20 alkyl. In some embodiments, R 26 is branched C 12 -C 18 alkyl. In some embodiments, R 26 is branched C 12 alkyl. In some embodiments, R 26 is branched C 13 alkyl. In some embodiments, R 26 is branched C 14 alkyl. In some embodiments, R 26 is branched C 15 alkyl. In some embodiments, R 26 is branched C 16 alkyl. In some embodiments, R 26 is branched C 17 alkyl. In some embodiments, R 26 is branched C 18 alkyl.

[0280] In some embodiments, R 21 and R 22 each independently have one of the following structures:

Chemical formula

Chem.

Chem.

Chem.

Chem.

Chem.

[0281] In one embodiment, the compound has one of the following structures shown in Table A below.

Math.

[0282] In one embodiment, the compound has the structure of formula (XVIII) below, or a stereoisomer, salt, or tautomer thereof:

Chemical formula

[0283] In one embodiment, the compound is one of the following structures of formulas (XVIIIA) to (XVIIIC):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0284] In one embodiment, R 27 is H, C1-C6 alkyl, or C1-C6 heteroalkyl. In some embodiments, R 27 is H, C1-C3 alkyl, or C1-C5 heteroalkyl. In some embodiments, R 27 is C1-C3 alkyl, C2-C4 alkyl alcohol, or C5 alkylamine. In some specific embodiments, R 27 has one of the following structures:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0285] In one embodiment, G 3 and G 4 are each independently -OC(=O)R 28 , -C(=O)OR 28 , -CH(CH2OC(=O)R 28 )(CH2OC(=O)R 28 )3, -PhOC(=O)R 28 , -Ph(OC(=O)R 28 )2, -OC(=O)Ph(OC(=O)R 28 )2, or -C(=O)OCH2C(OC(=O)R 28 )(CH2OC(=O)R 28 ). In some embodiments, G 3 and G 4 each have one of the following structures:

Chemical formula

Chemical formula

Chem.

Chem.

Chem.

Chem.

Chem.

[0286] In one embodiment, R 28 is, independently of each other, a linear C6-C 12 alkyl, a branched C 10 -C 40 alkyl, a linear C 15 -C 20 alkenyl, or a branched C 20 -C 40 alkenyl. In some embodiments, R 28is, independently of each other, linear C7-C 10 alkyl, branched C 14 -C 40 alkyl, linear C 15 -C 20 alkenyl, or branched C 30 -C 40 alkenyl. In some embodiments, R 28 is, independently of each other, linear C7 alkyl. In some embodiments, R 28 is, independently of each other, linear C8 alkyl. In some embodiments, R 28 is, independently of each other, linear C9 alkyl. In some embodiments, R 28 is, independently of each other, linear C 10 alkyl. In some embodiments, R 28 is, independently of each other, branched C 14 -alkyl. In some embodiments, R 28 is, independently of each other, branched C 15 alkyl. In some embodiments, R 28 is, independently of each other, branched C 16 alkyl. In some embodiments, R 28 is, independently of each other, branched C 17 alkyl. In some embodiments, R 28 is, independently of each other, branched C 18 alkyl. In some embodiments, R 28 is, independently of each other, branched C 19 alkyl. In some embodiments, R 28 is, independently of each other, branched C 20 alkyl. In some embodiments, R 28 is, independently of each other, branched C 21 alkyl. In some embodiments, R 28 is, independently of each other, branched C 22 alkyl. In some embodiments, R 28 is, independently of each other, branched C 23 alkyl. In some embodiments, R 28 is, independently of each other, branched C 24is alkyl. In some embodiments, R 28 is, independently of each other, branched C 25 alkyl. In some embodiments, R 28 is, independently of each other, branched C 26 alkyl. In some embodiments, R 28 is, independently of each other, branched C 27 alkyl. In some embodiments, R 28 is, independently of each other, branched C 28 alkyl. In some embodiments, R 28 is, independently of each other, branched C 29 alkyl. In some embodiments, R 28 is, independently of each other, branched C 30 alkyl. In some embodiments, R 28 is, independently of each other, branched C 31 alkyl. In some embodiments, R 28 is, independently of each other, branched C 32 alkyl. In some embodiments, R 28 is, independently of each other, branched C 33 alkyl. In some embodiments, R 28 is, independently of each other, branched C 34 alkyl. In some embodiments, R 28 is, independently of each other, branched C 35 alkyl. In some embodiments, R 28 is, independently of each other, branched C 36 alkyl. In some embodiments, R 28 is, independently of each other, branched C 37 alkyl. In some embodiments, R 28 is, independently of each other, branched C 38 alkyl. In some embodiments, R 28 is, independently of each other, branched C 39 alkyl. In some embodiments, R 28 is, independently of each other, branched C 40 alkyl. In some embodiments, R 28 is, independently of each other, linear C 15is alkenyl. In some embodiments, R 28 is, independently of each other, linear C 16 is alkenyl. In some embodiments, R 28 is, independently of each other, linear C 17 is alkenyl. In some embodiments, R 28 is, independently of each other, linear C 18 is alkenyl. In some embodiments, R 28 is, independently of each other, linear C 19 is alkenyl. In some embodiments, R 28 is, independently of each other, linear C 20 is alkenyl. In some embodiments, R 28 is, independently of each other, branched C 30 is alkenyl. In some embodiments, R 28 is, independently of each other, branched C 31 is alkenyl. In some embodiments, R 28 is, independently of each other, branched C 32 is alkenyl. In some embodiments, R 28 is, independently of each other, branched C 33 is alkenyl. In some embodiments, R 28 is, independently of each other, branched C 34 is alkenyl. In some embodiments, R 28 is, independently of each other, branched C 35 is alkenyl. In some embodiments, R 28 is, independently of each other, branched C 36 is alkenyl. In some embodiments, R 28 is, independently of each other, branched C 37 is alkenyl. In some embodiments, R 28 is, independently of each other, branched C 38 is alkenyl. In some embodiments, R 28 is, independently of each other, branched C 39 is alkenyl. In some embodiments, R 28 is, independently of each other, branched C 40 is alkenyl.

[0287] In some specific embodiments, R 28 each independently has one of the following structures:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0288] In one embodiment, m 1 and m 2 are each independently an integer from 1 to 6. In some embodiments, m 1 and m 2 are each independently an integer from 1 to 4. In some specific embodiments, m 1 or m 2 is 1. In some specific embodiments, m 1 or m 2 is 2. In some specific embodiments, m 1 or m 2 is 3. In some specific embodiments, m 1 or m 2 is 4. In some specific embodiments, m 1 or m 2 is 5. In some specific embodiments, m 1 or m 2 is 6.

[0289] In one embodiment, the compound has one of the following structures shown in Table B below.

Number

[0290] In one embodiment, the compound has the structure of the following formula (XIX), or a stereoisomer, salt, or tautomer thereof:

Chemistry

[0291] In one embodiment, R 29a and R 29b are each independently a linear C6-C 10 alkyl or a linear C 12 -C 20 alkylene. In some embodiments, R 29a and R 29b are each linear C6-C 10 alkyl. In some embodiments, R 29a and R 29b are each linear C 12 -C 20 alkylene. In some specific embodiments, R 29a and R 29b each independently have one of the following structures:

Chemical formula

Chemical formula

Chemical formula

[0292] In one embodiment, R 30is an aryl or a C3-C6 heterocyclic ring, and the aryl or C3-C6 heterocyclic ring is substituted with -OC(=O)R 31 , C1-C4 alkyl, or C1-C4 heteroalkyl. In some embodiments, the aryl of R 30 is phenyl or naphthalene. In some embodiments, the C3-C6 heterocyclic ring of R 30 is azetidine, pyrrolidine, imidazolidine, pyrazolidine, piperidine, diazinan, triazinan, or azepane. In some embodiments, R 30 is aryl substituted with -OC(=O)R 31 , or a C3-C6 heterocyclic ring substituted with C1-C4 alkyl, or C1-C4 heteroalkyl. In some embodiments, it is phenyl substituted with -OC(=O)R 31 , or a C3-C6N-heterocyclic phenyl substituted with C1-C4 alkyl, or C1-C4 heteroalkyl. In some embodiments, R 30 has one of the following structures:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0293] . In some embodiments, n 1 is an integer from 1 to 4. In some embodiments, n 1 is the integer 1. In some embodiments, n 1 is the integer 2. In some embodiments, n 1 is the integer 3. In some embodiments, n 1 is the integer 4. In some embodiments, n 1 is the integer 1 or 4.

[0294] In one embodiment, the compound has one of the following structures shown in Table C below. [Math.]

[0295] In one embodiment, an ionizable lipid of formula (XXI), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: [Chem.] wherein L 1 is C1-C6 alkylene, L 1’ is C1-C6 alkylene, L 2 is C1-C8 alkylene, L 2’ is C1-C8 alkylene, L 3is C1-C8 alkylene, L 3’ is C1-C8 alkylene, R 3 and R 4 are each independently H, C1-C4 alkyl, -CH2-cyclopropyl, or -(CH2) n OH, R 6 is C4-C 20 alkyl, R 6’ is C4-C 20 alkyl, R 7 is C4-C 20 alkyl, R 7’ is C4-C 20 alkyl, R 8 ’ is C4-C 20 alkyl, R 8’ is C4-C 20 alkyl, R 10 is C4-C 20 alkyl, R 10’ is C4-C 20 alkyl, n is 2, 3, or 4, and m is 1, 2, 3, 4, or 5.

[0296] In one embodiment, the compound has one of the following structures shown in Table D below.

Number

[0297] In one embodiment, the compound is the structure of formula (XX) below, or a stereoisomer, salt, or tautomer thereof:

Chemical formula

[0298] In one embodiment, G 1 and G 2 are each independently -OC(=O)- or -NR 25 C(=O)-. In some embodiments, G 1 and G 2 are each -OC(=O)-. In some embodiments, G 1 and G 2 are each -NR 25 C(=O)-. In some embodiments, one of G 1 or G 2 is -NR 25 C(=O)- and the other of G 1 or G 2 is -OC(=O)-.

[0299] In one embodiment, the compound has one of the structures of the following formulas (XXA)-(XXB):

Chemical formula

Chemical formula

Chemical formula

[0300] In one embodiment, Y is O. In other embodiments, Y is NR 32 wherein R 32 is H or C1-C4 alkyl. In some specific embodiments, Y is NR 32 where R 32 is H. In some other embodiments, Y is NR 32 where R 32 is C1-C4 alkyl. In some embodiments, the C1-C4 alkyl of R 32 is methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, or tert-butyl.

[0301] In one embodiment, the compound has one of the structures of the following formulas (XXA-1) to (XXB-2), or a stereoisomer, salt or tautomer thereof:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0302] In one embodiment, R 24 is C1-C6 heteroalkyl. In some embodiments, R 24 is C3-C6 heteroalkyl. In some embodiments, R 24 is C3 heteroalkyl. In some embodiments, R 24 is C4 heteroalkyl. In some embodiments, R 24 is C5 heteroalkyl. In some embodiments, R 24 is C6 heteroalkyl. In some embodiments, R 24 is C5 alkylamine. In some specific embodiments, R 24 is

Chemical formula

[0303] In one embodiment, R 24 is aryl. In some embodiments, the aryl of R 24 is substituted with C1-C6 heteroalkyl. In some embodiments, the aryl of R 24 is phenyl or naphthalene. In some specific embodiments, the aryl of R 24 is phenyl. In some other embodiments, the aryl of R 24 is substituted with C2-C4 heteroalkyl. In some embodiments, for example, the C2-C4 heteroalkyl is a substituted amine. In some embodiments, R 24 is phenyl substituted with C2-C4 heteroalkyl. In some embodiments, R 24is phenyl substituted with C3 heteroalkyl. In some specific embodiments, R 24 the substituted aryl is

Chemical Structure

[0304] In one embodiment, R 24 is C1-C4 alkyl substituted with 4- to 8-membered heterocycloalkyl. In some embodiments, R 24 is C1-C3 alkyl substituted with 4- to 6-membered heterocycloalkyl. In some embodiments, for example, the C1-C3 alkyl of R 24 is methyl, ethyl, or n-propyl. In some embodiments, for example, the 4- to 6-membered heterocycloalkyl of R 24 is azetidine, oxetane, phosphetane, thietane, diazetidine, dioxetane, dithietane, pyrrolidine, tetrahydrofuran, phosphorane, tetrahydrothiophene, imidazolidine, pyrazolidine, oxathiolidine, isoxathiolidine, oxazolidine, isoxazolidine, thiazolidine, dioxolane, dithiolane, piperidine, oxane, phosphinan, thiane, diazinan, morpholine, oxathiane, dioxane, or dithiane. In some specific embodiments, the 4- to 6-membered heterocycloalkyl of R 24 is azetidine or 1,4-diazinan. In some embodiments, R 24 has one of the following structures:

Chemical Structure

Chemical Structure

Chem.

[0305] In one embodiment, R 25 is H or C1-C4 alkyl. In some embodiments, R 25 is H. In some embodiments, R 25 is C1-C4 alkyl. In some embodiments, R 25 is C1 alkyl. In some embodiments, R 25 is C2 alkyl. In some embodiments, R 25 is C3 alkyl. In some embodiments, R 25 is C4 alkyl. In some specific embodiments, R 25 is -CH3. In some other specific embodiments, R 25 is -CH2CH3.

[0306] In one embodiment, R 21 and R 22 are each independently C1-C6 alkyl, linear C 10 -C 20 alkyl, linear C 10 -C 20 alkenyl, or branched C 10 -C 35 alkenyl, and C1-C6 alkyl is substituted with -OC(=O)R 26 . In some embodiments, R 21 and R 22 are each independently C2-C5 alkyl substituted with -OC(=O)R 26 linear C 12 -C 18 alkyl, linear C 12 -C 18 alkenyl, or branched C 14 -C 32 alkenyl. In some embodiments, one of R 21 or R 22 is linear C12 -C 18 is alkyl, and R 21 or R 22 the other of which is linear C 12 -C 18 is alkenyl. In some embodiments, R 21 or R 22 one of which is C2-C5 alkyl substituted with -OC(=O)R 26 , and the other of R 21 or R 22 is linear C 12 -C 18 is alkyl. In some embodiments, R 21 or R 22 one of which is linear C 12 -C 18 is alkyl, and the other of R 21 or R 22 is branched C 14 -C 32 is alkenyl. In some embodiments, R 21 and R 22 are each C2-C5 alkyl substituted with -OC(=O)R 26 . In some embodiments, R 21 and R 22 are each C2 alkyl substituted with -OC(=O)R 26 . In some embodiments, R 21 and R 22 are each C3 alkyl substituted with -OC(=O)R 26 . In some embodiments, R 21 and R 22 are each C4 alkyl substituted with -OC(=O)R 26 . In some embodiments, R 21 and R 22 are each C5 alkyl substituted with -OC(=O)R 26 . In some embodiments, R 21 and R 22 are each linear C 12 -C 18 is alkenyl. In some embodiments, R 21 and R 22is a linear C 12 alkenyl. In some embodiments, R 21 and R 22 are each linear C 13 alkenyl. In some embodiments, R 21 and R 22 are each linear C 14 alkenyl. In some embodiments, R 21 and R 22 are each linear C 15 alkenyl. In some embodiments, R 21 and R 22 are each linear C 16 alkenyl. In some embodiments, R 21 and R 22 are each linear C 17 alkenyl. In some embodiments, R 21 and R 22 are each linear C 18 alkenyl.

[0307] In one embodiment, R 26 is branched C 10 -C 30 alkyl. In some embodiments, R 26 is branched C 10 -C 20 alkyl. In some embodiments, R 26 is branched C 12 -C 18 alkyl. In some embodiments, R 26 is branched C 12 alkyl. In some embodiments, R 26 is branched C 13 alkyl. In some embodiments, R 26 is branched C 14 alkyl. In some embodiments, R 26 is branched C 15 alkyl. In some embodiments, R 26 is branched C 16 alkyl. In some embodiments, R 26 is branched C 17is alkyl. In some embodiments, R 26 is branched C 18 alkyl.

[0308] In some embodiments, R 21 and R 22 each independently have the following structure:

Chemical formula

[0309] In one embodiment, the compound has one of the following structures shown in Table E below.

Number

[0310] III. Nucleic Acids In one aspect, the ionizable lipids of the present disclosure are useful for the delivery of nucleic acids. Nucleic acids include, but are not limited to, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), miRNA inhibitors (antagomir / antimir), Dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), multivalent RNA, and mixtures thereof. In some aspects, the nucleic acid or mRNA includes self-amplifying RNA (saRNA), polycistronic RNA, circular RNA, and mixtures thereof.

[0311] In one aspect, the nucleic acid includes interfering RNA molecules such as, for example, siRNA, aiRNA, miRNA, or mixtures thereof. In certain other aspects, the nucleic acid includes one or more mRNA molecules (e.g., a cocktail).

[0312] Interfering RNA molecules include "small interfering RNA" or "siRNA", e.g., interfering RNA having a length of about 15-60, 15-50, or 15-40 (double-stranded) nucleotides, more typically having a length of about 15-30, 15-25, or 19-25 (double-stranded) nucleotides, preferably having a length of about 20-24, 21-22, or 21-23 (double-stranded) nucleotides (e.g., each complementary sequence of the double-stranded siRNA has a length of 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides, preferably having a length of about 20-24, 21-22, or 21-23 nucleotides, and the double-stranded siRNA has a length of about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs, preferably having a length of about 18-22, 19-20, or 19-21 base pairs). The siRNA duplex may include a 3' overhang of about 1 to about 4 nucleotides or about 2 to about 3, and a 5' phosphate terminus.

[0313] Examples of siRNA include double-stranded polynucleotide molecules assembled from two separate linear molecules, where one strand is the sense strand and the other is the complementary antisense strand; double-stranded polynucleotide molecules assembled from a single-stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; double-stranded polynucleotide molecules having a hairpin secondary structure with self-complementary sense and antisense regions; and circular single-stranded polynucleotide molecules having two or more loop structures and a stem with self-complementary sense and antisense regions, where the circular polynucleotide can be processed in vivo or in vitro to generate an active double-stranded siRNA molecule, but are not limited thereto.

[0314] siRNA can also be chemically synthesized. siRNA can also be generated by cleavage of longer dsRNA (e.g., dsRNA longer than about 25 nucleotides) by E. coli RNase III or Dicer. These enzymes process dsRNA into biologically active siRNA (see, for example, Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002), Calegari et al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002), Byrom et al., Ambion TechNotes, 10(l):4-6 (2003), Kawasaki et al., Nucleic Acids Res., 31:981-987 (2003), Knight et al., Science, 293:2269-2271 (2001), and Robertson et al., Biol. Chem., 243:82 (1968)). Preferably, the dsRNA is at least 50 nucleotides in length up to about 100, 200, 300, 400, or 500 nucleotides. The dsRNA can be on the order of 1000, 1500, 2000, 5000 nucleotides, or longer. The dsRNA can encode an entire gene transcript or a partial gene transcript. In certain cases, the siRNA can be encoded by a plasmid (e.g., transcribed as a sequence that automatically folds into a duplex with a hairpin loop).

[0315] In one embodiment, the nucleic acid comprises siRNA. In one embodiment, the siRNA molecule comprises a double-stranded region that is about 15 to about 60 nucleotides in length (e.g., about 15 to 60, 15 to 50, 15 to 40, 15 to 30, 15 to 25, or 19 to 25 nucleotides in length, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). The siRNA molecule can silence the expression of a target sequence in vitro and / or in vivo.

[0316] In other embodiments, the siRNA molecule comprises, but is not limited to, modified nucleotides including 2'-O-methyl (2'OMe) nucleotides, 2'-deoxy-2'-fluoro (2'F) nucleotides, 2'-deoxynucleotides, 2'-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA) nucleotides, and mixtures thereof. In some other embodiments, the siRNA comprises 2'-OMe nucleotides (e.g., 2'-OMe purine and / or pyrimidine nucleotides) such as, for example, 2'-OMe-guanosine nucleotides, 2'-OMe-uridine nucleotides, 2'OMe-adenosine nucleotides, 2'-OMe-cytosine nucleotides, and mixtures thereof. In certain instances, the siRNA does not comprise 2'-OMe-cytosine nucleotides. In other embodiments, the siRNA comprises a hairpin loop structure.

[0317] The nucleic acids can be prepared according to any available technique. For mRNA, the primary methodology for preparation is, but is not limited to, enzymatic synthesis (also called in vitro transcription) to produce long sequence-specific mRNA. In vitro transcription describes the process of template-directed synthesis of RNA molecules from an engineered DNA template consisting of an upstream bacteriophage promoter sequence (including, but not limited to, those from T7, T3, and SP6 E. coli phages) ligated to a downstream sequence encoding the gene of interest.

[0318] Transcription of RNA occurs in vitro using a linearized DNA template under conditions that support polymerase activity, in the presence of the corresponding RNA polymerase and the ribonucleoside triphosphates (rNTPs) adenosine, guanosine, uridine, and cytidine, while minimizing potential degradation of the resulting mRNA transcript. In vitro transcription can be performed using commercially available kits, including, but not limited to, the RiboMax Large Scale RNA Production System (Promega), the MegaScript Transcription Kit (Life Technologies), as well as commercially available reagents containing RNA polymerase and rNTPs. Methodologies for in vitro transcription of mRNA are well known in the art. (See, e.g., Losick, R., 1972, In vitro transcription, Ann Rev Biochem v.41 409-46; Kamakaka, R.T. and Kraus, W.L. 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology v.703 (Neilson, H. Ed), New York, N.Y. Humana Press, 2010; Brunelle, J.L. and Green, R., 2013, Chapter Five-In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v.530, 101-114, each of which is incorporated herein by reference in its entirety).

[0319] Next, the desired in vitro transcribed mRNA is purified from unwanted components of the transcription or related reactions (including unincorporated rNTPs, protein enzymes, salts, short RNA oligos, etc.). Techniques for the isolation of mRNA transcripts are well known in the art. Well-known procedures include phenol / chloroform extraction or precipitation with any alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Additional non-limiting examples of purification procedures that can be used include size exclusion chromatography (Lukaysky, P.J. and Puglisi, J.D., 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA v.10, 889-893), silica-based affinity chromatography, and polyacrylamide gel electrophoresis (Bowman, J.C., Azizi, B., Lenz, T.K., Ray, P., and Williams, L.D. in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v.941 Conn G.L. (ed), New York, N.Y. Humana Press, 2012). Purification can be performed using a variety of commercially available kits including, but not limited to, the SV Total Isolation System (Promega) and the In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0320] A variety of significant modifications have been described in the art for altering specific properties of in vitro transcribed mRNA and improving its utility. These include, but are not limited to, modifications to the 5' and 3' termini of the mRNA. Endogenous eukaryotic mRNA typically contains a cap structure at the 5' terminus of the mature molecule that plays an important role in mediating the binding of mRNA cap-binding protein (CBP), which in turn is involved in enhancing mRNA stability and the efficiency of mRNA translation in the cell. Thus, the highest levels of protein expression are achieved with capped mRNA transcripts. The 5' cap contains a 5'-5'-triphosphate bond between the most 5'-terminal nucleotide and a guanine nucleotide. Additional modifications include methylation of the most 5'-terminal and second nucleotides at the 2'-hydroxyl group.

[0321] Other components of mRNA that can be modified to provide benefits in terms of translatability and stability include the 5' and 3' untranslated regions (UTRs). Optimization of the UTRs (preferred 5′ and 3′ UTRs can be obtained from cellular or viral RNA) has been shown to increase the mRNA stability and translation efficiency of in vitro transcribed mRNA, either both or independently (see, for example, Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, P.H. Ed), 2013).

[0322] In some embodiments, the nucleic acid is a monosistronic or polycistronic RNA, and the polycistronic RNA has 2, 3, 4, 5, 6, or more different proteins or peptides, or different subunits or fragments of one or more proteins or peptides, and has 2, 3, 4, 5, 6, or more coding sequences. In some aspects, the nucleic acid is a cocktail of multiple mRNAs, each of which encodes at least one protein or peptide, or a subunit or fragment of a protein or peptide. In some aspects, the nucleic acid is a cocktail of multiple mRNAs, each of which encodes at least one reprogramming factor such as, but not limited to, OCT, SOX, KLF, Lin, Nanog, Myc, or GLis1. In some embodiments, the reprogramming factor is LIN28 or NANOG. In some aspects, the reprogramming factors are Yamanaka factors such as OCT4, SOX2, c-Myc, and KLF4. In some embodiments, the reprogramming factor is a combination of OCT4, SOX2, and c-Myc. In some embodiments, the reprogramming factor is a combination of OCT4, SOX2, KLF4, and c-Myc. In some embodiments, the reprogramming factor is a combination of OCT4, SOX2, KLF4, Lin28, Nanog, and c-Myc. In some embodiments, the reprogramming factor is a combination of OCT4, SOX2, KLF4, Lin28, Nanog, and c-Myc. In some embodiments, c-Myc in any of the above combinations is replaced by Glis1. In some embodiments, each mRNA encodes one reprogramming factor. In some embodiments, each mRNA encodes two or more reprogramming factors. In some embodiments, each mRNA encodes two reprogramming factors. In some embodiments, each mRNA encodes three reprogramming factors. In some embodiments, the nucleic acid is a single mRNA molecule encoding 2, 3, 4, 5, 6, or more reprogramming factors.In some embodiments, the nucleic acid is a single mRNA molecule encoding three reprogramming factors. In some embodiments, the nucleic acid is a single mRNA molecule encoding four reprogramming factors. In some embodiments, the nucleic acid is a single mRNA molecule encoding five reprogramming factors. In some embodiments, the nucleic acid is a single mRNA molecule encoding six reprogramming factors.

[0323] In some embodiments, the RNA is a polycistronic RNA encoding one or more reprogramming factors such as, but not limited to, OCT, SOX, KLF, Lin, Nanog, Myc, or GLis1. In some embodiments, the reprogramming factor is LIN28 or NANOG. In some aspects, the reprogramming factors are Yamanaka factors such as OCT4, SOX2, c-Myc, and KLF4. In some embodiments, the reprogramming factors are a combination of OCT4, SOX2, and c-Myc. In some embodiments, the reprogramming factors are a combination of OCT4, SOX2, KLF4, and c-Myc. In some embodiments, the reprogramming factors are a combination of OCT4, SOX2, KLF4, Lin28, Nanog, and c-Myc. In some embodiments, the reprogramming factors are a combination of OCT4, SOX2, KLF4, Lin28, Nanog, and c-Myc. In some embodiments, c-Myc in any of the above combinations is replaced by Glis1.

[0324] In some embodiments, the RNA is self-replicating RNA. Self-replicating constructs are described, for example, in U.S. Patent Publications Nos. 2018 / 0216079 and 2021 / 0108179, which are incorporated herein by reference. In some embodiments, the self-replicating RNA has an increased half-life in a mammal, such as a human. In some aspects, the self-amplifying RNA is polycistronic. In some aspects, the self-amplifying RNA is trans-amplifying RNA, and the amplifying polymerase is encoded by an RNA strand that is different from the strand or strands targeted for amplification.

[0325] In some embodiments, the RNA is a circular polynucleotide, or circular RNA. A circular polynucleotide, or circular RNA, is a polynucleotide that forms a circular structure via a covalent or non-covalent bond. In some embodiments, the circular polynucleotide is non-immunogenic in a mammal, such as a human. In some embodiments, the circular polynucleotide has an increased half-life in a mammal, such as a human. In some embodiments, the circular polynucleotide can replicate or replicates intracellularly. In some aspects, the circular RNA is polycistronic.

[0326] In some embodiments, the RNA comprises regulatory elements, such as sequences that modify the expression of the coding sequences within the RNA. The regulatory elements can include sequences that are located adjacent to the coding sequences encoding the expression products. The regulatory elements can be operably linked to the adjacent sequences. The regulatory elements can increase the amount of the expressed product as compared to the amount of the product expressed in the absence of the regulatory elements. In addition, one regulatory element can increase the amount of the products expressed from a plurality of expression sequences linked in series. Thus, one regulatory element can enhance the expression of one or more expression sequences. Multiple regulatory elements are well known to those skilled in the art. In some embodiments, the regulatory element is an IRES or a 2A element. In some embodiments, the IRES or 2A element is present upstream of the coding sequence. In some embodiments, the sequence of the IRES or 2A element is modified or optimized to achieve the desired expression profile. In some embodiments of the polycistronic RNA, each coding sequence is separately regulated by a different IRES or 2A element, i.e., each gene that is expressed has its own IRES or 2A element.

[0327] In some embodiments, one or more of the RNA molecules encode b18r, b19r, E3, K3, or other "decoy molecules" to neutralize the type I interferon gamma response to the transfected RNA, thereby blunting the cellular immune response to the transfected RNA and resulting in increased translation of the therapeutic molecules encoded by the RNA, such as the reprogramming factors described above.

[0328] In some embodiments, the RNA has a length of about 100 to about 300 nucleotides, about 300 to about 1,000 nucleotides, about 1,000 to about 3,000 nucleotides, about 3,000 to about 5,000 nucleotides, about 5,000 to about 7,000 nucleotides, about 7,000 to about 10,000 nucleotides, about 10,000 to about 13,000 nucleotides, or about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9000, 9100, 9200, 9300, 9400, 9500, 9600, 9700, 9800, 9900, 10000, 10100, 10200, 10300, 10400, 10500, 10600, 10700, 10800, 10900, 11000, 11100, 11200, 11300, 11400, 11500, 11600, 11700, 11800, 11900, 12000, 12100, 12200, 12300, 12400, 12500, 12600, 12700, 12800, 12900, 13000 base pairs.

[0329] In some embodiments, the nucleic acid (RNA or DNA) is cloned into a vector. In some aspects, the vector is an RNA vector that produces a monocistronic mRNA or a polycistronic mRNA, and the vector is linear or circular. In one embodiment, the vector is an mRNA production vector that produces mRNA by in vitro transcription of a DNA vector. The DNA vector can be monocistronic or polycistronic (having two, three, four, five, six, or more DNA sequences encoding reprogramming factors).

[0330] In some embodiments, the RNA vector is a polycistronic RNA vector. Such a polycistronic RNA vector can encode one or more reprogramming factors such as, but not limited to, OCT, SOX, KLF, Lin, Nanog, Myc, or Glis1. In some embodiments, one or more reprogramming factors include LIN28 or NANOG. In some aspects, one or more reprogramming factors include Yamanaka factors such as OCT4, SOX2, c-Myc, and KLF4. In some embodiments, the polycistronic RNA vector encodes OCT4, SOX2, and c-Myc. In some embodiments, the polycistronic RNA vector encodes OCT4, SOX2, KLF4, and c-Myc. In some embodiments, the polycistronic RNA vector encodes OCT4, SOX2, KLF4, c-Myc, LIN28, and NANOG. In any of the above embodiments, c-Myc can be replaced by Glis1.

[0331] In some embodiments, the RNA vector is a self-replicating vector. In some embodiments, the self-replicating vector has an on-switch. Self-replicating and polycistronic constructs, and those having an on / off switch are described, for example, in U.S. Patent Publication Nos. 2018 / 0216079 and 2021 / 0108179, which are incorporated herein by reference, or in U.S. Patent Application Nos. 17 / 812,709, 17 / 812,711, and 17 / 812,710, which are incorporated herein by reference.

[0332] In some embodiments, the RNA vector is a circular polynucleotide, or circular RNA. A circular polynucleotide, or circular RNA, is a polynucleotide that forms a circular structure via a covalent or non-covalent bond. In some embodiments, the circular polynucleotide is non-immunogenic in mammals, such as humans. In some embodiments, the circular polynucleotide can replicate or replicates intracellularly.

[0333] In some embodiments, the circular polynucleotide comprises a regulatory element, such as a sequence that modifies the expression of an expression sequence within the circular polynucleotide. The regulatory element can include a sequence located adjacent to an expression sequence encoding an expression product. The regulatory element can be operably linked to the adjacent sequence. The regulatory element can increase the amount of product expressed as compared to the amount of product expressed in the absence of the regulatory element. Additionally, one regulatory element can increase the amount of product expressed from a plurality of expression sequences linked in series. Thus, one regulatory element can enhance the expression of one or more expression sequences. Multiple regulatory elements are well known to those of skill in the art.

[0334] IV. Lipid-Nanoparticle Compositions The present disclosure features ionizable lipids and compositions containing the same. Such compositions can be, but are not limited to, nanoparticle compositions. The lipid-nanoparticle compositions of the present disclosure can include an ionizable lipid of any one of formulas (I)-(XI), as well as additional lipids such as helper lipids, stabilizing lipids, and / or structural lipids.

[0335] Without wishing to be bound by any theory, these lipid nanoparticles are thought to protect nucleic acids from degradation in serum and provide effective delivery of nucleic acids to cells in vitro and in vivo.

[0336] The lipid-nanoparticle compositions of the present disclosure can further include nucleic acids such as RNA as therapeutic and / or prophylactic and / or diagnostic agents for delivery to mammalian cells or organs to regulate polypeptide, protein, or gene expression.

[0337] The lipid compositions can be prepared by mixing an ionizable lipid of formulas (I)-(XIX), or a combination thereof, with a helper lipid or a combination thereof, a stabilizing lipid and / or a structural lipid or a combination thereof, in a solvent such as ethanol and water to obtain a desired molar ratio.

[0338] Helper lipids for use in the lipid-nanoparticle compositions of the present disclosure can include lipids that can assemble into one or more lipid bilayers. Helper lipids for use in the lipid-nanoparticle compositions of the present disclosure can include lipids that increase the stability or delivery efficiency of the lipid nanoparticles.

[0339] Exemplary examples of helper lipids that can also be used in the lipid-nanoparticle compositions of the present disclosure can include, but are not limited to:

Chemical formula

[0340] Other exemplary examples of helper lipids that can be used in the lipid nanoparticle compositions of the present disclosure can include, but are not limited to:

Chemical formula

[0341] Such exemplary helper lipids can be prepared using the methods described in J. Org. Chem. 1994, Vol. 59, 4805 - 4820, Org. Lett. 2005, Vol. 7, 2063 - 2065, and Tet. Lett. 1993, Vol. 34, 6881 - 6884.

[0342] In yet another embodiment, helper lipids useful in the composition can be selected from the group of phospholipids consisting of, but not limited to, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-didodecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and mixtures thereof.

[0343] Stabilizing lipids for use in lipid-nanoparticle compositions can include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG) and have an average PEG molecular weight of 2000.

[0344] The lipid component of the lipid-nanoparticle composition may include one or more structural lipids. The structural lipids can be selected from the group consisting of, but not limited to, cholesterol, fucosterol, sitosterol (including beta-sitosterol), ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipids include cholesterol and corticosteroids (such as prednisone, dexamethasone, prednisolone, and hydrocortisone), or combinations thereof.

[0345] Structural lipids can also include natural and synthetic cholesterol derivatives. Some examples of natural cholesterol derivatives include, but are not limited to, (7β-OHC, 22(R)-hydroxycholesterol (22R-OHC), 24(S)-hydroxycholesterol (24(S)-OHC)). Synthetic cholesterol derivatives include, but are not limited to, (22(R)-hydroxy-Δ9-cholestanol (22R-ISO-OHC), ((23-(4-methylfuran-2,5-dione)-3α-hydroxy-24-nor-5β-cholan (LITHO 1a), 23-(4-methylfuran-2,5-dione)-3α,7α-dihydroxy-24-nor-5β-cholan (CHENO 1b), 23-(4-methyl-1H-pyrrole-2,5-dione)-3α-hydroxy-24-nor-5β-cholan (LITOMAL 7a), 23-(4-methyl-1H-pyrrole-2,5-dione)-3α,7α,12α-trihydroxy-24-nor-5β-cholan (COLMAL 7f), and ethanol mimetic derivatives of lithocholic acid and chenodeoxycholic acid (LITOMET, CHENOMET))(146,147). The systematic name of LITOMET is (23-((2-hydroxyethyl)-4-methyl-1H-pyrrole-2,5-dione)-3α-hydroxy-24-nor-5β-cholan), and the systematic name of CHENOMET is (23-((2-hydroxyethyl)-4-methyl-1H-pyrrole-2,5-dione)-3α,7α-dihydroxy-24-nor-5β-cholan).

[0346] The lipid-nanoparticle compositions of the present disclosure include nucleic acids such as, but not limited to, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), miRNA inhibitors (antagomir / antimir), dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), multivalent RNA, and mixtures thereof.

[0347] The nucleic acid or mRNA can be self-amplifying RNA, polycistronic RNA, self-amplifying polycistronic RNA, or circular RNA. In some embodiments, the nucleic acid or mRNA expresses a protein or peptide. In some embodiments, the protein or peptide expressed from the nucleic acid or mRNA is an antibody, human antibody, camelid antibody, nanobody, humanized antibody, bispecific antibody, enzyme, genome editing enzyme or nuclease, growth factor, cytokine, chemokine, small molecule mimetic peptide, transcription factor, structural molecule, signaling molecule, reprogramming factor, vaccine antigen, or a combination thereof. In some embodiments, the mRNA encodes a protein or peptide that acts intracellularly. In some embodiments, the mRNA encodes at least one reprogramming factor.

[0348] The protein or peptide expressed from the nucleic acid or mRNA of the present technology can be at least one extracellular matrix protein such as collagen, laminin, elastin, fibronectin, integrin, tenascin, proteoglycan, fibrin, or a combination thereof. Collagen can be collagen I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XXII, XXIII, XXIV, XXV, XXVI, XXVII, XXVIII, or a combination thereof. In some embodiments, the collagen is collagen VII. In some embodiments, collagen VII is used in a method of rejuvenating, treating, remodeling, or improving the skin or extracellular matrix. In some embodiments, collagen VII is used in a method of wound healing.

[0349] The proteins or peptides expressed from the nucleic acids or mRNAs of the present technology can also be growth factors, cytokines, or combinations thereof, such as EGF, FGF, NGF, CNTF, PDGF, VEGF, IGF, GMCSF, GCSF, TGF, erythropoietin, ephrin, GDNF, GDF9, KGF, angiopoietin, TPO, BMP, HGF, BDNF, GDF, HGH (somatotropin), neurotrophin, MSF, SGF, GDF (including GDF11), TGF (including TGF-β), or combinations thereof. In some embodiments, the cytokine is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, TNF-α, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, CXCL8 (formerly IL-18), IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, or combinations thereof.

[0350] The protein or peptide expressed from the nucleic acid or mRNA of the present technology can be a human antibody, a humanized antibody, a camelid antibody, a companion animal antibody, or a nanobody. In some embodiments, the protein or peptide expressed from the nucleic acid or mRNA is an enzyme such as a nuclease, for example, a nuclease used in genome editing. In some aspects, the protein or peptide expressed from the nucleic acid or mRNA acts intracellularly. In some aspects, the protein or peptide expressed from the nucleic acid or mRNA is secreted. In some embodiments, the antibody is trastuzumab, golimumab, miliximab, mirvetuximab, nirsevimab, tremelimumab, teclistamab, donanemab, spesolimab, lecanemab, tislelizumab, pemtumumab, sintilimab, teprotumumab, tripalimumab, omburtamab, retifanlimab, ublituximab, inolimomab, oportuzumab, narsoplimab, mosunetuzumab, tixagevimab, cilgavimab, relatorlimab, tebentafusp, faricimab, stimulimab, sotrovimab, leguvimab, casirivimab, imdevimab, tezepelumab, chisotuzumab, amivantamab, anifrolumab, loncastuximab, bimekizumab, tralokinumab, evinacumab, sacituzumab, teprotumumab, isatuximab, eptinezumab, dostarlimab, ansuvimab, margetuximab, naxitamab, atorlivimab, mafutivimal, odesivimab, belantamab, tafasitamab, satralizumab, inebilizumab, enfuvirtide, crisantaspase, polatuzumab, lisankizumab, romosozumab, caplacizumab, labralizumab, emapalumab, semiprimab, fremantamab, moxetumomab, galcanezumab, ranalizumab, mogamulizumab, elenumab, tiludakizumab, ibalizumab, brosimumab, durvalumab, emicizumab, benralizumab, ocrelizumab, guselkumab, inotuzumab, sartalizumab, dupilumab, abemaciclib, brodalumab, atezolizumab, bezlotoxumab, orlatumumab, reslizumab, obinutuzumab, ixekizumab, daratumumab, elotuzumab, nesvacumab, idarucizumab, alirocumab, mepolizumab, evolocumab, dinutuximab, secukinumab,It is at least one of nivolumab, blinatumomab, pembrolizumab, ramucirumab, vedolizumab, siltuximab, obinutuzumab, lirilumab, pertuzumab, brentuximab, belimumab, ipilimumab, denosumab, tocilizumab, ofatumumab, canakinumab, golimumab, ustekinumab, certolizumab, catumaxomab, eculizumab, ranibizumab, panitumumab, natalizumab, bevacizumab, cetuximab, efalizumab, omalizumab, tositumomab, ibritumomab, adalimumab, alemtuzumab, gemtuzumab, infliximab, palivizumab, basiliximab, daclizumab, rituximab, abciximab, edrecolomab, nebacumab, or muromonab, or is substantially similar thereto. In some embodiments, the protein or peptide is used in a method of treating a human or veterinary disease.

[0351] The lipid-nanoparticle compositions of the present disclosure can be prepared by a mixing process such as microfluidics and T-junction mixing of two fluid streams, one of which contains nucleic acid and the other has a lipid component, but is not limited thereto. Such a mixing process induces nanoprecipitation and particle formation.

[0352] The lipid-nanoparticle compositions of the present disclosure can be characterized using a zetasizer to determine particle size, polydispersity index (PDI), and zeta potential. In some embodiments, the zeta potential of the lipid-nanoparticle composition can be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0353] The concentration of nucleic acids in the nanoparticle composition can be determined using ultraviolet-visible spectroscopy.

[0354] The lipid-nanoparticle composition can induce the expression of a desired protein both in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more of the ionizable lipids described herein, and the lipid nanoparticles are encapsulated with or associated with a nucleic acid (e.g., mRNA) that expresses to produce the desired protein.

[0355] The lipid-nanoparticle composition can reduce the expression of a target gene and protein both in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more of the ionizable lipids described herein, and the lipid nanoparticles are encapsulated with or associated with a nucleic acid (e.g., siRNA) that reduces target gene expression.

[0356] The lipid-nanoparticle composition can downregulate or silence the expression of a target gene and protein both in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more of the ionizable lipids described herein, and the lipid nanoparticles are encapsulated with or associated with a nucleic acid that downregulates or silences target gene expression.

[0357] The methods and compositions provided herein are applicable to cells, tissues, or organs of the nervous system, muscular system, respiratory system, cardiovascular system, skeletal system, genital system, integumentary system, lymphatic system, excretory system, immune system, endocrine system (e.g., endocrine and exocrine), or digestive system. As described herein, any type of cell can potentially be rejuvenated, including, but not limited to, epithelial cells (e.g., squamous epithelial cells, cuboidal epithelial cells, columnar epithelial cells, and pseudostratified epithelial cells), endothelial cells (e.g., venous endothelial cells, arterial endothelial cells, and lymphatic endothelial cells), and cells of connective tissue, muscle, and the nervous system. Such cells include, but are not limited to, epidermal cells, fibroblasts, chondrocytes, skeletal muscle cells, satellite cells, cardiomyocytes, smooth muscle cells, keratinocytes, basal cells, ameloblasts, exocrine cells, myoepithelial cells, osteoblasts, osteoclasts, neurons (e.g., sensory neurons, motor neurons, and interneurons), glial cells (e.g., oligodendrocytes, astrocytes, ependymal cells, microglia, Schwann cells, and satellite cells), columnar cells, adipocytes, pericytes, stellate cells, lung cells, blood and immune system cells (e.g., erythrocytes, monocytes, dendritic cells, macrophages, neutrophils, eosinophils, mast cells, T cells, B cells, natural killer cells), hormone-secreting cells, germ cells, stromal cells, lens cells, photoreceptor cells, taste receptor cells, and olfactory cells; and cells and / or tissues from the kidney, liver, pancreas, stomach, spleen, gallbladder, intestine, bladder, lung, prostate, breast, urogenital tract, pituitary cells, oral cavity, esophagus, skin, hair, nails, thyroid, parathyroid, adrenal, eye, nose, or brain may be included, but are not limited to these.

[0358] Cells that can be treated according to the present technique can be selected from fibroblasts, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells. In embodiments, the cells are fibroblasts. In embodiments, the cells are endothelial cells. In embodiments, the cells are chondrocytes. In embodiments, the cells are skeletal muscle stem cells. In embodiments, the cells are keratinocytes. In embodiments, the cells are mesenchymal stem cells. In embodiments, the cells are corneal epithelial cells.

[0359] The methods and compositions of the present technology can also be applied to immune cells including, but not limited to, lymphocytes, granulocytes, monocytes, macrophages, microglia, or dendritic cells. In some embodiments, the lymphocytes are T cells, B cells, or natural killer (NK) cells. In some embodiments, the lymphocytes are tumor-infiltrating lymphocytes.

[0360] The methods and compositions of the present technology can also be applied to lymphocytes in which the lymphocytes are T cells. In some embodiments, the T cells are cytotoxic T cells (CD8+), helper T cells (CD4+), suppressor or regulatory T cells (Tregs), memory T cells, natural killer T cells (NKT cells), or gamma delta T cells. In other embodiments, the helper T cells are Th1, Th2, Th17, Th9, or Tfh T cells. In some embodiments, the memory T cells are central memory T cells, effector memory T cells, tissue-resident memory T cells, or virtual memory T cells. In some embodiments, the suppressor or regulatory T cells of the present technology are FOXP3+ T cells or FOXP3- T cells. In some embodiments, the NKT cells are a subset of CD1d-restricted T cells.

[0361] The methods and compositions of the present technology can also be applied to granulocytes in which the granulocytes are neutrophils, eosinophils, basophils, or mast cells.

[0362] The methods and compositions of the present technology can also be applied to lymphocytes in which the lymphocytes are B cells such as memory B cells or plasma cells.

[0363] The methods and compositions of the present technology can also be applied to immune cells in which the immune cells are monocytes, macrophages, microglia cells, or dendritic cells.

[0364] The methods and compositions described herein can be used where the cell is an immune cell such as a natural immune cell or an engineered immune cell. In some embodiments, the methods and compositions described herein are used in parallel with, or sequentially to, a method of engineering a cell, including an engineered immune cell, such that the method is performed before, during, and / or after the engineering of the cell. In some embodiments, the methods and compositions described herein are used to engineer a cell, including an engineered immune cell. In some embodiments, such engineering includes engineering the cell to express a chimeric antigen receptor such as an immune cell that expresses a chimeric antigen. In some embodiments, such chimeric antigen receptor targets at least one of CD19, CD20, CD22, CD30, CD33, CD123, FLT3, BCMA, GD2, HER2, MUC1, B7-H3, IL13Ra2, TAG72, MUC16, BCMA, or any other antigen suitable for immunotherapy. In some embodiments, such engineering includes engineering a cell including an immune cell to express other proteins or peptides such as growth factors and cytokines. In some embodiments, the cytokine includes IL-15. In some embodiments, such engineering of a cell such as an immune cell is performed ex vivo, for example, in the manufacture of a cell therapy product such as an autologous or allogeneic chimeric antigen receptor (CAR)-T, CAR-NK, CAR-M, or CAR-NKT cell. In some embodiments, the CAR-T cells provided herein target at least one of CD19, CD20, CD22, HER2, MUC1, CD30, CD33, CD123, FLT3, B7-H3, IL13Ra2, GD2, TAG72, MUC16, or BCMA by a chimeric antigen receptor. In some embodiments, the CAR-T cells provided herein target at least one of CD19 or BCMA by a chimeric antigen receptor. In some embodiments, the CAR-T cells provided herein target CD19 by a chimeric antigen receptor. In some embodiments, the CAR-T cells provided herein target BCMA by a chimeric antigen receptor.In some embodiments, the CAR-NK cells provided herein are targeted to at least one of CD19, FLT3, CD20, CD38, CD138, BCMS, CS1, CD3, CD5, CD7, NKG2D, HER2, EGFR, EpCAM, TF, B7-H6, HLA-G, CD24, CD44, CD133, mesothelin, or alphaFR by a chimeric antigen receptor. In some embodiments, the CAR-M cells provided herein are targeted to at least one of CD19, HER2, CD22, or ALK19 by a chimeric antigen receptor. In some embodiments, the CAR-NK cells provided herein are engineered to express IL-2 and / or IL-15. In some embodiments, the CAR-NKT cells provided herein are targeted to GD2 by a chimeric antigen receptor and engineered to express IL-15. In some embodiments, the immune cell rejuvenation method described herein is performed ex vivo during or after the manufacture of a cell therapy product. In other embodiments, such manipulation of cells and / or immune cells is performed ex vivo, for example, in the so-called "in situ" generation of CAR-engineered cells. In such embodiments, the CAR or RNA and / or mRNA encoding a growth factor or cytokine contained in the lipid-containing composition or lipid-nanoparticle composition of the present disclosure is injected in vivo into a subject or patient for CAR engineering in the body of the patient without the need to remove cells for ex vivo transfection, for example, in the "in situ", i.e., ex vivo, of immune cells of the patient such as T cells, NK cells, macrophages, tumor-infiltrating lymphocytes, dendritic cells, and / or NKT cells. In such embodiments, the immune cell rejuvenation method described herein is also performed in vivo, and the mRNA encoding a reprogramming factor or factors is injected into the patient before, simultaneously with, or after the mRNA encoding a CAR or other cell manipulation molecule. In some embodiments, the lipids and lipid-nanoparticles of the present disclosure are selected for in vivo targeted delivery to any cell, including immune cells such as T cells, NK cells, macrophages, tumor-infiltrating cells, dendritic cells, and / or NKT cells.In yet other embodiments, the in vivo treatment is performed in the absence of any other in vivo cell manipulation to enhance or restore the efficacy of the immune system, improve the effect of the immune system against cancer or infection, or treat diseases associated with immune dysfunction or dysregulation such as reducing inflammation.

[0365] The methods and compositions of the present technology can also be applied to the rejuvenation of immune cells where the rejuvenating immune cells are non-adherent cells such as non-adherent immune cells. In some embodiments, non-adherent cells, including non-adherent immune cells, are processed, transiently reprogrammed, rejuvenated, or manufactured by means such that the cells remain non-adherent without adhering to a tissue culture substrate or forming or giving rise to cells or colonies of cells that adhere to the tissue culture substrate. In some embodiments, the reprogramming intervals and factors are selected such that the cells remain non-adherent without adhering to a tissue culture substrate or forming or giving rise to cells or colonies of cells that adhere to the tissue culture substrate and the cells rejuvenate with retention of cell identity. Thus, in some embodiments, the present technology provides lipid-containing compositions and lipid-nanoparticle compositions used to deliver mRNA encoding at least one reprogramming factor for cell rejuvenation, and cells comprising any non-adherent cells and / or non-adherent immune cells (e.g., non-adherent T cells, NK cells, macrophages, tumor infiltrating cells, dendritic cells, and / or NKT cells) are reprogrammed by means such that the cells rejuvenate with retention of cell identity, the cells remain in suspension and are non-adherent, and they do not become or give rise to adherent cells or form adherent colonies.

[0366] The methods described herein, including a method of rejuvenating immune cells, a method of reversing, preventing, or inhibiting exhaustion in immune cells, or a method of inducing proliferation in immune cells, comprise administering a lipid-containing composition or a lipid-nanoparticle composition of the present disclosure, which comprises providing to the immune cells mRNA encoding at least one reprogramming factor about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times over a period of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. For example, the mRNA can be administered once on the first or second day of a 5- or 6-day period, or it can be administered once on the first and third days of a 5- or 6-day period, or it can be administered once in a 1-day period. In some embodiments, the mRNA is administered to the immune cells 1, 2, 3, 4, 5, or 6 times over a period of 1, 2, 3, 4, 5, or 6 days. In some embodiments, the mRNA is administered after an immune cell activation step. In some embodiments, the immune cell activation step comprises activating the immune cells for 1, 2, or 3 days. In some embodiments, the immune cell activation step comprises activating the immune cells using at least one of CD3, CD28, and IL-2. In some embodiments, the immune cells are activated with CD3 and CD28. In some embodiments, the mRNA administration period occurs immediately after the immune cell activation step. In some embodiments, the mRNA administration period occurs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the immune cell activation step. In some embodiments, the administration of the mRNA encoding the reprogramming factor reverses immune cell exhaustion caused by the immune cell activation step. In some embodiments, the administration of the mRNA encoding the reprogramming factor reverses immune cell exhaustion in immune cells from an elderly patient or donor. In some embodiments, the administration of the mRNA is performed during a manufacturing process to produce immune cells for transplantation, such as CAR-T, CAR-M, or CAR-NK cells.

[0367] The use of the lipids or lipid nanoparticles of the present technology for the delivery of mRNA provides enhanced rejuvenation, proliferation, recovery from or prevention of exhaustion, therapeutic effect, anti-pathogenic effect, anti-cancer effect, anti-immunogenic effect, or anti-inflammatory effect in cells that are treated or rejuvenated using the methods and compositions herein, as compared to using different delivery mechanisms for mRNA. In some embodiments, such enhanced rejuvenation, proliferation, recovery from or prevention of exhaustion, therapeutic effect, anti-pathogenic effect, anti-cancer effect, anti-immunogenic effect, or anti-inflammatory effect is due to lower toxicity, immunogenicity, and / or lower physiological impact on the cells as compared to different delivery mechanisms. In some embodiments, the different delivery mechanism is electroporation, and the use of the lipids or lipid-nanoparticle compositions of the present disclosure for the delivery of mRNA results in enhanced rejuvenation, proliferation, recovery from or prevention of exhaustion, therapeutic effect, anti-pathogenic effect, anti-cancer effect, anti-immunogenic effect, or anti-inflammatory effect in the treated or rejuvenated cells as compared to using electroporation. This improvement as compared to electroporation may be due to reduced toxicity or reduced physiological impact on the cells as compared to electroporation.

[0368] The lipid or lipid-nanoparticle compositions of the present technology can be used in methods for delivering a therapeutic or diagnostic agent to the skin, such as by administering a lipid-containing composition or lipid-nanoparticle composition comprising at least one therapeutic or diagnostic agent. In some embodiments, the lipid or lipid-nanoparticle compositions of the present technology provide delivery of a therapeutic or diagnostic agent, such as a reprogramming factor, as a means of achieving transient reprogramming of cells such as skin cells or immune cells. In some embodiments, transient reprogramming of the cells provides transient expression of a therapeutic or diagnostic agent such as a reprogramming factor, and the agent is expressed in the cells for a period sufficient to reprogram and / or rejuvenate without changing the identity of the cells, i.e., to rejuvenate the skin or immune cells while maintaining the identity of the skin or immune cells to exhibit characteristics or younger skin cells or immune cells.

[0369] The therapeutic agent of the present technology is, in some cases, the mRNA disclosed herein. In some embodiments, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for treating or preventing a dermatological disease or condition, treating or preventing a disease or condition of the skin, or for cosmetic application on the skin, and comprises administering a lipid-containing composition or lipid-nanoparticle composition comprising at least one therapeutic or diagnostic agent. In some embodiments, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for rejuvenating the skin and comprises administering a lipid-containing composition or lipid-nanoparticle composition comprising a therapeutic or diagnostic agent. In some embodiments, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for rejuvenating the skin and comprises administering a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor. In such methods, the mRNA can be bound to the lipid or contained within the lipid-nanoparticle. In some embodiments, such methods further comprise transfecting skin cells with the lipid-containing composition or lipid-nanoparticle composition to deliver the mRNA. In some embodiments, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for rejuvenating the skin and comprises administering a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor to achieve skin rejuvenation while maintaining cell identity. In such methods, the mRNA can be bound to the lipid or contained within the lipid-nanoparticle. In some embodiments, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for rejuvenating the skin and comprises administering to skin cells a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor, wherein expression of at least one reprogramming factor in the skin cells results in an increase in fibroblast proliferation while maintaining skin cell identity. In such methods, the mRNA can be bound to the lipid or contained within the lipid-nanoparticle.

[0370] A lipid or lipid-nanoparticle composition can be used in a method of wound healing and involves administering a lipid-containing composition or lipid-nanoparticle composition that contains a therapeutic or diagnostic agent. In some embodiments, the lipid or lipid-nanoparticle composition is used in a method of wound healing and involves administering a lipid-containing composition or lipid-nanoparticle composition that contains mRNA encoding at least one reprogramming factor. In such methods, the mRNA can be bound to the lipid or contained within the lipid-nanoparticle. In some embodiments, the lipid or lipid-nanoparticle composition is used in a method of wound healing and the lipid-containing composition or lipid-nanoparticle composition delivers mRNA encoding at least one reprogramming factor to achieve wound healing while maintaining cell identity. In such methods, the mRNA can be bound to the lipid or contained within the lipid-nanoparticle. In some embodiments, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method of wound healing and involves administering a lipid-containing composition or lipid-nanoparticle composition that contains mRNA encoding at least one reprogramming factor to skin cells, and the expression of at least one reprogramming factor in the skin cells results in an increase in fibroblast proliferation while maintaining skin cell identity. In such methods, the mRNA can be bound to the lipid or contained within the lipid-nanoparticle.

[0371] A lipid or lipid-nanoparticle composition can be used in a method for treating or preventing a dermatological disease or condition, treating or preventing a disease or condition of the skin, or for cosmetic application on the skin, and includes administering a lipid-containing composition or a lipid-nanoparticle composition comprising a therapeutic agent to achieve reversal of at least one skin aging marker. In some embodiments, the lipid or lipid-nanoparticle composition is used in a method of rejuvenating the skin and includes administering a lipid-containing composition or a lipid-nanoparticle composition to deliver to skin cells an mRNA encoding at least one reprogramming factor to achieve reversal of at least one skin aging marker while maintaining cell identity. In some embodiments, the lipid or lipid-nanoparticle composition is used in a method of wound healing and includes administering a lipid-containing composition or a lipid-nanoparticle composition to deliver to skin cells an mRNA encoding at least one reprogramming factor to achieve reversal of at least one skin aging marker while maintaining cell identity. In some embodiments, reversal of at least one skin aging marker refers to producing rejuvenated cells that express at least one skin aging marker by means similar to the expression of that marker observed in youthful skin cells as compared to aged skin cells.

[0372] Markers that can be affected according to the present technology include mRNA or protein expression of IL6, CXCL8, CSF3, CXCL1, SERPINB2, LIF, IL11, CXCL2, IL24, PTGS2, MMP3, CCL2, TFPI2, IER3, ACKR3, PTGES, SLC16A6, TNFAIP6, PTPRN, IL1RN, IL1B, CXCL5, CXCL6, HAS1, HSD11B1, CH25H, ADGRD1, C3, RASD1, NR4A3, STC1, TCIM, SRGN, AC003092.1, LRRN3, CHI3L1, NR4A2, NAMPT, PRSS23, MMP1, SOD2, LOXL4, MMP11, ELN, CREG1, C15orf48, NFKBIZ, PID1, or any combination thereof. In some embodiments, the reversal of at least one skin aging marker is a down - regulation of mRNA or protein expression of IL6, CXCL8, CSF3, CXCL1, SERPINB2, LIF, IL11, CXCL2, IL24, PTGS2, MMP3, CCL2, TFPI2, IER3, ACKR3, PTGES, SLC16A6, TNFAIP6, PTPRN, IL1RN, IL1B, CXCL5, CXCL6, HAS1, HSD11B1, CH25H, ADGRD1, C3, RASD1, NR4A3, STC1, TCIM, SRGN, AC003092.1, LRRN3, CHI3L1, NR4A2, NAMPT, MMP1, SOD2, CREG1, C15orf48, NFKBIZ, PID1, or any combination thereof. In some embodiments, the reversal of at least one skin aging marker is an up - regulation of mRNA or protein expression of PRSS23, LOXL4, MMP11, ELN, or any combination thereof. In some embodiments, the reversal of at least one skin aging marker is an up - regulation of mRNA or protein expression of PRSS23. In some embodiments, the reversal of at least one skin aging marker is an up - regulation of mRNA or protein expression of LOXL4. In some embodiments, the reversal of at least one skin aging marker is an up - regulation of mRNA or protein expression of MMP11. In some embodiments, the reversal of at least one skin aging marker is an up - regulation of mRNA or protein expression of ELN.In some embodiments, reversal of at least one skin aging marker is downregulation of mRNA or protein expression of MMP3, MMP1, SOD2, or any combination thereof. In some embodiments, reversal of at least one skin aging marker is downregulation of mRNA or protein expression of MMP3. In some embodiments, reversal of at least one skin aging marker is downregulation of mRNA or protein expression of MMP1. In some embodiments, reversal of at least one skin aging marker is downregulation of mRNA or protein expression of SOD2. In some embodiments, reversal of at least one skin aging marker is upregulation of mRNA or protein expression of at least one of PRSS23, LOXL4, MMP11, or ELN; downregulation of mRNA or protein expression of at least one of MMP3, MMP1, SOD2; or any combination thereof.

[0373] A lipid or lipid-nanoparticle composition can be used in a method for treating or preventing a dermatological disease or condition, treating or preventing a disease or condition of the skin, or for cosmetic application to the skin, and includes administering a lipid-containing composition or lipid-nanoparticle composition comprising a therapeutic agent to achieve improvement of at least one skin quality marker. In some embodiments, the lipid or lipid-nanoparticle composition is used in a method for rejuvenating the skin and includes administering a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor to skin cells to achieve improvement of at least one skin quality marker. In some embodiments, the lipid or lipid-nanoparticle composition is used in a method for rejuvenating the skin and includes administering a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor to skin cells to achieve improvement of at least one skin quality marker while maintaining cell identity.

[0374] A lipid or lipid-nanoparticle composition can be used in a method of wound healing, comprising administering a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor to skin cells to achieve an improvement in at least one skin quality marker. In some embodiments, the marker is mRNA or protein expression of type I collagen, type III collagen, type V collagen, type VI collagen, type XI collagen, elastin, microfibril-associated protein 5, periostin, versican, connective tissue growth factor, lysyl oxidase, SPARC, secreted phosphoprotein 1, cartilage oligomeric matrix protein, MMP1, MMP3, MMP12, SOD2, or any combination thereof. In some embodiments, the improvement in at least one skin quality marker is a downregulation of mRNA or protein expression of MMP1, MMP3, MMP12, SOD2, or any combination thereof. In some embodiments, the improvement in at least one skin quality marker is an upregulation of mRNA or protein expression of type I collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type XI collagen, elastin, microfibril-associated protein 5, periostin, versican, connective tissue growth factor, lysyl oxidase, SPARC, secreted phosphoprotein 1, cartilage oligomeric matrix protein, or any combination thereof. In some embodiments, the improvement in at least one skin quality marker is an upregulation of mRNA or protein expression of collagen. In some embodiments, the improvement in at least one skin quality marker is an upregulation of mRNA or protein expression of type I collagen. In some embodiments, the improvement in at least one skin quality marker is an upregulation of mRNA or protein expression of type III collagen. In some embodiments, the improvement in at least one skin quality marker is an upregulation of mRNA or protein expression of type IV collagen. In some embodiments, the improvement in at least one skin quality marker is an upregulation of mRNA or protein expression of type V collagen. In some embodiments, the improvement in at least one skin quality marker is an upregulation of mRNA or protein expression of type VI collagen.In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of type XI collagen. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of type XI collagen. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of elastin. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of microfibril-associated protein 5. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of periostin. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of versican. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of connective tissue growth factor. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of lysyl oxidase. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of SPARC. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of secreted phosphoprotein 1. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of cartilage oligomeric matrix protein. In some embodiments, improvement of at least one skin quality marker is downregulation of the mRNA or protein expression of MMP3, MMP1, SOD2, or any combination thereof. In some embodiments, improvement of at least one skin quality marker is downregulation of the mRNA or protein expression of MMP3. In some embodiments, improvement of at least one skin quality marker is downregulation of the mRNA or protein expression of MMP1. In some embodiments, improvement of at least one skin quality marker is downregulation of the mRNA or protein expression of SOD2. In some embodiments, improvement of at least one skin quality marker is upregulation of the mRNA or protein expression of collagen VII and elastin.In some embodiments, improvement of at least one skin quality marker is an upregulation of at least one of the mRNAs or protein expressions of collagen VII and elastin; a downregulation of at least one of the mRNAs or protein expressions of MMP3, MMP1, and SOD2; or any combination thereof.

[0375] The lipid-nanoparticle composition or lipid-containing composition of the present disclosure can be topically administered to the skin. In some embodiments, the lipid-nanoparticle composition of the present disclosure or the composition containing the lipid of the present disclosure is administered to the skin in an ointment, cream, or plaster. In some embodiments, the lipid-nanoparticle composition of the present disclosure or the composition containing the lipid of the present disclosure is administered to the skin via intradermal or subcutaneous injection. In some embodiments, the lipid-nanoparticle composition of the present disclosure or the composition containing the lipid of the present disclosure is administered to the skin via a gel. In some embodiments, the lipid-nanoparticle composition of the present disclosure or the composition containing the lipid of the present disclosure is administered in vivo, in vitro, or ex vivo. In some embodiments, the lipid-nanoparticle composition of the present disclosure or the composition containing the lipid of the present disclosure is administered in vivo. In some embodiments, the lipid-nanoparticle composition of the present disclosure or the composition containing the lipid of the present disclosure is for human or animal subjects.

[0376] The lipid-nanoparticle composition or lipid-containing composition of the present disclosure can transfect skin cells to deliver at least one therapeutic or diagnostic agent to the skin cells. In some embodiments, the therapeutic agent is a nucleic acid. In some embodiments, the therapeutic agent is mRNA. In some embodiments, the therapeutic agent is a combination of mRNA and siRNA. In some embodiments, the therapeutic agent is a combination of mRNA and miRNA. In some embodiments, the skin cells are keratinocytes, melanocytes, Langerhans cells, follicular cells, fibroblasts, endothelial cells, smooth muscle cells, Merkel cells, basal cells, squamous epithelial cells, apocrine gland cells, eccrine gland cells, sebaceous gland cells, lymphatic endothelial cells, or a combination thereof. In some embodiments, the lipid or lipid-nanoparticle composition is selected to provide selective transfection to a particular cell type or cell types. In some embodiments, the lipid or lipid-nanoparticle composition is selected to provide diffusion within the skin or within at least one layer of the skin.

[0377] Diseases or conditions that can be treated or prevented include dermatological diseases or conditions, or skin diseases or conditions of the skin that can be treated or prevented using the lipid compositions or lipid-nanoparticle compositions of the present technology. For example, skin laxity or chronic wounds can be treated according to the present technology. In some embodiments, skin laxity or chronic wounds are diabetic, ischemic, and pressure ulcers. In some embodiments, the dermatological diseases or conditions, or skin diseases or conditions of the skin that are treated or prevented using the lipid compositions or lipid-nanoparticle compositions of the present disclosure are inflammatory skin diseases. In some embodiments, the inflammatory skin disease is psoriasis, atopic dermatitis, vitiligo, alopecia areata, or hidradenitis suppurativa. In some embodiments, the dermatological diseases or conditions, or skin diseases or conditions of the skin that are treated or prevented using the lipid compositions or lipid-nanoparticle compositions of the present disclosure are hair disorders. In some embodiments, the hair disorder is non-scarring or scarring alopecia, hair graying, hirsutism. In some embodiments, the hair disorder is non-scarring alopecia which is androgenetic alopecia. In some embodiments, the scarring alopecia is lichen planopilaris. In some embodiments, the dermatological diseases or conditions, or skin diseases or conditions of the skin that are treated or prevented using the lipid compositions or lipid-nanoparticle compositions of the present disclosure are skin cancers. In some embodiments, the skin cancer is basal cell carcinoma, squamous cell carcinoma, or actinic keratosis. In some embodiments, the dermatological diseases or conditions, or skin diseases or conditions of the skin that are treated or prevented using the lipid compositions or lipid-nanoparticle compositions of the present disclosure are prurigo nodularis, acne, rosacea, or solar lentigines. In some embodiments, a method of treating any of the above dermatological diseases or conditions or diseases comprises administering a lipid-containing composition of the present disclosure or a lipid-nanoparticle composition of the present disclosure comprising a therapeutic agent. In some embodiments, the therapeutic agent is mRNA. In some embodiments, the therapeutic agent is an antibody, a human antibody, a humanized antibody, a nanobody, a camelid antibody, a bispecific antibody, an enzyme, a genome editing enzyme or nuclease, a growth factor, a cytokine, a chemokine, a transcription factor, a structural molecule, a signaling molecule, an mRNA encoding a reprogramming factor. In some embodiments, the therapeutic agent is an mRNA encoding a protein or peptide that acts intracellularly.In some embodiments, the therapeutic agent is an mRNA encoding at least one reprogramming factor.

[0378] The lipid composition or lipid-nanoparticle composition can be used in a method of wound healing where the wound is a skin laxity or a chronic wound. In some embodiments, the skin laxity or chronic wound is a diabetic, ischemic, and pressure ulcer. In some embodiments, the lipid composition or lipid-nanoparticle composition of the present disclosure is used in a method of wound healing where the wound is a lesion from skin cancer. In some embodiments, the skin cancer is basal cell carcinoma, squamous cell carcinoma, or actinic keratosis. In some embodiments, any of the above wound healing methods comprises administering a composition containing the lipid of the present disclosure or a lipid-nanoparticle composition comprising a therapeutic agent. In some embodiments, the therapeutic agent is an mRNA. In some embodiments, the therapeutic agent is an antibody, a human antibody, a humanized antibody, a nanobody, a camelid antibody, a bispecific antibody, an enzyme, a genome editing enzyme or nuclease, a growth factor, a cytokine, a chemokine, a transcription factor, a structural molecule, a signaling molecule, an mRNA encoding a reprogramming factor. In some embodiments, the therapeutic agent is an mRNA encoding a protein or peptide that acts intracellularly. In some embodiments, the therapeutic agent is an mRNA encoding at least one reprogramming factor.

[0379] The lipid-nanoparticle composition can contain a lipid of formula (II) and has a higher transfection efficiency in skin cells compared to other lipid-nanoparticle compositions containing a lipid of formula (II) or other lipid-nanoparticle compositions containing other lipids described herein. Such a composition containing a lipid of formula (II), when used to transfect at least one reprogramming factor, can result in a greater improvement in at least one muscle marker compared to other formulations or compared to lipid-nanoparticle compositions containing other lipids described herein.

[0380] In embodiments, a lipid-nanoparticle composition containing lipid formula (I-B) provides a higher transfection efficiency compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, a lipid-nanoparticle composition containing lipid formula (I-B) provides a higher transfection efficiency compared to lipid-nanoparticle compositions containing lipid formula (I) having the same head group. In embodiments, a lipid-nanoparticle composition containing lipid formula (I-B) provides a higher transfection efficiency in skin cells compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, a lipid-nanoparticle composition containing lipid formula (I-B) provides a higher transfection efficiency in skin cells compared to lipid-nanoparticle compositions containing lipid formula (I) having the same head group. In embodiments, a lipid-nanoparticle composition containing lipid formula (I-B) provides a higher transfection efficiency in fibroblasts compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, a lipid-nanoparticle composition containing lipid formula (I-B) provides a higher transfection efficiency in fibroblasts compared to lipid-nanoparticle compositions containing lipid formula (I) having the same head group. In embodiments, a lipid-nanoparticle composition containing lipid formula (I-B) provides a higher transfection efficiency in immune cells compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, a lipid-nanoparticle composition containing lipid formula (I-B) provides a higher transfection efficiency in immune cells compared to lipid-nanoparticle compositions containing lipid formula (I) having the same head group. In embodiments, a lipid-nanoparticle composition containing lipid formula (I-B) provides a higher transfection efficiency in T cells compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, a lipid-nanoparticle composition containing lipid formula (I-B) provides a higher transfection efficiency in T cells compared to lipid-nanoparticle compositions containing lipid formula (I) having the same head group.In embodiments, the tail structure of lipid formula (I-B) provides increased transfection efficiency for lipid-nanoparticle compositions. In embodiments, the tail structure of lipid formula (I-B) provides increased transfection efficiency as compared to an unbranched alkyl or alkenyl tail structure in a lipid-nanoparticle composition. In embodiments, the tail structure of lipid formula (I-B) provides increased transfection efficiency for lipid-nanoparticle compositions as compared to the tail structure of lipid formula (I) having the same head group. In embodiments, the ester group adjacent to the branch of the alkyl group in the tail structure of the lipids described herein provides increased transfection efficiency for lipid-nanoparticle compositions. In embodiments, the ester group adjacent to the branch of the alkyl group in the tail structure of the lipids described herein provides increased transfection efficiency for lipid-nanoparticle compositions as compared to an alkyl or alkenyl tail structure having no branch.

[0381] In embodiments, a lipid-nanoparticle composition containing lipid formula (I-B) provides a higher survival rate compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, a lipid-nanoparticle composition containing lipid formula (I-B) provides a higher survival rate compared to a lipid-nanoparticle composition containing lipid formula (I) having the same head group. In embodiments, a lipid-nanoparticle composition containing lipid formula (I-B) provides a higher survival rate in skin cells compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, a lipid-nanoparticle composition containing lipid formula (I-B) provides a higher survival rate in skin cells compared to a lipid-nanoparticle composition containing lipid formula (I) having the same head group. In embodiments, a lipid-nanoparticle composition containing lipid formula (I-B) provides a higher survival rate in fibroblasts compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, a lipid-nanoparticle composition containing lipid formula (I-B) provides a higher survival rate in fibroblasts compared to a lipid-nanoparticle composition containing lipid formula (I) having the same head group. In embodiments, a lipid-nanoparticle composition containing lipid formula (I-B) provides a higher survival rate in immune cells compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, a lipid-nanoparticle composition containing lipid formula (I-B) provides a higher survival rate in immune cells compared to a lipid-nanoparticle composition containing lipid formula (I) having the same head group. In embodiments, a lipid-nanoparticle composition containing lipid formula (I-B) provides a higher survival rate in T cells compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, a lipid-nanoparticle composition containing lipid formula (I-B) provides a higher survival rate in T cells compared to a lipid-nanoparticle composition containing lipid formula (I) having the same head group. In embodiments, the tail structure of lipid formula (I-B) provides an increased survival rate for the lipid-nanoparticle composition. In embodiments, the tail structure of lipid formula (I-B) provides an increased survival rate for the lipid-nanoparticle composition compared to an unbranched alkyl or alkenyl tail structure.In embodiments, the tail structure of lipid formula (I-B) provides increased viability for the lipid-nanoparticle composition as compared to the tail structure of lipid formula (I). In embodiments, the ester groups adjacent to the branching of the alkyl groups in the tail structure of the lipids described herein provide increased viability for the lipid-nanoparticle composition. In embodiments, the ester groups adjacent to the branching of the alkyl groups in the tail structure of the lipids described herein provide increased viability for the lipid-nanoparticle composition as compared to an alkyl or alkenyl tail structure having no branching. In embodiments, together with the branching of R6, R7, R8, and R10 in the tail structure of lipid formula (I-B), the ester groups adjacent to R6 and R7 and to R8 and R10 provide increased viability for the lipid-nanoparticle composition. In embodiments, together with the branching of R6, R7, R8, and R10 in the tail structure of lipid formula (I-B), the ester groups adjacent to R6 and R7 and to R8 and R10 provide increased viability for the lipid-nanoparticle composition as compared to an alkyl or alkenyl tail structure having no branching.

[0382] In embodiments, the lipid-nanoparticle composition containing lipid formula (I-B) provides both a higher survival rate and a higher transfection efficiency as compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, the lipid-nanoparticle composition containing lipid formula (I-B) provides both a higher survival rate and a higher transfection efficiency as compared to lipid-nanoparticle compositions containing lipid formula (I) having the same head group. In embodiments, the lipid-nanoparticle composition containing lipid formula (I-B) provides both a higher survival rate and a higher transfection efficiency in skin cells as compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, the lipid-nanoparticle composition containing lipid formula (I-B) provides both a higher survival rate and a higher transfection efficiency in skin cells as compared to lipid-nanoparticle compositions containing lipid formula (I) having the same head group. In embodiments, the lipid-nanoparticle composition containing lipid formula (I-B) provides both a higher survival rate and a higher transfection efficiency in fibroblasts as compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, the lipid-nanoparticle composition containing lipid formula (I-B) provides both a higher survival rate and a higher transfection efficiency in fibroblasts as compared to lipid-nanoparticle compositions containing lipid formula (I) having the same head group. In embodiments, the lipid-nanoparticle composition containing lipid formula (I-B) provides both a higher survival rate and a higher transfection efficiency in immune cells as compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, the lipid-nanoparticle composition containing lipid formula (I-B) provides both a higher survival rate and a higher transfection efficiency in immune cells as compared to lipid-nanoparticle compositions containing lipid formula (I) having the same head group. In embodiments, the lipid-nanoparticle composition containing lipid formula (I-B) provides both a higher survival rate and a higher transfection efficiency in T cells as compared to lipid-nanoparticle compositions containing other lipids described herein.In an embodiment, a lipid-nanoparticle composition containing lipid formula (I-B) provides both a higher survival rate and a higher transfection efficiency in T cells as compared to a lipid-nanoparticle composition containing lipid formula (I) having the same head group. In an embodiment, the tail structure of lipid formula (I-B) provides both an increased transfection efficiency and an increased survival rate. In an embodiment, the tail structure of lipid formula (I-B) provides both an increased transfection efficiency and an increased survival rate as compared to an unbranched alkyl or alkenyl tail structure. In an embodiment, the tail structure of lipid formula (I-B) provides both an increased transfection efficiency and an increased survival rate for the lipid-nanoparticle composition as compared to the tail structure of lipid formula (I). In an embodiment, the ester group adjacent to the branch of the alkyl group in the tail structure of the lipid described herein provides both an increased transfection efficiency and an increased survival rate for the lipid-nanoparticle composition. In an embodiment, together with the branches of R6, R7, R8, and R10 in the tail structure of lipid formula (I-B), the ester groups adjacent to R6 and R7 and to R8 and R10 provide both an increased transfection efficiency and an increased survival rate for the lipid-nanoparticle composition. In an embodiment, together with the branches of R6, R7, R8, and R10 in the tail structure of lipid formula (I-B), the ester groups adjacent to R6 and R7 and to R8 and R10 provide both an increased transfection efficiency and an increased survival rate for the lipid-nanoparticle composition as compared to an unbranched alkyl or alkenyl tail structure.

[0383] When such a composition containing the lipid of formula (I-B) is used to transfect at least one reprogramming factor into skin cells, it can result in a greater improvement in at least one skin rejuvenation marker or skin quality marker as compared to a lipid-nanoparticle composition containing other lipids described herein. When such a composition containing the lipid of formula (I-B) is used to transfect at least one reprogramming factor into skin cells, it can result in a greater improvement in at least one skin rejuvenation marker or skin quality marker as compared to a lipid-nanoparticle composition containing the lipid of formula (I) having the same head group. When such a composition containing the lipid of formula (I-B) is used to transfect at least one reprogramming factor into immune cells, it can result in a greater improvement in at least one immune cell rejuvenation marker or immune cell stemness marker as compared to a lipid-nanoparticle composition containing other lipids described herein. When such a composition containing the lipid of formula (I-B) is used to transfect at least one reprogramming factor into immune cells, it can result in a greater improvement in at least one immune cell rejuvenation marker or stemness rejuvenation marker as compared to a lipid-nanoparticle composition containing the lipid of formula (I) having the same head group.

[0384] In an embodiment, a lipid-nanoparticle composition containing lipid formula (I) with q1 absent and q2 being 1 provides a higher transfection efficiency compared to a lipid-nanoparticle composition containing other lipids described herein. In an embodiment, a lipid-nanoparticle composition containing lipid formula (I) with q1 absent and q2 being 1 provides a higher transfection efficiency compared to a lipid-nanoparticle composition containing lipid formula (I) with q1 being 1 and q2 absent. In an embodiment, a lipid-nanoparticle composition containing lipid formula (I) with q1 absent and q2 being 1 provides a higher transfection efficiency in skin cells compared to a lipid-nanoparticle composition containing other lipids described herein. In an embodiment, a lipid-nanoparticle composition containing lipid formula (I) with q1 absent and q2 being 1 provides a higher transfection efficiency in skin cells compared to a lipid-nanoparticle composition containing lipid formula (I) with q1 being 1 and q2 absent. In an embodiment, a lipid-nanoparticle composition containing lipid formula (I) with q1 absent and q2 being 1 provides a higher transfection efficiency in fibroblasts compared to a lipid-nanoparticle composition containing other lipids described herein. In an embodiment, a lipid-nanoparticle composition containing lipid formula (I) with q1 absent and q2 being 1 provides a higher transfection efficiency in fibroblasts compared to a lipid-nanoparticle composition containing lipid formula (I) with q1 being 1 and q2 absent. In an embodiment, a lipid-nanoparticle composition containing lipid formula (I) with q1 absent and q2 being 1 provides a higher transfection efficiency in immune cells compared to a lipid-nanoparticle composition containing other lipids described herein. In an embodiment, a lipid-nanoparticle composition containing lipid formula (I) with q1 absent and q2 being 1 provides a higher transfection efficiency in immune cells compared to a lipid-nanoparticle composition containing lipid formula (I) with q2 absent and q1 being 1. In an embodiment, a lipid-nanoparticle composition containing lipid formula (I) with q1 absent and q2 being 1 provides a higher transfection efficiency in T cells compared to a lipid-nanoparticle composition containing other lipids described herein.In an embodiment, a lipid-nanoparticle composition containing lipid of formula (I) and having q1 absent and q2 being 1 provides a higher transfection efficiency in T cells as compared to a lipid-nanoparticle composition containing lipid of formula (I) and having q1 being 1 and q2 absent.

[0385] In embodiments, a lipid-nanoparticle composition containing lipid formula (I) with q1 absent and q2 being 1 provides a higher survival rate compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, a lipid-nanoparticle composition containing lipid formula (I) with q1 absent and q2 being 1 provides a higher survival rate compared to a lipid-nanoparticle composition containing lipid formula (I) with q1 being 1 and q2 absent. In embodiments, a lipid-nanoparticle composition containing lipid formula (I) with q1 absent and q2 being 1 provides a higher survival rate in skin cells compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, a lipid-nanoparticle composition containing lipid formula (I) with q1 absent and q2 being 1 provides a higher survival rate in skin cells compared to a lipid-nanoparticle composition containing lipid formula (I) with q1 being 1 and q2 absent. In embodiments, a lipid-nanoparticle composition containing lipid formula (I) with q1 absent and q2 being 1 provides a higher survival rate in fibroblasts compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, a lipid-nanoparticle composition containing lipid formula (I) with q1 absent and q2 being 1 provides a higher survival rate in fibroblasts compared to a lipid-nanoparticle composition containing lipid formula (I) with q1 being 1 and q2 absent. In embodiments, a lipid-nanoparticle composition containing lipid formula (I) with q1 absent and q2 being 1 provides a higher survival rate in immune cells compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, a lipid-nanoparticle composition containing lipid formula (I) with q1 absent and q2 being 1 provides a higher survival rate in immune cells compared to a lipid-nanoparticle composition containing lipid formula (I) with q1 being 1 and q2 absent. In embodiments, a lipid-nanoparticle composition containing lipid formula (I) with q1 absent and q2 being 1 provides a higher survival rate in T cells compared to lipid-nanoparticle compositions containing other lipids described herein.In an embodiment, a lipid-nanoparticle composition containing lipid of formula (I) with q1 absent and q2 being 1 provides a higher survival rate in T cells as compared to a lipid-nanoparticle composition containing lipid of formula (I) with q1 being 1 and q2 absent.

[0386] In embodiments, a lipid-nanoparticle composition containing lipid of formula (I) with q1 absent and q2 being 1 provides both a higher survival rate and a higher transfection efficiency as compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, a lipid-nanoparticle composition containing lipid of formula (I) with q1 absent and q2 being 1 provides both a higher survival rate and a higher transfection efficiency as compared to a lipid-nanoparticle composition containing lipid of formula (I) with q1 being 1 and q2 absent. In embodiments, a lipid-nanoparticle composition containing lipid of formula (I) with q1 absent and q2 being 1 provides both a higher survival rate and a higher transfection efficiency in skin cells as compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, a lipid-nanoparticle composition containing lipid of formula (I) with q1 absent and q2 being 1 provides both a higher survival rate and a higher transfection efficiency in skin cells as compared to a lipid-nanoparticle composition containing lipid of formula (I) with q1 being 1 and q2 absent. In embodiments, a lipid-nanoparticle composition containing lipid of formula (I) with q1 absent and q2 being 1 provides both a higher survival rate and a higher transfection efficiency in fibroblasts as compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, a lipid-nanoparticle composition containing lipid of formula (I) with q1 absent and q2 being 1 provides both a higher survival rate and a higher transfection efficiency in fibroblasts as compared to a lipid-nanoparticle composition containing lipid of formula (I) with q1 being 1 and q2 absent. In embodiments, a lipid-nanoparticle composition containing lipid of formula (I) with q1 absent and q2 being 1 provides both a higher survival rate and a higher transfection efficiency in immune cells as compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, a lipid-nanoparticle composition containing lipid of formula (I) with q1 absent and q2 being 1 provides both a higher survival rate and a higher transfection efficiency in immune cells as compared to a lipid-nanoparticle composition containing lipid of formula (I) with q1 being 1 and q2 absent.In embodiments, a lipid-nanoparticle composition containing lipid formula (I-B) provides both a higher survival rate and a higher transfection efficiency in T cells as compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, a lipid-nanoparticle composition containing lipid formula (I) with q1 absent and q2 being 1 provides both a higher survival rate and a higher transfection efficiency in T cells as compared to a lipid-nanoparticle composition containing lipid formula (I) with q1 being 1 and q2 absent.

[0387] Such a composition containing lipid formula (I) with q1 absent and q2 being 1, when used to transfect at least one reprogramming factor into skin cells, can result in a greater improvement in at least one skin rejuvenation marker or skin quality marker as compared to lipid-nanoparticle compositions containing other lipids described herein. Such a composition containing lipid formula (I) with q1 absent and q2 being 1, when used to transfect at least one reprogramming factor into skin cells, can result in a greater improvement in at least one skin rejuvenation marker or skin quality marker as compared to a lipid-nanoparticle composition containing lipid formula (I) with q1 being 1 and q2 absent. Such a composition containing lipid formula (I) with q1 absent and q2 being 1, when used to transfect at least one reprogramming factor into immune cells, can result in a greater improvement in at least one immune cell rejuvenation marker or immune cell stemness marker as compared to lipid-nanoparticle compositions containing other lipids described herein. Such a composition containing lipid formula (I) with q1 absent and q2 being 1, when used to transfect at least one reprogramming factor into immune cells, can result in a greater improvement in at least one immune cell rejuvenation marker or stemness rejuvenation marker as compared to a lipid-nanoparticle composition containing lipid formula (I) with the same head group, q1 being 1 and q2 absent.

[0388] The methods provided herein involve using a lipid or lipid-nanoparticle composition to transfect a cell with one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors, thereby producing rejuvenated cells. The cells to be rejuvenated can be of any cell type. In embodiments, the cells are contacted with, exposed to, or transfected with the mRNA for a period of about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1 day, or less than 1 day. In embodiments, the cells are contacted with, exposed to, or transfected with the mRNA for a period of about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1 day, or less than 1 day. In embodiments, the cells are contacted with, exposed to, or transfected with the mRNA for a period of about 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1 day, or less than 1 day. In embodiments, the cells are contacted with, exposed to, or transfected with the mRNA for a period of about 7, 6, 5, 4, 3, 2, or less than 1 day, or less than 1 day. In embodiments, the cells are contacted with, exposed to, or transfected with the mRNA for a period of about 5, 4, 3, 2, or less than 1 day, or less than 1 day. In embodiments, at least one reprogramming factor is expressed from the transfected mRNA within the cell, or the cell is exposed to at least one reprogramming factor expressed from the transfected mRNA for a period of about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1 day, or less than 1 day. In embodiments, at least one reprogramming factor is expressed from the transfected mRNA within the cell, or the cell is exposed to at least one reprogramming factor expressed from the transfected mRNA for a period of about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1 day, or less than 1 day.In embodiments, at least one reprogramming factor is expressed from transfected mRNA within the cell, or the cell is exposed to at least one reprogramming factor expressed from transfected mRNA for at least about 2 days and not more than about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days. In embodiments, at least one reprogramming factor is expressed from transfected mRNA within the cell, or the cell is exposed to at least one reprogramming factor expressed from transfected mRNA for not more than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or for less than 1 day. In embodiments, at least one reprogramming factor is expressed from transfected mRNA within the cell, or the cell is exposed to at least one reprogramming factor expressed from transfected mRNA for at least about 2 days and not more than about 10, 9, 8, 7, 6, 5, 4, 3, or 2 days. In embodiments, at least one reprogramming factor is expressed from transfected mRNA within the cell, or the cell is exposed to at least one reprogramming factor expressed from transfected mRNA for not more than about 7, 6, 5, 4, 3, 2, or 1 day, or for less than 1 day. In embodiments, at least one reprogramming factor is expressed from transfected mRNA within the cell, or the cell is exposed to at least one reprogramming factor expressed from transfected mRNA for at least about 2 days and not more than about 7, 6, 5, 4, 3, or 2 days. In embodiments, at least one reprogramming factor is expressed from transfected mRNA within the cell, or the cell is exposed to at least one reprogramming factor expressed from transfected mRNA for not more than about 5, 4, 3, 2, or 1 day, or for less than 1 day.In embodiments, at least one reprogramming factor is expressed from transfected mRNA within the cell, or the cell is exposed to at least one reprogramming factor expressed from transfected mRNA for at least about 2 days and not more than about 5, 4, 3, or 2 days. In embodiments, the rejuvenated cells have a phenotype or activity profile similar to that of young cells. The phenotype or activity profile includes one or more of a transcriptome profile, gene expression of one or more nuclear and / or epigenetic markers, proteolytic activity, mitochondrial integrity and function, SASP cytokine expression, and methylation landscape.

[0389] The rejuvenated cells described herein may have a transcriptome profile that is even more similar to the transcriptome profile of young cells. In embodiments, the transcriptome profile of the rejuvenated cells includes an increase in gene expression of one or more genes selected from RPL37, RHOA, SRSF3, EPHB4, ARHGAP18, RPL31, FKBP2, MAP1LC3B2, Elfl, Phf8, Pol2s2, Tafl, and Sin3a.

[0390] The rejuvenated cells described herein may also exhibit increased gene expression of one or more nuclear markers and / or epigenetic markers as compared to a reference value. In embodiments, the one or more nuclear markers and / or epigenetic markers are selected from Hpl gamma, H3K9me3, lamin support protein LAP2 alpha, and SIRTl protein. In embodiments, the rejuvenated cells have proteolytic activity similar to that of young cells. In embodiments, the proteolytic activity is measured as increased cellular autophagosome formation, increased chymotrypsin-like proteasome activity, or a combination thereof. In embodiments, the rejuvenated cells exhibit improved mitochondrial health and function as compared to a reference value. In embodiments, the improved mitochondrial health and function are measured as increased mitochondrial membrane potential, decreased reactive oxygen species (ROS), or a combination thereof.

[0391] The rejuvenated cells described herein may also exhibit decreased expression of one or more SASP cytokines as compared to a reference value. In embodiments, the one or more SASP cytokines include IL18, ILIA, GROA, IL22, and IL9. In embodiments, the rejuvenated cells exhibit a reversal of the methylation landscape. In embodiments, the reversal of the methylation landscape is measured by Horvath clock estimation. In some embodiments, the reference value is obtained from aged cells.

[0392] As described herein, cells can be rejuvenated by transient reprogramming with mRNA encoding one or more cell reprogramming factors transfected into the cells using the lipids or lipid-nanoparticle compositions of the disclosure. Transient reprogramming is achieved, in some embodiments, by transfecting the cells with non-integrating mRNA for a period not exceeding about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day. Transient reprogramming is achieved, in some embodiments, by transfecting the cells with non-integrating mRNA for a period not exceeding about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day. Transient reprogramming is achieved, in some embodiments, by transfecting the cells with non-integrating mRNA for a period not exceeding about 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day. Transient reprogramming is achieved, in some embodiments, by transfecting the cells with non-integrating mRNA for a period not exceeding about 6, 5, 4, 3, 2, or 1 day, or less than 1 day. Transient reprogramming is achieved, in some embodiments, by expressing at least one reprogramming factor from non-integrating mRNA transfected into the cells, or by exposing the cells to at least one reprogramming factor expressed from non-integrating mRNA transfected into the cells for a period not exceeding about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day. Transient reprogramming is achieved, in some embodiments, by expressing at least one reprogramming factor from non-integrating mRNA transfected into the cells, or by exposing the cells to at least one reprogramming factor expressed from non-integrating mRNA transfected into the cells for at least 2 days and not exceeding about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days.Transient reprogramming is, in some embodiments, achieved by expressing at least one reprogramming factor from non-integrating mRNA transfected intracellularly, or by exposing cells to at least one reprogramming factor expressed from non-integrating mRNA transfected intracellularly for a period not exceeding about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day. Transient reprogramming is, in some embodiments, achieved by expressing at least one reprogramming factor from non-integrating mRNA transfected intracellularly, or by exposing cells to at least one reprogramming factor expressed from non-integrating mRNA transfected intracellularly for at least 2 days and not exceeding about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days. Transient reprogramming is, in some embodiments, achieved by expressing at least one reprogramming factor from non-integrating mRNA transfected intracellularly, or by exposing cells to at least one reprogramming factor expressed from non-integrating mRNA transfected intracellularly for a period not exceeding about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day. Transient reprogramming is, in some embodiments, achieved by expressing at least one reprogramming factor from non-integrating mRNA transfected intracellularly, or by exposing cells to at least one reprogramming factor expressed from non-integrating mRNA transfected intracellularly for at least 2 days and not exceeding about 10, 9, 8, 7, 6, 5, 4, 3, or 2 days. Transient reprogramming is, in some embodiments, achieved by expressing at least one reprogramming factor from non-integrating mRNA transfected intracellularly, or by exposing cells to at least one reprogramming factor expressed from non-integrating mRNA transfected intracellularly for a period not exceeding about 7, 6, 5, 4, 3, 2, or 1 day, or less than 1 day.Transient reprogramming is, in some embodiments, achieved by expressing at least one reprogramming factor from non-integrating mRNA transfected intracellularly, or by exposing cells to at least one reprogramming factor expressed from non-integrating mRNA transfected intracellularly for at least 2 days and not exceeding about 7, 6, 5, 4, 3, or 2 days. Transient reprogramming is, in some embodiments, achieved by expressing at least one reprogramming factor from non-integrating mRNA transfected intracellularly, or by exposing cells to at least one reprogramming factor expressed from non-integrating mRNA transfected intracellularly for not exceeding about 5, 4, 3, 2, or 1 day, or for less than 1 day. Transient reprogramming is, in some embodiments, achieved by expressing at least one reprogramming factor from non-integrating mRNA transfected intracellularly, or by exposing cells to at least one reprogramming factor expressed from non-integrating mRNA transfected intracellularly for at least 2 days and not exceeding about 5, 4, 3, or 2 days. In embodiments, transient reprogramming of cells eliminates various features of aging while avoiding complete dedifferentiation of the cells into stem cells.

[0393] The methods and compositions provided herein achieve cellular rejuvenation, or youthful return, by transient overexpression of one or more mRNAs encoding cell reprogramming factors delivered by the lipid or lipid-nanoparticle compositions of the present disclosure. Such cell reprogramming factors can include transcription factors, epigenetic remodelers, or small molecules that affect mitochondrial function, proteolytic activity, heterochromatin levels, histone methylation, nuclear lamina polypeptides, cytokine secretion, or aging. In embodiments, the cell reprogramming factors include one or more of OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG. In embodiments, the cell reprogramming factors are OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG in different molar ratios, such as a:b:c:d:e:f molar ratio, where a, b, c, d, e, and f are all the same number (e.g., 1:1:1:1:1:1), some of the same number and some different numbers (e.g., 3:1:1:1:1:1, 2:1:1:1:1:1, 2:2:1:1:1:1, 2:2:2:1:1:1, 2:2:2:2:1:1, 2:2:2:2:2:1, 3:3:3:3:2:2), or all different numbers (e.g., 6:4:5:3:2:1), and are applied in an a:b:c:d:e:f molar ratio. In embodiments, a, b, c, d, e, and / or f are each 1 to 7, i.e., 1 to 7:1 to 7:1 to 7:1 to 7:1 to 7:1 to 7 (or in the case of combinations with fewer than six factors, 1 to 7:1 to 7:1 to 7:1 to 7:1 to 7, 1 to 7:1 to 7:1 to 7:1 to 7, 1 to 7:1 to 7:1 to 7, 1 to 7:1 to 7, or 1 to 7:1).In an embodiment, the cell reprogramming factors are OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG at a different weight ratio, for example, a weight ratio of a:b:c:d:e:f, where a, b, c, d, e, and f can each be the same (for example, 1:1:1:1:1:1), and each of a, b, c, d, e, and f can be the same or different numbers (for example, 3:1:1:1:1:1, 2:1:1:1:1:1, 2:2:1:1:1:1, 2:2:2:1:1:1, 2:2:2:2:1:1, 2:2:2:2:2:1, 3:3:3:3:2:2) or each can be different numbers (for example, 6:4:5:3:2:1), and are applied as OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG at a weight ratio of a:b:c:d:e:f. In an embodiment, a, b, c, d, e, and / or f are each 1 to 7, that is, 1 to 7:1 to 7:1 to 7:1 to 7:1 to 7:1:7 (or in the case of a combination of fewer than six factors, 1 to 7:1 to 7:1 to 7:1 to 7:1 to 7, 1 to 7:1 to 7:1 to 7:1 to 7, 1 to 7:1 to 7:1 to 7, 1 to 7:1 to 7, or 1 to 7:1).

[0394] The methods and compositions provided herein can be applied to any type of cell, tissue, or organ in need of rejuvenation. The methods and compositions of the disclosure can be used to rejuvenate cells in culture (e.g., ex vivo or in vitro) to improve function and efficacy for use in cell therapy. The cells used for treating a patient can be autologous or allogeneic. The cells can be derived from a patient or a matched donor, or they can be obtained from a cell bank or be derived from iPS cells. For example, in autologous ex vivo therapy, the cells can be obtained directly from the patient to be treated, transfected with the mRNA encoding the cell reprogramming factors described herein, and re-transplanted into the patient. Such cells can be obtained, for example, from a biopsy or surgical procedure performed on the patient. Alternatively, in allogeneic ex vivo therapy, the cells can be obtained from a cell bank or a cell line derived from iPS cells, transfected with the mRNA encoding the cell reprogramming factors as described herein, and re-transplanted into the patient. Alternatively, the cells in need of rejuvenation can be directly transfected in vivo using the mRNA encoding the cell reprogramming factors.

[0395] The lipid-containing composition or lipid-nanoparticle composition of the disclosure is optimized such that the reprogramming factor reduces any induced immune response against the protein / polypeptide and increases the stability of the protein / polypeptide, altering the protein / polypeptide activity such as increased activity compared to the wild-type reprogramming factor, and can be used for delivery of mRNA expressing the reprogramming factor that provides a more robust cell rejuvenation.

[0396] The methods provided herein include administering to a cell or subject a lipid-containing composition or lipid-nanoparticle composition of the disclosure that contains RNA, or treating or transfecting a cell with a lipid-containing composition or lipid-nanoparticle composition that contains RNA, for consecutive days with an administration interval not exceeding 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days. In some embodiments, the administration interval is 1 day. In some embodiments, the composition is administered once. In some embodiments, administration of the lipid-containing composition or lipid-nanoparticle composition that contains RNA is performed at least once a day during the administration interval period. In some embodiments, administration is performed at a less frequent rate of less than once a day during the administration interval, e.g., once every 2 days, once every 3 days, once every 4 days, once every x days (where x is a number from 4 to 25). Thus, in such embodiments, for example, administering a lipid-containing composition or lipid-nanoparticle composition that contains RNA once every 5 days at a 5-day administration interval means that the RNA is administered once during the interval, i.e., once during the total treatment period of 5 days, whereas administering the RNA twice a day at a 5-day administration interval means that the RNA is administered 10 times during the interval, i.e., 10 times in 5 days. In some embodiments, the methods include administering to a cell or subject a lipid-containing composition or lipid-nanoparticle composition that contains RNA, or treating or transfecting a cell with a lipid-containing composition or lipid-nanoparticle composition that contains RNA, for consecutive days with an administration interval not exceeding 21, 18, 14, 10, 7, or 5 days. In some embodiments, the methods include administering to a cell or subject a lipid-containing composition or lipid-nanoparticle composition that contains RNA, or treating or transfecting a cell with a lipid-containing composition or lipid-nanoparticle composition that contains RNA, for consecutive days with an administration interval not exceeding 18 days.In some embodiments, the method comprises administering a lipid-containing composition or lipid-nanoparticle composition comprising RNA to a cell or a subject, or treating or transfecting a cell with the lipid-containing composition or lipid-nanoparticle composition comprising RNA of the present disclosure for no more than 14 consecutive days. In some embodiments, the method comprises administering a lipid-containing composition or lipid-nanoparticle composition comprising RNA to a cell or a subject, or treating or transfecting a cell with the lipid-containing composition or lipid-nanoparticle composition comprising RNA for no more than 10 consecutive days. In some embodiments, the method comprises administering a lipid-containing composition or lipid-nanoparticle composition comprising RNA to a cell or a subject, or treating or transfecting a cell with the lipid-containing composition or lipid-nanoparticle composition comprising RNA for no more than 7 consecutive days. In some embodiments, the method comprises administering a lipid-containing composition or lipid-nanoparticle composition comprising RNA to a cell or a subject, or treating or transfecting a cell with the lipid-containing composition or lipid-nanoparticle composition comprising RNA for no more than 5 consecutive days. In other embodiments, the exposing comprises interrupting the exposing and repeating the exposing after the interrupting. In some embodiments, the exposing, treating, transfecting, expressing, or administering comprises exposing, treating, transfecting, expressing, or administering an immune cell for about 2 - 5 consecutive days, about 5 - 7 consecutive days, about 7 - 10 consecutive days, about 10 - 12 consecutive days, about 12 - 14 consecutive days, about 14 - 17 consecutive days, about 17 - 19 consecutive days, or about 19 - 21 consecutive days, and in some embodiments, further comprises interrupting the exposing and repeating the exposing after the interrupting.

[0397] The duration of exposure is controlled by mechanisms such as self-amplifying RNAs, circular RNAs, B18R and other decoys, and / or on / off switches such as L7Ae or its family members. In some embodiments, the repeating is performed any number of times, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, or up to 20 times, or up to 30 times, or more. In in vivo applications, the repeating can continue for any period, such as until the disease is successfully treated or cured, or throughout the life of the subject or patient. In some embodiments, the repeating is performed at any time after the interrupting, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, up to 20, up to 30 days, up to 3 months, up to 6 months, or up to 1 year after the interrupting. One exposure period is considered to be an administration interval, for example, such that the order of exposure-interrupt-repeat exposure includes two administration intervals.

[0398] Pharmaceutical composition The present disclosure also provides a pharmaceutical composition comprising the nanoparticle composition described herein and a pharmaceutically acceptable carrier thereof. The pharmaceutical composition is particularly useful for delivering nucleic acids to a patient (e.g., a human) or to cells for treating a particular disease or a targeted medical condition. Appropriate concentrations and dosages can be readily determined by those skilled in the art.

[0399] A pharmaceutical composition containing the returned cells can be obtained by transfecting the cells with the lipid-containing composition or lipid-nanoparticle composition of the present disclosure containing one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors for no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive days to transiently reprogram the cells for rejuvenation. Treating the cells by transfecting them with the lipid-containing composition or lipid-nanoparticle composition of the present disclosure containing therapeutic mRNA is also contemplated, and in embodiments, the transfection is performed once to obtain treated cells such as returned cells. A single transfection step is contemplated, for example, for self-replicating RNA constructs. In another aspect, provided herein is a pharmaceutical composition comprising returned cells obtained by transfecting the cells with the lipid-containing composition or lipid-nanoparticle composition of the present disclosure containing one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors for no more than 4, 5, 6, or 7 consecutive days to transiently reprogram the cells for rejuvenation.

[0400] The pharmaceutical compositions of the present disclosure can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administering such pharmaceutical compositions include, but are not limited to, topical, oral, local, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. As used herein, the term parenteral includes subcutaneous injection, intravenous, intramuscular, intradermal, intracardiac injection or infusion techniques. The pharmaceutical compositions of the present disclosure are formulated such that upon administration of the composition to a patient, the active ingredient contained therein becomes bioavailable. The composition administered to a subject or patient takes the form of one or more dosage units. For example, a tablet can be a single dosage unit, and a container of a compound of the present disclosure in aerosol form can hold a plurality of dosage units. The actual methods of preparing such dosage forms are known to those skilled in the art or will be apparent, and reference may be made, for example, to Remington: The Science and Practice of Pharmacy, 20th edition (Philadelphia College of Pharmacy and Science, 2000). The composition administered contains a therapeutically effective amount of the nanoparticle composition for the treatment of the targeted disease or condition.

[0401] The pharmaceutical compositions of the present disclosure can be in solid or liquid form. In one aspect, the carrier(s) is / are particulate such that the composition is in the form of, for example, a tablet or powder. The carrier(s) can be liquid and the composition can be, for example, an oral syrup, an injectable liquid, or an aerosol, which is useful, for example, in inhalation administration.

[0402] When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, and semi-solid, semi-liquid, suspension, and gel forms are included within the forms considered herein as either solid or liquid.

[0403] As a solid composition for oral administration, the pharmaceutical composition can be formulated in the form of powders, granules, compressed tablets, pills, capsules, chewing gums, wafers, etc. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch, lactose, or dextrin; disintegrants such as alginic acid, sodium alginate, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate, or orange flavor; and coloring agents.

[0404] When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain a liquid carrier such as polyethylene glycol or an oil in addition to the materials of the above types.

[0405] The pharmaceutical composition can be in the form of a liquid, for example, an elixir, syrup, solution, emulsion, or suspension. The liquid can be, as two examples, for delivery by oral administration or injection. When intended for oral administration, the preferred composition contains, in addition to the compound, one or more of a sweetening agent, a preservative, a dye / coloring agent, and a flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffering agent, a stabilizing agent, and an isotonic agent can be included.

[0406] The liquid pharmaceutical composition of the present disclosure, whether in the form of a solution, suspension, or other similar form, includes the following adjuvants: water for injection, physiological saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides that can act as solvents or suspension media, polyethylene glycol, glycerin, propylene glycol, or other sterile diluents such as solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetic acid, citric acid, or phosphoric acid, and agents for adjusting osmotic pressure such as sodium chloride or dextrose; and may include one or more of agents that act as cryoprotectants such as sucrose or trehalose. The parenteral preparation can be enclosed in an ampoule made of glass or plastic, a disposable syringe, or a multi-dose vial. Physiological saline is a preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.

[0407] The liquid pharmaceutical composition of the pres...

Claims

1. Compounds having the structure of the following formula (XVII), or their stereoisomers, salts, or tautomers: 【Chemistry 41】 During the ceremony, G １ and G ２ However, each operates independently as -OC (=O)-, -NR ２５ C(=O)-, or -CH=CH-, R ２１ and R ２２ are each independently C １ -C ６ alkyl, linear C １０ -C ２０ alkyl, linear C １０ -C ２０ alkenyl, or branched C １０ -C ３５ alkenyl, and the C １ -C ６ alkyl is substituted with -OC(=O)R ２６ and R ２３ However, H, OH, or OCH ３ And, R ２４ However, C １ -C ８ It is heteroalkyl, R ２５ However, H or C １ -C ４ It is alkyl, R ２６ However, branch C １０ -C ３０ It is alkyl, I understand １ and m ２ However, each of them is independently an integer of either 0 or 1.

2. G １ and G ２ The compound according to claim 1, wherein each is -OC(=O)-.

3. G １ or G ２ One of them is -NR ２５ C (= O) - and G １ or G ２ The compound according to claim 1, wherein the other of the two is -CH=CH-.

4. I understand ２ The compound according to claim 1, wherein the coefficient is 0.

5. The compound according to claim 1, wherein the compound has the structure of the following formulas (XVIIA) to (XVIIB), or one of its stereoisomers, salts, or tautomers: 【Chemistry 42】

6. R ２４ However, C ４ Alkylamine, C ５ Alkylamine, C ６ Alkylamine, or C ７ The compound according to claim 1, which is an alkylamine.

7. R ２４ but, 【Chemistry 43】 The compound according to claim 1.

8. R ２４ C １ -C ８ The compound according to claim 1, wherein the heteroalkyl group is further substituted with a cycloalkyl group.

9. R ２４ but, 【Chemistry 44】 The compound according to claim 8.

10. R ２１ and R ２２ However, each independently, -OC(=O)R ２６ C replaced by ２ -C ５ Alkyl, linear C １２ -C １８ Alkyl, linear C １２ -C １８ Alkenyl, or branched C １４ -C ３２ The compound according to claim 1, wherein the compound is an alkenyl.

11. R ２６ However, branch C １０ -C ２０ The compound according to claim 1, wherein it is alkyl.

12. R ２１ and R ２２ However, each operates independently, with the following structure: 【Chemistry 45】 The compound according to claim 1, having one of the following.

13. The compound has the following structure: 【Chemistry 46】 【change】 【change】 【change】 【change】 The compound according to claim 1, having one of the following.

14. A pharmaceutical composition comprising a compound according to any one of claims 1 to 13 and a therapeutic agent containing nucleic acid.

15. The pharmaceutical composition according to claim 14, further comprising a helper lipid, a stabilizing lipid, or a structural lipid.

16. The helper lipids are 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-diundecane. Noyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 dietherPC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 The pharmaceutical composition according to claim 15, selected from the group consisting of PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and mixtures thereof.

17. The pharmaceutical composition according to claim 15, wherein the stabilizing lipid is 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG) having an average PEG molecular weight of 2000.

18. The pharmaceutical composition according to claim 15, wherein the structural lipid is selected from the group consisting of cholesterol, cholesterol derivatives, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof.

19. The pharmaceutical composition according to claim 14, wherein the nucleic acid is selected from small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), or messenger RNA (mRNA).

20. The pharmaceutical composition according to claim 19, wherein the nucleic acid is mRNA.

21. Lipid nanoparticles comprising a compound according to any one of claims 1 to 13 and a therapeutic agent containing nucleic acid.

22. The lipid nanoparticles according to claim 21, wherein the therapeutic agent is mRNA.

23. Compounds having the structure of the following formula (XX), or their stereoisomers, salts, or tautomers: 【Transformation 63】 During the ceremony, G １ and G ２ However, each is independent of -OC (=O)- or -NR ２５ C (= O) - R ２１ and R ２２ However, each is independent, C １ -C ６ Alkyl, linear C １０ -C ２０ Alkyl, linear C １０ -C ２０ Alkenyl, or branched C １０ -C ３５ It is an alkenyl, and the C １ -C ６ Alkyl is -OC(=O)R ２６ It has been replaced with, R ２４ However, C １ -C ６ C substituted with heteroalkyl, aryl, or 4- to 8-membered heterocycloalkyl groups １ -C ４ It is alkyl, R ２５ However, H or C １ -C ４ It is alkyl, R ２６ is a branched C １０ -C ３０ alkyl, Y is O or NR ３２ And R ３２ However, H or C １ -C ４ It is alkyl.

24. The compound according to claim 23, wherein Y is O.

25. R ２４ is C ３ -C ６ heteroalkyl, the compound according to claim 23.

26. R ２４ However, C is substituted with a 4- to 8-membered heterocycloalkyl group. １ -C ４ The compound according to claim 23, wherein it is alkyl.

27. R ２１ and R ２２ However, each independently, -OC(=O)R ２６ C replaced by ２ -C ５ Alkyl, linear C １２ -C １８ Alkyl, linear C １２ -C １８ Alkenyl, or branched C １４ -C ３２ The compound according to claim 23, which is an alkenyl.

28. The compound has the following structure: 【Transformation 70】 The compound according to claim 23, having one of the following.

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