Lipid structures and compositions comprising the same

Abstract

The present disclosure relates to ionizable lipids and compositions comprising the ionizable lipids. Lipid-nanoparticle compositions consisting of ionizable lipids, 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 for delivery to mammalian cells or organs.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] 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 incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to ionizable lipids and helper lipids, which can be used in combination with other lipid components such as stabilizing lipids and structural lipids. The present disclosure also provides lipid-nanoparticle compositions comprising such lipids for delivery of therapeutic molecules, particularly therapeutic nucleic acids. Background Art

[0004] In recent years, nucleic acid-based therapies have attracted attention because they have great potential for treating diseases by targeting the genetic blueprint of diseases in vivo. Nucleic acid-based therapies can achieve lasting or even curative effects via gene inhibition, addition, replacement or editing. However, the clinical transformation of nucleic acid drugs and other therapeutic molecules depends on the delivery technology that improves stability, promotes internalization and / or increases target affinity.

[0005] Delivery systems based on lipids, such as, but not limited to, lipid nanoparticles (LNPs), can provide a method for stabilizing and delivering nucleic acids and other therapeutic molecules, and there is still a significant need to improve the technology. Design features, such as optimal particle size, encapsulation efficiency, robust manufacturing processes, different lipophilicity and appropriate surface charge, can be further improved to provide effective lipid-based delivery systems for nucleic acids and other therapeutic molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 is a schematic representation of the experimental design used to rejuvenate human adult dermal fibroblasts.

[0007] Figure 2 Data from immunocytochemistry experiments are shown, which show downregulation of SOD2 and MMP1 (markers of skin aging and impaired skin quality) after transient reprogramming of human adult dermal fibroblasts using lipid nanoparticles containing lipid formula (II) and a mixture of mRNAs in which each mRNA molecule encodes OCT4 (O), SOX2 (S), KLF4 (K), Lin28 (L), cMyc (M), or Nanog (N).

[0008] Figure 3Data from immunocytochemistry experiments are shown, which show downregulation of SOD2, MMP1, MMP2 and p16INK4A (markers of skin aging, impaired skin quality and senescence) after transient reprogramming of human adult dermal fibroblasts using lipid nanoparticles containing lipid formula (II) and a mixture of mRNAs in which each mRNA molecule encodes O, S, K, L, M or N.

[0009] Figure 4 Data are shown demonstrating increased proliferation of human adult dermal fibroblasts following transient reprogramming treatment using lipid nanoparticles containing lipid formula (II) and a mixture of mRNAs wherein each mRNA molecule encodes O, S, K, L, M or N.

[0010] Figure 5 is a schematic representation of the experimental design for rejuvenating human aged skin explants using the compositions and methods herein for epigenetic reprogramming of age (ERA).

[0011] Figure 6 Data from immunohistochemistry experiments are shown, which show that after transient reprogramming of human adult dermal fibroblasts using lipid nanoparticles containing lipid formula (II) and a mixture of mRNAs in which each mRNA molecule encodes O, S, K, L, M or N, there is downregulation of SOD2 (a marker of skin aging and oxidative stress) and upregulation of collagen VII (a marker of skin quality) compared to untreated conditions.

[0012] Figures 7A to 7B Transfection efficiency data and transfection efficacy are shown for lipid nanoparticle formulations comprising lipids of formula (I) or (II).

[0013] Figures 8A to 8D The results are shown in Figure 2. TM Pan T cells were transfected with enhanced green fluorescent protein (eGFP) mRNA in the lipid nanoparticle composition ( Fig. 8A and 8C ) or Jurkat cells ( Figure 8B and 8D ) after 24 hours of transfection efficiency ( Figures 8A to 8B ) and cell viability ( Figures 8C to 8D ) in a bar chart.

[0014] Figures 9A to 9B It is shown that the expression of proteins is regulated by electroporation (EP) or by Lipofectamine TMBar graph of the results after 24 hours of transfection of Pan T cells with enhanced green fluorescent protein (eGFP) mRNA in a lipid nanoparticle composition, where the transfection efficiency is Fig. 9A The median fluorescence intensity of eGFP and the percentage cell viability are shown in Fig. 9B Shown in.

[0015] Figures 10A to 10C is a bar graph showing the results 18-24 hours after transfection of Pan T cells with enhanced green fluorescent protein (eGFP) mRNA in a lipid nanoparticle composition comprising an ionizable lipid disclosed herein, wherein cell viability is Fig. 10A As shown in Fig. 10B The transfection efficiency is shown in Fig. 10C Shown in. Summary of the invention

[0016] The following aspects and embodiments thereof described below are intended to be exemplary and illustrative, not limiting in scope.

[0017] In one aspect, the present disclosure relates to an ionizable lipid of formula (I)

[0018]

[0019] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0020] L 1 is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 2 C6-C 20 Alkyl; R 3 and R 4 are each independently H or C1-C3 alkyl; q1 is absent or is 1; and q2 is absent or is 1.

[0021] In other aspects, the ionizable lipid of formula (I) has one of the following structures:

[0022]

[0023] In one aspect, the present disclosure relates to an ionizable lipid of formula (IA)

[0024]

[0025] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0026] L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; R2 C6-C 20 Alkyl; R 3 and R 4 Each independently represents H or C1-C3 alkyl; R 7 C4-C 20 alkyl; and R 8 C4-C 20 alkyl.

[0027] In other aspects, the ionizable lipid of formula (IA) has the following structure:

[0028]

[0029] In one aspect, the present disclosure relates to an ionizable lipid of formula (IB)

[0030]

[0031] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0032] L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; L 3 is C1-C8 alkylene; R 3 and R 4 Each independently represents H or C1-C3 alkyl; R 6 C4-C 20 Alkyl; R 7 C4-C 20 Alkyl; R 8 C4-C 20 alkyl; and R 10 C4-C 20 alkyl.

[0033] In other aspects, the ionizable lipid of formula (IB) has the following structure:

[0034]

[0035] In one aspect, the present disclosure relates to an ionizable lipid of formula (IB)

[0036]

[0037] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; L 3 is C1-C8 alkylene; R 3 and R 4are independently H, C1-C4 alkyl, -CH2-cyclopropyl, -(CH2) n OH, or R 3 With R 4 Together they form an N-heterocyclic ring; R 6 C4-C 20 Alkyl; R 7 C4-C 20 Alkyl; R 8 C4-C 20 Alkyl; R 10 C4-C 20 alkyl; and n is 2, 3 or 4.

[0038] In other aspects, the ionizable lipid of formula (IB) has one of the following structures:

[0039]

[0040]

[0041] In one aspect, the present disclosure relates to an ionizable lipid of formula (II)

[0042] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0043] L 1 is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 2 C6-C 20 Alkyl; R 3 and R 4 are each independently H or C1-C3 alkyl; and R 5 It is H or C1-C3 alkyl.

[0044] In other aspects, the ionizable lipid of formula (II) has one of the following structures:

[0045]

[0046] In one aspect, the present disclosure relates to an ionizable lipid of formula (III)

[0047]

[0048] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0049] L 1 is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 2 C6-C20 Alkyl; R 2 ' is C6-C 20 Alkyl; R 3 and R 4 are each independently H or C1-C3 alkyl; and R 5 It is H or C1-C3 alkyl.

[0050] In other aspects, the ionizable lipid of formula (III) has one of the following structures:

[0051]

[0052]

[0053] In one aspect, the present disclosure relates to an ionizable lipid of formula (IV)

[0054]

[0055] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0056] L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; R 2 C6-C 20 Alkyl; R 3 and R 4 Each independently represents H or C1-C3 alkyl; R 5 is H or C1-C3 alkyl; R 7 C4-C 20 alkyl; and R 8 C4-C 20 alkyl.

[0057] In other aspects, the ionizable lipid of formula (IV) has one of the following structures:

[0058]

[0059] In one aspect, the present disclosure relates to an ionizable lipid of formula (V)

[0060]

[0061] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0062] L 1 is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 3 and R 4 are each independently H or C1-C3 alkyl; and R12 C6-C 20 Alkenyl.

[0063] In other aspects, the ionizable lipid of formula (V) has the following structure:

[0064]

[0065] In one aspect, the present disclosure relates to an ionizable lipid of formula (VI)

[0066]

[0067] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0068] L 1 is C1-C6 alkylene; L 1 ' is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 1 ' is C6-C 20 Alkenyl; R 9 H, C1-C6 alkyl or –(CH2) n OH; R 12 C6-C 20 Alkenyl; R 12 ' is C6-C 20 and n is 2, 3 or 4.

[0069] In other aspects, the ionizable lipid of formula (VI) has one of the following structures:

[0070]

[0071] In one aspect, the present disclosure relates to an ionizable lipid of formula (VII)

[0072]

[0073] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0074] L 1 is C1-C6 alkylene; L 1 ' is C1-C6 alkylene; R 2 C6-C 20 Alkyl; R 2 ' is C6-C 20 Alkyl; R 9 H, C1-C6 alkyl or –(CH2) n OH; R 11 H or –CH2)OC(=O)R 16 ; R 14C6-C 20 Alkyl; R 14 ' is C6-C 20 Alkyl; R 15 C6-C 20 Alkyl; R 16 C6-C 20 alkyl; and n is 2, 3 or 4.

[0075] In other aspects, the ionizable lipid of formula (VII) has one of the following structures:

[0076]

[0077]

[0078] In one aspect, the present disclosure relates to an ionizable lipid of formula (VIII)

[0079]

[0080] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0081] L 1 is C1-C6 alkylene; L 1 ' is C1-C6 alkylene; R 2 C6-C 20 Alkyl; R 2 ' is C6-C 20 Alkyl; R 9 H, C1-C6 alkyl or –(CH2) n OH; R 14 C6-C 20 Alkyl; R 14 ' is C6-C 20 Alkyl; R 15 C6-C 20 alkyl; and n is 2, 3 or 4.

[0082] In other aspects, the ionizable lipid of formula (VIII) has the following structure:

[0083]

[0084] In one aspect, the present disclosure relates to an ionizable lipid of formula (IX)

[0085]

[0086] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0087] R 1 C6-C 20 Alkenyl; R9 H, C1-C6 alkyl or –(CH2) n OH; R 12 C6-C 20 and n is 2, 3 or 4.

[0088] In other aspects, the ionizable lipid of formula (IX) has one of the following structures:

[0089]

[0090] In one aspect, the present disclosure relates to an ionizable lipid of formula (X)

[0091]

[0092] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0093] L 4 L is absent or is C1-C6 alkylene; 5 does not exist or is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 2 C6-C 20 Alkyl; R 2 ' is C6-C 20 Alkyl; R 9 H, C1-C6 alkyl or –(CH2) n OH; R 12 C6-C 20 and n is 2, 3 or 4.

[0094] In other aspects, the ionizable lipid of formula (X) has one of the following structures:

[0095]

[0096] In one aspect, the present disclosure relates to an ionizable lipid of formula (XI)

[0097]

[0098] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0099] L 4 L is absent or is C1-C6 alkylene; 5 does not exist or is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 2 C6-C 20 Alkyl; R 2 ' is C6-C20 Alkyl; R 12 C6-C 20 Alkenyl; R 13 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.

[0100] In other aspects, the ionizable lipid of formula (XI) has the following structure:

[0101]

[0102] In one aspect, the present disclosure relates to an ionizable lipid of formula (XII)

[0103]

[0104] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0105] L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 7 C4-C 20 Alkyl; R 7 ' is C4-C 20 Alkyl; R 8 C4-C 20 Alkyl; R 8 ' is C4-C 20 Alkyl; R 13 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.

[0106] In other aspects, the ionizable lipid of formula (XII) has the following structure:

[0107]

[0108] In one aspect, the present disclosure relates to an ionizable lipid of formula (XIII)

[0109]

[0110] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0111] L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 7 C4-C 20Alkyl; R 7 ' is C4-C 20 Alkyl; R 8 C4-C 20 Alkyl; R 8 ' is C4-C 20 Alkyl; R 13 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.

[0112] In some embodiments, L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 7 C4-C 20 Alkyl; R 7 ' is a C4-C20 alkyl group; R8 is a C4-C20 alkyl group; R8' is a C4-C20 alkyl group; and in formula (XIII), R13 is H.

[0113] In some embodiments, 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 in formula (XIII), R13 is methyl.

[0114] In some embodiments, 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 in formula (XIII), n is 2.

[0115] In some embodiments, 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 in formula (XIII), q is 3.

[0116] Illustrative examples of ionizable lipids of formula (XIII) may include, but are not limited to, the following:

[0117]

[0118] In one embodiment, the ionizable lipid has formula (XIV)

[0119]

[0120] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0121] 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.

[0122] In some embodiments, L1 is C1-C6 alkylene; L2 is C1-C8 alkylene; R3 is methyl; R4 is methyl; R6 is C4-C20 alkyl; and in formula (XIV), R7 is C4-C20 alkyl.

[0123] Illustrative examples of ionizable lipids of formula (XIV) may include, but are not limited to, the following

[0124]

[0125] In one embodiment, the ionizable lipid has formula (XV)

[0126]

[0127] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0128] 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 is 1; p2 is absent or is 1; and q is 2, 3 or 4.

[0129] In some embodiments, 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 does not exist; and in formula (XV), p2 does not exist.

[0130] In some embodiments, 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 in formula (XV), p2 is 1.

[0131] In some embodiments, 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 does not exist; and in formula (XV), p2 does not exist.

[0132] In some embodiments, 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 in formula (XV), p2 is 1.

[0133] In some embodiments, 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 not present; and in formula (XV), p2 is not present.

[0134] In some embodiments, 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 in formula (XV), p2 is 1.

[0135] Illustrative examples of ionizable lipids of formula (XV) may include, but are not limited to, the following:

[0136]

[0137] In one embodiment, the ionizable lipid has formula (XVI)

[0138]

[0139] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0140] 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, C1-C6 alkyl, –(CH2)nOH or –(CH2)qN(CH3)2; n is 2, 3 or 4; p1 is absent or is 1; p2 is absent or is 1; and q is 2, 3 or 4.

[0141] In one embodiment, the ionizable lipid has formula (XVI-A):

[0142]

[0143] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0144] L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 6 C4-C 20 Alkyl; R 6 ' is C4-C 20 Alkyl; R 7 C4-C 20 Alkyl; R 7 ' is C4-C 20 Alkyl; R 13 H, C1-C6 alkyl, –(CH2) n OH or –(CH2) q N(CH3)2; n is 2, 3 or 4; p1 is absent or is 1; p2 is absent or is 1; and q is 2, 3 or 4.

[0145] In some embodiments, L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 6 C4-C 20 Alkyl; R 6 ' is C4-C 20 Alkyl; R 7 C4-C 20 Alkyl; R7 ' is C4-C 20 Alkyl; R 13 is H; p1 is absent or is 1; and in formula (XVI) and formula (XVI-A), p2 is absent or is 1.

[0146] In some embodiments, L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 6 C4-C 20 Alkyl; R 6 ' is C4-C 20 Alkyl; R 7 C4-C 20 Alkyl; R 7 ' is C4-C 20 Alkyl; R 13 is methyl; p1 is absent or is 1; and in Formula (XVI) and Formula (XVI-A), p2 is absent or is 1. In other embodiments, L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 6 C4-C 20 Alkyl; R 6 ' is C4-C 20 Alkyl; R 7 C4-C 20 Alkyl; R 7 ' is C4-C 20 Alkyl; R 13 for –(CH2) n OH; n is 4; p1 is absent; and in formula (XVI) and formula (XVI-A), p2 is absent.

[0147] In some embodiments, L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 6 C4-C 20 Alkyl; R 6 ' is C4-C 20 Alkyl; R 7 C4-C 20 Alkyl; R 7 ' is C4-C 20 Alkyl; R 13 for –(CH2) n OH; n is 4; p1 is 1; and in Formula (XVI) and Formula (XVI-A), p2 is 1.

[0148] Illustrative examples of ionizable lipids of formula (XVI) and formula (XVI-A) may include, but are not limited to, the following:

[0149]

[0150] In one embodiment, the ionizable lipid of formula (IC):

[0151]

[0152] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; L 3 is C1-C8 alkylene; R 3 and R 4 Each is independently H, C1-C4 alkyl or –(CH2) n OH; R 6 C4-C 20 Alkyl; R 7 C4-C 20 Alkyl; R 8 C4-C 20 Alkyl; R 10 C4-C 20 alkyl; and n is 2, 3 or 4. In some embodiments, the ionizable lipid has the following structure:

[0153]

[0154] In one embodiment, the ionizable lipid of formula (XXII):

[0155]

[0156] or a pharmaceutically acceptable salt or stereoisomer thereof, 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 3 is C1-C8 alkylene; L 3 ' is C1-C8 alkylene; R 3 and R 4 Each is independently H, C1-C4 alkyl, -CH2-cyclopropyl or –(CH2) n OH; R 6 C4-C 20 Alkyl; R 6 ' is C4-C 20 Alkyl; R 7 C4-C 20 Alkyl; R 7 ' is C4-C20 Alkyl; R 8 C4-C 20 Alkyl; R 8 ' is C4-C 20 Alkyl; R 10 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.

[0157] In some embodiments, the ionizable lipid has the structure:

[0158]

[0159] In one aspect, the present disclosure relates to a lipid-nanoparticle composition comprising an ionizable lipid of any one of formula (I) to formula (XXII). Throughout the disclosure herein, a "lipid-nanoparticle composition" may refer to a composition comprising lipid nanoparticles or to the lipid nanoparticles themselves. The lipid-nanoparticle composition may further comprise a helper lipid, a stabilizing lipid, a structural lipid, and an active agent, wherein the active agent is a nucleic acid, a small molecule, a protein or a peptide, or a combination thereof. In an embodiment, the lipid-nanoparticle composition does not include a stabilizing lipid. In an embodiment, the lipid-nanoparticle composition does not include a lipid conjugated to polyethylene glycol (PEG-lipid).

[0160] In some aspects, 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-distearo ... choline (DSPC), 1,2-di(undecanoyl)-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), l-oleoyl-2-cholesterol hemisuccinyl-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-di(docosahexaenoyl)-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dialinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG) and mixtures thereof.

[0161] In some aspects, the stabilizing lipid in the lipid-nanoparticle composition is 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG) having an average PEG molecular weight of about 2000 Daltons. In some aspects, the stabilizing lipid is a polysarcosine-lipid conjugate. In some embodiments, the polysarcosine-lipid conjugate does not associate with the 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 does not associate with the RNA to form RNA particles in the lipid-nanoparticle composition.

[0162] In some aspects, the structural lipid in the lipid-nanoparticle composition is selected from the group consisting of cholesterol, cholesterol derivatives, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof.

[0163] In some aspects, the nucleic acid is selected from the group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), micro RNA (miRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), guide RNA (gRNA), plasmid DNA (pDNA), antisense oligodeoxynucleotides (ODN), RNA or DNA vaccines, and mixtures thereof.

[0164] In some aspects, nucleic acid or mRNA is self-amplification RNA. In some aspects, nucleic acid or mRNA is polycistronic RNA. In some aspects, nucleic acid or mRNA is self-amplification polycistronic RNA. In some aspects, nucleic acid or mRNA is circular RNA. In some aspects, nucleic acid or mRNA expresses protein or peptide. In some aspects, the protein or peptide expressed from nucleic acid or mRNA is antibody, human antibody, camel antibody, nano antibody, humanized antibody, bispecific antibody, enzyme, genome editing enzyme or nuclease, growth factor, cytokine, chemokine, small molecule-simulation peptide, transcription factor, structural molecule, signal molecule, reprogramming factor, vaccine antigen or their combination. In some aspects, mRNA encodes a protein or peptide that works in the cell. In some aspects, mRNA encodes at least one reprogramming factor.

[0165] In some aspects, the protein or peptide expressed from nucleic acid or mRNA is at least one extracellular matrix protein. In certain embodiments, the extracellular matrix protein is collagen, laminin, elastin, fibronectin, integrin, tenascin, proteoglycan, fibrin or a combination thereof. In certain embodiments, 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 certain embodiments, collagen is collagen VII. In certain embodiments, collagen VII is used in a method for restoring, treating, remodeling or improving skin or extracellular matrix. In certain embodiments, collagen VII is used in a method for wound healing.

[0166] In some aspects, the protein or peptide expressed from nucleic acid or mRNA is a growth factor, a 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, angiogenin, TPO, BMP, HGF, BDNF, GDF, HGH (growth hormone), neurotrophin, MSF, SGF, GDF (including GDF11), TGF (including TGF-b), 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.

[0167] In some aspects, the protein or peptide expressed from nucleic acid or mRNA is a human antibody, a humanized antibody, a camel antibody, a companion animal antibody or a nanobody. In some embodiments, the protein or peptide expressed from 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 nucleic acid or mRNA works in the cell. In some aspects, the protein or peptide expressed from nucleic acid or mRNA is secreted.In some embodiments, the antibody is at least one of or is substantially similar to trastuzumab, grofituzumab, miliguzumab, sometuximab, nesecvir, tremelimumab, terituzumab, doramuzumab, sepelizumab, lecanezumab, tislelizumab, panplizumab, sintilimab, teplizumab, toripalizumab, obotuzumab, retivanlimab, ubrituximab, inomucomab, opotuzumab, nasolizumab, motuzumab, tesagvir, celagavir, relalizumab, tebentafusip, faricizumab, sutilizumab, sotovir, rendavimab, casprevir, edema Midevimab, tezelumab, tesofumab, ervantumab, anifrumumab, lantuximab, bimegizumab, trorocirumab, evuximab, saccitumomab, tetumumab, isatuximab, epinephrine, dotalimumab, ansvirmab, makituximab, nasituximab, atevimab, mateviromab, ociviromab, mabetuximab, tancitumomab, satelizumab, inbilizumab, enfortuzumab, rizanlizumab, bucitumomab, polotuzumab, risankizumab, romozumab, calicizumab, revulizumab, imatinib, cemiplizumab, remanezumab, mosetumab , gacanezumab, ranarumab, moglizumab, erenumab, tirizizumab, ibalizumab, brosuzumab, durvalumab, emicizumab, benralizumab, ocrelizumab, guselkumab, intuzumab, salizumab, dupilumab, avelumab, bodalumab, atezolizumab, belotuzumab, olaratumumab, reslizumab, otuximab, ixekizumab, datuximab, elotuzumab, necituzumab, idarucizumab, alirocumab, mepolizumab, evolocumab, datuximab, secukinumab, nivolumab, belintumumab, pembrolizumab, Ramuciumab, vedolizumab, cetuximab, obotuzumab, ranikumab, pertuzumab, brentuximab, belimumab, ipilimumab, denosumab, tocilizumab, ofatumumab, canakinumab, golimumab, ustekinumab, certolizumab pegol, catuximab, eculizumab, ranibizumab, panitumumab, natalizumab, bevacizumab, cetuximab, efalizumab, omalizumab, tositumomab, ibrutinib, adalimumab, alemtuzumab, gemtuzumab, infliximab, palivizumab, basiliximab, daclizumab, rituximab, abciximab, edrecolomab, nebacumab or muromonab. In some embodiments, the protein or peptide is used in a method of treating a human or veterinary disease.

[0168] 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 within the cell.

[0169] In some aspects, the protein or peptide is an antibody, a humanized antibody, a bispecific antibody, an enzyme, a genome editing enzyme or a nuclease, a growth factor, a cytokine, a chemokine, a small molecule mimetic peptide, a transcription factor, a structural molecule, a signal transduction molecule, a reprogramming factor, a vaccine antigen, or a combination thereof. In some aspects, the protein or peptide works in a cell.

[0170] In some aspects, the small molecules, proteins or peptides incorporated into the lipid nanoparticles and / or delivered by the lipid nanoparticle compositions are components of an "artificial niche" for maintaining the quiescence of progenitor cells. In some embodiments, the artificial niche components are selected from the group consisting of: elcatonin; MGCD-265, JNJ-7706621, forskolin, fomastatin, SB203580, SU5402, TGF-β, insulin-transferrin-selenium and combinations thereof. These and other artificial niche groups are disclosed in U.S. Pat. No. 10,688,136, which is incorporated herein by reference.

[0171] In some aspects, the present disclosure relates to a pharmaceutical composition comprising a lipid-nanoparticle composition and a pharmaceutically acceptable carrier thereof.

[0172] In some aspects, 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, reproductive 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 restored, including but not limited to epithelial cells (e.g., squamous, cuboidal, columnar and pseudostratified epithelial cells), endothelial cells (e.g., venous, arterial and lymphatic endothelial cells) and cells of the connective tissue, muscle and nervous system. Such cells may 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. cells and / or tissues from the kidney, liver, pancreas, stomach, spleen, gall bladder, intestine, bladder, lung, prostate, breast, genitourinary tract, pituitary cells, oral cavity, esophagus, skin, hair, nails, thyroid, parathyroid glands, adrenal glands, eyes, nose, or brain.

[0173] In some aspects, the cell is selected from fibroblasts, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells. In embodiments, the cell is a fibroblast. In embodiments, the cell is an endothelial cell. In embodiments, the cell is a chondrocyte. In embodiments, the cell is a skeletal muscle stem cell. In embodiments, the cell is a keratinocyte. In embodiments, the cell is a mesenchymal stem cell. In embodiments, the cell is a corneal epithelial cell.

[0174] In some aspects, 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, lymphocytes are T cells, B cells or natural killer (NK) cells. In some embodiments, lymphocytes are tumor infiltrating lymphocytes.

[0175] In some embodiments, lymphocytes are T cells. In some embodiments, T cells are cytotoxic T cells (CD8+), helper T cells (CD4+), suppressive or regulatory T cells (Treg), memory T cells, natural killer T cells (NKT cells) or γδT cells. In other embodiments, helper T cells are Th1, Th2, Th17, Th9 or Tfh T cells. In some embodiments, 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 suppression or regulation T cells of the present technology are FOXP3+T cells or FOXP3-T cells. In some embodiments, NKT cells are subsets of CD1d-restricted T cells.

[0176] In some embodiments, the granulocytes of the present technology are neutrophils, eosinophils, basophils, or mast cells.

[0177] 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.

[0178] In other embodiments, the immune cell is a monocyte, a macrophage, a microglia, or a dendritic cell.

[0179] In some embodiments, the methods and compositions described herein can be used, wherein 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 or continuously with the methods of engineered cells (including engineered immune cells) so that these methods are carried out before, during and / or after the cells are engineered. In some embodiments, the methods and compositions described herein are used to engineer cells (including engineered immune cells). In some embodiments, such engineering includes engineering so that cells express chimeric antigen receptors, such as immune cells expressing chimeric antigens. In some embodiments, such chimeric antigen receptors target CD19, CD30, CD33, CD123, FLT3, BCMA, GD2 or any other antigen suitable for immunotherapy. In some embodiments, such engineering includes engineering cells (including immune cells) to express other proteins or peptides, such as growth factors and cytokines. In some embodiments, the cytokines include IL-15. In some embodiments, such engineering of cells (such as immune cells) is performed in vitro, for example, in the manufacture of cell therapy products (such as autologous or allogeneic chimeric antigen receptor (CAR)-T, CAR-NK, CAR-M or CAR-NKT cells). In some embodiments, the CAR-NKT cells provided herein target GD2 via a chimeric antigen receptor and are engineered to express IL-15. In such embodiments, the immune cell recovery methods described herein are performed in vitro during or after the manufacture of cell therapy products. In other embodiments, such engineering of cells and / or immune cells is performed in vitro, for example, in the so-called "in situ" production of CAR engineered cells. In such embodiments, RNA and / or mRNA encoding CAR or growth factors or cytokines contained in lipid compositions or lipid-nanoparticle compositions disclosed herein are injected into a subject or patient, for example, for CAR engineering of immune cells (such as T cells, NK cells, macrophages, tumor infiltrating lymphocytes, dendritic cells and / or "in situ" NKT cells) of the patient, i.e., in the patient, in vitro transfection can be performed without removing cells. In such embodiments, the immune cell recovery method described herein is also performed in vivo, wherein the mRNA encoding one or more reprogramming factors is injected into the patient before, at the same time or after mRNA encoding CAR or other cell engineering molecules. In some embodiments, lipids and lipid-nanoparticle compositions disclosed herein are selected for targeted delivery to any cell in the body, including immune cells, such as T cells, NK cells, macrophages, tumor infiltrating cells, dendritic cells and / or NKT cells in vivo.In other embodiments, in vivo treatment is performed in the absence of any other in vivo cell engineering to enhance or restore the effectiveness of the immune system and treat diseases associated with immune dysfunction or disorder, such as improving the effectiveness of the immune system against cancer or infection or reducing inflammation.

[0180] In some embodiments, the immune cells to be restored are non-adherent cells, such as non-adherent immune cells. In some embodiments, non-adherent cells, including non-adherent immune cells, are treated, transiently reprogrammed, restored or manufactured in a manner where the cells remain non-adherent and do not adhere to a tissue culture matrix or form or produce cells or cell colonies that adhere to a tissue culture matrix. In some embodiments, the reprogramming interval and factors are selected so that the cells are restored and the cell characteristics are retained, where the cells remain non-adherent and do not adhere to a tissue culture matrix or form or produce cells or cell colonies that adhere to a tissue culture matrix. Thus, in some embodiments, the present technology provides lipid-containing compositions and lipid-nanoparticle compositions for delivering mRNA encoding at least one reprogramming factor for cell rejuvenation, wherein the cells, including 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 wherein the cells are rejuvenated and retain cellular properties and wherein the cells remain in suspension without adhering, nor do they become or generate adherent cells, become adherent or form adherent colonies.

[0181] In some embodiments, the methods described herein (including methods for restoring immune cells; methods for reversing, preventing or inhibiting the exhaustion of immune cells; or inducing the proliferation of immune cells) include applying 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times of the present disclosure to immune cells within a period of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. The mRNA containing the mRNA encoding at least one reprogramming factor or lipid-nanoparticle composition. For example, mRNA can be applied once on the first or second day of a five-day or six-day period, or can be applied once on the first day of a five-day or six-day period and once on the third day, or can be applied once within a one-day period. In some embodiments, 1, 2, 3, 4, 5 or 6 times of mRNA are applied to immune cells within a period of 1, 2, 3, 4, 5 or 6 days. In some embodiments, mRNA is applied after the immune cell activation step. In some embodiments, the immune cell activation step includes activating immune cells for 1, 2 or 3 days. In some embodiments, the immune cell activation step includes activating immune cells using at least one of CD3, CD28 and IL-2 to activate immune cells. In some embodiments, immune cells are activated with CD3 and CD28. In some embodiments, the mRNA administration phase occurs immediately after the immune cell activation step. In some embodiments, the mRNA administration phase 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 mRNA encoding the reprogramming factor is used to reverse the immune cell failure caused by the immune cell activation step. In some embodiments, the mRNA encoding the reprogramming factor is used to reverse the immune cell failure in the immune cells from the elderly patient or the donor. In some embodiments, mRNA is used during the manufacturing process for the manufacture of immune cells (e.g., CAR-T, CAR-M or CAR-NK cells) for transplantation.

[0182] In some embodiments, compared with using different mRNA delivery mechanisms, using lipids or lipid nanoparticles of the present disclosure for delivering mRNA results in enhanced recovery, proliferation, exhaustion recovery or prevention, therapeutic effect, antipathogenic effect, anticancer effect, anti-immunogenic effect or anti-inflammatory effect in cells treated or recovered using the methods or compositions herein. In some embodiments, such enhanced recovery, proliferation, exhaustion recovery or prevention, therapeutic effect, antipathogenic effect, anticancer effect, anti-immunogenic effect or anti-inflammatory effect are attributed to lower toxicity, immunogenicity and / or lower physiological effects on cells when compared with different delivery mechanisms. In some embodiments, different delivery mechanisms are electroporation, so that compared with using electroporation, using lipids or lipid-nanoparticle compositions of the present disclosure for delivering mRNA results in enhanced recovery, proliferation, exhaustion recovery or prevention, therapeutic effect, antipathogenic effect, anticancer effect, anti-immunogenic effect or anti-inflammatory effect in cells treated or recovered using the methods or compositions herein. Such improvements compared with electroporation can be attributed to reduced toxicity compared with electroporation or reduced physiological effects on cells.

[0183] In some aspects, the lipid or lipid-nanoparticle composition of the present disclosure is 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 composition of the present technology provides delivery of a therapeutic or diagnostic agent (such as a reprogramming factor) in a manner that achieves transient reprogramming of cells (such as skin cells or immune cells). In some embodiments, transient reprogramming of cells provides transient expression of a therapeutic or diagnostic agent (such as a reprogramming factor), wherein the agent is expressed in the cell for a duration sufficient to reprogram and / or restore without changing the properties of the cell, i.e., restore the skin or immune cell to exhibit characteristics or younger skin or immune cells while retaining the properties of the skin or immune cell.

[0184] In some aspects, the therapeutic agent is mRNA as disclosed herein. In some aspects, the lipid or lipid-nanoparticle composition of the present disclosure is used in the treatment or prevention of dermatological diseases or disorders, the treatment or prevention of diseases or disorders of the skin, or for cosmetic applications in the skin, including the application of a lipid-containing composition or lipid-nanoparticle composition comprising at least one therapeutic agent or diagnostic agent. In some aspects, the lipid or lipid-nanoparticle composition of the present disclosure is used in the treatment or prevention of dermatological diseases or disorders or diseases or disorders of the skin, including the application of a lipid-containing composition or lipid-nanoparticle composition comprising at least one therapeutic agent or diagnostic agent. In some aspects, the therapeutic agent is mRNA as disclosed herein. In some aspects, mRNA encodes at least one reprogramming factor. In some aspects, the lipid or lipid-nanoparticle composition of the present disclosure is used for cosmetic applications in the skin, including the application of a lipid-containing composition or lipid-nanoparticle composition comprising at least one therapeutic agent or diagnostic agent. In some aspects, the therapeutic agent is mRNA as disclosed herein. In some aspects, mRNA encodes at least one reprogramming factor. In some aspects, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for restoring skin, including applying a lipid-containing composition or lipid-nanoparticle composition comprising a therapeutic agent or a diagnostic agent. In some aspects, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for restoring skin, including applying a lipid-containing composition or lipid-nanoparticle composition comprising an mRNA encoding at least one reprogramming factor. In such methods, mRNA can be bound to lipids or contained in lipid nanoparticles. In some aspects, such methods further include transfecting skin cells with a lipid-containing composition or lipid-nanoparticle composition to deliver mRNA. In some aspects, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for restoring skin, including applying a lipid-containing composition or lipid-nanoparticle composition comprising an mRNA encoding at least one reprogramming factor, to achieve restoration of skin while retaining cell properties. In such methods, mRNA can be bound to lipids or contained in lipid nanoparticles. In some aspects, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for restoring skin, comprising applying to skin cells a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor, wherein expressing at least one reprogramming factor in skin cells results in increased fibroblast proliferation and retains skin cell properties. In some aspects, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for increasing skin thickness, comprising applying a lipid-containing composition or lipid-nanoparticle composition comprising a therapeutic agent or diagnostic agent. In some aspects, the therapeutic agent is mRNA.In some aspects, lipids or lipid-nanoparticle compositions of the present disclosure are used in methods for increasing skin thickness, including applying to skin cells a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor, wherein expressing at least one reprogramming factor in skin cells causes increased skin thickness and retains skin cell properties. In some aspects, lipids or lipid-nanoparticle compositions of the present disclosure are used in methods for increasing skin elasticity, including applying a lipid-containing composition or lipid-nanoparticle composition comprising a therapeutic agent or diagnostic agent. In some aspects, the therapeutic agent is mRNA. In some aspects, lipids or lipid-nanoparticle compositions of the present disclosure are used in methods for increasing skin elasticity, including applying to skin cells a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor, wherein expressing at least one reprogramming factor in skin cells causes increased skin elasticity and retains skin cell properties. In such methods, mRNA can be bound to lipids or contained in lipid nanoparticles.

[0185] In some aspects, lipids or lipid-nanoparticle compositions of the present disclosure are used in methods of wound healing, including administering lipid-containing compositions or lipid-nanoparticle compositions comprising therapeutic agents or diagnostic agents. In some aspects, lipids or lipid-nanoparticle compositions of the present disclosure are used in methods of wound healing, including administering lipid-containing compositions or lipid-nanoparticle compositions comprising mRNA encoding at least one reprogramming factor. In such methods, mRNA can be bound to lipids or contained in lipid nanoparticles. In some aspects, lipids or lipid-nanoparticle compositions of the present disclosure are used in methods of wound healing, wherein lipid-containing compositions or lipid-nanoparticle compositions deliver mRNA encoding at least one reprogramming factor to achieve wound healing while retaining cell properties. In such methods, mRNA can be bound to lipids or contained in lipid nanoparticles. In some aspects, lipids or lipid-nanoparticle compositions of the present disclosure are used in methods of wound healing, including administering lipid-containing compositions or lipid-nanoparticle compositions comprising mRNA encoding at least one reprogramming factor to skin cells, wherein expressing at least one reprogramming factor in skin cells results in increased fibroblast proliferation. In such methods, the mRNA can be bound to a lipid or contained within a lipid nanoparticle.

[0186] In some aspects, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for treating or preventing a dermatological disease or disorder, treating or preventing a skin disease or disorder, or for cosmetic applications in the skin, including applying a lipid-containing composition or a lipid nano-particle composition comprising a therapeutic agent to achieve the reversal of at least one skin aging marker. In some aspects, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for restoring the skin, including applying a lipid-containing composition or a lipid-nanoparticle composition to deliver an mRNA encoding at least one reprogramming factor to the skin, thereby achieving the reversal of at least one skin aging marker, while retaining cell properties. In some aspects, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for wound healing, including applying a lipid-containing composition or a lipid-nanoparticle composition to deliver an mRNA encoding at least one reprogramming factor to skin cells, thereby achieving the reversal of at least one skin aging marker, while retaining cell properties. In some embodiments, the reversal of at least one skin aging marker refers to the generation of restored cells that express at least one skin aging marker in a manner similar to the expression of the marker seen in young skin cells compared to aged skin cells.

[0187] In some aspects, 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 aspects, 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 aspects, the reversal of at least one skin aging marker is an upregulation of mRNA or protein expression of PRSS23, LOXL4, MMP11, ELN, or any combination thereof. In some aspects, the reversal of at least one skin aging marker is an upregulation of mRNA or protein expression of PRSS23. In some aspects, the reversal of at least one skin aging marker is an upregulation of mRNA or protein expression of LOXL4. In some aspects, the reversal of at least one skin aging marker is an upregulation of mRNA or protein expression of MMP11. In some aspects, the reversal of at least one skin aging marker is an upregulation of mRNA or protein expression of ELN. In some aspects, the reversal of at least one skin aging marker is a downregulation of mRNA or protein expression of MMP3, MMP1, SOD2, or any combination thereof. In some aspects, the reversal of at least one skin aging marker is a downregulation of mRNA or protein expression of MMP3. In some aspects, the reversal of at least one skin aging marker is a downregulation of mRNA or protein expression of MMP1.In some aspects, the reversal of at least one skin aging marker is downregulation of mRNA or protein expression of SOD2. In some aspects, the 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.

[0188] In some aspects, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for treating or preventing a dermatological disease or disorder, treating or preventing a skin disease or disorder, or for cosmetic applications in the skin, including applying 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 aspects, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for increasing skin thickness, including applying to skin cells a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor, wherein expressing at least one reprogramming factor in skin cells results in increased skin thickness and retains skin cell properties. In some aspects, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for increasing skin elasticity, including applying to skin cells a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor, wherein expressing at least one reprogramming factor in skin cells results in increased skin elasticity and retains skin cell properties. In some aspects, the lipid or lipid-nanoparticle composition of the present disclosure is used in a method for restoring skin, including applying 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 lipid or lipid-nanoparticle composition of the present disclosure is used in a method for restoring skin, including applying 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 retaining cell properties. In some aspects, at least one skin quality marker includes skin thickness. In some aspects, epidermal thickness increases. In some aspects, at least one skin quality marker is skin elasticity. In some aspects, skin elasticity increases. In some aspects, 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 skilled in the art.

[0189] In some aspects, lipid or lipid-nanoparticle composition of the present disclosure is used in the method for wound healing, including applying to skin cells a lipid composition or lipid-nanoparticle composition comprising the mRNA encoding at least one reprogramming factor, to achieve the improvement of at least one skin quality marker. In some aspects, 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, secretory phosphoprotein 1, cartilage oligomeric matrix protein, MMP1, MMP3, MMP12, SOD2 or any combination thereof. In some aspects, the improvement of at least one skin quality marker is the mRNA or protein expression of MMP1, MMP3, MMP12, SOD2 or any combination thereof down-regulation. In some aspects, the improvement of at least one skin quality marker is the increase 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, secretory phosphoprotein 1, cartilage oligomeric matrix protein or any combination thereof. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of collagen. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of type I collagen. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of type III collagen. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of type IV collagen. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of type V collagen. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of type VI collagen. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of type XI collagen. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of type XI collagen. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of elastin. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of microfibril-associated protein 5. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of periostin. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of versican. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of connective tissue growth factor.In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of lysyl oxidase. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of SPARC. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of secretory phosphoprotein 1. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of cartilage oligomeric matrix protein. In some aspects, the improvement of at least one skin quality marker is the decrease of mRNA or protein expression of MMP3, MMP1, SOD2 or any combination thereof. In some aspects, the improvement of at least one skin quality marker is the decrease of mRNA or protein expression of MMP3. In some aspects, the improvement of at least one skin quality marker is the decrease of mRNA or protein expression of MMP1. In some aspects, the improvement of at least one skin quality marker is the decrease of mRNA or protein expression of SOD2. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of collagen VII and elastin. In some aspects, the improvement in at least one skin quality marker is upregulation of mRNA or protein expression of at least one of collagen VII and elastin; downregulation of mRNA or protein expression of at least one of MMP3, MMP1, SOD2; or any combination thereof.

[0190] In some aspects, the lipid-nanoparticle composition of the present invention or the composition containing the lipid of the present invention is topically applied to the skin. In some aspects, the lipid-nanoparticle composition of the present invention or the composition containing the lipid of the present invention is applied to the skin in the form of an ointment, cream or salves. In some aspects, the lipid-nanoparticle composition of the present invention or the composition containing the lipid of the present invention is applied to the skin via dermal, intradermal or subcutaneous injection. In some aspects, the lipid-nanoparticle composition of the present invention or the composition containing the lipid of the present invention is applied to the skin via a gel. In some aspects, the lipid-nanoparticle composition of the present invention or the composition containing the lipid of the present invention is applied in vivo, in vitro or ex vivo. In some aspects, the lipid-nanoparticle composition of the present invention or the composition containing the lipid of the present invention is applied in vivo. In some aspects, the lipid-nanoparticle composition of the present invention or the composition containing the lipid of the present invention is used for human or animal subjects.

[0191] In some aspects, the lipid-nanoparticle composition of the present disclosure or the composition containing the lipid of the present disclosure transfects skin cells to deliver at least one therapeutic agent or diagnostic agent to skin cells. In some aspects, the therapeutic agent is a nucleic acid. In some aspects, the therapeutic agent is an mRNA. In some aspects, the therapeutic agent is a combination of mRNA and siRNA. In some aspects, the therapeutic agent is a combination of mRNA and miRNA. In some aspects, the skin cells are keratinocytes, melanocytes, Langerhans cells, follicle cells, fibroblasts, endothelial cells, smooth muscle cells, Merkel cells, basal cells, squamous cells, apocrine cells, eccrine cells, sebaceous gland cells, lymphatic endothelial cells or their combinations. In some aspects, select lipids or lipid-nanoparticle compositions to provide selective transfection of specific cell types or multiple cell types. In some aspects, select lipids or lipid-nanoparticle compositions to provide diffusion in the skin or in at least one layer of the skin.

[0192] In some aspects, the dermatological diseases or disorders or diseases or disorders of the skin treated or prevented using the lipid compositions or lipid-nanoparticle compositions of the present disclosure are dermatoporosis or chronic wounds. In some aspects, dermatoporosis or chronic wounds are diabetic ulcers, ischemic ulcers and pressure sores. In some aspects, the dermatological diseases or disorders or diseases or disorders of the skin treated or prevented using the lipid compositions or lipid-nanoparticle compositions of the present disclosure are inflammatory skin diseases. In some aspects, inflammatory skin diseases are psoriasis, atopic dermatitis, vitiligo, alopecia areata or hidradenitis suppurativa. In some aspects, the dermatological diseases or disorders or diseases or disorders of the skin treated or prevented using the lipid compositions or lipid-nanoparticle compositions of the present disclosure are hair disorders. In some aspects, hair disorders are non-scarring or scarring alopecia, graying of hair, hirsutism. In some aspects, hair disorders are non-scarring alopecia, which is androgenic alopecia. In some aspects, scarring alopecia is lichen planus. In some aspects, the dermatological diseases or disorders or skin diseases or disorders treated or prevented using lipid compositions or lipid-nanoparticle compositions of the present disclosure are skin cancer. In some aspects, skin cancer is basal cell carcinoma, squamous cell carcinoma or actinic keratosis. In some aspects, the dermatological diseases or disorders or skin diseases or disorders treated or prevented using lipid compositions or lipid-nanoparticle compositions of the present disclosure are nodular prurigo, acne, rosacea or solar lentigo. In some aspects, the method for treating any one of dermatological diseases or disorders or diseases includes applying a composition containing a lipid of the present disclosure or a lipid-nanoparticle composition containing a therapeutic agent of the present disclosure. In some aspects, the therapeutic agent is mRNA. In some aspects, the therapeutic agent is mRNA encoding antibodies, human antibodies, humanized antibodies, nanobodies, camel antibodies, bispecific antibodies, enzymes, genome editing enzymes or nucleases, growth factors, cytokines, chemokines, transcription factors, structural molecules, signaling molecules, reprogramming factors. In some aspects, the therapeutic agent is an mRNA encoding a protein or peptide that functions within a cell. In some aspects, the therapeutic agent is an mRNA encoding at least one reprogramming factor.

[0193] In some aspects, the lipid composition or lipid-nanoparticle composition of the present disclosure is used in a method for wound healing, wherein the wound is dermatoporosis or a chronic wound. In some aspects, dermatoporosis or a chronic wound is a diabetic ulcer, an ischemic ulcer, and a pressure sore. In some aspects, the lipid composition or lipid-nanoparticle composition of the present disclosure is used in a method for wound healing, wherein the wound is a lesion from skin cancer. In some aspects, skin cancer is basal cell carcinoma, squamous cell carcinoma, or actinic keratosis. In some aspects, any wound healing method in the above-mentioned wound healing method includes applying a composition containing a lipid of the present disclosure or a lipid-nanoparticle composition containing a therapeutic agent of the present disclosure. In some aspects, the therapeutic agent is mRNA. In some aspects, the therapeutic agent is an mRNA encoding an antibody, a human antibody, a humanized antibody, a nanobody, a camel antibody, a bispecific antibody, an enzyme, a genome editing enzyme or a nuclease, a growth factor, a cytokine, a chemokine, a transcription factor, a structural molecule, a signaling molecule, a reprogramming factor. In some aspects, the therapeutic agent is an mRNA encoding a protein or peptide that works in a cell. In some aspects, the therapeutic agent is mRNA encoding at least one reprogramming factor.

[0194] In some aspects, some lipid-nanoparticle compositions of the present disclosure containing lipid formula (II) have higher transfection efficiency in skin cells and produce greater improvement in at least one skin quality marker when used to transfect at least one reprogramming factor compared to other lipid-nanoparticle compositions containing lipid formula (II) or to other lipid-nanoparticle compositions containing other lipids of the present disclosure. In some aspects, at least one skin quality marker is skin thickness, skin elasticity, transepidermal water loss, or any combination thereof.

[0195] In some aspects, lipid-nanoparticle compositions of the present disclosure or compositions containing lipids of the present disclosure transfect skin cells to deliver at least one therapeutic agent or diagnostic agent to eye cells. In some aspects, the therapeutic agent is a nucleic acid. In some aspects, the therapeutic agent is an mRNA. In some aspects, the therapeutic agent is a combination of mRNA and siRNA. In some aspects, the therapeutic agent is a combination of mRNA and miRNA. In some aspects, the eye cells are retinal pigment epithelial cells, ganglion cells, photoreceptor cells, rod cells, cone cells, choroid cells, keratocytes, conjunctival cells, corneal epithelial cells, ocular muscle cells or a combination thereof. In some aspects, select lipids or lipid-nanoparticle compositions to provide selective transfection of specific cell types or multiple cell types. In some aspects, select lipids or lipid-nanoparticle compositions to provide diffusion in the eye (such as in the vitreous humor or in the retina).

[0196] In some aspects, the lipid or lipid-nanoparticle compositions of the present disclosure are used in a method of treating or preventing an ocular disease or disorder or treating or preventing a disease or disorder of the eye comprising administering to the eye a lipid-containing composition or lipid-nanoparticle composition comprising a therapeutic agent.

[0197] In some aspects, the eye disease or condition treated or prevented using the lipid composition or lipid-nanoparticle composition of the present disclosure or the disease or condition of the eye treated is age-related macular degeneration, glaucoma, cataract, dry eye, diabetic retinopathy, vision loss, myopia, presbyopia, dry macular degeneration or wet macular degeneration. In some aspects, the method for treating any one of an eye disease or condition or disease includes administering a composition containing a lipid of the present disclosure or a lipid-nanoparticle composition of the present disclosure comprising a therapeutic agent. In some aspects, the therapeutic agent is mRNA. In some aspects, the therapeutic agent is an mRNA encoding an antibody, a human antibody, a humanized antibody, a nanobody, a camel antibody, a bispecific antibody, an enzyme, a genome editing enzyme or a nuclease, a growth factor, a cytokine, a chemokine, a transcription factor, a structural molecule, a signaling molecule, a reprogramming factor. In some aspects, the therapeutic agent is an mRNA encoding a protein or peptide that works in a cell. In some aspects, the therapeutic agent is an mRNA encoding at least one reprogramming factor.

[0198] Methods provided herein include using lipids or lipid-nanoparticle compositions to transfect cells with one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors, thereby producing recovered cells. The cells to be recovered can have any cell type. In an embodiment, cells are contacted with mRNA, exposed to mRNA, or transfected with mRNA for no more than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day or continue to be less than 1 day. In an embodiment, cells are contacted with mRNA, exposed to mRNA, or transfected with mRNA for no more than about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day or continue to be less than one day. In an embodiment, cells are contacted with mRNA, exposed to mRNA, or transfected with mRNA for no more than about 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day or continue to be less than one day. In an embodiment, the cell is contacted with mRNA, exposed to mRNA, or transfected with mRNA for no more than about 7, 6, 5, 4, 3, 2, or 1 day or for less than one day. In an embodiment, the cell is contacted with mRNA, exposed to mRNA, or transfected with mRNA for no more than about 5, 4, 3, 2, or 1 day or for less than one day. In an embodiment, at least one reprogramming factor is expressed from the transfected mRNA in the cell or the cell is exposed to at least one reprogramming factor expressed from the transfected mRNA, and it lasts no more than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day or for less than 1 day. In an embodiment, at least one reprogramming factor is expressed from the transfected mRNA in the cell or the cell is exposed to at least one reprogramming factor expressed from the transfected mRNA, and it lasts no more than about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day or for less than one day. In an embodiment, at least one reprogramming factor is expressed from a transfected mRNA in a cell or a cell is exposed to at least one reprogramming factor expressed from a transfected mRNA for at least about 2 days and no more than about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 days. In an embodiment, at least one reprogramming factor is expressed from a transfected mRNA in a cell or a cell is exposed to at least one reprogramming factor expressed from a transfected mRNA for no more than about 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day or for less than one day. In an embodiment, at least one reprogramming factor is expressed from a transfected mRNA in a cell or a cell is exposed to at least one reprogramming factor expressed from a transfected mRNA for at least about 2 days and no more than about 10, 9, 8, 7, 6, 5, 4, 3 or 2 days.In an embodiment, at least one reprogramming factor is expressed from a transfected mRNA in a cell, or a cell is exposed to at least one reprogramming factor expressed from a transfected mRNA, for no more than about 7, 6, 5, 4, 3, 2 or 1 day or for less than one day. In an embodiment, at least one reprogramming factor is expressed from a transfected mRNA in a cell, or a cell is exposed to at least one reprogramming factor expressed from a transfected mRNA, for at least about 2 days and no more than about 7, 6, 5, 4, 3 or 2 days. In an embodiment, at least one reprogramming factor is expressed from a transfected mRNA in a cell or a cell is exposed to at least one reprogramming factor expressed from a transfected mRNA, for no more than about 5, 4, 3, 2 or 1 day or for less than one day. In an embodiment, at least one reprogramming factor is expressed from a transfected mRNA in a cell or a cell is exposed to at least one reprogramming factor expressed from a transfected mRNA for at least about 2 days and no more than about 5, 4, 3 or 2 days. In an embodiment, the rejuvenated cell has a phenotype or activity spectrum similar to that of a young cell. The phenotypic or activity profile includes one or more of a transcriptomic profile, gene expression of one or more nuclear and / or epigenetic markers, proteolytic activity, mitochondrial health and function, SASP cytokine expression, and methylation landscape.

[0199] In some embodiments, the rejuvenated cells have a transcriptome profile that is 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, Elf1, Phf8, Pol2s2, Taf1, and Sin3a.

[0200] In some embodiments, the rejuvenated cells show increased gene expression compared to a reference value for one or more nuclear and / or epigenetic markers. In an embodiment, one or more nuclear and / or epigenetic markers are selected from Hplgamma, H3K9me3, lamina support protein LAP2α and SIRT1 protein. In an embodiment, the rejuvenated cells have a proteolytic activity more similar to the proteolytic activity of young cells. In an embodiment, the proteolytic activity is measured as increased cell autophagosome formation, increased chymosin-like proteasome activity or a combination thereof. In an embodiment, the rejuvenated cells show improved mitochondrial health and function compared to a reference value. In an embodiment, improved mitochondrial health and function are measured as increased mitochondrial membrane potential, reduced reactive oxides (ROS) or a combination thereof.

[0201] In some embodiments, the rejuvenated cells exhibit reduced expression of one or more SASP cytokines 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 an aged cell.

[0202] In an embodiment, the cell is restored by transient reprogramming with mRNA encoding one or more cell reprogramming factors, and the mRNA is transfected into the cell using lipids or lipid-nanoparticle compositions disclosed herein. In certain embodiments, transient reprogramming is completed by transfecting cells with non-integrative mRNA for no more than 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 certain embodiments, transient reprogramming is completed by transfecting cells with non-integrative mRNA 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. In certain embodiments, transient reprogramming is completed by transfecting cells with non-integrative mRNA for no more than about 9, 8, 7, 6, 5, 4, 3, 2 or 1 day or less than 1 day. In some embodiments, transient reprogramming is completed by transfecting cells with non-integrative mRNA for no more than about 6, 5, 4, 3, 2 or 1 days or less than 1 day. In some embodiments, transient reprogramming is completed by expressing at least one reprogramming factor from a non-integrative mRNA transfected in a cell or exposing the cell to at least one reprogramming factor expressed from a non-integrative mRNA transfected in a cell for no more than 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 some embodiments, transient reprogramming is completed by expressing at least one reprogramming factor from a non-integrative mRNA transfected in a cell or exposing the cell to at least one reprogramming factor expressed from a non-integrative mRNA transfected in a cell for at least 2 days and no more than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 days. In some embodiments, transient reprogramming is performed by expressing at least one reprogramming factor from a non-integrating mRNA transfected in a cell or exposing the cell to at least one reprogramming factor expressed from a non-integrating mRNA transfected in a cell 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. In some embodiments, transient reprogramming is performed by expressing at least one reprogramming factor from a non-integrating mRNA transfected in a cell or exposing the cell to at least one reprogramming factor expressed from a non-integrating mRNA transfected in a cell 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. In some embodiments, transient reprogramming is performed by expressing at least one reprogramming factor from a non-integrating mRNA transfected in a cell or exposing the cell to at least one reprogramming factor expressed from a non-integrating mRNA transfected in a cell for no more than about 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day or less than 1 day.In some embodiments, transient reprogramming is performed by expressing at least one reprogramming factor from a non-integrated mRNA transfected in a cell or exposing the cell to at least one reprogramming factor expressed from a non-integrated mRNA transfected in a cell for at least 2 days and no more than about 10, 9, 8, 7, 6, 5, 4, 3 or 2 days. In some embodiments, transient reprogramming is performed by expressing at least one reprogramming factor from a non-integrated mRNA transfected in a cell or exposing the cell to at least one reprogramming factor expressed from a non-integrated mRNA transfected in a cell for no more than about 7, 6, 5, 4, 3, 2 or 1 day or less than 1 day. In some embodiments, transient reprogramming is performed by expressing at least one reprogramming factor from a non-integrated mRNA transfected in a cell or exposing the cell to at least one reprogramming factor expressed from a non-integrated mRNA transfected in a cell for at least 2 days and no more than about 7, 6, 5, 4, 3 or 2 days. In some embodiments, transient reprogramming is performed by expressing at least one reprogramming factor from a non-integrating mRNA transfected in a cell or exposing the cell to at least one reprogramming factor expressed from a non-integrating mRNA transfected in a cell for no more than about 5, 4, 3, 2 or 1 day or less than 1 day. In some embodiments, transient reprogramming is performed by expressing at least one reprogramming factor from a non-integrating mRNA transfected in a cell or exposing the cell to at least one reprogramming factor expressed from a non-integrating mRNA transfected in a cell for at least 2 days and no more than about 5, 4, 3 or 2 days. In an embodiment, the transient reprogramming of cells eliminates various aging markers while avoiding complete dedifferentiation of cells into stem cells.

[0203] In the embodiments of the methods and compositions provided herein, cell age reversal or restoration is achieved by transient overexpression of one or more mRNAs encoding cell reprogramming factors delivered by lipids or lipid-nanoparticle compositions disclosed herein. Such cell reprogramming factors may include transcription factors, epigenetic remodeling agents, or small molecules that affect mitochondrial function, proteolytic activity, heterochromatin levels, histone methylation, laminar polypeptides, cytokine secretion, or aging. In an embodiment, cell reprogramming factors include one or more of OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG. In an embodiment, the cell reprogramming factors are applied in different molar ratios, for example, OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG are applied in a molar ratio of a:b:c:d:e:f, wherein a, b, c, d, e, and f can be all the same number (e.g., 1:1:1:1:1:1), some the same and some different numbers (e.g., 3:1:1:1:1:1, 2:1:1:1:1:1, 2:2:1:1:1:1, 2:2:2:1:1:1:1, 2:2: 2:2:1:1, 2:2:2:2:2:1, 3:3:3:3:2:2) or all different numbers (for example 6:4:5:3:2:1), and wherein a, b, c, d, e and f are each 1-7, i.e. 1-7:1-7:1-7:1-7:1-7:1-7 (or 1-7:1-7:1-7:1-7:1-7:1-7, 1-7:1-7:1-7:1-7, 1-7:1-7:1-7, 1-7:1-7 or 1-7:1 (in the case of combinations of less than 6 factors).

[0204] In an embodiment, the methods and compositions provided herein can be applied to any type of cells, tissues or organs that need to be restored. The methods and compositions disclosed herein can be used to restore cells in culture (e.g., in vitro or in vitro) to improve the function and efficacy for use in cell therapy. The cells used in the treatment of patients can be autologous or allogeneic. The cells can be derived from patients or matching donors, or they can be obtained from cell banks or derived from iPS cells. For example, in autologous ex vivo therapy, cells can be obtained directly from patients to be treated, transfected with mRNA encoding cell reprogramming factors, as described herein, and re-implanted in patients. Such cells can be obtained, for example, from biopsies or surgical operations performed on patients. Alternatively, in allogeneic ex vivo therapy, cells can be obtained from cell banks or cell lines derived from iPS cells, transfected with mRNA encoding cell reprogramming factors, as described herein, and re-implanted in patients. Alternatively, mRNA encoding cell reprogramming factors can be used to directly transfect cells that need to be restored in vivo.

[0205] In another aspect, provided herein are pharmaceutical compositions comprising rejuvenating cells obtained by transiently reprogramming cells for rejuvenating cells obtained by transfecting cells with lipid-containing compositions or lipid-nanoparticle compositions comprising one or more non-integrating messenger RNA encoding one or more cell reprogramming factors disclosed herein for no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive days. In another aspect, provided herein are pharmaceutical compositions comprising rejuvenating cells obtained by transiently reprogramming cells for rejuvenating cells obtained by transfecting cells with lipid-containing compositions or lipid-nanoparticle compositions comprising one or more non-integrating messenger RNA encoding one or more cell reprogramming factors disclosed herein for no more than 4, 5, 6, or 7 consecutive days.

[0206] In some embodiments, the lipid-containing compositions or lipid-nanoparticle compositions of the present disclosure are used to deliver mRNA expressing reprogramming factors, which provide more robust cell restoration because the reprogramming factors have been optimized to reduce any triggered immune response to the protein / polypeptide, increase the stability of the protein / polypeptide, and alter the protein / polypeptide activity, such as increased activity compared to the wild-type reprogramming factor.

[0207] In some embodiments, the methods of the present disclosure include administering the lipid-containing composition or lipid-nanoparticle composition comprising RNA of the present disclosure to a cell or 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 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 consecutive days of dosing interval. In some embodiments, the administration of the lipid-containing composition or lipid-nanoparticle composition comprising RNA of the present disclosure is performed at least once a day during the dosing interval. In some embodiments, administration is performed at a frequency less than once a day during the dosing interval, for example, once every two days, once every three days, once every four days, once every x days, where x is a number from 4 to 25. Thus, in such embodiments, for example, administration of a RNA-containing lipid-containing composition or lipid-nanoparticle composition of the present disclosure once every 5 days within a 5-day dosing interval means that the RNA is administered once within the interval, i.e., once within a total treatment period of 5 days, and administration of the RNA twice daily within a 5-day dosing interval means that the RNA is administered 10 times within the interval, i.e., 10 times within 5 days. In some embodiments, the methods of the present disclosure comprise administering a RNA-containing lipid-containing composition or lipid-nanoparticle composition of the present disclosure to a cell or subject, or treating or transfecting a cell with a RNA-containing lipid-containing composition or lipid-nanoparticle composition of the present disclosure for no more than 21, 18, 14, 10, 7, or 5 consecutive days. In some embodiments, the methods of the present disclosure include administering to cells or subjects a lipid-containing composition or lipid-nanoparticle composition comprising RNA of the present disclosure, or treating or transfecting cells with a lipid-containing composition or lipid-nanoparticle composition comprising RNA of the present disclosure. In some embodiments, the methods of the present disclosure include administering to cells or subjects a lipid-containing composition or lipid-nanoparticle composition comprising RNA of the present disclosure, or treating or transfecting cells with a lipid-containing composition or lipid-nanoparticle composition comprising RNA of the present disclosure. For no more than 14 consecutive days. In some embodiments, the methods of the present disclosure include administering to cells or subjects a lipid-containing composition or lipid-nanoparticle composition comprising RNA of the present disclosure, or treating or transfecting cells with a lipid-containing composition or lipid-nanoparticle composition comprising RNA of the present disclosure. For no more than 10 consecutive days. In some embodiments, the methods of the present disclosure include administering to cells or subjects a lipid-containing composition or lipid-nanoparticle composition comprising RNA of the present disclosure, or treating or transfecting cells with a lipid-containing composition or lipid-nanoparticle composition comprising RNA of the present disclosure. For no more than 7 consecutive days.In some embodiments, the methods of the present disclosure include administering to cells or subjects a lipid composition or lipid-nanoparticle composition comprising RNA of the present disclosure, or treating or transfecting cells with a lipid composition or lipid-nanoparticle composition comprising RNA of the present disclosure for no more than 5 consecutive days. In other embodiments, the exposure includes interrupting the exposure and repeating the exposure after the interruption. In some embodiments, the exposure, treatment, transfection, expression or administration includes exposure, treatment, transfection, expression or administration for continuous continuous between about 2-5 days, continuous between about 5-7 days, continuous between about 7-10 days, continuous between about 10-12 days, continuous between about 12-14 days, continuous between about 14-17 days, continuous between about 17-19 days or continuous between about 19-21 days, and in some embodiments, further includes interrupting the exposure and repeating the exposure after the interruption.

[0208] In some embodiments, the duration of exposure is controlled by a mechanism such as self-amplifying RNA, circular RNA, B18R and other decoys and / or an on / off switch such as L7Ae or its family members. In some embodiments, the repetition 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 times. For in vivo applications, the repetition can continue for any duration, for example, until the disease is successfully treated or cured, or throughout the life of the subject or patient. In some embodiments, the repetition is performed at any time after the interruption, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, up to 20 days, up to 30 days, up to 3 months, up to 6 months or up to 1 year after the interruption. An exposure cycle is considered to be a dosing interval, so that, for example, a series of exposure-interruption-repeat exposures comprises two dosing intervals.

[0209] In one embodiment, a compound having the structure of formula (XVII) is provided:

[0210]

[0211] or a stereoisomer, salt or tautomer thereof, G 1 , G 2 , R 21 , R 22 , R 23 , R 24 、m 1 and m 2 As defined herein.

[0212] In one embodiment, a compound having the structure of formula (XVIII) is provided:

[0213]

[0214] or a stereoisomer, salt or tautomer thereof, G 3 , G 4 , R 27 、m 1 and m 2 As defined herein.

[0215] In one embodiment, a compound having the structure of formula (XIX) is provided:

[0216]

[0217] or a stereoisomer, salt or tautomer thereof, R 29a , R 29b , R 30 and n are as defined herein.

[0218] Also provided are pharmaceutical compositions comprising one or more of the compounds of the foregoing formulae (XVII) to (XIX) and a therapeutic agent.

[0219] In other embodiments, provided is a method of treating a disease by administering a compound of the aforementioned formula (I) or a pharmaceutical composition comprising a compound of formula (XVII) to (XIX) to a subject in need thereof.

[0220] In one embodiment, a lipid nanoparticle is also provided, comprising one or more compounds of the aforementioned formulae (XVII) to (XIX) and a therapeutic agent comprising a nucleic acid.

[0221] Various aspects and embodiments will now be described more fully below. Such aspects and embodiments may take many different forms, and the exemplary aspects and embodiments disclosed herein should not be construed as limiting; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey its scope to those skilled in the art. DETAILED DESCRIPTION

[0222] The present disclosure relates, at least in part, to ionizable lipids that may provide certain advantages when used in nanoparticle compositions to deliver active or therapeutic agents, such as nucleic acids, into cells.

[0223] Ionizable lipids of the present disclosure comprise an ionizable group, one or more ester groups and one or more hydrophobic tail groups. The ionizable group is an amine-containing head group with an acid dissociation constant (pKa) higher than 7. Without wishing to be bound by any theory, it is believed that high pKa allows ionizable lipids to be positively charged at acidic pH (<6.0) and neutral at physiological pH (7.4). This in turn leads to a high encapsulation efficiency of nucleic acids at acidic pH. Ionizable lipids and other auxiliary lipids also interact with the negatively charged membrane of the endosome, which causes its membrane to rupture and therefore promotes the release of nucleic acids.

[0224] One or more ester groups in the ionizable lipids of the present disclosure confer biodegradability, as it is a key feature of the clinical transformation of ionizable lipids. The ester bond is included in the ionizable group to reduce accumulation and potential side effects, thereby causing the ionizable lipid to be degraded into non-toxic metabolites after successful delivery of intracellular cargo (e.g., nucleic acids, small molecules or peptides or proteins).

[0225] One or more hydrophobic tail groups are composed of aliphatic carbon chains. The aliphatic carbon chain may further include unsaturated bonds, such as double bonds or triple bonds. The hydrophobic tail may further include a branched tail. It is believed that the tail length and tail saturation can greatly affect the mobility and delivery efficiency of ionizable lipids. The molecular hypothesis is that due to the increase in the cross-section of the tail region, it is expected that the ionizable lipids of the present invention will produce a cone structure with enhanced endosome destruction ability. Since the ionizable lipids are protonated at the endosomal pH value, this in turn promotes endosomal release. The nanoparticle composition comprising ionizable lipids of the present invention is very useful for systemic delivery applications because they can show an extended circulation life and can mediate the expression of transfected genes or the silencing of target gene expression in vivo. The nanoparticle composition comprising ionizable lipids of the present invention is very useful for local delivery applications because their characteristics such as but not limited to ionization constant, size and surface charge can be adjusted for delivery and transfection to specific tissues.

[0226] The design of ionizable lipids is inspired by lipid-based natural products such as phospholipids and sphingomyelins, as well as other common compounds such as glycerides, to achieve better in vivo delivery and subsequent clearance. Such lipids are also believed to help improve transfection efficiency and cell selectivity.

[0227] The introduction of aromatic rings into the hydrophobic chains can introduce a more organized structure through π stacking, which can lead to better packing and stability of the LNPs. The aromatic rings also provide a facile approach for other structural modifications to change the cone shape by introducing more aliphatic chains and / or by changing the attachment points at different positions.

[0228] The use of heterocycles in the lipid head group is intended to provide more defined tapered lipids and better nucleic acid encapsulation.

[0229] I. definition

[0230] For convenience, certain terms employed in the specification, examples, and claims are collected here. 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 belongs.

[0231] Where a range of values ​​is provided, it is intended that each intervening value between the upper and lower limits of the range and any other stated or intervening value in the stated range be encompassed within the disclosure. For example, if a range of 1 mg to 8 mg is specified, it is intended that 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, and 7 mg, as well as a range of values ​​greater than or equal to 1 mg and a range of values ​​less than or equal to 8 mg, are expressly disclosed.

[0232] As used herein, the term "Cn-m alkyl" refers to a saturated hydrocarbon group that can be straight or branched. The alkyl group formally corresponds to an alkane in which one C-H bond is 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, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, etc.; higher homologues such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, etc. 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 halogen groups. In some embodiments, the alkyl group is optionally substituted with 1, 2, 3 or more hetero groups.

[0233] As used herein, the term "Cn-m alkenyl" refers to an unsaturated hydrocarbon group, which can be linear or branched, corresponding to an alkyl group with one or more carbon-carbon double bonds. The alkenyl group formally corresponds to an alkene, in which a CH bond is replaced by the connection point of the alkenyl group to the rest of the compound. The term "Cn-m alkenyl" refers to an alkenyl group with n to m carbons. Examples of alkenyl groups include, but are not limited to, chemical groups such as vinyl, propenyl, isopropenyl, n-butenyl, sec-butenyl. In some embodiments, the alkenyl 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 alkenyl group is optionally substituted by 1, 2, 3 or more halogen groups. In some embodiments, the alkenyl group is optionally substituted by 1, 2, 3 or more hetero groups.

[0234] As used herein, the term "Cn-m alkylene" refers to a divalent alkyl linking group. The alkylene group formally corresponds to an alkane, in which two CH bonds are replaced by the connection points of the alkylene group to the rest of the compound. The term "Cn-m alkylene" refers to an alkylene group having n to m carbon atoms. Examples of alkylene groups include, but are not limited to, second-1,2-diyl, third-1,3-diyl, third-1,2-diyl, fourth-1,4-diyl, fourth-1,3-diyl, fourth-1,2-diyl, 2-methyl-third-1,3-diyl, etc. In some embodiments, the alkylene group is optionally substituted by 1, 2, 3 or more halogen groups. In some embodiments, the alkylene group is optionally substituted by 1, 2, 3 or more hetero groups.

[0235] As used herein, the terms "approximately" and "about" refer to values ​​similar to the reference value. In certain embodiments, the terms "approximately" or "about" refer to a range of values ​​that are 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 than or less than) the reference value, unless otherwise specified or obvious from the context (unless the number will exceed 100% of a possible value). For example, when used in the context of the amount of the lipid component of the nanoparticle composition, "about" can mean + / -10% of the listed value. For example, a nanoparticle composition comprising a lipid component with about 40% of a given lipid can include 30%-50% of lipid.

[0236] As used herein, the terms "disease" and "disorder" may be used interchangeably or may be different in that a particular malady or disorder may have no known causative agent (and thus an etiology has not been determined), and therefore the particular malady or disorder has not been recognized as a disease, but only as an undesirable condition or syndrome in which clinicians have more or less identified a specific set of symptoms.

[0237] 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), with 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 "complete encapsulation" means that the nucleic acid in 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 achieved by Determine by measurement. is an ultrasensitive fluorescent nucleic acid stain for quantifying RNA in solution (available from Invitrogen Corporation; Carlsbad, California). "Fully encapsulated" also indicates that the lipid particles are serum stable, i.e., they do not rapidly break down into their component parts after 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.

[0238] As used herein, the term "halo" or "halogen" refers to F, Cl, Br or I.

[0239] As used herein, the term "hetero" refers to a heteroatom selected from oxygen, nitrogen and sulfur.

[0240] As used herein, the term "interfering RNA" refers to a single-stranded RNA (e.g., mature miRNA, circular RNA, guide RNA) or double-stranded RNA (i.e., duplex 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 when the interfering RNA and the target gene or sequence are located in the same cell (e.g., by mediating the degradation of an mRNA complementary to the interfering RNA sequence or inhibiting its translation). Therefore, interfering RNA refers to a single-stranded RNA complementary to a target mRNA sequence or a double-stranded RNA formed by two complementary chains or by a single self-complementary chain. Interfering RNA may have substantial or complete identity with a target gene or sequence, or may contain a mismatch region (i.e., a mismatch motif). The sequence of the interfering RNA may correspond to a full-length target gene or a subsequence thereof.

[0241] Interfering RNA includes "small interfering RNA" or "siRNA", for example, an interfering RNA of about 15-60, 15-50 or 15-40 (duplex) nucleotides in length, more usually about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, and preferably about 20-24, 21-22 or 21-23 (duplex) nucleotides in length (e.g., each complementary sequence of a double-stranded siRNA is about 100 nt long). The siRNA duplex may comprise a 3' overhang of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and a 5' phosphate terminus. Examples of siRNA include, but are not limited to, double-stranded polynucleotide molecules assembled from two separate strand molecules, one of which is a sense strand and the other is a complementary antisense strand; double-stranded polynucleotide molecules assembled from single-stranded molecules, wherein the sense region and the antisense region are connected by a nucleic acid-based or non-nucleic acid-based linker; double-stranded polynucleotide molecules having a hairpin secondary structure, which have self-complementary sense and antisense regions; and circular single-stranded polynucleotide molecules having two or more loop structures and a stem having self-complementary sense and antisense regions, wherein the circular polynucleotide can be treated in vivo or in vitro to produce active double-stranded siRNA molecules.

[0242] siRNA can also be chemically synthesized. siRNA can also be produced by cleaving longer dsRNAs (eg, dsRNAs greater than about 25 nucleotides in length) with E. coli RNase III or Dicer. These enzymes process dsRNA into biologically active siRNA (see, e.g., 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 Tech Notes, 10(1):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., J. Biol. Chem., 243:82 (1968)). Preferably, the dsRNA is at least 50 nucleotides to about 100, 200, 300, 400 or 500 nucleotides in length. The length of dsRNA can be up to 1000, 1500, 2000, 5000 nucleotides or longer. dsRNA can encode the entire gene transcript or a portion of the gene transcript. In some cases, siRNA can be encoded by a plasmid (e.g., transcribed as a sequence that automatically folds into a duplex with a hairpin loop).

[0243] As used herein, the term "in vitro" refers to occurring in an artificial environment, eg, a test tube or reaction vessel, a cell culture medium, a petri dish, etc. rather than within an organism (eg, an animal, plant, or microorganism).

[0244] As used herein, the term "in vivo" refers to events that occur within an organism (eg, an animal, plant, or microorganism, or a cell or tissue thereof).

[0245] As used herein, the term "restored cell" refers to an aging cell that has been treated or transiently reprogrammed with one or more cell reprogramming factors so that the cell has a transcriptome profile of a younger cell while still retaining one or more cell characteristic markers. In some embodiments, the treated cell is restored and reprogrammed to express markers and transcriptome profiles of younger cells while still retaining cell characteristic markers, such as a restored cell is reprogrammed to express at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75% or more than 75% increase / improvement in expression of a restoration marker compared to an untreated cell.

[0246] As used herein, the terms "subject," "individual," and "patient" are used interchangeably herein and refer to any vertebrate subject, including, but not limited to, humans and other primates, including non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; rodents such as mice, rats, rabbits, hamsters, and guinea pigs; birds, including domestic, wild, and game birds such as chickens, turkeys, and other Gallidae, ducks, geese, and the like. In some cases, the methods of the present disclosure can be used in experimental animals, veterinary applications, and the development of animal models of disease. The term does not denote a particular age. Thus, adults, adolescents, and neonatal individuals are intended to be covered.

[0247] As used herein, the term "transfection" refers to the uptake of exogenous DNA or RNA by a cell. A cell has been "transfected" when the exogenous DNA or RNA has been introduced into the cell membrane.

[0248] As used herein, the term "transfection efficiency" refers to the degree to which transfected cells take up exogenous DNA or RNA. Transfection efficiency can be measured as, for example, the percentage of cells expressing the gene product of the transfected exogenous DNA or RNA in a sample. The percentage of cells expressing the gene product of the transfected exogenous DNA or RNA in a sample can be measured by using flow cytometry to determine the percentage of cells expressing a reporter gene (such as green fluorescent protein) in a sample 18-24 hours after transfection according to methods known to those skilled in the art.

[0249] As used herein, the term "transfection efficacy" refers to the average expression level of the gene product of the exogenous DNA or RNA transfected per cell in a sample. The average expression level of the gene product of the exogenous DNA or RNA transfected per cell in a sample can be measured 18-24 hours after transfection by using flow cytometry to determine the total fluorescence intensity of cells expressing a reporter gene (such as green fluorescent protein) in the sample, divided by the number of cells in the sample according to methods well known to those skilled in the art.

[0250] As used herein, the term "viability" refers to the extent to which cells remain viable after transfection and is measured as the percentage of cells that remain viable in a sample subjected to transfection. Viability can be measured 18-24 hours after transfection as the percentage of cells that are positive for propidium iodide staining in a sample using flow cytometry according to methods well known to those skilled in the art.

[0251] As used herein, the term "transient reprogramming" refers to exposing cells to cell reprogramming factors for a period of time sufficient to rejuvenate the cells (i.e., eliminate all or some signs of aging), but not sufficient to cause dedifferentiation into stem cells. Such transient reprogramming results in rejuvenated cells and retention of their characteristics (i.e., differentiated cell types).

[0252] The term "treat" is used herein, for example, in relation to a method of treating a cell, tissue or subject, and generally includes administering a compound or composition that reduces the frequency or delays the onset of symptoms of aging or a medical condition in a subject relative to a subject that has not received the compound or composition. This can include reversing, reducing or preventing the symptoms, clinical signs and underlying pathology of a condition in a manner that improves or stabilizes the condition in the subject.

[0253] As used herein, the term "isomer" refers to any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer or diastereomer of a compound. Compounds may include one or more chiral centers and / or double bonds and may therefore exist as stereoisomers, such as double bond isomers (i.e., geometric E / Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-) or cis / trans isomers). The present disclosure encompasses any and all isomers of the compounds described herein, including stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) as well as enantiomeric and stereoisomeric mixtures, e.g., racemates. Enantiomers and stereoisomeric mixtures of compounds and methods of resolving them into their component enantiomers or stereoisomers are well known.

[0254] In the present disclosure, for convenience, in some cases, the structural formula of lipid can represent a certain isomer, but the present disclosure includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers, etc., and it should be understood that not all isomers can have the same level of activity. In addition, the lipid represented by the formula can have crystal polymorphism. It should be noted that any crystal form, crystal form mixture, or anhydride or hydrate thereof is included in the scope of the present disclosure.

[0255] 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.

[0256] As used herein, the term "lipid nanoparticle" or "LNP" refers to a nanoparticle formed by at least one lipid or a nanoparticle comprising at least one lipid. A "lipid-nanoparticle composition" refers to a composition comprising lipid nanoparticles or refers to the lipid nanoparticle itself. In some embodiments, the lipid nanoparticle is a lipid-nucleic acid particle or a nucleic acid-lipid particle (e.g., a stable nucleic acid-lipid particle). LNP can be a particle formed by or comprising an ionizable lipid of the present disclosure and a cargo encapsulated in the lipid. In some embodiments, the cargo is a nucleic acid.

[0257] The LNPs can generally 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.

[0258] As used herein, the term "mammal" includes both humans and livestock animals, such as laboratory animals and domestic pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), and non-livestock animals, such as wild animals, etc.

[0259] As used herein, the term "nanoparticle composition" refers to a composition comprising one or more lipids. The size of the nanoparticle composition is generally micrometers or smaller orders of magnitude, and may include a lipid bilayer. The nanoparticle composition includes lipid nanoparticles (LNP), liposomes (e.g., lipid vesicles), polymer complexes, and lipid complexes. For example, the nanoparticle composition can be a lipid nanoparticle having a diameter of 500 nm or less.

[0260] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, and includes DNA and RNA. DNA can be, for example, an antisense molecule, plasmid DNA, precondensed DNA, PCR product, carrier (PI, PAC, BAC, YAC, artificial chromosome), expression cassette, chimeric sequence, 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 main chain residues or bonds, which are synthetic, naturally occurring and non-naturally occurring, and have binding properties similar to reference nucleic acids. 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, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methyl ribonucleotides and peptide-nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids. Unless otherwise indicated, specific nucleic acid sequences also implicitly encompass variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences and sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more (or all) selected codons is replaced by mixed bases and / or deoxyinosine residues (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)). "Nucleotide" comprises the sugar deoxyribose (DNA) or ribose (RNA), a base and a phosphate group. The nucleotides are linked together by the phosphate group. "Bases" include: purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine and natural analogs; and synthetic derivatives of purines and pyrimidines, which include but are not limited to modifications that place new reactive groups (such as but not limited to amines, alcohols, thiols, carboxylates and alkyl halides).

[0261] As used herein, the term "optionally substituted" means unsubstituted or substituted. The term "substituted" means that a hydrogen atom is removed and replaced with a substituent. It is understood that substitution at a given atom is limited by valence.

[0262] As used herein, the term "pharmaceutically acceptable" refers to those compounds, salts, compositions, dosage forms, etc. that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and / or other animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio. In some aspects, "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals (e.g., animals), and more particularly in humans.

[0263] As used herein, the term "pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier that has been approved by the U.S. Food and Drug Administration as acceptable for use in humans or livestock animals. In some aspects, the "pharmaceutically acceptable carrier" or carrier, adjuvant, excipient is a component of an "artificial niche" for maintaining the quiescence of progenitor cells. In some embodiments, the artificial niche component is selected from the group consisting of: calcitonin; MGCD-265, JNJ-7706621, forskolin, fomastatin, 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. When used as excipients, such artificial niche components may be encapsulated by the lipid nanoparticles, present in the shell of the lipid nanoparticles, or included in the formulation separately from the lipid nanoparticles.

[0264] As used herein, the term "pharmaceutically acceptable salts" includes acid addition salts and base addition salts.

[0265] As used herein, the term "pharmaceutical composition" refers to nanoparticle compositions and vehicles, such as pharmaceutically acceptable carriers, generally accepted in the art for delivering biologically active compounds to mammals.

[0266] As used herein, the term "subject" or "patient" refers to any organism to which a composition according to the present disclosure can be administered, e.g., for experimental, diagnostic, preventive and / or therapeutic purposes. Typical subjects include animals, such as, but not limited to, mammals, such as, but not limited to, mice, rats, rabbits, non-human primates, and humans.

[0267] As used herein, the term "systemic delivery" refers to the delivery of therapeutic products that can cause active agents to be widely exposed in vivo. Some administration techniques can cause systemic delivery of certain drugs, but cannot cause systemic delivery of other drugs. Systemic delivery means that a useful amount of a preferably therapeutic amount of a medicament is exposed to most parts of the body. Systemic delivery of compositions of the present disclosure (e.g., lipid nanoparticles) can be carried out by any means known in the art, including, for example, intravenous, intra-arterial, subcutaneous, and intraperitoneal delivery. In certain embodiments, systemic delivery of lipid nanoparticles is carried out by intravenous delivery.

[0268] 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 location, tissue, organ, or cell type within a living organism. Some ionized lipid compositions (including some lipid nanoparticles) can result in local delivery of certain agents, but not other agents. Local delivery means exposing a useful amount, preferably a therapeutic amount, of an agent to a specific location, tissue, organ, or cell type within a living organism. Local delivery of the compositions of the present disclosure (e.g., lipid nanoparticles) can be performed by any means known in the art, including, for example, intravenous, intraarterial, subcutaneous, intradermal, dermal, topical, intratissue, intraorgan, and intraperitoneal delivery. In some embodiments, local delivery of the compositions of the present disclosure (e.g., lipid nanoparticles) is performed by intravenous delivery, wherein the characteristics of the nanoparticles (such as size, shape, ionization constant, and surface charge) determine the specific location, tissue, organ, or cell type targeted by the LNC. In some embodiments, the local delivery of compositions (e.g., lipid nanoparticles) of the present disclosure is carried out by local injection into a target location, tissue or organ, wherein the compositions (e.g., lipid nanoparticles) of the present disclosure can then be transfected in the local environment. In some embodiments, the local delivery of compositions (e.g., lipid nanoparticles) of the present disclosure is carried out by dermal, intradermal or subcutaneous injection, wherein the compositions (e.g., lipid nanoparticles) of the present disclosure can then be transfected in the local environment. In some embodiments, the local delivery of compositions (e.g., lipid nanoparticles) of the present disclosure is carried out by topical administration, wherein the compositions (e.g., lipid nanoparticles) of the present disclosure can then be transfected in the local environment.

[0269] As used herein, the term "therapeutic agent" or "prophylactic agent" refers to any agent that has a therapeutic, diagnostic and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. Therapeutic agents are also referred to as "active substances" or "active agents".

[0270] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a nanoparticle composition that is sufficient to achieve treatment of 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 specific lipid nanoparticles used), the disorder and its severity, the mode of administration, and the age of the subject to be treated, but can be routinely determined by one of ordinary skill in the art based on his or her own knowledge and the present disclosure.

[0271] As used herein, the terms "treating" or "treatment" encompass the treatment of a disease or condition of interest in a subject (preferably a mammal, more preferably a human) suffering from the disease or condition of interest, and includes preventing the disease or condition from occurring, particularly when such subject is susceptible to the condition but has not yet been diagnosed with the condition; inhibiting the disease or condition, i.e., arresting its development; relieving the disease or condition, i.e., causing regression of the disease or condition; or alleviating symptoms caused by the disease or condition, e.g., relieving pain without addressing the underlying disease or condition.

[0272] As used herein, the term "zeta potential" refers to the zeta potential of lipids in a nanoparticle composition or the zeta potential of a nanoparticle, such as a lipid nanoparticle.

[0273] The compositions of the present disclosure may comprise, consist essentially of, or consist of the components described herein.

[0274] All percentages, parts and ratios are based upon the total weight of the composition and all measurements made are at about 25°C, unless otherwise specified.

[0275] Less than the entirety of the disclosure may be claimed for any reason by reserving the right to limit or exclude any individual member of any such group that may be claimed by range or in any similar manner, including any subrange or combination of subranges within said group. In addition, less than the entirety of the disclosure may be claimed for any reason by reserving the right to limit or exclude any individual substituent, analog, compound, ligand, structure, or group thereof or any member of a claimed group.

[0276] Throughout this disclosure, various patents, patent applications and publications are cited. The disclosures of these patents, patent applications and publications are incorporated into this disclosure by reference in their entirety to more fully describe the state of the art known to those skilled in the art as of the date of this disclosure. If there is any inconsistency between the cited patents, patent applications and publications and this disclosure, the present disclosure shall prevail.

[0277] For convenience, certain terms employed in the specification, examples, and claims are collected here. 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 belongs.

[0278] II. Ionizable Lipids

[0279] Ionizable lipids disclosed herein are positively charged at acidic pH to condense nucleic acids (such as RNA) into lipid nanoparticles (LNPs). Ionizable lipids are neutral at physiological pH to minimize toxicity. They can be protonated in acidic endosomes after cellular uptake and interact with anionic endosomal phospholipids to form conical ion pairs that are incompatible with the double layer. These cation-anion lipid pairs drive the transition of the double layer structure to the inverted hexagonal HII phase, thereby promoting membrane fusion / destruction, endosomal escape, and cargo release into the cytosol (Semple, SC et al., Nat. Biotechnol. 2010, 28, 172-176).

[0280] The pKa of the ionizable lipids disclosed herein is about 8.5 to about 9.5. The lipid nanoparticles comprising one or more of the ionizable lipids described herein may have a pKa of about 6.5 to about 7.5.

[0281] The present disclosure provides ionizable lipids of Formula (I) to Formula (XVI). These lipids may be positively charged or partially positively charged at physiological pH.

[0282] In one embodiment, the ionizable lipid has formula (I):

[0283]

[0284] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0285] L 1 is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 2 C6-C 20 Alkyl; R 3 and R 4 are each independently H or C1-C3 alkyl; q1 is absent or is 1; and q2 is absent or is 1.

[0286] In some embodiments, 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 in formula (I), q2 is absent.

[0287] In some embodiments, 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 in formula (I), q2 is 1.

[0288] In some embodiments, 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 in formula (I), q2 is 1.

[0289] In some embodiments, 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 in formula (I), q2 is absent.

[0290] Illustrative examples of ionizable lipids of formula (I) may include, but are not limited to, the following:

[0291]

[0292]

[0293] In one embodiment, the ionizable lipid has formula (IA):

[0294]

[0295] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0296] L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; R 2 C6-C 20 Alkyl; R 3 and R 4 Each independently represents H or C1-C3 alkyl; R 7 C4-C 20 alkyl; and R 8 C4-C 20 alkyl.

[0297] In some embodiments, L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; R 2 C6-C 20 Alkyl; R 3 is methyl; R 4 is methyl; R 7 C4-C 20 alkyl; and in formula (IA), R 8 C4-C 20alkyl.

[0298] Illustrative examples of ionizable lipids of formula (IA) may include, but are not limited to, the following:

[0299]

[0300] In one embodiment, the ionizable lipid has formula (IB):

[0301]

[0302] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0303] L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; L 3 is C1-C8 alkylene; R 3 and R 4 Each independently represents H or C1-C3 alkyl; R 6 C4-C 20 Alkyl; R 7 C4-C 20 Alkyl; R 8 C4-C 20 alkyl; and R 10 C4-C 20 alkyl.

[0304] In some embodiments, L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; L 3 is C1-C8 alkylene; R 3 is methyl; R 4 is methyl; R 6 C4-C 20 Alkyl; R 7 C4-C 20 Alkyl; R 8 C4-C 20 alkyl; and in formula (IB), R 10 C4-C 20 alkyl.

[0305] Illustrative examples of ionizable lipids of formula (IB) may include, but are not limited to, the following:

[0306]

[0307] In one embodiment, the ionizable lipid has formula (II):

[0308]

[0309] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0310] L 1 is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 2 C6-C 20 Alkyl; R 3 and R 4 are each independently H or C1-C3 alkyl; and R 5 It is H or C1-C3 alkyl.

[0311] In some embodiments, L 1 is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 2 C6-C 20 Alkyl; R 3 is methyl; R 4 is methyl; and in formula (II), R 5 For H.

[0312] In some embodiments, L 1 is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 2 C6-C 20 Alkyl; R 3 is methyl; R 4 is methyl; and in formula (II), R 5 It is methyl.

[0313] Illustrative examples of ionizable lipids of formula (II) may include, but are not limited to, the following:

[0314]

[0315] In one embodiment, the ionizable lipid has formula (III):

[0316]

[0317] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0318] L 1 is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 2 C6-C 20 Alkyl; R 2 ' is C6-C 20 Alkyl; R 3and R 4 are each independently H or C1-C3 alkyl; and R 5 It is H or C1-C3 alkyl.

[0319] In some embodiments, L 1 is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 2 C6-C 20 Alkyl; R 2 ' is C6-C 20 Alkyl; R 3 is methyl; R 4 is methyl; and in formula (III), R 5 For H.

[0320] In some embodiments, L 1 is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 2 C6-C 20 Alkyl; R 2 ' is C6-C 20 Alkyl; R 3 is methyl; R 4 is methyl; and in formula (III), R 5 It is methyl.

[0321] Illustrative examples of ionizable lipids of formula (III) may include, but are not limited to, the following:

[0322]

[0323] In one embodiment, the ionizable lipid has formula (IV):

[0324]

[0325] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0326] L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; R 2 C6-C 20 Alkyl; R 3 and R 4 Each independently represents H or C1-C3 alkyl; R 5 is H or C1-C3 alkyl; R 7 C4-C 20 alkyl; and R 8 C4-C 20 alkyl.

[0327] In some embodiments, L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; R 2 C6-C 20 Alkyl; R 3 is methyl; R 4 is methyl; R 5 H; R 7 C4-C 20 alkyl; and in formula (IV), R 8 C4-C 20 alkyl.

[0328] In some embodiments, L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; R 2 C6-C 20 Alkyl; R 3 is methyl; R 4 is methyl; R 5 is methyl; R 7 C4-C 20 alkyl; and in formula (IV), R 8 C4-C 20 alkyl.

[0329] Illustrative examples of ionizable lipids of formula (IV) may include, but are not limited to, the following:

[0330]

[0331] In one embodiment, the ionizable lipid has formula (V):

[0332]

[0333] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0334] L 1 is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 3 and R 4 are each independently H or C1-C3 alkyl; and R 12 C6-C 20 Alkenyl.

[0335] In some embodiments, L 1 is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 3 is methyl; R4 is methyl; and in formula (V), R 12 C6-C 20 Alkenyl.

[0336] Illustrative examples of ionizable lipids of formula (V) may include, but are not limited to, the following:

[0337]

[0338] In one embodiment, the ionizable lipid has formula (VI):

[0339]

[0340] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0341] L 1 is C1-C6 alkylene; L 1 ' is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 1 ' is C6-C 20 Alkenyl; R 9 H, C1-C6 alkyl or –(CH2) n OH; R 12 C6-C 20 Alkenyl; R 12 ' is C6-C 20 and n is 2, 3 or 4.

[0342] In some embodiments, L 1 is C1-C6 alkylene; L 1 ' is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 1 ' is C6-C 20 Alkenyl; R 9 H; R 12 C6-C 20 alkenyl; and in formula (VI), R 12 ' is C6-C 20 Alkenyl.

[0343] In some embodiments, L 1 is C1-C6 alkylene; L 1 ' is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 1 ' is C6-C 20 Alkenyl; R 9 is methyl; R 12 C6-C20 alkenyl; and in formula (VI), R 12 ' is C6-C 20 Alkenyl.

[0344] In some embodiments, L 1 is C1-C6 alkylene; L 1 ' is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 1 ' is C6-C 20 Alkenyl; R 9 for –(CH2) n OH; R 12 C6-C 20 Alkenyl; R 12 ' is C6-C 20 and in formula (VI), n is 2, 3 or 4.

[0345] In some embodiments, L 1 is C1-C6 alkylene; L 1 ' is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 1 ' is C6-C 20 Alkenyl; R 9 for –(CH2) n OH; R 12 C6-C 20 Alkenyl; R 12 ' is C6-C 20 and in formula (VI), n is 2.

[0346] Illustrative examples of ionizable lipids of formula (VI) may include, but are not limited to, the following:

[0347]

[0348] In one embodiment, the ionizable lipid has formula (VII):

[0349]

[0350] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0351] L 1 is C1-C6 alkylene; L 1 ' is C1-C6 alkylene; R 2 C6-C 20 Alkyl; R 2 ' is C6-C 20 Alkyl; R 9H, C1-C6 alkyl or –(CH2) n OH; R 11 H or –CH2)OC(=O)R 16 ; R 14 C6-C 20 Alkyl; R 14 ' is C6-C 20 Alkyl; R 15 C6-C 20 Alkyl; R 16 C6-C 20 alkyl; and n is 2, 3 or 4.

[0352] In some embodiments, L 1 is C1-C6 alkylene; L 1 ' is C1-C6 alkylene; R 2 C6-C 20 Alkyl; R 2 ' is C6-C 20 Alkyl; R 9 H; R 11 H; R 14 C6-C 20 Alkyl; R 14 ' is C6-C 20 alkyl; and in formula (VII), R 15 C6-C 20 alkyl.

[0353] In some embodiments, L 1 is C1-C6 alkylene; L 1 ' is C1-C6 alkylene; R 2 C6-C 20 Alkyl; R 2 ' is C6-C 20 Alkyl; R 9 is methyl; R 11 H; R 14 C6-C 20 Alkyl; R 14 ' is C6-C 20 alkyl; and in formula (VII), R 15 C6-C 20 alkyl.

[0354] In some embodiments, 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 in formula (VII), n is 2.

[0355] Illustrative examples of ionizable lipids of formula (VII) may include, but are not limited to, the following:

[0356]

[0357]

[0358] In one embodiment, the ionizable lipid has formula (VIII):

[0359]

[0360] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0361] L 1 is C1-C6 alkylene; L 1 ' is C1-C6 alkylene; R 2 C6-C 20 Alkyl; R 2 ' is C6-C 20 Alkyl; R 9 H, C1-C6 alkyl or –(CH2) n OH; R 14 C6-C 20 Alkyl; R 14 ' is C6-C 20 Alkyl; R 15 C6-C 20 alkyl; and n is 2, 3 or 4.

[0362] In some embodiments, L1 is C1-C6 alkylene; L1' is C1-C6 alkylene; R2 is C6-C20 alkyl; R2' is C6-C20 alkyl; R9 is H; R14 is C6-C20 alkyl; R14' is C6-C20 alkyl; and in formula (VIII), R15 is C6-C20 alkyl.

[0363] In some embodiments, 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 in formula (VIII), R15 is C6-C20 alkyl.

[0364] In some embodiments, 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 in formula (VIII), n is 2.

[0365] Illustrative examples of ionizable lipids of formula (VIII) may include, but are not limited to, the following:

[0366]

[0367] In one embodiment, the ionizable lipid has formula (IX):

[0368]

[0369] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0370] R 1 C6-C 20 Alkenyl; R 9 H, C1-C6 alkyl or –(CH2) n OH; R 12 C6-C 20 and n is 2, 3 or 4.

[0371] In some embodiments, R1 is C6-C20 alkenyl; R9 is H; and in formula (IX), R12 is C6-C20 alkenyl.

[0372] In some embodiments, R1 is C6-C20 alkenyl; R9 is methyl; and in formula (IX), R12 is C6-C20 alkenyl.

[0373] In some embodiments, R1 is C6-C20 alkenyl; R9 is -(CH2)nOH; R12 is C6-C20 alkenyl; and in formula (IX), n is 2.

[0374] Illustrative examples of ionizable lipids of formula (IX) may include, but are not limited to, the following:

[0375]

[0376]

[0377] In one embodiment, the ionizable lipid has formula (X):

[0378]

[0379] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0380] L 4 L is absent or is C1-C6 alkylene; 5 does not exist or is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 2 C6-C 20 Alkyl; R 2 ' is C6-C 20 Alkyl; R 9 H, C1-C6 alkyl or –(CH2) n OH; R 12 C6-C 20 and n is 2, 3 or 4.

[0381] In some embodiments, 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 in formula (X), n is 2, 3 or 4.

[0382] In some embodiments, 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 in formula (X), n is 2, 3 or 4.

[0383] In some embodiments, 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 in formula (X), n is 2, 3 or 4.

[0384] In some embodiments, 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 in formula (X), n is 2, 3 or 4.

[0385] In some embodiments, L4 is absent or is C1-C6 alkylene; L5 is absent or is C1-C6 alkylene; R1 is C6-C20 alkenyl; R2 is C6-C20 alkyl; R2' is C6-C20 alkyl; R9 is H; and in formula (X), R12 is C6-C20 alkenyl.

[0386] In some embodiments, L 4 L is absent or is C1-C6 alkylene; 5 does not exist or is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 2 C6-C 20 Alkyl; R 2 ' is C6-C 20 Alkyl; R 9 is methyl; and in formula (X), R 12 C6-C 20 Alkenyl.

[0387] In some embodiments, L 4 L is absent or is C1-C6 alkylene; 5 does not exist or is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 2 C6-C 20 Alkyl; R 2 ' is C6-C 20 Alkyl; R 9 for –(CH2) n OH; R 12 C6-C 20 and in formula (X), n is 2.

[0388] Illustrative examples of ionizable lipids of formula (X) may include, but are not limited to, the following:

[0389]

[0390]

[0391] In one embodiment, the ionizable lipid has formula (XI):

[0392]

[0393] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0394] L 4 L is absent or is C1-C6 alkylene; 5 does not exist or is C1-C6 alkylene; R 1 C6-C 20 Alkenyl; R 2 C6-C 20 Alkyl; R 2 ' is C6-C 20 Alkyl; R 12C6-C 20 Alkenyl; R 13 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.

[0395] In some embodiments, 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 in formula (XI), q is 2, 3 or 4.

[0396] In some embodiments, 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 in formula (XI), q is 2, 3 or 4.

[0397] In some embodiments, 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 in formula (XI), q is 2, 3 or 4.

[0398] In some embodiments, 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 in formula (XI), q is 2, 3 or 4.

[0399] In some embodiments, 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; and in formula (XI), R13 is H.

[0400] In some embodiments, 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 C6-C 20 alkenyl; and in formula (XI), R 13 It is methyl.

[0401] In some embodiments, 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 in formula (XI), n is 2.

[0402] In some embodiments, 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 in formula (XI), q is 3.

[0403] Illustrative examples of ionizable lipids of formula (XI) may include, but are not limited to, the following:

[0404]

[0405] In one embodiment, the ionizable lipid has formula (XII):

[0406]

[0407] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0408] L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 7 C4-C 20 Alkyl; R 7 ' is C4-C 20 Alkyl; R 8 C4-C 20 Alkyl; R 8 ' is C4-C 20 Alkyl; R 13 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.

[0409] In some embodiments, 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 in formula (XII), R13 is H.

[0410] In some embodiments, 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 in formula (XII), R13 is methyl.

[0411] In some embodiments, 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 in formula (XII), n is 4.

[0412] Illustrative examples of ionizable lipids of formula (XII) may include, but are not limited to, the following:

[0413]

[0414] In one embodiment, the ionizable lipid has formula (XIII):

[0415]

[0416] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0417] L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 7 C4-C 20 Alkyl; R 7 ' is C4-C 20 Alkyl; R 8 C4-C 20 Alkyl; R 8 ' is C4-C 20 Alkyl; R 13 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.

[0418] In some embodiments, 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 in formula (XIII), R13 is H.

[0419] In some embodiments, 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 in formula (XIII), R13 is methyl.

[0420] In some embodiments, 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 in formula (XIII), n is 2.

[0421] In some embodiments, 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 in formula (XIII), q is 3.

[0422] Illustrative examples of ionizable lipids of formula (XIII) may include, but are not limited to, the following:

[0423]

[0424] In one embodiment, the ionizable lipid has formula (XIV):

[0425]

[0426] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0427] L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; R 3 and R 4 Each independently represents H or C1-C3 alkyl; R 6 C4-C 20 alkyl; and R 7 C4-C 20 alkyl.

[0428] In some embodiments, L 1 is C1-C6 alkylene; L 2is C1-C8 alkylene; R 3 is methyl; R 4 is methyl; R 6 C4-C 20 alkyl; and in formula (XIV), R 7 C4-C 20 alkyl.

[0429] Illustrative examples of ionizable lipids of formula (XIV) may include, but are not limited to, the following:

[0430]

[0431] In one embodiment, the ionizable lipid has formula (XV):

[0432]

[0433] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0434] L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 6 C4-C 20 Alkyl; R 6 ' is C4-C 20 Alkyl; R 7 C4-C 20 Alkyl; R 7 ' is C4-C 20 Alkyl; R 8 C4-C 20 Alkyl; R 8 ' is C4-C 20 Alkyl; R 10 C4-C 20 Alkyl; R 10 ' is C4-C 20 Alkyl; R 13 H, C1-C6 alkyl, –(CH2) n OH or –(CH2) q N(CH3)2; n is 2, 3 or 4; p1 is absent or is 1; p2 is absent or is 1; and q is 2, 3 or 4.

[0435] In some embodiments, L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 6 C4-C 20 Alkyl; R 6 ' is C4-C 20 Alkyl; R 7 C4-C20 Alkyl; R 7 ' is C4-C 20 Alkyl; R 8 C4-C 20 Alkyl; R 8 ' is C4-C 20 Alkyl; R 10 C4-C 20 Alkyl; R 10 ' is C4-C 20 Alkyl; R 13 is H; p1 does not exist; and in formula (XV), p2 does not exist.

[0436] In some embodiments, L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 6 C4-C 20 Alkyl; R 6 ' is C4-C 20 Alkyl; R 7 C4-C 20 Alkyl; R 7 ' is C4-C 20 Alkyl; R 8 C4-C 20 Alkyl; R 8 ' is C4-C 20 Alkyl; R 10 C4-C 20 Alkyl; R 10 ' is C4-C 20 Alkyl; R 13 is H; p1 is 1; and in formula (XV), p2 is 1.

[0437] In some embodiments, 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 does not exist; and in formula (XV), p2 does not exist.

[0438] In some embodiments, 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 in formula (XV), p2 is 1.

[0439] In some embodiments, 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 not present; and in formula (XV), p2 is not present.

[0440] In some embodiments, 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 in formula (XV), p2 is 1.

[0441] Illustrative examples of ionizable lipids of formula (XV) may include, but are not limited to, the following:

[0442]

[0443] In one embodiment, the ionizable lipid has formula (XVI):

[0444]

[0445] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0446] L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 6 C4-C 20 Alkyl; R 6 ' is C4-C 20 Alkyl; R 7 C4-C 20 Alkyl; R 7 ' is C4-C 20Alkyl; R 13 H, C1-C6 alkyl, –(CH2) n OH or –(CH2) q N(CH3)2; n is 2, 3 or 4; p1 is absent or is 1; p2 is absent or is 1; and q is 2, 3 or 4.

[0447] In one embodiment, the ionizable lipid has formula (XVI-A):

[0448]

[0449] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0450] L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 6 C4-C 20 Alkyl; R 6 ' is C4-C 20 Alkyl; R 7 C4-C 20 Alkyl; R 7 ' is C4-C 20 Alkyl; R 13 H, C1-C6 alkyl, –(CH2) n OH or –(CH2) q N(CH3)2; n is 2, 3 or 4; p1 is absent or is 1; p2 is absent or is 1; and q is 2, 3 or 4.

[0451] In some embodiments, 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 is 1; and in Formula (XVI) and Formula (XVI-A), p2 is absent or is 1.

[0452] In some embodiments, 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 is 1; and in formula (XVI) and formula (XVI-A), p2 is absent or is 1.

[0453] L2 is C1-C8 alkylene; L2' is C1-C8 alkylene; R6 is C4-C20 alkyl; R6' is C4-C20 alkyl;

[0454] R7 is a C4-C20 alkyl group; R7' is a C4-C20 alkyl group; R13 is -(CH2)nOH; n is 4; p1 does not exist;

[0455] Also, in formula (XVI) and formula (XVI-A), p2 does not exist.

[0456] In some embodiments, 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 in Formula (XVI) and Formula (XVI-A), p2 is 1.

[0457] Illustrative examples of ionizable lipids of formula (XVI) and formula (XVI-A) may include, but are not limited to, the following:

[0458]

[0459] In one embodiment, the compound has the following structure:

[0460]

[0461] or a stereoisomer, salt or tautomer thereof, wherein: G 1 and G 2 are independently -OC(=O)-, -NR 25 C(=O)- or -CH=CH-; R 21 and R 22 Each independently is a C1-C6 alkyl, a straight chain C 10 -C 20 Alkyl, straight chain C 10 -C 20 Alkenyl or branched C 10 -C 35 alkenyl, wherein the C1-C6 alkyl is replaced by -OC(=O)R 26 Replacement; R 23 is H, OH or OCH3; R 24 is C1-C8 heteroalkyl; R 25 is H or C1-C4 alkyl; R 26 For branched chain C 10 -C 30 alkyl; and m 1 and m 2 Each independently represents an integer of 0 or 1.

[0462] In one embodiment, G 1 and G 2 are independently -OC(=O)-, -NR25 C(═O)— or —CH═CH—. In some embodiments, G 1 and G 2 Each is -OC(=O)-. In some embodiments, G 1 or G 2 One of them is -NR 25 C(=O)- and G 1 or G 2 The other one of them is -CH=CH-.

[0463] In one embodiment, m 2 is 0. In some embodiments, m 2 is 1.

[0464] In one embodiment, the compound has one of the following structures (XVIIA) to (XVIIB):

[0465]

[0466] or a stereoisomer, salt or tautomer thereof. In some embodiments, the compound is In some embodiments, the compound is

[0467] In one embodiment, R24 is a C1-C8 heteroalkyl. In some embodiments, R24 is a C3-C7 heteroalkyl. In some embodiments, R24 is a C3 heteroalkyl. In some embodiments, R24 is a C4 heteroalkyl. In some embodiments, R24 is a C5 heteroalkyl. In some embodiments, R24 is a C6 heteroalkyl. In some embodiments, R24 is a C7 heteroalkyl. In some embodiments, R24 is a C4 alkylamine. In some embodiments, R24 is a C5 alkylamine. In some embodiments, R24 is a C6 alkylamine. In some embodiments, R24 is a C7 alkylamine. In some certain embodiments, R24 is In some embodiments, R24 is In some embodiments, R24 is In some embodiments, R24 is In some embodiments, R24 is In some embodiments, R24 is In some embodiments, R24 is In some embodiments, R24 is In some embodiments, R24 is

[0468] In some embodiments, the C1-C8 heteroalkyl of R24 is further substituted by a cycloalkyl. In some embodiments, R24 is In some embodiments, R24 is In some embodiments, R 24 for

[0469] In one embodiment, R 25 is H or C1-C4 alkyl. In some embodiments, R 25 is H. In some embodiments, R 25 is a C1-C4 alkyl group. 25 is a C1 alkyl group. 25 is a C2 alkyl group. In some embodiments, R 25 is a C3 alkyl group. In some embodiments, R 25 is a C4 alkyl group. In some certain embodiments, R 25 In some other certain embodiments, R 25 It is -CH2CH3.

[0470] In one embodiment, R 21 and R 22 Each independently is a C1-C6 alkyl, a straight chain C 10 -C 20 Alkyl, straight chain C 10 -C 20 Alkenyl or branched C 10 -C 35 alkenyl, wherein the C1-C6 alkyl is replaced by -OC(=O)R 26 In some embodiments, R 21 and R 22 Each independently is -OC(=O)R 26 Substituted C2-C5 alkyl, straight chain C 12 -C 18 Alkyl, straight chain C 12 -C 18 Alkenyl or branched C 14 -C 32 In some embodiments, R 21 or R 22 One of them is a straight chain C 12 -C 18 Alkyl, and R 21 or R 22 The other one is a straight chain C 12 -C 18 In some embodiments, R 21 or R 22 One of them is -OC(=O)R 26substituted C2-C5 alkyl, and R 21 or R 22 The other one is a straight chain C 12 -C 18 In some embodiments, R 21 or R 22 One of them is a straight chain C 12 -C 18 Alkyl, and R 21 or R 22 The other one is a branched chain C 14 -C 32 In some embodiments, R 21 and R 22 Each is -OC(=O)R 26 In some embodiments, R 21 and R 22 Each is -OC(=O)R 26 In some embodiments, R 21 and R 22 Each is -OC(=O)R 26 In some embodiments, R 21 and R 22 Each is -OC(=O)R 26 In some embodiments, R 21 and R 22 Each is -OC(=O)R 26 In some embodiments, R 21 and R 22 Each is a straight chain C 12 -C 18 In some embodiments, R 21 and R 22 Each is a straight chain C 12 In some embodiments, R 21 and R 22 Each is a straight chain C 13 In some embodiments, R 21 and R 22 Each is a straight chain C 14 In some embodiments, R 21 and R 22 Each is a straight chain C 15 In some embodiments, R 21 and R 22 Each is a straight chain C 16 In some embodiments, R 21 and R 22 Each is a straight chain C17 In some embodiments, R 21 and R 22 Each is a straight chain C 18 Alkenyl.

[0471] In one embodiment, R 26 For branched chain C 10 -C 30 In some embodiments, R 26 For branched chain C 10 -C 20 In some certain embodiments, R 26 For branched chain C 12 -C 18 In some certain embodiments, R 26 For branched chain C 12 In some certain embodiments, R 26 For branched chain C 13 In some certain embodiments, R 26 For branched chain C 14 In some certain embodiments, R 26 For branched chain C 15 In some certain embodiments, R 26 For branched chain C 16 In some certain embodiments, R 26 For branched chain C 17 In some certain embodiments, R 26 For branched chain C 18 alkyl.

[0472] In some embodiments, R 21 and R 22 Each independently has one of the following structures:

[0473] In some embodiments, R 21 or R 22 for In some embodiments, R 21 or R 22 for In some embodiments, R 21 or R 22 for In some embodiments, R 21 or R 22 for In some embodiments, R 21 or R 22 for

[0474] In one embodiment, the compound has one of the following structures shown in Table A below.

[0475] Table A: List of compounds (XVII) to (XVIIB)

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482] In one embodiment, the compound has the following structure:

[0483]

[0484] or a stereoisomer, salt or tautomer thereof, wherein: G 3 and G 4 Each is independently -OC(=O)R 28 、-C(=O)OR 28 、-CH(CH2OC(=O)R 28 )2, -C(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 );R 27 is H, C1-C6 alkyl or C1-C6 heteroalkyl; R 28 Each independently is a straight chain C6-C 12 Alkyl, branched chain C 10 -C 40 Alkyl, straight chain C 15 -C 20 Alkenyl or branched C 20 -C 40 Alkenyl; m 1 and m 2 Each independently is an integer from 1 to 6; is a direct key or does not exist; and It is a single bond or a double bond.

[0485] In one embodiment, the compound has one of the following structures (XVIIIA) to (XVIIIC):

[0486]

[0487]

[0488] or a stereoisomer, salt or tautomer thereof. In some embodiments, the compound is In some embodiments, the compound is In some embodiments, the compound is

[0489] In one embodiment, R 27 is H, C1-C6 alkyl or C1-C6 heteroalkyl. 27 is H, C1-C3 alkyl or C1-C5 heteroalkyl. 27 is a C1-C3 alkyl group, a C2-C4 alkyl alcohol or a C5 alkyl amine. 27 Has one of the following structures: In some embodiments, R 27 for In some embodiments, R 27 for In some embodiments, R 27 for In some embodiments, R 27 for In some embodiments, R 27 for

[0490] In one embodiment, G 3 and G 4 Each is independently -OC(=O)R 28 、-C(=O)OR 28 、-CH(CH2OC(=O)R 28 )2, -C(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 G4 Each has one of the following structures: In some embodiments, G 3 or G 4 have In some embodiments, G 3 or G 4 have In some embodiments, G 3 or G 4 have In some embodiments, G 3 or G 4 have In some embodiments, G 3 or G 4 have In some embodiments, G 3 or G 4 have

[0491] In one embodiment, R 28 Each independently is a straight chain C6-C 12 Alkyl, branched chain C 10 -C 40 Alkyl, straight chain C 15 -C 20 Alkenyl or branched C 20 -C 40 In some embodiments, R 28 Each independently is a straight chain C7-C 10 Alkyl, branched chain C 14 -C 40 Alkyl, straight chain C 15 -C 20 Alkenyl or branched C 30 -C 40 In some embodiments, R 28 Each is independently a straight chain C7 alkyl group. 28 Each is independently a straight chain C8 alkyl. 28 Each is independently a straight chain C9 alkyl group. 28 Each independently is a straight chain C 10 In some embodiments, R 28 Each independently is a branched C 14 In some embodiments, R 28 Each independently is a branched C 15 In some embodiments, R 28 Each independently is a branched C 16In some embodiments, R 28 Each independently is a branched C 17 In some embodiments, R 28 Each independently is a branched C 18 In some embodiments, R 28 Each independently is a branched C 19 In some embodiments, R 28 Each independently is a branched C 20 In some embodiments, R 28 Each independently is a branched C 21 In some embodiments, R 28 Each independently is a branched C 22 In some embodiments, R 28 Each independently is a branched C 23 In some embodiments, R 28 Each independently is a branched C 24 In some embodiments, R 28 Each independently is a branched C 25 In some embodiments, R 28 Each independently is a branched C 26 In some embodiments, R 28 Each independently is a branched C 27 In some embodiments, R 28 Each independently is a branched C 28 In some embodiments, R 28 Each independently is a branched C 29 In some embodiments, R 28 Each independently is a branched C 30 In some embodiments, R 28 Each independently is a branched C 31 In some embodiments, R 28 Each independently is a branched C 32 In some embodiments, R 28 Each independently is a branched C 33 In some embodiments, R 28 Each independently is a branched C 34 In some embodiments, R 28 Each independently is a branched C 35 In some embodiments, R 28 Each independently is a branched C 36 In some embodiments, R 28 Each independently is a branched C 37 In some embodiments, R28 Each independently is a branched C 38 In some embodiments, R 28 Each independently is a branched C 39 In some embodiments, R 28 Each independently is a branched C 40 In some embodiments, R 28 Each independently is a straight chain C 15 In some embodiments, R 28 Each independently is a straight chain C 16 In some embodiments, R 28 Each independently is a straight chain C 17 In some embodiments, R 28 Each independently is a straight chain C 18 In some embodiments, R 28 Each independently is a straight chain C 19 In some embodiments, R 28 Each independently is a straight chain C 20 In some embodiments, R 28 Each independently is a branched C 30 In some embodiments, R 28 Each independently is a branched C 31 In some embodiments, R 28 Each independently is a branched C 32 In some embodiments, R 28 Each independently is a branched C 33 In some embodiments, R 28 Each independently is a branched C 34 In some embodiments, R 28 Each independently is a branched C 35 In some embodiments, R 28 Each independently is a branched C 36 In some embodiments, R 28 Each independently is a branched C 37 In some embodiments, R 28 Each independently is a branched C 38 In some embodiments, R 28 Each independently is a branched C 39 In some embodiments, R 28 Each independently is a branched C 40 Alkenyl.

[0492] In some certain embodiments, R 28 Each independently has one of the following structures: In some embodiments, R 28 for In some embodiments, R 28 for In some embodiments, R 28 for In some embodiments, R 28 for In some embodiments, R 28 for In some embodiments, R 28 for In some embodiments, R 28 for

[0493] In one embodiment, m 1 and m 2 Each independently represents an integer from 1 to 6. In some embodiments, m 1 and m 2 Each is independently an integer from 1 to 4. In some certain embodiments, m 1 or m 2 is 1. In some certain embodiments, m 1 or m 2 is 2. In some certain embodiments, m 1 or m 2 is 3. In some certain embodiments, m 1 or m 2 is 4. In some certain embodiments, m 1 or m 2 is 5. In some certain embodiments, m 1 or m 2 is 6.

[0494] In one embodiment, the compound has one of the following structures shown in Table B below.

[0495] Table B: List of compounds (XVIII) to (XVIIIC)

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504] In one embodiment, the compound has the following structure:

[0505]

[0506] or a stereoisomer, salt or tautomer thereof, wherein: R 29a and R 29b Each independently is a straight chain C6-C 10 Alkyl or straight chain C 12 -C 20 Alkylene; R 30 is an aryl group or a C3-C6 heterocycle, wherein the aryl group or the C3-C6 heterocycle is replaced by -OC(=O)R 31 , C1-C4 alkyl or C1-C4 heteroalkyl substituted; R 31 is C1-C6 heteroalkyl; and n 1 An integer from 1 to 6.

[0507] In one embodiment, R 29a and R 29b Each independently is a straight chain C6-C 10 Alkyl or straight chain C 12 -C 20 In some embodiments, R 29a and R 29b Each is a straight chain C6-C 10 In some embodiments, R 29a and R 29b Each is a straight chain C 12 -C 20 In some certain embodiments, R 29a and R 29b Each independently has one of the following structures: In some embodiments, R 29a or R 29b for In some embodiments, R 29a or R 29b for In some embodiments, R 29a or R 29b for In some embodiments, R 29a or R 29b for In some embodiments, R 29a or R 29b for In some embodiments, R 29a or R 29b for In some certain embodiments, R 29a and R 29b Each In some other certain embodiments, R 29a and R 29b Each

[0508] In one embodiment, R 30 is an aryl group or a C3-C6 heterocycle, wherein the aryl group or the C3-C6 heterocycle is replaced by -OC(=O)R 31 , C1-C4 alkyl or C1-C4 heteroalkyl. In some embodiments, R 30 The aryl group is phenyl or naphthalene. In some embodiments, R 30 The C3-C6 heterocycle is azetidine, pyrrolidine, imidazolidine, pyrazolidine, piperidine, diazine, triazine or azepane. In some embodiments, R 30 -OC(=O)R 31 substituted aryl, or C3-C6 heterocycle substituted by C1-C4 alkyl, or C1-C4 heteroalkyl. 31 In some embodiments, R 30 Has one of the following structures: In some embodiments, R 30 for In some embodiments, R 30 for In some embodiments, R 30 for In some embodiments, R 30 for In some embodiments, R 30 for In some embodiments, R 30 for In some embodiments, R 30 for In some embodiments, R 30 for In some embodiments, R 30 for In some embodiments, R 30 for In some embodiments, R 30 for In some embodiments, R 30 for In some embodiments, R 30 for In some embodiments, R 30 for In some embodiments, R 30 for In some embodiments, R 30 for In some embodiments, R 30 for In some embodiments, R 30 In some embodiments, R 30 for In some embodiments, R 30 for In some embodiments, R 30 for In some embodiments, R 30 for

[0509] In some embodiments, n 1 is an integer from 1 to 4. In some embodiments, n 1 is an integer of 1. In some embodiments, n 1 is an integer of 2. In some embodiments, n 1 is an integer of 3. In some embodiments, n 1 is an integer of 4. In some embodiments, n 1 An integer of 1 or 4.

[0510] In one embodiment, the compound has one of the following structures shown in Table C below.

[0511] Table C: List of (XIX) compounds

[0512]

[0513] In one embodiment, the ionizable lipid of formula (XXI):

[0514]

[0515] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0516] 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 3 is C1-C8 alkylene; L 3 ' is C1-C8 alkylene; R 3 and R 4 Each is independently H, C1-C4 alkyl, -CH2-cyclopropyl or –(CH2) n OH; R 6 C4-C 20 Alkyl; R 6 ' is C4-C 20 Alkyl; R 7 C4-C 20 Alkyl; R 7 ' is C4-C 20 Alkyl; R 8 C4-C 20 Alkyl; R 8 ' is C4-C 20 Alkyl; R 10 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.

[0517] In one embodiment, the compound has one of the following structures shown in Table D below.

[0518] Table D: List of (XXI) compounds In one embodiment, the compound has the following structure:

[0519]

[0520] or a stereoisomer, salt or tautomer thereof, wherein: G 1 and G 2 Each is independently -OC(=O)- or -NR 25 C(=O)-;R 21 and R 22 Each independently is a C1-C6 alkyl, a straight chain C 10 -C 20 Alkyl, straight chain C 10 -C 20 Alkenyl or branched C 10 -C 35 alkenyl, wherein the C1-C6 alkyl is replaced by -OC(=O)R 26 Replacement; R 24 is C1-C6 heteroalkyl, aryl or C1-C4 alkyl substituted by 4- to 8-membered heterocycloalkyl; R 25is H or C1-C4 alkyl; R 26 For branched chain C 10 -C 30 alkyl; and Y is O or NR 32 , where R 32 It is H or C1-C4 alkyl.

[0521] In one embodiment, G 1 and G 2 Each is independently -OC(=O)- or -NR 25 C(=O)-. In some embodiments, G 1 and G 2 Each is -OC(=O)-. In some embodiments, G 1 and G 2 Each is -NR 25 C(=O)-. In some embodiments, G 1 or G 2 One of them is -NR 25 C(=O)- and G 1 or G 2 The other one of them is -OC(=O)-.

[0522] In one embodiment, the compound has one of the following structures (XXA) to (XXB):

[0523]

[0524] or a stereoisomer, salt or tautomer thereof. In some embodiments, the compound is In some embodiments, the compound is

[0525] In one embodiment, Y is O. In other embodiments, Y is NR 32 , where R 32 is H or C1-C4 alkyl. In some certain embodiments, Y is NR 32 And R 32 is H. In some embodiments, Y is NR 32 And R 32 is a C1-C4 alkyl group. 32 The C1-C4 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl or tert-butyl.

[0526] In one embodiment, the compound has one of the following structures (XXA-1) to (XXB-2):

[0527]

[0528] or a stereoisomer, salt or tautomer thereof. In one embodiment, the compound is In some embodiments, the compound is In some embodiments, the compound is In some embodiments, the compound is

[0529] In one embodiment, R 24 is a C1-C6 heteroalkyl group. 24 is a C3-C6 heteroalkyl group. 24 is a C3 heteroalkyl group. 24 is a C4 heteroalkyl group. 24 is a C5 heteroalkyl group. 24 is a C6 heteroalkyl group. 24 is a C5 alkylamine. In some certain embodiments, R 24 for

[0530] In one embodiment, R 24 In some embodiments, R 24 The aryl group is substituted with a C1-C6 heteroalkyl group. 24 The aryl group is phenyl or naphthalene. In some certain embodiments, R 24 In some other embodiments, R 24 In some embodiments, for example, the C2-C4 heteroalkyl group is a substituted amine. In some embodiments, R 24 is a phenyl group substituted with a C2-C4 heteroalkyl group. 24 is phenyl substituted by C3 heteroalkyl. 24 The substituted aryl group is

[0531] In one embodiment, R 24 is a C1-C4 alkyl group substituted with a 4- to 8-membered heterocycloalkyl group. 24 is a C1-C3 alkyl group substituted with a 4- to 6-membered heterocycloalkyl group. 24 The C1-C3 alkyl group is methyl, ethyl or n-propyl. In some embodiments, for example, R 24The 4- to 6-membered heterocycloalkyl of is azetidine, oxetane, phosphatane, thietane, diazetidine, dioxetane, dithietane, pyrrolidine, tetrahydrofuran, phospholane, tetrahydrothiophene, imidazolidine, pyrazolidine, thietane, isothiol, oxazolidine, isoxazolidine, thiazolidine, dioxolane, dithiolane, piperidine, oxane, phosphine, thiazane, diazines, morpholine, thioxane, dioxane or dithiane. In some certain embodiments, R 24 The 4- to 6-membered heterocycloalkyl of is azetidine or 1,4-diazinane. In some embodiments,

[0532] R 24 Has one of the following structures: In some embodiments, R 24 for In some embodiments, R 24 for

[0533] In one embodiment, R 25 is H or C1-C4 alkyl. In some embodiments, R 25 is H. In some embodiments, R 25 is a C1-C4 alkyl group. 25 is a C1 alkyl group. 25 is a C2 alkyl group. In some embodiments, R 25 is a C3 alkyl group. In some embodiments, R 25 is a C4 alkyl group. In some certain embodiments, R 25 In some other certain embodiments, R 25 It is -CH2CH3.

[0534] In one embodiment, R 21 and R 22 Each independently is a C1-C6 alkyl, a straight chain C 10 -C 20 Alkyl, straight chain C 10 -C 20 Alkenyl or branched C 10 -C 35 alkenyl, wherein the C1-C6 alkyl is replaced by -OC(=O)R 26 In some embodiments, R 21 and R 22 Each independently is -OC(=O)R 26 Substituted C2-C5 alkyl, straight chain C 12 -C 18 Alkyl, straight chain C 12 -C 18 Alkenyl or branched C14 -C 32 In some embodiments, R 21 or R 22 One of them is a straight chain C 12 -C 18 Alkyl, and R 21 or R 22 The other one is a straight chain C 12 -C 18 In some embodiments, R 21 or R 22 One of them is -OC(=O)R 26 substituted C2-C5 alkyl, and R 21 or R 22 The other one is a straight chain C 12 -C 18 In some embodiments, R 21 or R 22 One of them is a straight chain C 12 -C 18 Alkyl, and R 21 or R 22 The other one is a branched chain C 14 -C 32 In some embodiments, R 21 and R 22 Each is -OC(=O)R 26 In some embodiments, R 21 and R 22 Each is -OC(=O)R 26 In some embodiments, R 21 and R 22 Each is -OC(=O)R 26 In some embodiments, R 21 and R 22 Each is -OC(=O)R 26 In some embodiments, R 21 and R 22 Each is -OC(=O)R 26 In some embodiments, R 21 and R 22 Each is a straight chain C 12 -C 18 In some embodiments, R 21 and R 22 Each is a straight chain C 12 In some embodiments, R 21 and R 22 Each is a straight chain C13 In some embodiments, R 21 and R 22 Each is a straight chain C 14 In some embodiments, R 21 and R 22 Each is a straight chain C 15 In some embodiments, R 21 and R 22 Each is a straight chain C 16 In some embodiments, R 21 and R 22 Each is a straight chain C 17 In some embodiments, R 21 and R 22 Each is a straight chain C 18 Alkenyl.

[0535] In one embodiment, R 26 For branched chain C 10 -C 30 In some embodiments, R 26 For branched chain C 10 -C 20 In some certain embodiments, R 26 For branched chain C 12 -C 18 In some certain embodiments, R 26 For branched chain C 12 In some certain embodiments, R 26 For branched chain C 13 In some certain embodiments, R 26 For branched chain C 14 In some certain embodiments, R 26 For branched chain C 15 In some certain embodiments, R 26 For branched chain C 16 In some certain embodiments, R 26 For branched chain C 17 In some certain embodiments, R 26 For branched chain C 18 alkyl.

[0536] In some embodiments, R 21 and R 22 Each independently has the following structure:

[0537]

[0538] In one embodiment, the compound has one of the following structures shown in Table E below.

[0539] Table E: List of compounds (XX) to (XXB-2)

[0540]

[0541]

[0542] III. Nucleic Acids

[0543] In one aspect, the ionizable lipids disclosed herein can be used 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 (antagomirs / antimirs), dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), multivalent RNA and mixtures thereof. In some aspects, nucleic acids or mRNA include self-amplification RNA (saRNA), polycistronic RNA, circular RNA and mixtures thereof.

[0544] In one aspect, the nucleic acid comprises an interfering RNA molecule, such as siRNA, aiRNA, miRNA, or a mixture thereof. In certain other aspects, the nucleic acid comprises one or more mRNA molecules (e.g., a mixture).

[0545] Interfering RNA molecules include "small interfering RNA" or "siRNA", e.g., interfering RNA of about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, and preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (e.g., the length of each complementary sequence of a double-stranded siRNA The length of the double-stranded siRNA is about 15-60, 15-50, 15-40, 15-30, 15-25 or 19-25 nucleotides, preferably, the length is about 20-24, 21-22 or 21-23 nucleotides, and the length of the double-stranded siRNA is about 15-60, 15-50, 15-40, 15-30, 15-25 or 19-25 base pairs, preferably, the length is about 18-22, 19-20 or 19-21 base pairs). The siRNA duplex can include a 3' overhang of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and a 5' phosphate terminus.

[0546] Examples of siRNA include, but are not limited to, double-stranded polynucleotide molecules assembled from two separate strand molecules, one of which is a sense strand and the other is a complementary antisense strand; double-stranded polynucleotide molecules assembled from single-stranded molecules, wherein the sense region and the antisense region are connected by a nucleic acid-based or non-nucleic acid-based linker; double-stranded polynucleotide molecules having a hairpin secondary structure, which have self-complementary sense and antisense regions; and circular single-stranded polynucleotide molecules having two or more loop structures and a stem having self-complementary sense and antisense regions, wherein the circular polynucleotide can be treated in vivo or in vitro to produce active double-stranded siRNA molecules.

[0547] siRNA can also be chemically synthesized. siRNA can also be produced by cleaving longer dsRNAs (eg, dsRNAs greater than about 25 nucleotides in length) with E. coli RNase III or Dicer. These enzymes process dsRNA into biologically active siRNA (see, e.g., 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 Tech Notes, 10(1):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., J. Biol. Chem., 243:82 (1968)). Preferably, the dsRNA is at least 50 nucleotides to about 100, 200, 300, 400 or 500 nucleotides in length. The length of dsRNA can be up to 1000, 1500, 2000, 5000 nucleotides or longer. dsRNA can encode the entire gene transcript or a portion of the gene transcript. In some cases, siRNA can be encoded by a plasmid (e.g., transcribed as a sequence that automatically folds into a duplex with a hairpin loop).

[0548] In one embodiment, nucleic acid comprises siRNA. In one embodiment, siRNA molecule comprises a double-stranded region of about 15 to about 60 nucleotides (e.g., a length of about 15-60, 15-50, 15-40, 15-30, 15-25 or 19-25 nucleotides or a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides). siRNA molecule can silence the expression of target sequence in vitro and / or in vivo.

[0549] In other embodiments, the siRNA molecule comprises modified nucleotides, including but not limited to 2'-O-methyl (2'OMe) nucleotides, 2'-deoxy-2'-fluoro (2'F) nucleotides, 2'-deoxy nucleotides, 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 some cases, the siRNA does not comprise 2'-OMe-cytosine nucleotides. In other embodiments, the siRNA comprises a hairpin loop structure.

[0550] Nucleic acids can be prepared according to any available technology. For mRNA, the main preparation method is but not limited to enzymatic synthesis (also known as in vitro transcription) for producing long sequence-specific mRNA. In vitro transcription describes the process of template-directed synthesis of RNA molecules from an engineered DNA template, which consists of an upstream phage promoter sequence (e.g., including but not limited to promoter sequences from T7, T3 and SP6 coliphages) connected to a downstream sequence encoding a gene of interest.

[0551] RNA transcription is performed in vitro using a linearized DNA template in the presence of a corresponding RNA polymerase and adenosine, guanosine, uridine and cytidine triphosphate ribonucleosides (rNTPs) under conditions that support polymerase activity, while minimizing potential degradation of the resulting mRNA transcript. In vitro transcription can be performed using a variety of commercially available kits, including but not limited to the RiboMax large-scale RNA production system (Promega), MegaScript transcription kit (Life Technologies) and commercially available reagents, including RNA polymerase and rNTPs. Methods for in vitro transcription of mRNA are well known in the art. (See, e.g., Losick, R., 1972, In vitro transcription, Ann Rev Biochem 41st ed., 409-46; Kamakaka, RT and Kraus, WL 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 Methods in Molecular Biology 703rd ed. (Neilson, H. ed.), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter 5 - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology 530th ed., 101-114; all of which are incorporated herein by reference).

[0552] The desired in vitro transcribed mRNA is then purified from unwanted components of the transcription or related reactions (including unincorporated rNTPs, proteases, salts, short RNA oligonucleotides, etc.). Techniques for isolating mRNA transcripts are well known in the art. Well-known methods 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 methods that can be used include size exclusion chromatography (Lukaysky, PJ and Puglisi, JD, 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA 10th edition, 889-893), silica-based affinity chromatography and polyacrylamide gel electrophoresis (Bowman, JC, Azizi, B., Lenz, TK, Ray, P. and Williams, LD in RNA in vitro transcription and RNA purification by denaturing PAGE, which is contained in Recombinant and in vitro RNA syntheses Methods 941st edition Conn GL (ed.), New York, NY Humana Press, 2012). Purification can be performed using a variety of commercially available kits, including but not limited to SV Total Isolation System (Promega) and In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0553] Various modifications have been described in the art that are used to change the specific properties of in vitro transcribed mRNA and improve its effectiveness. These include, but are not limited to, modifications to the 5' and 3' ends of the mRNA. Endogenous eukaryotic mRNAs typically contain a cap structure at the 5' end of the mature molecule, which plays an important role in mediating the binding of mRNA cap binding protein (CBP), which in turn is responsible for enhancing the stability of mRNA in cells and mRNA translation efficiency. Therefore, the highest level of protein expression can be achieved by capping mRNA transcripts. The 5'-cap contains a 5'-5'-triphosphate bond between the 5'-most nucleotide and the guanine nucleotide. Additional modifications include methylation of the last and penultimate 5'-nucleotides on the 2'-hydroxyl group.

[0554] Other components of mRNA that can be modified to provide benefits in terms of translatability and stability include 5' and 3' untranslated regions (UTRs). Optimization of UTRs (favorable 5' and 3' UTRs can be obtained from cells or viral RNAs), both or independently, has been shown to improve mRNA stability and translation efficiency of in vitro transcribed mRNAs (see, e.g., Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of longRNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology 969th Edition (Rabinovich, PH editor), 2013).

[0555] In some embodiments, nucleic acid is a monocistronic or polycistronic RNA, wherein the polycistronic RNA has 2, 3, 4, 5, 6 or more coding sequences, which express 2, 3, 4, 5, 6 or more different proteins or peptides or different subunits or fragments of one or more proteins or peptides. In some aspects, nucleic acid is a mixture of multiple mRNAs, each mRNA encoding at least one protein or peptide or a subunit or fragment of a protein or peptide. In some aspects, nucleic acid is a mixture of multiple mRNAs, each mRNA encoding 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 factor is a Yamanaka factor, 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 combination of the above combinations is replaced by Glis1. In some embodiments, each mRNA encodes a 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 two, three, four, five, six 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.

[0556] In some embodiments, 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 factor is a Yamanaka factor, 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, the reprogramming factor is a combination of OCT4, SOX2, KLF4, Lin28, Nanog and c-Myc. In some embodiments, c-Myc in any combination of the above combinations is replaced by Glis1.

[0557] In some embodiments, RNA is self-replicating RNA.Self-replicating constructs are described in, for example, U.S. Patent Publication Nos. 2018 / 0216079 and 2021 / 0108179, which are incorporated herein by reference. In some embodiments, self-replicating RNA has an increased half-life in mammals, such as humans. In some aspects, self-amplifying RNA is a polycistronic. In some aspects, self-amplifying RNA is a trans-amplified RNA, in which the amplification polymerase is encoded by an RNA chain different from one or more chains targeted by the amplification.

[0558] In certain embodiments, RNA is cyclic polyribonucleotide or circular RNA. Circular polyribonucleotide or circular RNA are polyribonucleotides that form annular structure by covalent bond or non-covalent bond. In certain embodiments, cyclic polyribonucleotide is non-immunogenic in mammals such as humans. In certain embodiments, cyclic polyribonucleotide has the half-life of increase in mammals such as humans. In certain embodiments, cyclic polyribonucleotide can be replicated in cells or replicated in cells. In some aspects, circular RNA is polycistronic.

[0559] In certain embodiments, RNA comprises regulatory elements, for example, sequences that modify the expression of coding sequences in RNA. Regulatory elements may include sequences adjacent to coding sequences encoding expression products. Regulatory elements may be operably connected to adjacent sequences. Compared with the amount of products expressed when there is no regulatory element, regulatory elements may increase the amount of products expressed. In addition, a regulatory element may increase the amount of products expressed for multiple expressed sequences connected in series. Therefore, a regulatory element may enhance the expression of one or more expressed sequences. Multiple regulatory elements are well known to those of ordinary skill in the art. In certain embodiments, regulatory elements are IRES or 2A elements. In certain embodiments, IRES or 2A elements are present in the upstream of coding sequences. In certain embodiments, the sequences of IRES or 2A elements are modified or optimized to achieve desired expression profiles. In some embodiments of polycistronic RNA, each coding sequence is regulated by different IRES or 2A elements respectively, that is, each gene expressed has its own IRES or 2A elements.

[0560] 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 attenuating the cell's immune response to the transfected RNA and resulting in increased translation of the therapeutic molecule encoded by the RNA (e.g., the reprogramming factors described above).

[0561] In some embodiments, the RNA has 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 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, 970 0, 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 in length.

[0562] In some embodiments, 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, wherein 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 a monocistronic or polycistronic (DNA sequence with 2, 3, 4, 5, 6 or more encoding reprogramming factors).

[0563] In some embodiments, the RNA vector is a polycistronic RNA vector. Such polycistronic RNA vectors 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 expresses 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.

[0564] In some aspects, RNA vector is a self-replicating vector. In some aspects, self-replicating vector has a switch off. Self-replication and polycistronic constructs and constructs with start / shutdown are described in, for example, U.S. Patent Publication No. 2018 / 0216079 and No. 2021 / 0108179 (these patents are incorporated herein by reference), or described in U.S. Patent Application Nos. 17 / 812,709, 17 / 812,711 and 17 / 812,710 (these patents are incorporated herein by reference).

[0565] In certain embodiments, RNA carrier is cyclic polyribonucleotide or circular RNA. Circular polyribonucleotide or circular RNA are polyribonucleotides that form cyclic structure by covalent bond or non-covalent bond. In certain embodiments, cyclic polyribonucleotide is non-immunogenic in mammals such as humans. In certain embodiments, cyclic polyribonucleotide can be replicated in cells or replicated in cells.

[0566] In certain embodiments, the cyclic polyribonucleotide comprises an adjusting element, for example, modifies the sequence of expression of an expressed sequence in the cyclic polyribonucleotide. An adjusting element can include a sequence adjacent to the expressed sequence of the encoded expression product. An adjusting element can be operably connected to an adjacent sequence. Compared with the amount of the product expressed when there is no adjusting element, an adjusting element can increase the amount of the product expressed. In addition, an adjusting element can increase the amount of the product expressed for a plurality of expressed sequences connected in series. Therefore, an adjusting element can strengthen the expression of one or more expressed sequences. A plurality of adjusting elements are well-known to those of ordinary skill in the art.

[0567] IV. Lipid-nanoparticle compositions

[0568] The present disclosure features ionizable lipids and compositions comprising 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) to (XI), and additional lipids such as helper lipids, stabilizing lipids, and / or structural lipids.

[0569] Without wishing to be bound by any theory, it is believed that these lipid nanoparticles protect nucleic acids from degradation in serum and provide efficient delivery of nucleic acids to cells in vitro and in vivo.

[0570] The lipid-nanoparticle compositions of the present disclosure may further include nucleic acids (such as RNA) as therapeutic and / or prophylactic and / or diagnostic agents for delivery to mammalian cells or organs to modulate polypeptide, protein or gene expression.

[0571] The lipid composition can be prepared by mixing the ionizable lipids of Formula (I) to (XIX) or a combination thereof with helper lipids or a combination thereof, stabilizing lipids and / or structural lipids or a combination thereof in a solvent such as ethanol and water to produce the desired molar ratio.

[0572] Helper lipids for use in lipid-nanoparticle compositions of the present disclosure may include lipids that can assemble into one or more lipid bilayers. Helper lipids for use in lipid-nanoparticle compositions of the present disclosure may include lipids that increase the stability or delivery efficiency of lipid nanoparticles.

[0573] Illustrative examples of helper lipids that may also be used in the lipid nanoparticle compositions of the present disclosure may include, but are not limited to, the following:

[0574]

[0575] Other illustrative examples of helper lipids that can be used in the lipid nanoparticle compositions of the present disclosure can include, but are not limited to, the following:

[0576]

[0577] 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.

[0578] In yet another embodiment, the auxiliary lipid that can be used 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-di(undecanoyl)-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), l-oleoyl-2-cholesterol hemisuccinyl-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-di(docosahexaenoyl)-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dialinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG) and mixtures thereof.

[0579] Stabilizing lipids for use in lipid-nanoparticle compositions may include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG) having an average PEG molecular weight of 2,000.

[0580] The lipid component of lipid-nanoparticle composition can include one or more structural lipids. The structural lipid can be selected from the group consisting of but not limited to cholesterol, coprostanol, sitosterol (including beta sitosterol), ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, alpha-tocopherol and mixtures thereof. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid includes cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone and hydrocortisone) or a combination thereof.

[0581] Structured lipids may 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 may 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-cholane (LITHO 1a), 23-(4-methylfuran-2,5-dione)-3α,7α-dihydroxy-24-nor-5β-cholane (CHENO 1b), 23-(4-methyl-1H-pyrrole-2,5-dione)-3α-hydroxy-24-nor-5β-cholane (LITOMAL 7a), 23-(4-methyl-1H-pyrrole-2,5-dione)-3α,7α,12α-trihydroxy-24-nor-5β-cholane (COLMAL 7b). 7f) and ethanol maleimide 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-cholane), and the systematic name of CHENOMET is (23-((2-hydroxyethyl)-4-methyl-1H-pyrrole-2,5-dione)-3α,7α-dihydroxy-24-n-5β-cholane).

[0582] 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), micro RNA (miRNA), miRNA inhibitors (antagomirs / antimirs), dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), multivalent RNA, and mixtures thereof.

[0583] Nucleic acid or mRNA can be self-amplification RNA, polycistronic RNA, self-amplification polycistronic RNA or circular RNA.In some aspects, nucleic acid or mRNA expresses protein or peptide.In some aspects, the protein or peptide expressed from nucleic acid or mRNA is antibody, human antibody, camel antibody, nano antibody, humanized antibody, bispecific antibody, enzyme, genome editing enzyme or nuclease, growth factor, cytokine, chemokine, small molecule-mimetic peptide, transcription factor, structural molecule, signal molecule, reprogramming factor, vaccine antigen or their combination.In some aspects, mRNA encodes protein or peptide that works in cell.In some aspects, mRNA encodes at least one reprogramming factor.

[0584] The protein or peptide expressed by 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, collagen is collagen VII. In some embodiments, collagen VII is used in a method for restoring, treating, remodeling or improving skin or extracellular matrix. In some embodiments, collagen VII is used in a method for wound healing.

[0585] The protein or peptide expressed by the nucleic acid or mRNA of the present technology can also be a growth factor, a cytokine, or a combination thereof, such as EGF, FGF, NGF, CNTF, PDGF, VEGF, IGF, GMCSF, GCSF, TGF, erythropoietin, ephrin, GDNF, GDF9, KGF, angiogenin, TPO, BMP, HGF, BDNF, GDF, HGH (growth hormone), neurotrophin, MSF, SGF, GDF (including GDF11), TGF (including TGF-b), 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.

[0586] The protein or peptide expressed by the nucleic acid or mRNA of the present technology can be a human antibody, a humanized antibody, a camel 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 works in the cell. In some aspects, the protein or peptide expressed from the nucleic acid or mRNA is secreted.In some embodiments, the antibody is at least one of or is substantially similar to trastuzumab, grofituzumab, miliguzumab, sometuximab, nesecvir, tremelimumab, terituzumab, doramuzumab, sepelizumab, lecanezumab, tislelizumab, panplizumab, sintilimab, teplizumab, toripalizumab, obotuzumab, retivanlimab, ubrituximab, inomucomab, opotuzumab, nasolizumab, motuzumab, tesagvir, celagavir, relalizumab, tebentafusip, faricizumab, sutilizumab, sotovir, rendavimab, casprevir, edema Midevimab, tezelumab, tesofumab, ervantumab, anifrumumab, lantuximab, bimegizumab, trorocirumab, evuximab, saccitumomab, tetumumab, isatuximab, epinephrine, dotalimumab, ansvirmab, makituximab, nasituximab, atevimab, mateviromab, ociviromab, mabetuximab, tancitumomab, satelizumab, inbilizumab, enfortuzumab, rizanlizumab, bucitumomab, polotuzumab, risankizumab, romozumab, calicizumab, revulizumab, imatinib, cemiplizumab, remanezumab, mosetumab , gacanezumab, ranarumab, moglizumab, erenumab, tirizizumab, ibalizumab, brosuzumab, durvalumab, emicizumab, benralizumab, ocrelizumab, guselkumab, intuzumab, salizumab, dupilumab, avelumab, bodalumab, atezolizumab, belotuzumab, olaratumumab, reslizumab, otuximab, ixekizumab, datuximab, elotuzumab, necituzumab, idarucizumab, alirocumab, mepolizumab, evolocumab, datuximab, secukinumab, nivolumab, belintumumab, pembrolizumab, Ramuciumab, vedolizumab, cetuximab, obotuzumab, ranikumab, pertuzumab, brentuximab, belimumab, ipilimumab, denosumab, tocilizumab, ofatumumab, canakinumab, golimumab, ustekinumab, certolizumab pegol, catuximab, eculizumab, ranibizumab, panitumumab, natalizumab, bevacizumab, cetuximab, efalizumab, omalizumab, tositumomab, ibrutinib, adalimumab, alemtuzumab, gemtuzumab, infliximab, palivizumab, basiliximab, daclizumab, rituximab, abciximab, edrecolomab, nebacumab or muromonab. In some embodiments, the protein or peptide is used in a method of treating a human or veterinary disease.

[0587] The lipid-nanoparticle compositions of the present disclosure can be prepared by mixing processes such as, but not limited to, microfluidics and T-junction mixing of two fluid streams, one of which contains nucleic acids and the other has a lipid component. Such mixing processes result in nanoprecipitation and particle formation.

[0588] The lipid-nanoparticle compositions of the present disclosure can be characterized using a zeta sizer to determine particle size, polydispersity index (PDI), and zeta potential. In some embodiments, the zeta potential of the lipid-nanoparticle composition can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0589] UV-Vis spectroscopy can be used to determine the concentration of nucleic acids in nanoparticle compositions.

[0590] The lipid-nanoparticle compositions can induce expression of a desired protein in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more of the ionizable lipids described herein, wherein the lipid nanoparticles are encapsulated or associated with a nucleic acid (e.g., mRNA) that is expressed to produce the desired protein.

[0591] The lipid-nanoparticle compositions can reduce the expression of both target genes and proteins in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more of the ionizable lipids described herein, wherein the lipid nanoparticles are encapsulated or associated with a nucleic acid (e.g., siRNA) that reduces the expression of the target gene.

[0592] The lipid-nanoparticle compositions can downregulate or silence the expression of both target genes and proteins in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more of the ionizable lipids described herein, wherein the lipid nanoparticles are encapsulated or associated with nucleic acids that downregulate or silence the expression of the target gene.

[0593] 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, reproductive 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 restored, including but not limited to epithelial cells (e.g., squamous, cuboidal, columnar and pseudostratified epithelial cells), endothelial cells (e.g., venous, arterial and lymphatic endothelial cells) and cells of the connective tissue, muscle and nervous system. Such cells may 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. cells and / or tissues from the kidney, liver, pancreas, stomach, spleen, gall bladder, intestine, bladder, lung, prostate, breast, genitourinary tract, pituitary cells, oral cavity, esophagus, skin, hair, nails, thyroid, parathyroid glands, adrenal glands, eyes, nose, or brain.

[0594] Cells that can be treated according to the present technology can be 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.

[0595] 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 certain embodiments, lymphocytes are T cells, B cells or natural killer (NK) cells. In certain embodiments, lymphocytes are tumor infiltrating lymphocytes.

[0596] The methods and compositions of the present technology can also be applied to lymphocytes, wherein the lymphocytes are T cells. In some embodiments, T cells are cytotoxic T cells (CD8+), helper T cells (CD4+), suppressive or regulatory T cells (Treg), memory T cells, natural killer T cells (NKT cells) or γδT cells. In other embodiments, helper T cells are Th1, Th2, Th17, Th9 or Tfh T cells. In some embodiments, 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 suppression or regulation T cells of the present technology are FOXP3+T cells or FOXP3-T cells. In some embodiments, NKT cells are subsets of CD1d-restricted T cells.

[0597] The methods and compositions of the present technology can also be applied to granulocytes, wherein the granulocytes are neutrophils, eosinophils, basophils, or mast cells.

[0598] The methods and compositions of the present technology can also be applied to lymphocytes, wherein the lymphocytes are B cells, such as memory B cells or plasma cells.

[0599] The methods and compositions of the present technology can also be applied to immune cells, wherein the immune cells are monocytes, macrophages, microglia or dendritic cells.

[0600] The methods and compositions described herein can be used, wherein the cells are immune cells, such as natural immune cells or engineered immune cells. In some embodiments, the methods and compositions described herein are used in parallel or continuously with the methods of engineered cells (including engineered immune cells), so that these methods are performed before, during and / or after the cells are engineered. In some embodiments, the methods and compositions described herein are used to engineer cells (including engineered immune cells). In some embodiments, such engineering includes engineering so that cells express chimeric antigen receptors, such as immune cells expressing chimeric antigens. In some embodiments, such chimeric antigen receptors target CD19, CD20, CD22, CD30, CD33, CD123, FLT3, BCMA, GD2, HER2, MUC1, B7-H3, IL13Ra2, TAG72, MUC16, BCMA or at least one of any other antigens suitable for immunotherapy. In some embodiments, such engineering includes engineering cells (including immune cells) to express other proteins or peptides, such as growth factors and cytokines. In some embodiments, the cytokines include IL-15. In some embodiments, such engineering of cells (such as immune cells) is performed in vitro, for example, in the manufacture of cell therapy products (such as autologous or allogeneic chimeric antigen receptor (CAR)-T, CAR-NK, CAR-M or CAR-NKT cells). 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 via a chimeric antigen receptor. In some embodiments, the CAR-T cells provided herein target at least one of CD19 or BCMA via a chimeric antigen receptor. In some embodiments, the CAR-T cells provided herein target CD19 via a chimeric antigen receptor. In some embodiments, the CAR-T cells provided herein target BCMA via a chimeric antigen receptor. In some embodiments, the CAR-NK cells provided herein target CD19, FLT3, CD20, CD38, CD138, BCMS, CS1, CD3, CD5, CD7, NKG2D, HER2, EGFR, EpCAM, TF, B7-H6, HLA-G, CD24, CD44, CD133, mesothelin, or at least one of αFR through a chimeric antigen receptor. In some embodiments, the CAR-M cells provided herein target CD19, HER2, CD22, or at least one of ALK19 through 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 target GD2 through a chimeric antigen receptor and are engineered to express IL-15. In some embodiments, the immune cell recovery method described herein is performed in vitro during or after the manufacture of a cell therapy product. In other embodiments, such engineering of cells and / or immune cells is performed in vitro, for example, in the so-called "in situ" production of CAR engineered cells. In such embodiments, RNA and / or mRNA encoding CAR or growth factors or cytokines contained in a lipid composition or lipid-nanoparticle composition disclosed herein are injected into a subject or patient, for example, for CAR engineering of immune cells (such as T cells, NK cells, macrophages, tumor infiltrating lymphocytes, dendritic cells and / or "in situ" NKT cells) of a patient, i.e., in a patient, cells can be transfected in vitro without removing cells. In such embodiments, the immune cell recovery method described herein is also performed in vivo, wherein mRNA encoding one or more reprogramming factors is injected into a patient before, at the same time, or after mRNA encoding CAR or other cell engineering molecules. In some embodiments, lipids and lipid-nanoparticle compositions of the present disclosure are selected for targeted delivery to any cell in the body, including immune cells, such as T cells, NK cells, macrophages, tumor infiltrating cells, dendritic cells and / or NKT cells in the body. In other embodiments, in vivo treatment is performed in the absence of any other in vivo cell engineering to enhance or restore the effectiveness of the immune system and treat diseases associated with immune dysfunction or disorders, such as improving the effect of the immune system against cancer or infection or reducing inflammation.

[0601] The methods and compositions of the present technology can also be applied to the recovery of immune cells, wherein the immune cells to be recovered are non-adherent cells, such as non-adherent immune cells. In some embodiments, non-adherent cells, including non-adherent immune cells, are treated, transiently reprogrammed, recovered or manufactured in a manner where the cells remain non-adherent and do not adhere to a tissue culture matrix or form or produce cells or cell colonies that adhere to a tissue culture matrix. In some embodiments, the reprogramming interval and factors are selected so that the cells are recovered and the cell characteristics are retained, wherein the cells remain non-adherent and do not adhere to a tissue culture matrix or form or produce cells or cell colonies that adhere to a tissue culture matrix. Thus, in some embodiments, the present technology provides lipid-containing compositions and lipid-nanoparticle compositions for delivering mRNA encoding at least one reprogramming factor for cell rejuvenation, wherein the cells, including 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 wherein the cells are rejuvenated and retain cellular properties and wherein the cells remain in suspension without adhering, nor do they become or generate adherent cells, become adherent or form adherent colonies.

[0602] The methods described herein (including methods for restoring immune cells; methods for reversing, preventing or inhibiting the exhaustion of immune cells; or inducing the proliferation of immune cells) include applying about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times of the present disclosure to immune cells within a period of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, including providing an mRNA encoding at least one reprogramming factor, containing a lipid composition or lipid-nanoparticle composition. For example, mRNA can be applied once on the first or second day of a five-day or six-day period, or can be applied once on the first day of a five-day or six-day period and once on the third day, or can be applied once within a one-day period. In some embodiments, 1, 2, 3, 4, 5 or 6 times of mRNA are applied to immune cells within a period of 1, 2, 3, 4, 5 or 6 days. In some embodiments, mRNA is applied after the immune cell activation step. In some embodiments, the immune cell activation step includes activating immune cells for 1, 2 or 3 days. In some embodiments, the immune cell activation step includes activating immune cells using at least one of CD3, CD28 and IL-2 to activate immune cells. In some embodiments, immune cells are activated with CD3 and CD28. In some embodiments, the mRNA administration phase occurs immediately after the immune cell activation step. In some embodiments, the mRNA administration phase 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 mRNA encoding the reprogramming factor is used to reverse the immune cell failure caused by the immune cell activation step. In some embodiments, the mRNA encoding the reprogramming factor is used to reverse the immune cell failure in the immune cells from the elderly patient or the donor. In some embodiments, mRNA is used during the manufacturing process for the manufacture of immune cells (e.g., CAR-T, CAR-M or CAR-NK cells) for transplantation.

[0603] Compared with using different mRNA delivery mechanisms, lipids or lipid nanoparticles using the present technology are used to deliver mRNA to provide enhanced recovery, proliferation, exhaustion recovery or prevention, therapeutic effect, antipathogenic effect, anticancer effect, anti-immunogenic effect or anti-inflammatory effect in cells treated or recovered using the methods or compositions herein. In certain embodiments, such enhanced recovery, proliferation, exhaustion recovery or prevention, therapeutic effect, antipathogenic effect, anticancer effect, anti-immunogenic effect or anti-inflammatory effect are attributed to lower toxicity, immunogenicity and / or lower physiological effects on cells when compared with different delivery mechanisms. In certain embodiments, different delivery mechanisms are electroporation, so that compared with using electroporation, lipids or lipid-nanoparticle compositions of the present disclosure are used to deliver mRNA to cause enhanced recovery, proliferation, exhaustion recovery or prevention, therapeutic effect, antipathogenic effect, anticancer effect, anti-immunogenic effect or anti-inflammatory effect in cells treated or recovered using the methods or compositions herein. Such improvements compared with electroporation can be attributed to reduced toxicity compared with electroporation or reduced physiological effects on cells.

[0604] The lipid or lipid-nanoparticle composition of the present technology is used in a method of delivering a therapeutic or diagnostic agent to the skin, such as 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 technology provides delivery of a therapeutic or diagnostic agent (such as a reprogramming factor) in a manner that achieves transient reprogramming of cells (such as skin cells or immune cells). In some embodiments, transient reprogramming of cells provides transient expression of a therapeutic or diagnostic agent (such as a reprogramming factor), wherein the agent is expressed in the cell for a duration sufficient to reprogram and / or restore without changing the properties of the cell, i.e., restore the skin or immune cells to exhibit characteristics or younger skin or immune cells while retaining the properties of the skin or immune cells.

[0605] In some cases, the therapeutic agent of the present technology is mRNA disclosed herein. In some aspects, the lipid or lipid-nanoparticle composition of the present disclosure is used in the method for treating or preventing dermatological diseases or disorders, treating or preventing diseases or disorders of the skin, or for cosmetic applications in the skin, including applying a lipid-containing composition or lipid-nanoparticle composition comprising at least one therapeutic agent or diagnostic agent. In some aspects, the lipid or lipid-nanoparticle composition of the present disclosure is used in the method for restoring skin, including applying a lipid-containing composition or lipid-nanoparticle composition comprising a therapeutic agent or diagnostic agent. In some aspects, the lipid or lipid-nanoparticle composition of the present disclosure is used in the method for restoring skin, including applying a lipid-containing composition or lipid-nanoparticle composition comprising an mRNA encoding at least one reprogramming factor. In such methods, mRNA can be bound to lipids or contained in lipid nanoparticles. In some aspects, such methods further include transfecting skin cells with a lipid-containing composition or lipid-nanoparticle composition to deliver mRNA. In some aspects, lipids or lipid-nanoparticle compositions of the present disclosure are used in methods for restoring skin, including applying a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor, to achieve restoration of skin while retaining cell properties. In such methods, mRNA can be bound to lipids or contained in lipid nanoparticles. In some aspects, lipids or lipid-nanoparticle compositions of the present disclosure are used in methods for restoring skin, including applying a lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor to skin cells, wherein expressing at least one reprogramming factor in skin cells causes increased fibroblast proliferation and retains skin cell properties. In such methods, mRNA can be bound to lipids or contained in lipid nanoparticles.

[0606] Lipid or lipid-nanoparticle composition can be used in the method of wound healing, including applying lipid-containing composition or lipid-nanoparticle composition comprising therapeutic agent or diagnostic agent. In some aspects, lipid or lipid-nanoparticle composition is used in the method of wound healing, including applying lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor. In such methods, mRNA can be bound to lipid or contained in lipid nanoparticles. In some aspects, lipid or lipid-nanoparticle composition is used in the method of wound healing, wherein lipid-containing composition or lipid-nanoparticle composition delivers mRNA encoding at least one reprogramming factor, to achieve wound healing, while retaining cell characteristics. In such methods, mRNA can be bound to lipid or contained in lipid nanoparticles. In some aspects, lipid or lipid-nanoparticle composition of the present disclosure is used in the method of wound healing, including applying lipid-containing composition or lipid-nanoparticle composition comprising mRNA encoding at least one reprogramming factor to skin cells, wherein expressing at least one reprogramming factor in skin cells causes increased fibroblast proliferation. In such methods, mRNA can be bound to lipid or contained in lipid nanoparticles.

[0607] Lipid or lipid-nanoparticle composition can be used in the method for treating or preventing dermatological diseases or disorders, treating or preventing skin diseases or disorders or for cosmetic applications in the skin, including applying a lipid-containing composition or lipid-nanoparticle composition comprising a therapeutic agent to achieve the reversal of at least one skin aging marker. In some aspects, lipid or lipid-nanoparticle composition is used in a method for restoring skin, including applying a lipid-containing composition or lipid-nanoparticle composition to deliver mRNA encoding at least one reprogramming factor to the skin, thereby achieving the reversal of at least one skin aging marker, while retaining cell characteristics. In some aspects, lipid or lipid-nanoparticle composition is used in a method for wound healing, including applying a lipid-containing composition or lipid-nanoparticle composition to deliver mRNA encoding at least one reprogramming factor to skin cells, thereby achieving the reversal of at least one skin aging marker, while retaining cell characteristics. In some embodiments, the reversal of at least one skin aging marker refers to the generation of restored cells that express at least one skin aging marker in a manner similar to the expression of the marker seen in young skin cells compared to aged skin cells.

[0608] Markers that may 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 aspects, 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 aspects, the reversal of at least one skin aging marker is an upregulation of mRNA or protein expression of PRSS23, LOXL4, MMP11, ELN, or any combination thereof. In some aspects, the reversal of at least one skin aging marker is an upregulation of mRNA or protein expression of PRSS23. In some aspects, the reversal of at least one skin aging marker is an upregulation of mRNA or protein expression of LOXL4. In some aspects, the reversal of at least one skin aging marker is an upregulation of mRNA or protein expression of MMP11. In some aspects, the reversal of at least one skin aging marker is an upregulation of mRNA or protein expression of ELN. In some aspects, the reversal of at least one skin aging marker is a downregulation of mRNA or protein expression of MMP3, MMP1, SOD2, or any combination thereof. In some aspects, the reversal of at least one skin aging marker is a downregulation of mRNA or protein expression of MMP3. In some aspects, the reversal of at least one skin aging marker is a downregulation of mRNA or protein expression of MMP1.In some aspects, the reversal of at least one skin aging marker is downregulation of mRNA or protein expression of SOD2. In some aspects, the 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.

[0609] The lipid or lipid-nanoparticle composition can be used in a method for treating or preventing a dermatological disease or condition, treating or preventing a skin disease or condition, or for cosmetic applications in the skin, including applying a lipid-containing composition or a lipid-nanoparticle composition comprising a therapeutic agent to achieve an improvement in at least one skin quality marker. In some aspects, the lipid or lipid-nanoparticle composition is used in a method for restoring the skin, including applying a lipid-containing composition or a lipid-nanoparticle composition comprising an 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 lipid or lipid-nanoparticle composition is used in a method for restoring the skin, including applying a lipid-containing composition or a lipid-nanoparticle composition comprising an mRNA encoding at least one reprogramming factor to achieve an improvement in at least one skin quality marker while retaining cell properties.

[0610] Lipid or lipid-nanoparticle composition can be used in the method for wound healing, including applying to skin cells the lipid composition or lipid-nanoparticle composition containing the mRNA encoding at least one reprogramming factor, to achieve the improvement of at least one skin quality marker. In some aspects, 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, secretory phosphoprotein 1, cartilage oligomeric matrix protein, MMP1, MMP3, MMP12, SOD2 or any combination thereof. In some aspects, the improvement of at least one skin quality marker is the mRNA or protein expression of MMP1, MMP3, MMP12, SOD2 or any combination thereof downward regulation. In some aspects, the improvement of at least one skin quality marker is the increase 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, secretory phosphoprotein 1, cartilage oligomeric matrix protein or any combination thereof. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of collagen. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of type I collagen. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of type III collagen. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of type IV collagen. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of type V collagen. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of type VI collagen. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of type XI collagen. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of type XI collagen. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of elastin. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of microfibril-associated protein 5. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of periostin. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of versican. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of connective tissue growth factor.In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of lysyl oxidase. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of SPARC. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of secretory phosphoprotein 1. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of cartilage oligomeric matrix protein. In some aspects, the improvement of at least one skin quality marker is the decrease of mRNA or protein expression of MMP3, MMP1, SOD2 or any combination thereof. In some aspects, the improvement of at least one skin quality marker is the decrease of mRNA or protein expression of MMP3. In some aspects, the improvement of at least one skin quality marker is the decrease of mRNA or protein expression of MMP1. In some aspects, the improvement of at least one skin quality marker is the decrease of mRNA or protein expression of SOD2. In some aspects, the improvement of at least one skin quality marker is the increase of mRNA or protein expression of collagen VII and elastin. In some aspects, the improvement in at least one skin quality marker is upregulation of mRNA or protein expression of at least one of collagen VII and elastin; downregulation of mRNA or protein expression of at least one of MMP3, MMP1, SOD2; or any combination thereof.

[0611] The lipid-nanoparticle compositions or lipid-containing compositions of the present disclosure can be topically applied to the skin. In some aspects, the lipid-nanoparticle compositions of the present disclosure or compositions containing lipids of the present disclosure are applied to the skin in the form of ointments, creams or salves. In some aspects, the lipid-nanoparticle compositions of the present disclosure or compositions containing lipids of the present disclosure are applied to the skin via dermal, intradermal or subcutaneous injections. In some aspects, the lipid-nanoparticle compositions of the present disclosure or compositions containing lipids of the present disclosure are applied to the skin via gels. In some aspects, the lipid-nanoparticle compositions of the present disclosure or compositions containing lipids of the present disclosure are applied in vivo, in vitro or ex vivo. In some aspects, the lipid-nanoparticle compositions of the present disclosure or compositions containing lipids of the present disclosure are applied in vivo. In some aspects, the lipid-nanoparticle compositions of the present disclosure or compositions containing lipids of the present disclosure are used for human or animal subjects.

[0612] The lipid-nanoparticle compositions of the present disclosure or lipid-containing compositions can transfect skin cells to deliver at least one therapeutic agent or diagnostic agent to skin cells. In some aspects, the therapeutic agent is a nucleic acid. In some aspects, the therapeutic agent is an mRNA. In some aspects, the therapeutic agent is a combination of mRNA and siRNA. In some aspects, the therapeutic agent is a combination of mRNA and miRNA. In some aspects, the skin cells are keratinocytes, melanocytes, Langerhans cells, follicle cells, fibroblasts, endothelial cells, smooth muscle cells, Merkel cells, basal cells, squamous cells, apocrine cells, eccrine cells, sebaceous gland cells, lymphatic endothelial cells or a combination thereof. In some aspects, select lipids or lipid-nanoparticle compositions to provide selective transfection of specific cell types or multiple cell types. In some aspects, select lipids or lipid-nanoparticle compositions to provide diffusion in the skin or in at least one layer of the skin.

[0613] Diseases or conditions that can be treated or prevented include dermatological diseases or conditions or diseases or conditions of the treated skin that can be treated or prevented using the lipid compositions or lipid-nanoparticle compositions of the present technology. For example, skin looseness or chronic wounds can be treated according to the present technology. In some aspects, skin looseness or chronic wounds are diabetic ulcers, ischemic ulcers and pressure sores. In some aspects, the dermatological diseases or conditions or diseases or conditions of the skin treated or prevented using the lipid compositions or lipid-nanoparticle compositions of the present disclosure are inflammatory skin diseases. In some aspects, inflammatory skin diseases are psoriasis, atopic dermatitis, vitiligo, alopecia areata or hidradenitis suppurativa. In some aspects, the dermatological diseases or conditions or diseases or conditions of the skin treated or prevented using the lipid compositions or lipid-nanoparticle compositions of the present disclosure are hair disorders. In some aspects, hair disorders are non-scarring or scarring alopecia, graying of hair, hirsutism. In some aspects, hair disorders are non-scarring alopecia, which is androgenic alopecia. In some aspects, scarring alopecia is lichen planus. In some aspects, the dermatological disease or disorder or skin disease or disorder treated or prevented using the lipid composition or lipid-nanoparticle composition of the present disclosure is skin cancer. In some aspects, skin cancer is basal cell carcinoma, squamous cell carcinoma or actinic keratosis. In some aspects, the dermatological disease or disorder or skin disease or disorder treated or prevented using the lipid composition or lipid-nanoparticle composition of the present disclosure is prurigo nodularis, acne, rosacea or solar lentigo. In some aspects, the method for treating any one of dermatological diseases or disorders or diseases includes applying a composition containing a lipid of the present disclosure or a lipid-nanoparticle composition containing a therapeutic agent of the present disclosure. In some aspects, the therapeutic agent is mRNA. In some aspects, the therapeutic agent is mRNA encoding antibodies, human antibodies, humanized antibodies, nanobodies, camel antibodies, bispecific antibodies, enzymes, genome editing enzymes or nucleases, growth factors, cytokines, chemokines, transcription factors, structural molecules, signaling molecules, reprogramming factors. In some aspects, the therapeutic agent is an mRNA encoding a protein or peptide that functions within a cell. In some aspects, the therapeutic agent is an mRNA encoding at least one reprogramming factor.

[0614] Lipid compositions or lipid-nanoparticle compositions can be used in methods for wound healing, wherein the wound is dermatoporosis or chronic wounds. In some aspects, dermatoporosis or chronic wounds are diabetic ulcers, ischemic ulcers and pressure sores. In some aspects, lipid compositions or lipid-nanoparticle compositions of the present disclosure are used in methods for wound healing, wherein the wound is a lesion from skin cancer. In some aspects, skin cancer is basal cell carcinoma, squamous cell carcinoma or actinic keratosis. In some aspects, any wound healing method in the above-mentioned wound healing methods includes applying a composition containing a lipid of the present disclosure or a lipid-nanoparticle composition containing a therapeutic agent. In some aspects, the therapeutic agent is mRNA. In some aspects, the therapeutic agent is an mRNA encoding an antibody, a human antibody, a humanized antibody, a nanobody, a camel antibody, a bispecific antibody, an enzyme, a genome editing enzyme or a nuclease, a growth factor, a cytokine, a chemokine, a transcription factor, a structural molecule, a signaling molecule, a reprogramming factor. In some aspects, the therapeutic agent is an mRNA encoding a protein or peptide that works in a cell. In some aspects, the therapeutic agent is mRNA encoding at least one reprogramming factor.

[0615] The lipid-nanoparticle composition can contain lipid formula (II) and 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 described herein. Such compositions comprising lipid formula (II) when used to transfect at least one reprogramming factor can produce a greater improvement in at least one skin quality marker compared to other formulations or to lipid-nanoparticle compositions containing other lipids described herein.

[0616] In embodiments, lipid-nanoparticle compositions containing lipid formula (IB) provide higher transfection efficiency compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, lipid-nanoparticle compositions containing lipid formula (IB) provide higher transfection efficiency compared to lipid-nanoparticle compositions containing lipid formula (I) with the same head group. In embodiments, lipid-nanoparticle compositions containing lipid formula (IB) provide higher transfection efficiency in skin cells compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, lipid-nanoparticle compositions containing lipid formula (IB) provide higher transfection efficiency in skin cells compared to lipid-nanoparticle compositions containing lipid formula (I) with the same head group. In embodiments, lipid-nanoparticle compositions containing lipid formula (IB) provide higher transfection efficiency in fibroblasts compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, lipid-nanoparticle compositions containing lipid formula (IB) provide higher transfection efficiency in fibroblasts compared to lipid-nanoparticle compositions containing lipid formula (I) with the same head group. In an embodiment, a lipid-nanoparticle composition containing lipid formula (IB) 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 (IB) provides a higher transfection efficiency in immune cells compared to a lipid-nanoparticle composition containing lipid formula (I) with the same head group. In an embodiment, a lipid-nanoparticle composition containing lipid formula (IB) 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 formula (IB) provides a higher transfection efficiency in T cells compared to a lipid-nanoparticle composition containing lipid formula (I) with the same head group. In an embodiment, the tail structure of lipid formula (IB) provides an increased transfection efficiency for the lipid-nanoparticle composition. In an embodiment, the tail structure of lipid formula (IB) provides an increased transfection efficiency compared to the non-branched alkyl or alkenyl tail structure of the lipid-nanoparticle composition. In an embodiment, the tail structure of lipid formula (IB) provides an increased transfection efficiency for lipid-nanoparticle compositions compared to the tail structure of lipid formula (I) having the same head group. In an embodiment, the ester group adjacent to the branching of the alkyl group in the tail structure of the lipid described herein provides an increased transfection efficiency for the lipid-nanoparticle composition. In an embodiment, the ester group adjacent to the branching of the alkyl group in the tail structure of the lipid described herein provides an increased transfection efficiency for the lipid-nanoparticle composition compared to an alkyl or alkenyl tail structure without branching.

[0617] In embodiments, lipid-nanoparticle compositions containing lipid formula (IB) provide higher viability compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, lipid-nanoparticle compositions containing lipid formula (IB) provide higher viability compared to lipid-nanoparticle compositions containing lipid formula (I) with the same head group. In embodiments, lipid-nanoparticle compositions containing lipid formula (IB) provide higher viability in skin cells compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, lipid-nanoparticle compositions containing lipid formula (IB) provide higher viability in skin cells compared to lipid-nanoparticle compositions containing lipid formula (I) with the same head group. In embodiments, lipid-nanoparticle compositions containing lipid formula (IB) provide higher viability in fibroblasts compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, lipid-nanoparticle compositions containing lipid formula (IB) provide higher viability in fibroblasts compared to lipid-nanoparticle compositions containing lipid formula (I) with the same head group. In an embodiment, a lipid-nanoparticle composition containing lipid formula (IB) provides higher activity in immune cells compared to lipid-nanoparticle compositions containing other lipids described herein. In an embodiment, a lipid-nanoparticle composition containing lipid formula (IB) provides higher activity in immune cells compared to lipid-nanoparticle compositions containing lipid formula (I) with the same head group. In an embodiment, a lipid-nanoparticle composition containing lipid formula (IB) provides higher activity in T cells compared to lipid-nanoparticle compositions containing other lipids described herein. In an embodiment, a lipid-nanoparticle composition containing lipid formula (IB) provides higher activity in T cells compared to lipid-nanoparticle compositions containing lipid formula (I) with the same head group. In an embodiment, the tail structure of lipid formula (IB) provides increased activity for lipid-nanoparticle compositions. In an embodiment, the tail structure of lipid formula (IB) provides increased activity for lipid-nanoparticle compositions compared to unbranched alkyl or alkenyl tail structures. In an embodiment, the tail structure of lipid formula (IB) provides increased activity for lipid-nanoparticle compositions compared to the tail structure of lipid formula (I). In an embodiment, the ester group adjacent to the branching of the alkyl group in the tail structure of lipids described herein provides increased activity for lipid-nanoparticle compositions. In an embodiment, the ester group adjacent to the branching of the alkyl group in the tail structure of lipids described herein provides increased activity for lipid-nanoparticle compositions compared to alkyl or alkenyl tail structures without branching.In an embodiment, the ester groups adjacent to R6 and R7 and to R8 and R10 in the tail structure of lipid formula (IB) together with the branching of R6, R7, R8 and R10 provide the lipid-nanoparticle composition with increased activity. In an embodiment, the ester groups adjacent to R6 and R7 and to R8 and R10 in the tail structure of lipid formula (IB) together with the branching of R6, R7, R8 and R10 provide the lipid-nanoparticle composition with increased activity compared to an alkyl or alkenyl tail structure without branching.

[0618] In an embodiment, a lipid-nanoparticle composition containing lipid formula (IB) provides both higher viability and higher transfection efficiency compared to lipid-nanoparticle compositions containing other lipids described herein. In an embodiment, a lipid-nanoparticle composition containing lipid formula (IB) provides both higher viability and higher transfection efficiency compared to lipid-nanoparticle compositions containing lipid formula (I) with the same head group. In an embodiment, a lipid-nanoparticle composition containing lipid formula (IB) provides both higher viability and higher transfection efficiency in skin cells compared to lipid-nanoparticle compositions containing other lipids described herein. In an embodiment, a lipid-nanoparticle composition containing lipid formula (IB) provides both higher viability and higher transfection efficiency in skin cells compared to lipid-nanoparticle compositions containing lipid formula (I) with the same head group. In an embodiment, a lipid-nanoparticle composition containing lipid formula (IB) provides both higher viability and higher transfection efficiency in fibroblasts compared to lipid-nanoparticle compositions containing other lipids described herein. In an embodiment, a lipid-nanoparticle composition containing lipid formula (IB) provides both higher viability and higher transfection efficiency in fibroblasts compared to a lipid-nanoparticle composition containing lipid formula (I) with the same head group. In an embodiment, a lipid-nanoparticle composition containing lipid formula (IB) provides both higher viability and 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 (IB) provides both higher viability and higher transfection efficiency in immune cells compared to a lipid-nanoparticle composition containing lipid formula (I) with the same head group. In an embodiment, a lipid-nanoparticle composition containing lipid formula (IB) provides both higher viability and 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 formula (IB) provides both higher viability and higher transfection efficiency in T cells compared to a lipid-nanoparticle composition containing lipid formula (I) with the same head group. In an embodiment, the tail structure of lipid formula (IB) provides both increased transfection efficiency and increased viability. In an embodiment, the tail structure of lipid formula (IB) provides both increased transfection efficiency and increased viability compared to an alkyl or alkenyl tail structure without branching. In an embodiment, the tail structure of lipid formula (IB) provides both increased transfection efficiency and increased viability for a lipid-nanoparticle composition compared to the tail structure of lipid formula (I).In an embodiment, the ester groups adjacent to the branching of the alkyl groups in the tail structure of the lipids described herein provide both increased transfection efficiency and increased activity for the lipid-nanoparticle composition. In an embodiment, the ester groups adjacent to R6 and R7 and to R8 and R10 in the tail structure of lipid formula (IB) provide both increased transfection efficiency and increased activity for the lipid-nanoparticle composition together with the branching of R6, R7, R8 and R10. In an embodiment, the ester groups adjacent to R6 and R7 and to R8 and R10 in the tail structure of lipid formula (IB) provide both increased transfection efficiency and increased activity for the lipid-nanoparticle composition compared to the alkyl or alkenyl tail structure without branching.

[0619] When used to transfect at least one reprogramming factor into skin cells, such compositions comprising lipid formula (IB) can produce greater improvement in at least one skin rejuvenation marker or skin quality marker compared to lipid-nanoparticle compositions containing other lipids as described herein. When used to transfect at least one reprogramming factor into skin cells, such compositions comprising lipid formula (IB) can produce greater improvement in at least one skin rejuvenation marker or skin quality marker compared to lipid-nanoparticle compositions containing lipid formula (I) with the same head group. When used to transfect at least one reprogramming factor into immune cells, such compositions comprising lipid formula (IB) can produce greater improvement in at least one immune cell rejuvenation marker or immune cell stemness marker compared to lipid-nanoparticle compositions containing other lipids as described herein. When used to transfect at least one reprogramming factor into immune cells, such compositions comprising lipid formula (IB) can produce greater improvement in at least one immune cell rejuvenation marker or stemness rejuvenation marker compared to lipid-nanoparticle compositions containing lipid formula (I) with the same head group.

[0620] In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide higher transfection efficiency than lipid-nanoparticle compositions containing other lipids described herein. In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide higher transfection efficiency than lipid-nanoparticle compositions containing (I) wherein q1 is 1 and q2 is absent. In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide higher transfection efficiency than lipid-nanoparticle compositions containing other lipids described herein. In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide higher transfection efficiency in skin cells than lipid-nanoparticle compositions containing other lipids described herein. In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide higher transfection efficiency in skin cells than lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is 1 and q2 is absent. In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide higher transfection efficiency in fibroblasts compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide higher transfection efficiency in fibroblasts compared to lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is 1 and q2 is absent. In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide higher transfection efficiency in immune cells compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide higher transfection efficiency in immune cells compared to lipid-nanoparticle compositions containing lipid formula (I) wherein q2 is absent and q1 is 1. In embodiments, a lipid-nanoparticle composition comprising a lipid of formula (I) wherein q1 is absent and q2 is 1 provides higher transfection efficiency in T cells compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, a lipid-nanoparticle composition comprising a lipid of formula (I) wherein q1 is absent and q2 is 1 provides higher transfection efficiency in T cells compared to lipid-nanoparticle compositions containing lipids of formula (I) wherein q1 is 1 and q2 is absent.

[0621] In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide higher viability than lipid-nanoparticle compositions containing other lipids described herein. In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide higher viability than lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is 1 and q2 is absent. In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide higher viability in skin cells than lipid-nanoparticle compositions containing other lipids described herein. In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide higher viability in skin cells than lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is 1 and q2 is absent. In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide higher viability in fibroblasts compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide higher viability in fibroblasts compared to lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is 1 and q2 is absent. In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide higher viability in immune cells compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide higher viability in immune cells compared to lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is 1 and q2 is absent. In embodiments, a lipid-nanoparticle composition comprising a lipid of formula (I) wherein q1 is absent and q2 is 1 provides higher viability in T cells compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, a lipid-nanoparticle composition comprising a lipid of formula (I) wherein q1 is absent and q2 is 1 provides higher viability in T cells compared to lipid-nanoparticle compositions containing lipids of formula (I) wherein q1 is 1 and q2 is absent.

[0622] In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide both higher viability and higher transfection efficiency compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide both higher viability and higher transfection efficiency compared to lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is 1 and q2 is absent. In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide both higher viability and higher transfection efficiency compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide both higher viability and higher transfection efficiency in skin cells compared to lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is 1 and q2 is absent. In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide both higher viability and higher transfection efficiency in skin cells compared to lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is 1 and q2 is absent. In an embodiment, a lipid-nanoparticle composition containing a lipid formula (I) in which q1 is absent and q2 is 1 provides both higher viability and higher transfection efficiency in fibroblasts compared to lipid-nanoparticle compositions containing other lipids described herein. In an embodiment, a lipid-nanoparticle composition containing a lipid formula (I) in which q1 is absent and q2 is 1 provides both higher viability and higher transfection efficiency in fibroblasts compared to lipid-nanoparticle compositions containing lipid formula (I) in which q1 is 1 and q2 is absent. In an embodiment, a lipid-nanoparticle composition containing a lipid formula (I) in which q1 is absent and q2 is 1 provides both higher viability and higher transfection efficiency in immune cells compared to lipid-nanoparticle compositions containing other lipids described herein. In an embodiment, a lipid-nanoparticle composition containing a lipid formula (I) in which q1 is absent and q2 is 1 provides both higher viability and higher transfection efficiency in immune cells compared to lipid-nanoparticle compositions containing lipid formula (I) in which q1 is 1 and q2 is absent. In embodiments, lipid-nanoparticle compositions containing lipid formula (IB) provide both higher viability and higher transfection efficiency in T cells compared to lipid-nanoparticle compositions containing other lipids described herein. In embodiments, lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is absent and q2 is 1 provide both higher viability and higher transfection efficiency in T cells compared to lipid-nanoparticle compositions containing lipid formula (I) wherein q1 is 1 and q2 is absent.

[0623] When used to transfect at least one reprogramming factor into skin cells, such compositions comprising lipid formula (I) in which q1 is absent and q2 is 1 can produce greater improvement in at least one skin rejuvenation marker or skin quality marker compared to lipid-nanoparticle compositions containing other lipids described herein. When used to transfect at least one reprogramming factor into skin cells, such compositions comprising lipid formula (I) in which q1 is absent and q2 is 1 can produce greater improvement in at least one skin rejuvenation marker or skin quality marker compared to nanoparticle compositions containing lipid formula (I) in which q1 is 1 and q2 is absent. When used to transfect at least one reprogramming factor into immune cells, such compositions comprising lipid formula (I) in which q1 is absent and q2 is 1 can produce greater improvement in at least one immune cell rejuvenation marker or immune cell stemness marker compared to lipid-nanoparticle compositions containing other lipids described herein. When used to transfect at least one reprogramming factor into immune cells, such compositions comprising a lipid formula (I) in which q1 is absent and q2 is 1 can produce greater improvement in at least one immune cell restoration marker or stemness restoration marker compared to a nanoparticle composition having the same head group containing a lipid formula (I) in which q1 is 1 and q2 is absent.

[0624] Methods provided herein include using lipids or lipid-nanoparticle compositions to transfect cells with one or more non-integrated messenger RNAs encoding one or more cell reprogramming factors, thereby producing recovered cells. The cells to be recovered can have any cell type. In an embodiment, cells are contacted with mRNA, exposed to mRNA, or transfected with mRNA for no more than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day or continue to be less than 1 day. In an embodiment, cells are contacted with mRNA, exposed to mRNA, or transfected with mRNA for no more than about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day or continue to be less than one day. In an embodiment, cells are contacted with mRNA, exposed to mRNA, or transfected with mRNA for no more than about 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day or continue to be less than one day. In an embodiment, the cell is contacted with mRNA, exposed to mRNA, or transfected with mRNA for no more than about 7, 6, 5, 4, 3, 2, or 1 day or for less than one day. In an embodiment, the cell is contacted with mRNA, exposed to mRNA, or transfected with mRNA for no more than about 5, 4, 3, 2, or 1 day or for less than one day. In an embodiment, at least one reprogramming factor is expressed from the transfected mRNA in the cell or the cell is exposed to at least one reprogramming factor expressed from the transfected mRNA, and it lasts no more than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day or for less than 1 day. In an embodiment, at least one reprogramming factor is expressed from the transfected mRNA in the cell or the cell is exposed to at least one reprogramming factor expressed from the transfected mRNA, and it lasts no more than about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day or for less than one day. In an embodiment, at least one reprogramming factor is expressed from a transfected mRNA in a cell or a cell is exposed to at least one reprogramming factor expressed from a transfected mRNA for at least about 2 days and no more than about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 days. In an embodiment, at least one reprogramming factor is expressed from a transfected mRNA in a cell or a cell is exposed to at least one reprogramming factor expressed from a transfected mRNA for no more than about 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day or for less than one day. In an embodiment, at least one reprogramming factor is expressed from a transfected mRNA in a cell or a cell is exposed to at least one reprogramming factor expressed from a transfected mRNA for at least about 2 days and no more than about 10, 9, 8, 7, 6, 5, 4, 3 or 2 days.In an embodiment, at least one reprogramming factor is expressed from a transfected mRNA in a cell, or a cell is exposed to at least one reprogramming factor expressed from a transfected mRNA, for no more than about 7, 6, 5, 4, 3, 2 or 1 day or for less than one day. In an embodiment, at least one reprogramming factor is expressed from a transfected mRNA in a cell, or a cell is exposed to at least one reprogramming factor expressed from a transfected mRNA, for at least about 2 days and no more than about 7, 6, 5, 4, 3 or 2 days. In an embodiment, at least one reprogramming factor is expressed from a transfected mRNA in a cell or a cell is exposed to at least one reprogramming factor expressed from a transfected mRNA, for no more than about 5, 4, 3, 2 or 1 day or for less than one day. In an embodiment, at least one reprogramming factor is expressed from a transfected mRNA in a cell or a cell is exposed to at least one reprogramming factor expressed from a transfected mRNA for at least about 2 days and no more than about 5, 4, 3 or 2 days. In an embodiment, the rejuvenated cell has a phenotype or activity spectrum similar to that of a young cell. The phenotypic or activity profile includes one or more of a transcriptomic profile, gene expression of one or more nuclear and / or epigenetic markers, proteolytic activity, mitochondrial health and function, SASP cytokine expression, and methylation landscape.

[0625] The rejuvenated cells described herein may have a transcriptome profile that is more similar to the transcriptome profile of young cells. In an embodiment, 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, Elf1, Phf8, Pol2s2, Taf1, and Sin3a.

[0626] The rejuvenated cells described herein can also be expressed with increased gene expression compared to a reference value by showing one or more nuclear and / or epigenetic markers. In an embodiment, one or more nuclear and / or epigenetic markers are selected from Hplgamma, H3K9me3, lamina support protein LAP2α and SIRT1 protein. In an embodiment, the rejuvenated cells have a proteolytic activity more similar to the proteolytic activity of young cells. In an embodiment, the proteolytic activity is measured as increased cell autophagosome formation, increased chymosin-like proteasome activity or a combination thereof. In an embodiment, the rejuvenated cells show improved mitochondrial health and function compared to a reference value. In an embodiment, improved mitochondrial health and function are measured as increased mitochondrial membrane potential, reduced reactive oxides (ROS) or a combination thereof.

[0627] The rejuvenated cells described herein may also exhibit reduced expression of one or more SASP cytokines 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 an aged cell.

[0628] As described herein, cells can be restored by transient reprogramming with mRNA encoding one or more cell reprogramming factors, and the mRNA is transfected into the cell using lipids or lipid-nanoparticle compositions disclosed herein. In certain embodiments, transient reprogramming is completed by transfecting cells with non-integrative mRNA for no more than 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 certain embodiments, transient reprogramming is completed by transfecting cells with non-integrative mRNA 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. In certain embodiments, transient reprogramming is completed by transfecting cells with non-integrative mRNA for no more than about 9, 8, 7, 6, 5, 4, 3, 2 or 1 day or less than 1 day. In some embodiments, transient reprogramming is completed by transfecting cells with non-integrative mRNA for no more than about 6, 5, 4, 3, 2 or 1 days or less than 1 day. In some embodiments, transient reprogramming is completed by expressing at least one reprogramming factor from a non-integrative mRNA transfected in a cell or exposing the cell to at least one reprogramming factor expressed from a non-integrative mRNA transfected in a cell for no more than 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 some embodiments, transient reprogramming is completed by expressing at least one reprogramming factor from a non-integrative mRNA transfected in a cell or exposing the cell to at least one reprogramming factor expressed from a non-integrative mRNA transfected in a cell for at least 2 days and no more than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 days. In some embodiments, transient reprogramming is performed by expressing at least one reprogramming factor from a non-integrating mRNA transfected in the cell or exposing the cell to at least one reprogramming factor expressed from a non-integrating mRNA transfected in the cell for no more than about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day or less. In some embodiments, transient reprogramming is performed by expressing at least one reprogramming factor from a non-integrating mRNA transfected in the cell or exposing the cell to at least one reprogramming factor expressed from a non-integrating mRNA transfected in the cell 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.In some embodiments, transient reprogramming is completed by expressing at least one reprogramming factor from a non-integrating mRNA transfected in a cell or exposing the cell to at least one reprogramming factor expressed from a non-integrating mRNA transfected in a cell for no more than about 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day or less than 1 day. In some embodiments, transient reprogramming is completed by expressing at least one reprogramming factor from a non-integrating mRNA transfected in a cell or exposing the cell to at least one reprogramming factor expressed from a non-integrating mRNA transfected in a cell for at least 2 days and no more than about 10, 9, 8, 7, 6, 5, 4, 3 or 2 days. In some embodiments, transient reprogramming is completed by expressing at least one reprogramming factor from a non-integrating mRNA transfected in a cell or exposing the cell to at least one reprogramming factor expressed from a non-integrating mRNA transfected in a cell for no more than about 7, 6, 5, 4, 3, 2 or 1 day or less than 1 day. In some embodiments, transient reprogramming is completed by expressing at least one reprogramming factor from a non-integrated mRNA transfected in a cell or exposing the cell to at least one reprogramming factor expressed from a non-integrated mRNA transfected in a cell for at least 2 days and no more than about 7, 6, 5, 4, 3 or 2 days. In some embodiments, transient reprogramming is completed by expressing at least one reprogramming factor from a non-integrated mRNA transfected in a cell or exposing the cell to at least one reprogramming factor expressed from a non-integrated mRNA transfected in a cell for no more than about 5, 4, 3, 2 or 1 day or less than 1 day. In some embodiments, transient reprogramming is completed by expressing at least one reprogramming factor from a non-integrated mRNA transfected in a cell or exposing the cell to at least one reprogramming factor expressed from a non-integrated mRNA transfected in a cell for at least 2 days and no more than about 5, 4, 3 or 2 days. In an embodiment, the transient reprogramming of a cell eliminates various aging marks while avoiding the complete dedifferentiation of the cell into a stem cell.

[0629] The methods and compositions provided herein are achieved by transient overexpression of one or more mRNAs encoding cell reprogramming factors delivered by lipids or lipid-nanoparticle compositions disclosed herein to reverse or restore cell age. Such cell reprogramming factors may include transcription factors, epigenetic remodeling agents, or small molecules that affect mitochondrial function, proteolytic activity, heterochromatin levels, histone methylation, laminar polypeptides, cytokine secretion, or aging. In an embodiment, the cell reprogramming factor includes one or more of OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG. In an embodiment, cell reprogramming factors are applied in different molar ratios, for example, OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG are applied in a molar ratio of a:b:c:d:e:f, where a, b, c, d, e and f can be all the same number (e.g., 1:1:1:1:1:1), some the same 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, 2:2:2:2:1:1, 3:3:3:3:2:2) or all different numbers (e.g., 6:4:5:3:2:1). In an embodiment, a, b, c, d, e and / or f are each between 1-7, i.e., 1-7:1-7:1-7:1-7:1-7:1-7 (or in the case of a combination of less than 6 factors, 1-7:1-7:1-7:1-7:1-7, 1-7:1-7:1-7:1-7, 1-7:1-7:1-7, 1-7:1-7:1-7, 1-7:1-7 or 1-7:1). In an embodiment, cell reprogramming factors are applied in different weight ratios, for example, OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG are applied in a weight ratio of a:b:c:d:e:f, wherein a, b, c, d, e and f can all be the same number (e.g., 1:1:1:1:1:1), each of a, b, c, d, e and f can be some same 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 each can be a different number (e.g., 6:4:5:3:2:1). In an embodiment, a, b, c, d, e and / or f are each between 1-7, i.e., 1-7:1-7:1-7:1-7:1-7:1-7 (or in the case of a combination of less than 6 factors, 1-7:1-7:1-7:1-7:1-7, 1-7:1-7:1-7:1-7, 1-7:1-7:1-7, 1-7:1-7:1-7, 1-7:1-7 or 1-7:1).

[0630] The methods and compositions provided herein can be applied to any type of cells, tissues or organs that need to be restored. The methods and compositions disclosed herein can be used to restore cells in culture (e.g., in vitro or in vitro) to improve the function and efficacy for use in cell therapy. The cells used in the treatment of patients can be autologous or allogeneic. The cells can be derived from patients or matching donors, or they can be obtained from cell banks or derived from iPS cells. For example, in autologous ex vivo therapy, cells can be obtained directly from patients to be treated, transfected with mRNA encoding cell reprogramming factors, as described herein, and re-implanted in patients. Such cells can be obtained, for example, from biopsies or surgical operations performed on patients. Alternatively, in allogeneic ex vivo therapy, cells can be obtained from cell banks or cell lines derived from iPS cells, transfected with mRNA encoding cell reprogramming factors, as described herein, and re-implanted in patients. Alternatively, mRNA encoding cell reprogramming factors can be used to directly transfect cells that need to be restored in vivo.

[0631] The lipid-containing compositions or lipid-nanoparticle compositions of the present disclosure can be used to deliver mRNA expressing reprogramming factors, which provide more robust cell restoration because the reprogramming factors have been optimized to reduce any triggered immune response to the protein / polypeptide, increase the stability of the protein / polypeptide, and alter the protein / polypeptide activity, such as increased activity compared to the wild-type reprogramming factor.

[0632] The methods provided herein include administering to cells or subjects a lipid-containing composition or lipid-nanoparticle composition comprising RNA of the present disclosure, or treating or transfecting cells with a lipid-containing composition or lipid-nanoparticle composition comprising RNA for a dosing interval of no more than 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 consecutive days. In some embodiments, the dosing interval is one day. In some embodiments, the composition is administered once. In some embodiments, the administration of a lipid-containing composition or lipid-nanoparticle composition comprising RNA is performed at least once a day during the dosing interval. In some embodiments, administration is performed at a frequency less than once a day during the dosing interval, for example, once every two days, once every three days, once every four days, once every x days, where x is a number from 4 to 25. Thus, in such embodiments, for example, administration of a lipid-containing composition or lipid-nanoparticle composition comprising RNA once every 5 days within a 5-day dosing interval means that the RNA is administered once within the interval, i.e., once within a total treatment period of 5 days, and administration of RNA twice a day within a 5-day dosing interval means that the RNA is administered 10 times within the interval, i.e., 10 times within 5 days. In some embodiments, the method comprises administering a lipid-containing composition or lipid-nanoparticle composition comprising RNA to a cell or subject, or treating or transfecting a cell with a lipid-containing composition or lipid-nanoparticle composition comprising RNA for no more than 21, 18, 14, 10, 7, or 5 consecutive days. In some embodiments, the method comprises administering a lipid-containing composition or lipid-nanoparticle composition comprising RNA to a cell or subject, or treating or transfecting a cell with a lipid-containing composition or lipid-nanoparticle composition comprising RNA for no more than 18 consecutive days. In some embodiments, the method comprises administering a lipid-containing composition or lipid-nanoparticle composition comprising RNA to a cell or subject, or treating or transfecting a cell with a 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 to a cell or subject a lipid-containing composition or lipid-nanoparticle composition comprising RNA, or treating or transfecting a cell with a lipid-containing composition or lipid-nanoparticle composition comprising RNA for no more than 10 consecutive days. In some embodiments, the method comprises administering to a cell or subject a lipid-containing composition or lipid-nanoparticle composition comprising RNA, or treating or transfecting a cell with a lipid-containing composition or lipid-nanoparticle composition comprising RNA for no more than 7 consecutive days. In some embodiments, the method comprises administering to a cell or subject a lipid-containing composition or lipid-nanoparticle composition comprising RNA, or treating or transfecting a cell with a lipid-containing composition or lipid-nanoparticle composition comprising RNA for no more than 5 consecutive days.In other embodiments, the exposure comprises interrupting the exposure and repeating the exposure after the interruption. In some embodiments, the exposure, treatment, transfection, expression or administration comprises exposure, treatment, transfection, expression or administration for between about 2-5 consecutive days, between about 5-7 consecutive days, between about 7-10 consecutive days, between about 10-12 consecutive days, between about 12-14 consecutive days, between about 14-17 consecutive days, between about 17-19 consecutive days or between about 19-21 consecutive days, and in some embodiments, further comprises interrupting the exposure and repeating the exposure after the interruption.

[0633] The duration of exposure is controlled by mechanisms such as self-amplifying RNA, circular RNA, B18R and other decoys and / or an on / off switch such as L7Ae or its family members. In some embodiments, the repetition 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 times. For in vivo applications, the repetition can continue for any duration, for example, until the disease is successfully treated or cured, or throughout the life of the subject or patient. In some embodiments, the repetition is performed at any time after the interruption, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, up to 20 days, up to 30 days, up to 3 months, up to 6 months or up to 1 year after the interruption. An exposure cycle is considered to be a dosing interval, so that, for example, a series of exposure-interruption-repeat exposure comprises two dosing intervals.

[0634] V. Pharmaceutical composition

[0635] The present invention 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 patients (e.g., humans) or cells to treat a particular disease or condition of interest. Suitable concentrations and dosages can be readily determined by those skilled in the art.

[0636] The pharmaceutical composition comprising recovered cells can be obtained by transfecting cells with lipid compositions or lipid-nanoparticle compositions comprising one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors disclosed herein for no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days to transiently reprogram cells for recovery. It is also contemplated that cells are treated by transfecting with lipid compositions or lipid-nanoparticle compositions comprising therapeutic mRNA disclosed herein, wherein, in an embodiment, a single transfection is performed to obtain treated cells, such as recovered cells. For example, a single transfection step is considered for self-replicating RNA constructs. On the other hand, a pharmaceutical composition is provided herein, comprising transfecting cells with lipid compositions or lipid-nanoparticle compositions comprising one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors disclosed herein for no more than 4, 5, 6 or 7 days to transiently reprogram cells for recovery to obtain recovered cells.

[0637] The pharmaceutical composition of the present disclosure can be formulated into a preparation in solid, semisolid, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, local, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal and intranasal administration. The term parenteral as used herein includes subcutaneous injection, intravenous injection, intramuscular injection, intradermal injection, intrasternal injection or infusion technology. The pharmaceutical composition of the present disclosure is formulated so that the active ingredient contained therein is bioavailable when the composition is administered to the patient. The composition administered to the subject or patient is in the form of one or more dosage units, wherein, for example, a tablet can be a single dosage unit, and the container of the compound in the aerosol form of the present disclosure can accommodate multiple dosage units. Actual methods of preparing such dosage forms are known or will be apparent to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will include a therapeutically effective amount of the nanoparticle composition for treating the disease or disorder of interest.

[0638] The pharmaceutical composition of the present disclosure can be in solid or liquid form. In one aspect, the carrier is granular so that the composition is, for example, in tablet or powder form. The carrier can be a liquid, wherein the composition is, for example, an oral syrup, an injection, or an aerosol, which can be used, for example, for administration by inhalation.

[0639] When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, with semi-solid, semi-liquid, suspension and gel forms included within the forms considered herein as solid or liquid.

[0640] As a solid composition for oral administration, the pharmaceutical composition can be formulated into the form of powder, granules, compressed tablets, pills, capsules, chewing gum, glutinous rice paper capsules, etc. Such solid compositions will generally contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: a binder such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth gum or gelatin; an excipient such as starch, lactose or dextrin, a disintegrant such as alginic acid, sodium alginate, Primogel, corn starch, etc.; a lubricant such as magnesium stearate or hydrogenated vegetable oil (Sterotex); a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; a flavoring agent such as mint, methyl salicylate or orange flavoring; and a coloring agent.

[0641] When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.

[0642] The pharmaceutical composition can be in liquid form, for example, elixir, syrup, solution, emulsion or suspension. As two examples, the liquid can be used for oral administration or for delivery by injection. When intended for oral administration, in addition to the compounds of this invention, the preferred composition also comprises one or more of a sweetener, a preservative, a dye / colorant and a flavor enhancer. In the composition intended to be administered by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersant, a suspending agent, a buffer, a stabilizer and an isotonic agent can be included.

[0643] Liquid pharmaceutical compositions of the present disclosure (whether they are solutions, suspensions or other similar forms) may include one or more of the following adjuvants: sterile diluents, such as water for injection, saline solutions, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils, such as synthetic monoglycerides or diglycerides, polyethylene glycol, glycerol, propylene glycol or other solvents that can be used as solvents or suspension media; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates and agents for adjusting tonicity, such as sodium chloride or glucose; agents that act as cryoprotectants, such as sucrose or trehalose. Parenteral preparations may be sealed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0644] Liquid pharmaceutical compositions of the present disclosure intended for parenteral or oral administration should contain an amount of a compound of the present disclosure so that a suitable dosage is obtained.

[0645] Pharmaceutical compositions of the present disclosure may be intended for topical administration, in which case the carrier may suitably include a solution, emulsion, ointment or gel matrix. For example, the matrix may include one or more of: vaseline, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickeners may be present in the pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or an iontophoresis device.

[0646] Pharmaceutical compositions of the present disclosure may be intended for rectal administration in the form of, for example, suppositories, which will melt and release the drug in the rectum. Compositions for rectal administration may contain an oleaginous base as a suitable non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycol.

[0647] Pharmaceutical compositions of the present disclosure may include various materials that change the physical form of solid or liquid dosage units. For example, the composition may include materials that form a coating shell around the active ingredient. The materials that form the coating shell are generally inert and may be selected from, for example, sugar, shellac and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

[0648] Pharmaceutical compositions of the present disclosure in solid or liquid form may contain agents that bind to the compounds of the present disclosure and thereby facilitate the delivery of the compounds. Suitable agents that may act in this capacity include monoclonal or polyclonal antibodies or proteins.

[0649] Pharmaceutical compositions of the present disclosure can be composed of dosage units that can be administered as aerosols. The term aerosol is used to represent various systems, ranging from systems of colloidal nature to systems consisting of pressurized packages. It can be delivered by liquefied gas or compressed gas or by a suitable pump system that distributes the active ingredient. Aerosols of compounds of the present disclosure can be delivered in a single-phase, two-phase or three-phase system to deliver the active ingredient. The delivery of an aerosol includes necessary containers, activators, valves, sub-containers, etc., which together can form a set of test kits. Those skilled in the art can determine preferred aerosols without excessive experimentation.

[0650] Pharmaceutical compositions of the present disclosure can be prepared by methods well known in the pharmaceutical field. For example, pharmaceutical compositions intended to be administered by injection can be prepared by combining lipid nanoparticles of the present disclosure with sterile distilled water, buffer or other carriers to form a solution. Surfactants can be added to promote the formation of uniform solutions or suspensions. Surfactants are compounds that non-covalently interact with compounds of the present disclosure to promote the dissolution or uniform suspension of the compound in an aqueous delivery system.

[0651] The compositions of the present disclosure containing the compounds described herein or pharmaceutically acceptable salts thereof are administered in a therapeutically effective amount, which amount will vary depending on a variety of factors, including the activity of the specific therapeutic agent employed; the metabolic stability and duration of action of the therapeutic agent; the age, weight, general health, sex and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disease or condition; and the subject being treated.

[0652] The compositions of the present disclosure may also be administered simultaneously, before or after the administration of one or more other therapeutic agents. Such combination therapies include administering a single pharmaceutical dosage formulation of the compositions of the present disclosure and one or more additional active agents, as well as administering the compositions of the present disclosure and each active agent in the form of its own separate pharmaceutical dosage formulation. For example, the compositions of the present disclosure and other active agents may be administered to the patient together in a single oral dosage composition (such as a tablet or capsule) or a single injection (such as a subcutaneous, intradermal or intravenous injection), or each agent may be administered in a separate oral dosage formulation or a separate injection. When a separate dosage formulation is used, the compounds of the present disclosure and one or more additional active agents may be administered substantially simultaneously (i.e., simultaneously or at separate staggered times, i.e., sequentially); combination therapies are understood to include all of these regimens.

[0653] VI. Examples

[0654] The present disclosure provides lipid-containing nanoparticle compositions to deliver biologically active substances such as nucleic acids (e.g., RNA) to cells and or intracellular compartments to facilitate the delivery of therapeutic / prophylactic agents. Such compositions generally include at least one ionizable lipid.

[0655] The ionizable lipids of the present invention can be prepared in a variety of ways by employing standard synthetic methods and procedures known to those skilled in the art or methods and procedures that will be apparent to those skilled in the art in light of the teachings herein using commercially available starting materials, compounds known in the literature, or from readily prepared intermediates.

[0656] Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be obtained from the relevant scientific literature or from standard textbooks in the field. Although not limited to any one or more sources, classical texts incorporated herein by reference, such as Smith, MB, March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition, John Wiley & Sons: New York, 2001; Greene, TW, Wuts, PGM, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons: New York, 1999; R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), are useful and recognized reference textbooks on organic synthesis known to those skilled in the art. The following descriptions of synthetic methods are intended to illustrate, but not to limit, general procedures for preparing compounds of the present disclosure.

[0657] It should be noted that the reaction scheme provided herein provides an exemplary method for preparing the ionizable lipids of the present disclosure. It will be apparent to those of ordinary skill in the art to use protecting groups and other modifications to the general reaction schemes mentioned in the following examples as required. The list of protecting groups and how to introduce and remove these groups can be found in Greene, TW, Wuts, PGM, Protective Groups in Organic Synthesis, the 3rd edition, John Wiley & Sons: New York, 1999.

[0658] In the reaction schemes described herein, a variety of stereoisomers can be produced. When no specific stereoisomer is indicated, it is understood to refer to all possible stereoisomers that can be produced by the reaction. One of ordinary skill in the art will recognize that the reaction can be optimized to give a preferential isomer, or a new scheme can be designed to produce a single isomer. If a mixture is produced, the isomers can be separated using techniques such as preparative thin layer chromatography, preparative HPLC, preparative chiral HPLC or preparative SFC.

[0659] The following examples of ionizable lipids of the present disclosure are illustrative in nature and are not intended to be limiting in any way.

[0660] Example 1: Synthesis of ionizable lipids of formula (I)

[0661] The ionizable lipids of formula (I) are based on phospholipid scaffolds. The synthesis of ionizable lipids of formula (I) is depicted in Scheme 1. These procedures and methods are well known in the literature and are described in Chem. Sci., 2021, 12, 2549-2557. Similar ionizable lipids of formula (I) based on phospholipids can be synthesized using methods well known in the art.

[0662]

[0663] Scheme 1: Synthesis of ionizable lipid 1.

[0664] Example 2: Synthesis of ionizable lipid of formula (II)

[0665] Formula (II) ionizable lipids are based on sphingomyelin scaffolds. The synthesis of formula (II) ionizable lipids is depicted in Scheme 2 and Scheme 3, respectively. These procedures and methods are well known in the literature and are described in Org. Lett. 2006, 8, 5569 (for Scheme 2) and Chem. Eur. J. 2011, 17, 8568-8575 (for Schemes 2 and 3). Similar ionizable lipids of formula (II) based on sphingomyelin can be synthesized using methods well known in the art.

[0666]

[0667] Scheme 2: Synthesis of ionizable lipid 2.

[0668]

[0669] Scheme 3: Synthesis of ionizable lipid 3.

[0670] Example 3: Synthesis of ionizable lipid of formula (III)

[0671] The ionizable lipids of formula (III) are based on sphingomyelin scaffolds. The synthesis of ionizable lipids of formula (III) is depicted in Scheme 4, respectively. These procedures and methods are well known in the literature and are described in Chem. Eur. J. 2011, 17, 8568-8575. Similar ionizable lipids of formula (III) based on sphingomyelin can be synthesized using methods well known in the art.

[0672]

[0673] Scheme 4: Synthesis of ionizable lipid 4.

[0674] Example 4: Synthesis of ionizable lipid of formula (IV)

[0675] The ionizable lipids of formula (IV) are based on sphingomyelin scaffolds. The synthesis of ionizable lipids of formula (IV) is depicted in Scheme 5 and Scheme 6, respectively. These procedures and methods are well known in the literature and are described in Chem. Eur. J. 2011, 17, 8568-8575 and WO 2017 / 075531. Similar ionizable lipids of formula (IV) based on sphingomyelin can be synthesized using methods well known in the art.

[0676]

[0677] Scheme 5 and Scheme 6: Synthesis of ionizable lipid 5.

[0678] Example 5: Synthesis of ionizable lipid of formula (V)

[0679] The ionizable lipids of formula (V) have a DOPE core. The synthesis of the ionizable lipids of formula (V) is depicted in Scheme 7, respectively. The procedures and methods are well known in the literature and are described in J. Med. Chem. 2005, 48, 7305-7314. Similar ionizable lipids of formula (V) based on a DOPE core can be synthesized using methods well known in the art.

[0680]

[0681] Scheme 7: Synthesis of ionizable lipid 6.

[0682] Example 6: Synthesis of ionizable lipid of formula (VI)

[0683] The ionizable lipid of formula (VI) has a DOPE core. The synthesis of the ionizable lipid of formula (VI) is depicted in Scheme 8 and Scheme 9, respectively. Similar ionizable lipids of formula (VI) based on a DOPE core can be synthesized using synthetic schemes well known in the art.

[0684]

[0685] Scheme 8: Synthesis of ionizable lipid 7.

[0686]

[0687] Scheme 9: Synthesis of ionizable lipid 8.

[0688] Example 7: Synthesis of ionizable lipid of formula (VII)

[0689] The ionizable lipid of formula (VII) is based on a triglyceride scaffold. The synthesis of the ionizable lipid of formula (VII) is depicted in Scheme 10 and Scheme 11, respectively. Similar triglyceride-based modifications of the ionizable lipid of formula (VII) can be synthesized using methods well known in the art.

[0690]

[0691] Scheme 10: Synthesis of ionizable lipid 9.

[0692]

[0693] Scheme 11: Synthesis of ionizable lipid 10.

[0694] Example 8: Synthesis of ionizable lipid of formula (VIII)

[0695] The ionizable lipids of formula (VIII) are based on a triglyceride scaffold. The synthesis of the ionizable lipids of formula (VIII) is depicted in Scheme 12. Similar triglyceride-based modifications of the ionizable lipids of formula (VIII) can be synthesized using methods well known in the art.

[0696]

[0697] Scheme 12: Synthesis of ionizable lipid 11.

[0698] Example 9: Synthesis of ionizable lipid of formula (IX)

[0699] The ionizable lipid of formula (IX) has a cyclic head group in the scaffold. The synthesis of the ionizable lipid of formula (IX) is depicted in Scheme 13 and Scheme 14, respectively. Similar cyclic head group-based modifications of the ionizable lipid of formula (IX) can be synthesized using methods well known in the art.

[0700]

[0701] Scheme 13: Synthesis of ionizable lipid 12.

[0702]

[0703] Scheme ...

Claims

1. An ionizable lipid of formula (I): or a pharmaceutically acceptable salt or stereoisomer thereof, in: L 1 is C1-C6 alkylene; R 1 C6-C 20 alkenyl; R 2 C6-C 20 alkyl; R 3 and R 4 Each independently is H or C1-C3 alkyl; q1 does not exist or is 1; and q2 does not exist or is 1.

2. The ionizable lipid according to claim 1, having a structure in the following structures:

3. An ionizable lipid of formula (IA): or a pharmaceutically acceptable salt or stereoisomer thereof, in: L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; R 2 C6-C 20 alkyl; R 3 and R 4 Each independently is H or C1-C3 alkyl; R 7 C4-C 20 Alkyl; and R 8 C4-C 20 alkyl.

4. The ionizable lipid according to claim 3, having the following structure:

5. An ionizable lipid of formula (IB): or a pharmaceutically acceptable salt or stereoisomer thereof, in: L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; L 3 is C1-C8 alkylene; R 3 and R 4 are independently H, C1-C4 alkyl, -CH2-cyclopropyl, -(CH2) n OH, or R 3 With R 4 together to form an N-heterocycle; R 6 C4-C 20 alkyl; R 7 C4-C 20 alkyl; R 8 C4-C 20 alkyl; R 10 C4-C 20 Alkyl; and n is 2, 3 or 4.

6. The ionizable lipid of claim 5, wherein the ionizable lipid has a structure in the following structures:

7. An ionizable lipid of formula (II): or a pharmaceutically acceptable salt or stereoisomer thereof, in: L 1 is C1-C6 alkylene; R 1 C6-C 20 alkenyl; R 2 C6-C 20 alkyl; R 3 and R 4 are each independently H or C1-C3 alkyl; and R 5 It is H or C1-C3 alkyl.

8. The ionizable lipid according to claim 7, having a structure in the following structures:

9. An ionizable lipid of formula (III): or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L 1 is C1-C6 alkylene; R 1 C6-C 20 alkenyl; R 2 C6-C 20 alkyl; R 2 ' is C6-C 20 alkyl; R 3 and R 4 are each independently H or C1-C3 alkyl; and R 5 It is H or C1-C3 alkyl.

10. The ionizable lipid according to claim 9, having a structure in the following structures:

11. An ionizable lipid of formula (IV): or a pharmaceutically acceptable salt or stereoisomer thereof, in: L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; R 2 C6-C 20 alkyl; R 3 and R 4 Each independently is H or C1-C3 alkyl; R 5 is H or C1-C3 alkyl; R 7 C4-C 20 Alkyl; and R 8 C4-C 20 alkyl.

12. The ionizable lipid according to claim 11, having a structure in the following structures:

13. An ionizable lipid of formula (V): or a pharmaceutically acceptable salt or stereoisomer thereof, in: L 1 is C1-C6 alkylene; R 1 C6-C 20 alkenyl; R 3 and R 4 are each independently H or C1-C3 alkyl; and R 12 C6-C 20 Alkenyl.

14. The ionizable lipid according to claim 13, having the following structure:

15. An ionizable lipid of formula (VI): or a pharmaceutically acceptable salt or stereoisomer thereof, in: L 1 is C1-C6 alkylene; L 1 ' is C1-C6 alkylene; R 1 C6-C 20 alkenyl; R 1 ' is C6-C 20 alkenyl; R 9 H, C1-C6 alkyl or –(CH2) n OH; R 12 C6-C 20 alkenyl; R 12 ' is C6-C 20 alkenyl; and n is 2, 3 or 4.

16. The ionizable lipid according to claim 15, having a structure in the following structures:

17. An ionizable lipid of formula (VII): or a pharmaceutically acceptable salt or stereoisomer thereof, in: L 1 is C1-C6 alkylene; L 1 ' is C1-C6 alkylene; R 2 C6-C 20 alkyl; R 2 ' is C6-C 20 alkyl; R 9 H, C1-C6 alkyl or –(CH2) n OH; R 11 H or –(CH2)OC(=O)R 16 ; R 14 C6-C 20 alkyl; R 14 ' is C6-C 20 alkyl; R 15 C6-C 20 alkyl; R 16 C6-C 20 Alkyl; and n is 2, 3 or 4.

18. The ionizable lipid according to claim 17, having a structure in the following structures:

19. An ionizable lipid of formula (VIII): or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L 1 is C1-C6 alkylene; L 1 ' is C1-C6 alkylene; R 2 C6-C 20 alkyl; R 2 ' is C6-C 20 alkyl; R 9 H, C1-C6 alkyl or –(CH2) n OH; R 14 C6-C 20 alkyl; R 14 ' is C6-C 20 alkyl; R 15 C6-C 20 Alkyl; and n is 2, 3 or 4.

20. The ionizable lipid according to claim 19, having the following structure:

21. An ionizable lipid of formula (IX): or a pharmaceutically acceptable salt or stereoisomer thereof, in: R 1 C6-C 20 alkenyl; R 9 H, C1-C6 alkyl or –(CH2) n OH; R 12 C6-C 20 alkenyl; and n is 2, 3 or 4.

22. The ionizable lipid of claim 21, having one of the following structures:

23. An ionizable lipid of formula (X): or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L 4 Not present or C1-C6 alkylene; L 5 Not present or C1-C6 alkylene; R 1 C6-C 20 alkenyl; R 2 C6-C 20 alkyl; R 2 ' is C6-C 20 alkyl; R 9 H, C1-C6 alkyl or –(CH2) n OH; R 12 C6-C 20 alkenyl; and n is 2, 3 or 4.

24. The ionizable lipid of claim 23, having a structure in the following structures:

25. An ionizable lipid of formula (XI): or a pharmaceutically acceptable salt or stereoisomer thereof, in: L 4 Not present or C1-C6 alkylene; L 5 Not present or C1-C6 alkylene; R 1 C6-C 20 alkenyl; R 2 C6-C 20 alkyl; R 2 ' is C6-C 20 alkyl; R 12 C6-C 20 alkenyl; R 13 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.

26. The ionizable lipid of claim 25, having the structure:

27. An ionizable lipid of formula (XII): or a pharmaceutically acceptable salt or stereoisomer thereof, in: L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 7 C4-C 20 alkyl; R 7 ' is C4-C 20 alkyl; R 8 C4-C 20 alkyl; R 8 ' is C4-C 20 alkyl; R 13 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.

28. The ionizable lipid of claim 27, having the structure:

29. An ionizable lipid of formula (XIII): or a pharmaceutically acceptable salt or stereoisomer thereof, in: L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 7 C4-C 20 alkyl; R 7 ' is C4-C 20 alkyl; R 8 C4-C 20 alkyl; R 8 ' is C4-C 20 alkyl; R 13 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.

30. The ionizable lipid of claim 29 having the structure:

31. An ionizable lipid of formula (XIV): or a pharmaceutically acceptable salt or stereoisomer thereof, in: L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; R 3 and R 4 Each independently is H or C1-C3 alkyl; R 6 C4-C 20 Alkyl; and R 7 C4-C 20 alkyl.

32. The ionizable lipid of claim 31 , having the structure:

33. An ionizable lipid of formula (XV): or a pharmaceutically acceptable salt or stereoisomer thereof, in: L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 6 C4-C 20 alkyl; R 6 ' is C4-C 20 alkyl; R 7 C4-C 20 alkyl; R 7 ' is C4-C 20 alkyl; R 8 C4-C 20 alkyl; R 8 ' is C4-C 20 alkyl; R 10 C4-C 20 alkyl; R 10 ' is C4-C 20 alkyl; R 13 H, C1-C6 alkyl, –(CH2) n OH or –(CH2) q N(CH3)2; n is 2, 3 or 4; p1 does not exist or is 1; p2 does not exist or is 1; and q is 2, 3 or 4.

34. The ionizable lipid of claim 33, having a structure in the following structures:

35. An ionizable lipid of formula (XVI): or a pharmaceutically acceptable salt or stereoisomer thereof, in: L 2 is C1-C8 alkylene; L 2 ' is C1-C8 alkylene; R 6 C4-C 20 alkyl; R 6 ' is C4-C 20 alkyl; R 7 C4-C 20 alkyl; R 7 ' is C4-C 20 alkyl; R 13 H, C1-C6 alkyl, –(CH2) n OH or –(CH2) q N(CH3)2; n is 2, 3 or 4; p1 does not exist or is 1; p2 does not exist or is 1; and q is 2, 3 or 4.

36. The ionizable lipid of claim 35, having a structure in the following structures:

37. An ionizable lipid of formula (IC): or a pharmaceutically acceptable salt or stereoisomer thereof, in: L 1 is C1-C6 alkylene; L 2 is C1-C8 alkylene; L 3 is C1-C8 alkylene; R 3 and R 4 Each is independently H, C1-C4 alkyl or –(CH2) n OH; R 6 C4-C 20 alkyl; R 7 C4-C 20 alkyl; R 8 C4-C 20 alkyl; R 10 C4-C 20 Alkyl; and n is 2, 3 or 4.

38. The ionizable lipid of claim 37, wherein the ionizable lipid has the structure:

39. An ionizable lipid of formula (XXII): or a pharmaceutically acceptable salt or stereoisomer thereof, in: 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 3 is C1-C8 alkylene; L 3 ' is C1-C8 alkylene; R 3 and R 4 Each is independently H, C1-C4 alkyl, -CH2-cyclopropyl or –(CH2) n OH; R 6 C4-C 20 alkyl; R 6 ' is C4-C 20 alkyl; R 7 C4-C 20 alkyl; R 7 ' is C4-C 20 alkyl; R 8 C4-C 20 alkyl; R 8 ' is C4-C 20 alkyl; R 10 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.

40. The ionizable lipid of claim 39, wherein the ionizable lipid has the structure:

41. A lipid-nanoparticle composition comprising the ionizable lipid according to any one of claims 1 to 40.

42. The lipid-nanoparticle composition of claim 41, further comprising a helper lipid, a stabilizing lipid, a structural lipid, and a nucleic acid.

43. The lipid-nanoparticle composition of claim 42, wherein the helper lipid 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-di(undecanoyl)-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), l-oleoyl-2-cholesterol hemisuccinyl-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-di(docosahexaenoyl)-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dialinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG) and mixtures thereof.

44. The lipid-nanoparticle composition of claim 42, wherein the stabilizing lipid is 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG) having an average PEG molecular weight of 2000.

45. The lipid-nanoparticle composition of claim 42, wherein the structured lipid is selected from the group consisting of cholesterol, cholesterol derivatives, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof.

46. ​​The lipid-nanoparticle composition of claim 42, wherein the nucleic acid is selected from the group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), micro RNA (miRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

47. A pharmaceutical composition comprising the lipid-nanoparticle composition according to claims 42 to 46 and a pharmaceutically acceptable carrier thereof.

48. A compound having the following structure: or a stereoisomer, salt or tautomer thereof, wherein: G 1 and G 2 are independently -OC(=O)-, -NR 25 C(═O)— or —CH═CH—; R 21 and R 22 Each independently is a C1-C6 alkyl, a straight chain C 10 -C 20 Alkyl, straight chain C 10 -C 20 Alkenyl or branched C 10 -C 35 alkenyl, wherein the C1-C6 alkyl is replaced by -OC(=O)R 26 replace; R 23 is H, OH or OCH3; R 24 is a C1-C8 heteroalkyl group; R 25 is H or C1-C4 alkyl; R 26 For branched chain C 10 -C 30 Alkyl; and m 1 and m 2 Each independently represents an integer of 0 or 1.

49. The compound according to claim 48, wherein G 1 and G 2 Each is -OC(=O)-.

50. A compound according to any one of claims 48 to 49, wherein G 1 or G 2 One of them is -NR 25 C(=O)- and G 1 or G 2 The other one of them is -CH=CH-.

51. according to the compound described in any one of claims 48 to 50, wherein m 2 is 0.

52. A compound according to any one of claims 48 to 51, wherein the compound has one of the following structures (XVIIA) to (XVIIB): or a stereoisomer, a salt or a tautomer thereof.

53. according to the compound described in any one of claim 48 to 52, wherein R 24 It is a C3-C7 heteroalkyl group.

54. according to the compound described in any one of claims 48 to 53, wherein R 24 It is C4 heteroalkyl, C5 heteroalkyl, C6 heteroalkyl or C7 heteroalkyl.

55. according to the compound described in any one of claims 48 to 54, wherein R 24 It is a C4 alkylamine, a C5 alkylamine, a C6 alkylamine or a C7 alkylamine.

56. according to the compound described in any one of claims 48 to 55, wherein R 24 for 57. according to the compound described in any one of claims 48 to 52, wherein R 24 The C1-C8 heteroalkyl group is further substituted by a cycloalkyl group.

58. The compound according to claim 57, wherein R 24 for 59. according to the compound described in any one of claims 48 to 58, wherein R 25 For H.

60. A compound according to any one of claims 48 to 59, wherein R 25 It is a C1-C4 alkyl group.

61. according to the compound described in any one of claims 48 to 60, wherein R 25 It is -CH3.

62. according to the compound described in any one of claims 48 to 61, wherein R 25 It is -CH2CH3.

63. according to the compound described in any one of claim 48 to 62, wherein R 21 and R 22 Each independently is -OC(=O)R 26 Substituted C2-C5 alkyl, straight chain C 12 -C 18 Alkyl, straight chain C 12 -C 18 Alkenyl or branched C 14 -C 32 Alkenyl.

64. according to the compound described in any one of claims 48 to 63, wherein R 21 or R 22 One of them is a straight chain C 12 -C 18 Alkyl and R 21 or R 22 The other one is a straight chain C 12 -C 18 Alkenyl.

65. according to the compound described in any one of claims 48 to 64, wherein R 21 or R 22 One of them is -OC(=O)R 26 Substituted C2-C5 alkyl and R 21 or R 22 The other one is a straight chain C 12 -C 18 alkyl.

66. according to the compound described in any one of claims 48 to 65, wherein R 21 or R 22 One of them is a straight chain C 12 -C 18 Alkyl and R 21 or R 22 The other one is a branched chain C 14 -C 32 Alkenyl.

67. A compound according to any one of claims 48 to 66, wherein R 21 and R 22 Each is -OC(=O)R 26 Substituted C2-C5 alkyl.

68. A compound according to any one of claims 48 to 67, wherein R 21 and R 22 Each is a straight chain C 12 -C 18 Alkenyl.

69. according to the compound described in any one of claims 48 to 68, wherein R 26 For branched chain C 10 -C 20 alkyl.

70. A compound according to any one of claims 48 to 69, wherein R 26 For branched chain C 12 -C 18 alkyl.

71. according to the compound described in any one of claims 48 to 70, wherein R 21 and R 22 Each independently has one of the following structures:

72. The compound of claim 48, wherein the compound has one of the following structures:

73. A compound having the following structure: or a stereoisomer, salt or tautomer thereof, wherein: G 3 and G 4 Each is independently -OC(=O)R 28 、-C(=O)OR 28 、-CH(CH2OC(=O)R 28 )2, -C(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 ); R 27 is H, C1-C6 alkyl or C1-C6 heteroalkyl; R 28 Each independently is a straight chain C6-C 12 Alkyl, branched chain C 10 -C 40 Alkyl, straight chain C 15 -C 20 Alkenyl or branched C 20 -C 40 alkenyl; m 1 and m 2 Each independently is an integer from 1 to 6; is a direct key or does not exist; and It is a single bond or a double bond.

74. The compound of claim 73, wherein the compound has one of the following structures (XVIIIA) to (XVIIIC): or a stereoisomer, a salt or a tautomer thereof.

75. according to the compound described in any one of claims 73 to 74, wherein R 27 It is H, C1-C3 alkyl or C1-C5 heteroalkyl.

76. according to the compound described in any one of claims 73 to 75, wherein R 27 It is a C1-C3 alkyl, a C2-C4 alkyl alcohol or a C5 alkyl amine.

77. A compound according to any one of claims 73 to 76, wherein R 27 Has one of the following structures:

78. A compound according to any one of claims 73 to 77, wherein G 3 and G 4 Each has one of the following structures:

79. according to the compound described in any one of claims 73 to 78, wherein R 28 Each independently is a straight chain C7-C 10 Alkyl, branched chain C 14 -C 40 Alkyl, straight chain C 15 -C 20 Alkenyl or branched C 30 -C 40 Alkenyl.

80. A compound according to any one of claims 73 to 79, wherein R 28 Each independently has one of the following structures:

81. according to the compound described in any one of claims 73 to 80, wherein m 1 and m 2 Each independently represents an integer from 1 to 4.

82. The compound of claim 73, wherein the compound has one of the following structures:

83. A compound having the following structure: or a stereoisomer, salt or tautomer thereof, wherein: R 29a and R 29b Each independently is a straight chain C6-C 10 Alkyl or straight chain C 12 -C 20 Alkylene; R 30 is an aryl group or a C3-C6 heterocycle, wherein the aryl group or the C3-C6 heterocycle is replaced by -OC(=O)R 31 , C1-C4 alkyl or C1-C4 heteroalkyl substitution; R 31 is a C1-C6 heteroalkyl group; and n 1 An integer from 1 to 6.

84. The compound according to claim 83, wherein R 29a and R 29b Each is a straight chain C6-C 10 alkyl.

85. according to the compound described in any one of claim 83 to 84, wherein R 29a and R 29b Each is a straight chain C 12 -C 20 Alkylene.

86. according to the compound described in any one of claims 83 to 85, wherein R 29a and R 29b Each independently has one of the following structures:

87. according to the compound described in any one of claims 83 to 86, wherein R 29a and R 29b Each 88. according to the compound described in any one of claims 83 to 87, wherein R 29a and R 29b Each 89. according to the compound described in any one of claims 83 to 88, wherein R 30 The aryl group is phenyl or naphthalene.

90. according to the compound described in any one of claims 83 to 89, wherein R 30 The C3-C6 heterocycle is azetidine, pyrrolidine, imidazolidine, pyrazolidine, piperidine, diazine, triazine or azepane.

91. according to the compound described in any one of claims 83 to 90, wherein R 30 -OC(=O)R 31 substituted aryl, or a C3-C6 heterocycle substituted by a C1-C4 alkyl, or a C1-C4 heteroalkyl.

92. according to the compound described in any one of claims 83 to 91, wherein R 30 -OC(=O)R 31 substituted phenyl, or a C3-C6 N-heterocycle substituted by a C1-C4 alkyl, or a C1-C4 heteroalkyl.

93. according to the compound described in any one of claims 83 to 92, wherein R 30 Has one of the following structures:

94. according to the compound described in any one of claims 83 to 93, wherein n 1 An integer from 1 to 4.

95. according to the compound described in any one of claims 83 to 94, wherein n 1 An integer of 1 or 4.

96. The compound of claim 83, wherein the compound has one of the following structures:

97. An ionizable lipid of formula (XXI): or a pharmaceutically acceptable salt or stereoisomer thereof, 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 3 is C1-C8 alkylene; L 3 ' is C1-C8 alkylene; R 3 and R 4 Each is independently H, C1-C4 alkyl, -CH2-cyclopropyl or –(CH2) n OH; R 6 C4-C 20 alkyl; R 6 ' is C4-C 20 alkyl; R 7 C4-C 20 alkyl; R 7 ' is C4-C 20 alkyl; R 8 C4-C 20 alkyl; R 8 ' is C4-C 20 alkyl; R 10 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.

98. The ionizable lipid of claim 97, wherein the ionizable lipid has the structure:

99. A pharmaceutical composition comprising a compound according to any one of claims 48 to 98 and a therapeutic agent comprising a nucleic acid.

100. The pharmaceutical composition of claim 99, further comprising a helper lipid, a stabilizing lipid, or a structural lipid.

101. The pharmaceutical composition of claim 100, wherein the helper lipid 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-di(undecanoyl)-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), l-oleoyl-2-cholesterol hemisuccinyl-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-di(docosahexaenoyl)-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dialinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG) and mixtures thereof.

102. The pharmaceutical composition of claim 100, wherein the stabilizing lipid is 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG) having an average PEG molecular weight of 2000.

103. The pharmaceutical composition of claim 100, wherein the structured lipid is selected from the group consisting of cholesterol, cholesterol derivatives, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof.

104. The pharmaceutical composition of claim 99, wherein the nucleic acid is selected from small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), micro RNA (miRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA) or messenger RNA (mRNA).

105. The pharmaceutical composition of claim 104, wherein the nucleic acid is mRNA.

106. A method for administering a therapeutic agent comprising a nucleic acid to a patient in need thereof, the method comprising administering to the patient the composition of claim 99.

107. A lipid nanoparticle comprising a compound according to any one of claims 48 to 98 and a therapeutic agent comprising a nucleic acid.

108. The lipid nanoparticle of claim 107, wherein the therapeutic agent is mRNA.

109. A compound having the following structure: or a stereoisomer, salt or tautomer thereof, wherein: G 1 and G 2 Each is independently -OC(=O)- or -NR 25 C(=O)-; R 21 and R 22 Each independently is a C1-C6 alkyl, a straight chain C 10 -C 20 Alkyl, straight chain C 10 -C 20 Alkenyl or branched C 10 -C 35 alkenyl, wherein the C1-C6 alkyl is replaced by -OC(=O)R 26 replace; R 24 is C1-C6 heteroalkyl, aryl or C1-C4 alkyl substituted by 4- to 8-membered heterocycloalkyl; R 25 is H or C1-C4 alkyl; R 26 For branched chain C 10 -C 30 Alkyl; and Y is O or NR 32 , where R 32 It is H or C1-C4 alkyl.

110. The compound according to claim 109, wherein G 1 and G 2 Each is -OC(=O)-.

111. The compound according to claim 109, wherein G 1 and G 2 Each is -NR 25 C(=O)-.

112. A compound according to any one of claims 109 to 111, wherein the compound has one of the following structures (XXA) to (XXB): or a stereoisomer, a salt or a tautomer thereof.

113. The compound of claim 112, wherein Y is O.

114. The compound according to claim 112, wherein Y is NR 32 .

115. A compound according to any one of claims 109 to 114, wherein the compound has one of the following structures (XXA-1) to (XXB-2): or a stereoisomer, a salt or a tautomer thereof.

116. A compound according to any one of claims 109 to 115, wherein R 24 It is a C3-C6 heteroalkyl group.

117. A compound according to any one of claims 109 to 116, wherein R 24 It is a C5 heteroalkyl group.

118. A compound according to any one of claims 109 to 117, wherein R 24 It is a C5 alkylamine.

119. A compound according to any one of claims 109 to 118, wherein R 24 for 120. A compound according to any one of claims 109 to 115, wherein R 24 It is an aromatic group.

121. The compound according to claim 120, wherein R 24 The aryl group is substituted with a C1-C6 heteroalkyl group.

122. The compound according to claim 121, wherein R 24 for 123. according to the compound described in any one of claims 109 to 115, wherein R 24 It is a C1-C4 alkyl group substituted by a 4- to 8-membered heterocycloalkyl group.

124. The compound according to claim 123, wherein R 24 It is a C1-C3 alkyl group substituted by a 4- to 6-membered heterocycloalkyl group.

125. A compound according to claim 123 or claim 124, wherein R 24 Has one of the following structures:

126. according to the compound described in any one of claims 109 to 125, wherein R 25 For H.

127. A compound according to any one of claims 109 to 125, wherein R 25 It is a C1-C4 alkyl group.

128. according to the compound described in any one of claims 109 to 125, wherein R 25 It is -CH3.

129. according to the compound described in any one of claims 109 to 125, wherein R 25 It is -CH2CH3.

130. A compound according to any one of claims 109 to 129, wherein R 21 and R 22 Each independently is -OC(=O)R 26 Substituted C2-C5 alkyl, straight chain C 12 -C 18 Alkyl, straight chain C 12 -C 18 Alkenyl or branched C 14 -C 32 Alkenyl.

131. according to the compound described in any one of claims 109 to 130, wherein R 21 or R 22 One of them is a straight chain C 12 -C 18 Alkyl and R 21 or R 22 The other one is a straight chain C 12 -C 18 Alkenyl.

132. according to the compound described in any one of claims 109 to 131, wherein R 21 or R 22 One of them is -OC(=O)R 26 Substituted C2-C5 alkyl and R 21 or R 22 The other one is a straight chain C 12 -C 18 alkyl.

133. according to the compound described in any one of claims 109 to 132, wherein R 21 or R 22 One of them is a straight chain C 12 -C 18 Alkyl and R 21 or R 22 The other one is a branched chain C 14 -C 32 Alkenyl.

134. according to the compound described in any one of claims 109 to 133, wherein R 21 and R 22 Each is -OC(=O)R 26 Substituted C2-C5 alkyl.

135. according to the compound described in any one of claims 109 to 134, wherein R 21 and R 22 Each is a straight chain C 12 -C 18 Alkenyl.

136. according to the compound described in any one of claims 109 to 135, wherein R 26 For branched chain C 10 -C 20 alkyl.

137. A compound according to any one of claims 109 to 136, wherein R 26 For branched chain C 12 -C 18 alkyl.

138. according to the compound described in any one of claims 109 to 137, wherein R 21 and R 22 Each independently has the following structure:

139. The compound of claim 109, wherein the compound has one of the following structures:

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