Cationic or ionizable lipids and uses thereof

Abstract

The present disclosure provides a compound of formula I: I or a pharmaceutically acceptable salt thereof, that is useful for forming particles (e.g., lipid nanoparticles) for delivery of nucleic acids. The present disclosure further provides particle compositions and suspensions comprising the compound of formula I, as well as uses thereof.

Description

[0001] Cationic or Ionizable Lipids and Uses Thereof

[0002] DESCRIPTION

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to International Application No. PCT / IB2025 / 051231, fded February 05, 2025, which is hereby incorporated by reference in its entirety.

[0005] BACKGROUND

[0006] Particles for delivery of nucleic acids have been the subject of much recent work. Certain particles, such as lipid nanoparticles (LNPs) are particularly useful for the transport of therapies such as nucleic acid therapies to cells. See Tenchov, et al., ACS Nano, 2021, 15, 11 16982–17015. LNPs comprise, among other things, cationic or ionizable lipids that, through electrostatic interaction, form stable complexes that encapsulate nucleic acids and thereby facilitate delivery into the cell.

[0007] SUMMARY

[0008] There remains a need for cationic or ionizable lipids that are capable of forming complexes with nucleic acids, but have improved manufacturability and improved properties (e.g., improved transfection of nucleic acids and improved stability) relative to previous lipid formulations. The present disclosure provides cationic or ionizable lipid compounds that avoid the problems associated with previous lipid compounds, while exhibiting improved properties (e.g., improved transfection) and ease of manufacture.

[0009] In some embodiments, the present disclosure provides a compound represented by formula I:

[0010] / L1— X1— T1

[0011] G-L3-N

[0012]

[0013] \2- X2- T2

[0014] I

[0015] or a pharmaceutically acceptable salt thereof, wherein:

[0016] L1and L2are each independently an optionally substituted C1-C30 aliphatic group;

[0017] L3is a bond or optionally substituted C1-C10 aliphatic group;

[0018] X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -S(O)2N(R’)-, -N(R1)S(O)2-, -S(O)-, -S(O)2-, -S(O)2C(R1)2-, -OC(S)C(R1)2-, -C(R1)2C(S)O-, -S-, -N(H)C(O)N(R1)-, -NHC(O)-, -C(O)N(R’)-, -NHS(O)2N(R1)-, and -C(O)-;

[0019] each R1is, independently, at each instance, optionally substituted C1-C20 aliphatic or H;

[0020] T1and T2are each independently an optionally substituted C3-C30 aliphatic;

[0021] G is -N(R2)C(S)N(R2)2, -OH, -N(R2)2, -N+(R3)3, -N(R5)C(O)R3, -N(R5)S(O)2R3, -N(R5)C(O)N(R3)2, –CH(N–R2), -R4, -S(O)2R3, S(O)R3, or -SR3;each R2is, independently, at each instance, selected from the group consisting of H, optionally substituted Ci-Ce aliphatic and OR3;

[0022] R3is selected from the group consisting of H and optionally substituted Ci-Cio aliphatic;

[0023] R4is optionally substituted 4- to 12-membered heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S, optionally substituted 4- to 12 membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S, optionally substituted Ce-Cn aryl, or optionally substituted C3-C12 cycloaliphatic; and each R5is independently selected from H and optionally substituted Ci-Ce aliphatic.

[0024] In some embodiments, the present disclosure provides a compound represented by Formula IA:

[0025] L1-X1-T1

[0026]

[0027] \2- X2— T2

[0028] IA

[0029] or a pharmaceutically acceptable salt thereof, wherein:

[0030] L1and L2are each independently an optionally substituted C1-C30 aliphatic group;

[0031] L3is a bond or optionally substituted C1-C10 aliphatic;

[0032] X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -S(O)2N(R’)-, -N(R1)S(O)2-, -S(O)-, -S(O)2-, -S(O)2C(R1)2-, -OC(S)C(R1)2-, -C(R1)2C(S)O-, -S-, -N(H)C(O)N(R1)-, -NHC(O)-, -C(O)N(R’)-, -NHS(O)2N(R1)-, and -C(O)-;

[0033] each R1is, independently, at each instance, optionally substituted C1-C20 aliphatic or H;

[0034] T1and T2are each independently an optionally substituted C3-C30 aliphatic;

[0035] G1is -S(O)R3or -SR3; and

[0036] R3is optionally substituted C1-C10 aliphatic.

[0037] In some embodiments, the present disclosure provides a compound represented by formula IB:

[0038] L1-X1-T1

[0039] G2-L3-N

[0040] 2- X2- T2

[0041] IB

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

[0043] L1and L2are each independently an optionally substituted C1-C30 aliphatic group;

[0044] L3is a bond or optionally substituted C1-C10 aliphatic;

[0045] X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -S(O)2N(R’)-, -N(R’)S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R1)2-, -OC(S)C(R1)2-, -C(R1)2C(S)O-, -S-, and -N(H)C(O)N(R1);

[0046] each R1is, independently, at each instance, optionally substituted C1-C20 aliphatic or H;

[0047] T1and T2are each independently an optionally substituted C3-C30 aliphatic;

[0048] G2is -S(O)2R3a;

[0049] R3ais -(CH2)0-6-R3bor Ci-Ce aliphatic;

[0050] R3bis -OH, -NH(CH3), or -N(CH3)2;provided that when R3ais -CH3, then X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -N(H)C(O)N(R1)-, -NHC(O)-, -C(O)N(R1)-, and -NHS(O)2N(R1)-.

[0051] In some embodiments, the present disclosure provides a compound represented by formula IC:

[0052] L1-X1-T1

[0053] G3-L3-N / /

[0054] \2- X2— T2

[0055] IC

[0056] or a pharmaceutically acceptable salt thereof, wherein:

[0057] L1and L2are each independently an optionally substituted C1-C30 aliphatic group;

[0058] L3is a bond or optionally substituted C1-C10 aliphatic;

[0059] X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -N(H)C(O)N(R1)-, -NHC(O)-, and -C(O)N(R1)-;

[0060] each R1is, independently, at each instance, optionally substituted C1-C20 aliphatic or H;

[0061] T1and T2are each independently an optionally substituted C3-C30 aliphatic group;

[0062]

[0063] each R30is independently selected from -SH,-OH, -NH2, -NH(CH3), -N(CH3)2, -C(O)NH2, and optionally substituted Ci-Ce aliphatic;

[0064] provided that when G3is -OH, -NH2, -NH(CH3), or -N(CH3)2, and X1or X2is a bond, -OC(O)-, or -C(O)O-, then L3is C2-Cio alkenyl, or C2-Cio alkynyl.

[0065] In some embodiments, the present disclosure provides a particle comprising a compound described herein and a nucleic acid. In some embodiments, the present disclosure provides a composition (e.g., a pharmaceutical composition) comprising particles described herein.

[0066] In some embodiments, the present disclosure provides a method of treating a disease, disorder, or condition in a subject comprising administering to the subject a composition comprising particles described herein.

[0067] BRIEF DESCRIPTION OF THE DRAWING

[0068] Figure 1 is a bar graph illustrating the particle size and PDI of LNPs comprising ionizable lipid compounds BL- 194, BNT-47, or BNT-91.

[0069] Figure 2 is a bar graph illustrating zeta potential of LNPs comprising ionizable lipid compounds BL- 194, BNT-47, or BNT-91.Figures 3A and 3B depict mRNA encapsulation efficiency of LNPs comprising ionizable lipid compounds BL- 194, BNT-47, or BNT-91 using accessibility (Figure 3 A) and AGE determination of free mRNA (Figure 3B).

[0070] Figure 4 is a bar graph illustrating mRNA integrity via capillary electrophoresis of LNPs comprising ionizable lipid compounds BL- 194, BNT-47, or BNT-91.

[0071] Figures 5A-5D are a set of bar graphs illustrating viability of cells (C2C12 cells - Figure 5A; HepG2 cells - Figure 5B; RAW cells - Figure 5C; HEK293T cells - Figure 5D) expressing luciferase mRNA from LNPs comprising ionizable lipid compounds BL- 194, BNT-47, or BNT-91. Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0072] Figures 6A-6D are a set of bar graphs illustrating in vitro expression of luciferase mRNA from LNPs comprising ionizable lipid compounds BL-194, BNT-47, or BNT-91 in a skeletal muscle cell line (C2C12; Figure 6A), a hepatocarcinoma cell line (HepG2; Figures 6B), a murine macrophage cell line (RAW; Figure 6C), and a human embryonic kidney cell line (HEK293T; Figure 6D). Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0073] Figure 7 is a bar graph illustrating the particle size and PDI of LNPs comprising ionizable lipid compounds BNT-101, BNT-102, or BNT-103.

[0074] Figure 8 is a bar illustrating zeta potential of LNPs comprising ionizable lipid compounds BNT-101, BNT-102, or BNT-103.

[0075] Figures 9A and 9B depict mRNA encapsulation efficiency of LNPs comprising ionizable lipid compounds BNT-101, BNT-102, or BNT-103 using accessibility (Figure 9A) and AGE determination of free mRNA (Figure 9B).

[0076] Figure 10 is a bar graph illustrating mRNA integrity via capillary electrophoresis of LNPs comprising ionizable lipid compounds BNT-101, BNT-102, or BNT-103.

[0077] Figures 11A-11C are a set of bar graphs illustrating viability of cells (C2C12 cells - Figure 11A; HepG2 cells - Figure 11B; RAW cells - Figure 11C) expressing luciferase mRNA from LNPs comprising ionizable lipid compounds BNT-101, BNT-102, or BNT-103. Firefly luciferase expression was quantified 24h postincubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0078] Figures 12A-12C are a set of bar graphs illustrating in vitro expression of luciferase mRNA from LNPs comprising ionizable lipid compounds BNT-101, BNT-102, or BNT-103 in a skeletal muscle cell line (C2C12; Figure 12A), a hepatocarcinoma cell line (HepG2; Figure 12B), and a murine macrophage cell line (RAW; Figure 12C). Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0079] Figures 13A-13B depict the hemolytic effect of LNPs and the effect of the LNPs on complement activation. Figure 13A is a bar graph illustrating hemolysis after incubation (in neutral pH) of whole human blood with LNPs comprising ionizable lipid compound BNT-101 and various controls. Figure 13B is a bar graph illustrating terminal complement complex (SC5b-9) formation of human serum with LNPs comprising ionizable lipid compound BNT-101 and various controls. The dashed horizontal line shows the level of SC5b-9 formation for PBS control.Figure 14 is a bar graph illustrating the particle size and PDI of various LNPs comprising ionizable lipid compound BNT-106.

[0080] Figure 15 is a bar illustrating zeta potential of various LNPs comprising ionizable lipid compound BNT-106.

[0081] Figure 16 is a bar graph illustrating RNA accessibility (%) and encapsulation efficiency (%) of various LNPs comprising ionizable lipid compound BNT-106.

[0082] Figure 17 is a bar graph illustrating mRNA integrity via capillary electrophoresis of various LNPs comprising ionizable lipid compound BNT-106.

[0083] Figures 18A-18C are a set of bar graphs illustrating viability of cells (C2C12 cells - Figure 18A; HepG2 cells - Figure 18B; RAW cells - Figure 18C) expressing luciferase mRNA from various LNPs comprising ionizable lipid compound BNT-106. Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0084] Figures 19A-19C are a set of bar graphs illustrating in vitro expression of luciferase mRNA from various LNPs comprising ionizable lipid compound BNT-106 in a skeletal muscle cell line (C2C12; Figure 19A), a hepatocarcinoma cell line (HepG2; Figures 19B), and a murine macrophage cell line (RAW; Figure 19C). Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0085] Figures 20A-20D depict the results of bioluminescence imaging (BLI) in mice administered with various LNPs comprising ionizable lipid compound BNT-106. Figure 20A depicts BLI of whole animals and organs at 6h and 24h timepoints. Figure 20B depicts the total flux of in vivo imaging at 6h and 24h timepoints. Figure 20C depicts the total flux of ex vivo organ imaging at 6h and 24h timepoints. Figure 20D depicts the percent of biodistribution of the LNPs in various organs.

[0086] Figure 21 is a bar graph illustrating the particle size and PDI of LNPs comprising ionizable lipid compounds BNT-129, BNT-110, BNT-130, or BNT-131.

[0087] Figure 22 is a bar illustrating zeta potential of LNPs comprising ionizable lipid compounds BNT-129, BNT- 110, BNT- 130, or BNT- 131.

[0088] Figure 23 is a bar graph illustrating RNA accessibility (%) and encapsulation efficiency (%) of LNPs comprising ionizable lipid compounds BNT-129, BNT-110, BNT-130, or BNT-131.

[0089] Figure 24 is a bar graph illustrating mRNA integrity via capillary electrophoresis of LNPs comprising ionizable lipid compounds BNT-129, BNT-110, BNT-130, or BNT-131.

[0090] Figures 25A-25D are a set of bar graphs illustrating viability of cells (C2C12 cells - Figure 25 A; HepG2 cells - Figure 25B; RAW cells - Figure 25C; HEK293T cells - Figure 25D) expressing luciferase mRNA from LNPs comprising ionizable lipid compounds BNT-129, BNT-110, BNT-130, or BNT-131. Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0091] Figures 26A-26D are a set of bar graphs illustrating in vitro expression of luciferase mRNA from LNPs comprising ionizable lipid compounds BNT-129, BNT-110, BNT-130, or BNT-131 in a skeletal muscle cell line (C2C12; Figure 26A), a hepatocarcinoma cell line (HepG2; Figures 26B), a murine macrophagecell line (RAW; Figure 26C), and a human embryonic kidney cell line (HEK293T; Figure 26D). Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0092] Figure 27 is a bar graph illustrating the particle size and PDI of LNPs comprising ionizable lipid compounds BNT-108, BNT-109, BNT-111, BNT-119, BNT-48, or BNT-121.

[0093] Figure 28 is a bar illustrating zeta potential of LNPs comprising ionizable lipid compounds BNT-108, BNT-109, BNT-111, BNT-119, BNT-48, or BNT-121.

[0094] Figure 29 is a bar graph illustrating RNA accessibility (%) and encapsulation efficiency (%) of LNPs comprising ionizable lipid compounds BNT-108, BNT-109, BNT-111, BNT-119, BNT-48, or BNT-121.

[0095] Figure 30 is a bar graph illustrating mRNA integrity via capillary electrophoresis of LNPs comprising ionizable lipid compounds BNT-108, BNT-109, BNT-111, BNT-119, BNT-48, or BNT-121.

[0096] Figures 31A-31C are a set of bar graphs illustrating viability of cells (C2C12 cells - Figure 31A; HepG2 cells - Figure 3 IB; RAW cells - Figure 31C) expressing luciferase mRNA from LNPs comprising ionizable lipid compounds BNT-108, BNT-109, BNT-111, BNT-119, BNT-48, or BNT-121. Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0097] Figures 32A-32C are a set of bar graphs illustrating in vitro expression of luciferase mRNA from LNPs comprising ionizable lipid compounds BNT-108, BNT-109, BNT-111, BNT-119, BNT-48, or BNT-121 in a skeletal muscle cell line (C2C12; Figure 32A), a hepatocarcinoma cell line (HepG2; Figures 32B), and a murine macrophage cell line (RAW; Figure 32C). Firefly luciferase expression was quantified 24h postincubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0098] Figure 33 is a bar graph illustrating the particle size and PDI of LNPs comprising ionizable lipid compounds BNT-114 or BNT-110.

[0099] Figure 34 is a bar illustrating zeta potential of LNPs comprising ionizable lipid compounds BNT-114 or BNT-110.

[0100] Figure 35 is a bar graph illustrating RNA accessibility (%) and encapsulation efficiency (%) of LNPs comprising ionizable lipid compounds BNT-114 or BNT-110.

[0101] Figure 36 is a bar graph illustrating mRNA integrity via capillary electrophoresis of LNPs comprising ionizable lipid compounds BNT-114 or BNT-110.

[0102] Figures 37A-37D are a set of bar graphs illustrating viability of cells (C2C12 cells - Figure 37A; HepG2 cells - Figure 37B; RAW cells - Figure 37C; HEK293T cells - Figure 37D) expressing luciferase mRNA from LNPs comprising ionizable lipid compounds BNT-114 or BNT-110. Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0103] Figures 38A-38D are a set of bar graphs illustrating in vitro expression of luciferase mRNA from LNPs comprising ionizable lipid compounds BNT-114 or BNT-110 in a skeletal muscle cell line (C2C12; Figure 38A), a hepatocarcinoma cell line (HepG2; Figures 38B), a murine macrophage cell line (RAW; Figure 38C), and a human embryonic kidney cell line (HEK293T; Figure 38D). Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.Figure 39 is a bar graph illustrating the particle size and PDI of LNPs comprising ionizable lipid compounds BNT-132, BNT-133, BNT-134, BNT-135, BNT-136, BNT-138, BNT-139, BNT-140, or BNT-141, and a benchmark LNP (BM).

[0104] Figure 40 is a bar illustrating zeta potential of LNPs comprising ionizable lipid compounds BNT-132, BNT-133, BNT-134, BNT-135, BNT-136, BNT-138, BNT-139, BNT-140, or BNT-141, and a benchmark LNP (BM).

[0105] Figure 41 is a bar graph illustrating RNA accessibility (%) and encapsulation efficiency (%) of LNPs comprising ionizable lipid compounds BNT-132, BNT-133, BNT-134, BNT-135, BNT-136, BNT-138, BNT-139, BNT-140, or BNT-141, and a benchmark LNP (BM).

[0106] Figure 42 is gel electrophoresis images of LNPs comprising ionizable lipid compounds BNT-132, BNT-133, BNT-134, BNT-135, BNT-136, BNT-138, BNT-139, BNT-140, or BNT-141, and a benchmark LNP (BM).

[0107] Figure 43 is a bar graph illustrating mRNA integrity via capillary electrophoresis of LNPs comprising ionizable lipid compounds BNT-132, BNT-133, BNT-134, BNT-135, BNT-136, BNT-138, BNT-139, BNT-140, or BNT-141, and a benchmark LNP (BM).

[0108] Figures 44A-44B are a set of bar graphs illustrating viability of cells (HepG2 cells - Figure 44A; HEK293T cells - Figure 44B) expressing luciferase mRNA from LNPs comprising ionizable lipid compounds BNT-132, BNT-133, BNT-134, BNT-135, BNT-136, BNT-138, BNT-139, BNT-140, or BNT-141, and a benchmark LNP (BM). Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0109] Figures 45A-45B are a set of bar graphs illustrating in vitro expression of luciferase mRNA from LNPs comprising ionizable lipid compounds BNT-132, BNT-133, BNT-134, BNT-135, BNT-136, BNT-138, BNT-139, BNT-140, or BNT-141, and a benchmark LNP (BM) in a hepatocarcinoma cell line (HepG2; Figures 45A), and a human embryonic kidney cell line (HEK293T; Figure 45B). Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs. Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0110] Figures 46 is a bar graph illustrating the particle size and PDI of LNPs comprising ionizable lipid compounds BL-222 or BL-223, and two benchmark LNPs (BM1 and BM2).

[0111] Figure 47 is a bar illustrating zeta potential of LNPs comprising ionizable lipid compounds BL-222 or BL-223, and two benchmark LNPs (BM1 and BM2).

[0112] Figure 48 is a bar graph illustrating RNA accessibility (%) and encapsulation efficiency (%) of LNPs comprising ionizable lipid compounds BL-222 or BL-223, and two benchmark LNPs (BM1 and BM2).

[0113] Figure 49 is gel electrophoresis images of LNPs comprising ionizable lipid compounds BL-222 or BL-223, and two benchmark LNPs (BM1 and BM2).

[0114] Figure 50 is a bar graph illustrating mRNA integrity via capillary electrophoresis of LNPs comprising ionizable lipid compounds BL-222 or BL-223, and two benchmark LNPs (BM1 and BM2).Figures 51A-51B are a set of bar graphs illustrating viability of cells (HepG2 cells - Figure 51 A; HEK293T cells - Figure 5 IB) expressing luciferase mRNA from LNPs comprising ionizable lipid compounds BL-222 or BL-223, and two benchmark LNPs (BM1 and BM2). Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0115] Figures 52A-52B are a set of bar graphs illustrating in vitro expression of luciferase mRNA from LNPs comprising ionizable lipid compounds BL-222 or BL-223, and two benchmark LNPs (BM1 and BM2). in a hepatocarcinoma cell line (HepG2; Figures 52A), and a human embryonic kidney cell line (HEK293T; Figure 52B). Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0116] Figure 53 is a bar graph illustrating the particle size and PDI of LNPs comprising ionizable lipid compounds BNT-93 or BNT-94.

[0117] Figure 54 is a bar illustrating zeta potential of LNPs comprising ionizable lipid compounds BNT-93 or BNT-94.

[0118] Figure 55 is a bar graph illustrating RNA accessibility (%) and encapsulation efficiency (%) of LNPs comprising ionizable lipid compounds BNT-93 or BNT-94.

[0119] Figure 56 is gel electrophoresis images of LNPs comprising ionizable lipid compounds BNT-93 or BNT-94.

[0120] Figure 57 is a bar graph illustrating mRNA integrity via capillary electrophoresis of LNPs comprising ionizable lipid compounds BNT-93 or BNT-94.

[0121] Figures 58A-58D are a set of bar graphs illustrating viability of cells (C2C12 cells - Figure 58A; HepG2 cells - Figure 58B; RAW cells - Figure 58C; HEK293T cells - Figure 58D) expressing luciferase mRNA from LNPs comprising ionizable lipid compounds BNT-93 or BNT-94. Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0122] Figures 59A-59D are a set of bar graphs illustrating in vitro expression of luciferase mRNA from LNPs comprising ionizable lipid compounds BNT-93 or BNT-94 in a skeletal muscle cell line (C2C12; Figure 59A), a hepatocarcinoma cell line (HepG2; Figures 59B), a murine macrophage cell line (RAW; Figure 59C), and a human embryonic kidney cell line (HEK293T; Figure 59D). Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0123] Figure 60 is a bar graph illustrating the particle size and PDI of LNPs comprising ionizable lipid compounds BNT-97 or BNT-98.

[0124] Figure 61 is a bar illustrating zeta potential of LNPs comprising ionizable lipid compounds BNT-97 or BNT-98.

[0125] Figure 62 is a bar graph illustrating RNA accessibility (%) and encapsulation efficiency (%) of LNPs comprising ionizable lipid compounds BNT-97 or BNT-98.

[0126] Figure 63 is gel electrophoresis images of LNPs comprising ionizable lipid compounds BNT-97 or BNT-98.

[0127] Figure 64 is a bar graph illustrating mRNA integrity via capillary electrophoresis of LNPs comprising ionizable lipid compounds BNT-97 or BNT-98.Figures 65A-65C are a set of bar graphs illustrating viability of cells (C2C12 cells - Figure 65 A; HepG2 cells - Figure 65B; RAW cells - Figure 65C) expressing luciferase mRNA from LNPs comprising ionizable lipid compounds BNT-97 or BNT-98. Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0128] Figures 66A-66C are a set of bar graphs illustrating in vitro expression of luciferase mRNA from LNPs comprising ionizable lipid compounds BNT-97 or BNT-98 in a skeletal muscle cell line (C2C12; Figure 66A), a hepatocarcinoma cell line (HepG2; Figures 66B), and a murine macrophage cell line (RAW; Figure 66C). Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0129] Figure 67 is a bar graph illustrating the particle size and PDI of LNPs comprising ionizable lipid compounds BNT-104, BNT-105, BNT-107 or BNT-122.

[0130] Figure 68 is a bar illustrating zeta potential of LNPs comprising ionizable lipid compounds BNT-104, BNT-105, BNT-107 or BNT-122.

[0131] Figure 69 is a bar graph illustrating RNA accessibility (%) and encapsulation efficiency (%) of LNPs comprising ionizable lipid compounds BNT-104, BNT-105, BNT-107 or BNT-122.

[0132] Figure 70 is a bar graph illustrating mRNA integrity via capillary electrophoresis of LNPs comprising ionizable lipid compounds BNT-104, BNT-105, BNT-107 or BNT-122.

[0133] Figures 71A-71C are a set of bar graphs illustrating viability of cells (C2C12 cells - Figure 71A; HepG2 cells - Figure 71B; RAW cells - Figure 71C) expressing luciferase mRNA from LNPs comprising ionizable lipid compounds BNT-104, BNT-105, BNT-107 or BNT-122. Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0134] Figures 72A-72C are a set of bar graphs illustrating in vitro expression of luciferase mRNA from LNPs comprising ionizable lipid compounds BNT-104, BNT-105, BNT-107 or BNT-122 in a skeletal muscle cell line (C2C12; Figure 72A), a hepatocarcinoma cell line (HepG2; Figures 72B), and a murine macrophage cell line (RAW; Figure 72C). Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0135] Figures 73A-73B depict the hemolytic effect of LNPs and the effect of the LNPs on complement activation. Figure 73A is a bar graph illustrating hemolysis after incubation (in neutral pH) of whole human blood with L LNPs comprising ionizable lipid compound BNT-105 and various controls. Figure 73B is a bar graph illustrating terminal complement complex (SC5b-9) formation of human serum with LNPs comprising ionizable lipid compound BNT-105 and various controls. The dashed horizontal line shows the level of SC5b-9 formation for PBS control.

[0136] Figures 74A-74E depict the results of bioluminescence imaging (BLI) in mice administered with LNPs comprising ionizable lipid compound BNT-105. Figure 74A depicts BLI of whole animals at 6h and 24h timepoints. Figure 74B depicts the total flux of in vivo imaging at 6h and 24h timepoints. Figure 74C depicts BLI of organs at the 24h timepoint. Figure 74D depicts the total flux of ex vivo or gain imaging at 6h and 24h timepoints. Figure 74E depicts the percent of biodistribution of the LNPs in various organs.Figure 75 is a bar graph illustrating the particle size and PDI of LNPs comprising ionizable lipid compounds BNT-116 or BNT-117.

[0137] Figure 76 is a bar illustrating zeta potential of LNPs comprising ionizable lipid compounds BNT-116 or BNT-117.

[0138] Figure 77 is a bar graph illustrating RNA accessibility (%) and encapsulation efficiency (%) of LNPs comprising ionizable lipid compounds BNT-116 or BNT-117.

[0139] Figure 78 is gel electrophoresis images of LNPs comprising ionizable lipid compounds BNT-116 or BNT-117.

[0140] Figure 79 is a bar graph illustrating mRNA integrity via capillary electrophoresis of LNPs comprising ionizable lipid compounds BNT-116 or BNT-117.

[0141] Figures 80A-80D are a set of bar graphs illustrating viability of cells (C2C12 cells - Figure 80A; HepG2 cells - Figure 80B; RAW cells - Figure 80C; HEK293T cells - Figure 80D) expressing luciferase mRNA from LNPs comprising ionizable lipid compounds BNT-116 or BNT-117. Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0142] Figures 81A-81D are a set of bar graphs illustrating in vitro expression of luciferase mRNA from LNPs comprising ionizable lipid compounds BNT-116 or BNT-117 in a skeletal muscle cell line (C2C12; Figure 80A), a hepatocarcinoma cell line (HepG2; Figures 80B), a murine macrophage cell line (RAW; Figure 80C), and a human embryonic kidney cell line (HEK293T; Figure 80D). Firefly luciferase expression was quantified 24h post-incubation with 12.5, 25, and 50 ng per well of mRNA-loaded LNPs.

[0143] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0144] The present disclosure provides, among other things, the surprising discovery of particular cationic or ionizable lipids. Such cationic or ionizable lipids exhibit improved properties (e.g., improved transfection to cells) relative to previous lipids. For example, as illustrated in the Examples provided herein, the lipid compounds of the present disclosure exhibit improvements over previous lipids, including, for example, improved transfection of cells with RNA. In some embodiments, particles prepared using lipid compounds described herein exhibit narrow size distribution (i.e., are substantially uniform in size), high encapsulation efficiency, low cytotoxicity, and improved biodistribution and efficacy relative to particles comprising previous lipid compounds.

[0145] Compounds and Definitions

[0146] Compounds of this disclosure include those described generally above and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of Elements, CAS version, Handbook of Chemistry and Physics, 75thEd. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March’s Advanced Organic Chemistry”, 5thEd.,Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0147] Unless otherwise stated, structures depicted herein are meant to include all stereoisomeric (e.g., enantiomeric or diastereomeric) forms of the structure, as well as all geometric or conformational isomeric forms of the structure. For example, the R and S configurations of each stereocenter are contemplated as part of the disclosure. Therefore, single stereochemical isomers, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of provided compounds are within the scope of the disclosure. For example, in some cases, Tables 1, 1A, 1B, and 1C show one or more stereoisomers of a compound, and unless otherwise indicated, represents each stereoisomer alone and / or as a mixture. Unless otherwise stated, all tautomeric forms of provided compounds are within the scope of the disclosure.

[0148] Unless otherwise indicated, structures depicted herein are meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including replacement of hydrogen by deuterium or tritium, or replacement of a carbon by13C-or14C-enriched carbon are within the scope of this disclosure.

[0149] About or approximately: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In general, those skilled in the art, familiar within the context, will appreciate the relevant degree of variance encompassed by "about" or "approximately" in that context. For example, in some embodiments, the term "approximately" or "about" may encompass a range of values that are within (i.e., ±) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0150] Administering: As used herein, the term "administering" or "administration" typically refers to the administration of a composition to a subject to achieve delivery of an agent that is, or is included in, a composition to a target site or a site to be treated. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may be parenteral. In some embodiments, administration may be oral. In some particular embodiments, administration may be intravenous. In some particular embodiments, administration may be subcutaneous. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time. In some embodiments, administration may comprisea prime -and-boost protocol. A prime -and-boost protocol can include administration of a first dose of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) followed by, after an interval of time, administration of a second or subsequent dose of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine). In the case of an immunogenic composition, a prime-and-boost protocol can result in an increased immune response in a patient.

[0151] Aliphatic: The term “aliphatic” refers to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “cycloaliphatic”), that has a single point or more than one points of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-12 aliphatic carbon atoms (e.g., C1-C12). In some embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms (e.g., Ci-Ce). In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms (e.g., C1-C5). In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms (e.g., C1--C4). In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms (e.g., C1-C3), and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms (e.g., C1-C2). Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, or alkynyl groups and hybrids thereof. A preferred aliphatic group is C1-6 alkyl.

[0152] Alkyl: The term “alkyl”, used alone or as part of a larger moiety, refers to a saturated, optionally substituted straight or branched chain hydrocarbon group having (unless otherwise specified) 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, or 1-2 carbon atoms (e.g., C1-C12, C1-C10, Ci-Cs, Ci-Ce, C1-C4, C1-C3, or C1-C2). Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, and heptyl.

[0153] Alkylene: The term “alkylene” refers to a bivalent alkyl group. In some embodiments, “alkylene” is a bivalent straight or branched alkyl group. In some embodiments, an "alkylene chain" is a polymethylene group, i.e., -(CH₂)ₙ-, wherein n is a positive integer, e.g., from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. An optionally substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms is optionally replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group and also include those described in the specification herein. It will be appreciated that two substituents of the alkylene group may be taken together to form a ring system. In certain embodiments, two substituents can be taken together to form a 3 - to 7-membered ring. The substituents can be on the same or different atoms. The suffix “-ene” or “-enyl” when appended to certain groups herein are intended to refer to a bifunctional moiety of said group. For example, “-ene” or “-enyl”, when appended to “cyclopropyl” becomes “cyclopropylene” or “cyclopropylenyl” and is

[0154] intended to refer to a bifunctional cyclopropyl group, e.g.,

[0155]

[0156] Alkenyl: The term “alkenyl”, used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain or cyclic hydrocarbon group having at least one double bond and having (unlessotherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms(e.g., C2-C12, C2-C10, C2-C8, C2-C6, C2-C4, or C2-C3). Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl. The term “cycloalkenyl” refers to an optionally substituted non -aromatic monocyclic or multicyclic ring system containing at least one carbon -carbon double bond and having about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0157] Alkynyl: The term “alkynyl”, used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain hydrocarbon group having at least one triple bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C2-C12, C2-C10, C2-C8, C2-C6, C2-C4, or C2-C3). Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl. Aryl: The term “aryl” refers to monocyclic and bicyclic ring systems having a total of six to fourteen ring members (e.g., Ce-Cu), wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. In some embodiments, an “aryl” group contains between six and twelve total ring members (e.g., Ce-Cn). The term “aryl” may be used interchangeably with the term “aryl ring”. In certain embodiments, “aryl” refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Unless otherwise specified, “aryl” groups are hydrocarbons. In some embodiments, an “aryl” ring system is an aromatic ring (e.g., phenyl) that is fused to a non-aromatic ring (e.g., cycloalkyl). Examples of aryl rings

[0158] include that are fused include

[0159]

[0160] Associated: Two events or entities are “associated” with one another, as that term is used herein, if the presence, level and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of and / or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non -covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0161] Bicyclic. The term “bicyclic ring” or “bicyclic ring system” refers to any bicyclic ring system, z.e., carbocyclic or heterocyclic, saturated or having one or more units of unsaturation, having one or more atoms in common between the two rings of the ring system. Thus, the term includes any permissible ring fusion, such as ort / io-fused or spirocyclic. As used herein, the term “heterobicyclic” is a subset of “bicyclic” that requires that one or more heteroatoms are present in one or both rings of the bicycle. Such heteroatoms may be present at ring junctions and are optionally substituted, and may be selected fromnitrogen (including N-oxides), oxygen, sulfur (including oxidized forms such as sulfones and sulfonates), phosphorus (including oxidized forms such as phosphates), boron, etc. In some embodiments, a bicyclic group has 7-12 ring members and 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. As used herein, the term “bridged bicyclic” refers to any bicyclic ring system, z.e., carbocyclic or heterocyclic, saturated or partially unsaturated, having at least one bridge. As defined by IUPAC, a “bridge” is an unbranched chain of atoms or an atom or a valence bond connecting two bridgeheads, where a “bridgehead” is any skeletal atom of the ring system which is bonded to three or more skeletal atoms (excluding hydrogen). In some embodiments, a bridged bicyclic group has 7-12 ring members and 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Such bridged bicyclic groups are well known in the art and include those groups set forth below where each group is attached to the rest of the molecule at any substitutable carbon or nitrogen atom. Unless otherwise specified, a bridged bicyclic group is optionally substituted with one or more substituents as set forth for aliphatic groups. Additionally or alternatively, any substitutable nitrogen of a bridged bicyclic group is optionally substituted. Exemplary bicyclic rings include:

[0162]

[0163] Exemplary bridged bicyclics include:

[0164]

[0165]

[0166] Biological sample: As used herein, the term “biological sample” typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell culture) of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a biological sample is or comprises biological tissue or fluid. In some embodiments, a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell -containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and / or excretions; and / or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, obtained cells are or include cells from an individual from whom the sample is obtained. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of certain components, etc.

[0167] Carrier: As used herein, the term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered. In some exemplary embodiments, carriers can include sterile liquids, such as, for example, water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil and the like. In some embodiments, carriers are or include one or more solid components.

[0168] Combination therapy: As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeuticagents or modality(ies)). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).

[0169] Comparable. As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0170] Composition: Those skilled in the art will appreciate that the term “composition” may be used to refer to a discrete physical entity that comprises one or more specified components. In general, unless otherwise specified, a composition may be of any form - e.g., gas, gel, liquid, solid, etc.

[0171] Cycloaliphatic. As used herein, the term “cycloaliphatic” refers to a monocyclic Cs-Cs hydrocarbon or a bicyclic Ce-Cio hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point or more than one points of attachment to the rest of the molecule.

[0172] Cycloalkyl. As used herein, the term “cycloalkyl” refers to an optionally substituted saturated ring monocyclic or polycyclic system of about 3 to about 10 ring carbon atoms. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0173] Deoxyribonucleic Acid (DNA). As used herein, the term “DNA” refers to a polymeric molecule of nucleotides that are typically double-stranded and comprise adenine, cytosine, guanine and thymine, and a deoxyribose sugar backbone structure as specified in the definition “Nucleic Acid / Polynucleotide.” In some embodiments, DNA is linear DNA, plasmid DNA, minicircle DNA, nanoplasmid DNA, doggybone DNA, or a transposon.Deoxyribonucleotide: As used herein, the term “deoxyribonucleotide” refers to unmodified and modified deoxyribonucleotides. For example, unmodified deoxyribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and thymine (T). Modified deoxyribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g., replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, and (d) intemucleoside linkage modifications, including modification or replacement of the phosphodiester linkages.

[0174] Dosage form or unit dosage form: Those skilled in the art will appreciate that the term “dosage form” may be used to refer to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject. Typically, each such unit contains a predetermined quantity of active agent. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen).

[0175] Dosing regimen or therapeutic regimen: Those skilled in the art will appreciate that the terms “dosing regimen” and “therapeutic regimen” may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0176] Effective Amount. The term “effective amount” refers to the amount of a compound sufficient to effect beneficial or desired results (e.g., a therapeutic, ameliorative, inhibitory, or preventative result). An effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or administration route.

[0177] Excipient: As used herein, the term “excipient” refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin,malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0178] Halogen: The term “halogen” or “halo” means F, Cl, Br, or I.

[0179] Heteroaryl. The terms “heteroaryl” and “heteroar-”, used alone or as part of a larger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refer to monocyclic or bicyclic ring groups having 5 to 10 ring atoms (e.g., 5- to 6-membered monocyclic heteroaryl or 9- to 10-membered bicyclic heteroaryl); having 6, 10, or 14 π-electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo[l,2-a]pyrimidinyl, imidazo[l,2-a]pyridyl, imidazo[4,5-b]pyridyl, imidazo[4,5-c]pyridyl, pyrrolopyridyl, pyrrolopyrazinyl, thienopyrimidinyl, triazolopyridyl, and benzoisoxazolyl. The terms “heteroaryl” and “heteroar-”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms). Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzotriazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H- quinolizinyl. carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3-b]-l,4-oxazin-3(4H)-one, 4H-thieno[3,2-b]pyrrole, and benzoisoxazolyl. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of which terms include rings that are optionally substituted.

[0180] Heteroatom: The term “heteroatom” as used herein refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen.

[0181] Heterocycle: As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclic radical”, and “heterocyclic ring” are used interchangeably and refer to a stable 3- to 8-membered monocyclic, a 6- to 10-membered bicyclic, or a 10- to 16-membered polycyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, such as one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR+(as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and thiamorpholinyl. A heterocyclyl group may be mono-, bi-, tri-, or polycyclic, preferably mono-, bi-, or tricyclic, more preferably mono- or bicyclic. A bicyclic heterocyclic ring also includes groupsin which the heterocyclic ring is fused to one or more aryl rings. Exemplary bicyclic heterocyclic groups include indolinyl, isoindolinyl, benzodioxolyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl, and tetrahydroquinolinyl. A bicyclic heterocyclic ring can also be a spirocyclic ring system (e.g., 7- to 11-membered spirocyclic fused heterocyclic ring having, in addition to carbon atoms, one or more heteroatoms as defined above (e.g., one, two, three or four heteroatoms)). A bicyclic heterocyclic ring can also be a bridged ring system (e.g., 7- to 11-membered bridged heterocyclic ring having one, two, or three bridging atoms.

[0182] Nanoparticle: As used herein, the term “nanoparticle” refers to a discrete entity of small size, e.g., typically having a longest dimension that is shorter than about 1000 nanometers (nm) and often is shorter than 500 nm, or even 100 nm or less. In many embodiments, a nanoparticle may be characterized by a longest dimension between about 1 nm and about 100 nm, or between about 1 μm and about 500 nm, or between about 1 nm and 1000 nm. In many embodiments, a population of microparticles is characterized by an average size (e.g., longest dimension) that is below about 1000 nm, about 500 nm, about 100 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, or about 10 nm and often above about 1 nm. In many embodiments, a microparticle may be substantially spherical (e.g., so that its longest dimension may be its diameter). In some embodiments, a nanoparticle has a diameter of less than 100 nm as defined by the National Institutes of Health. In some embodiments, nanoparticles are micelles in that they comprise an enclosed compartment, separated from the bulk solution by a micellar membrane, typically comprised of amphiphilic entities which surround and enclose a space or compartment (e.g., to define a lumen). In some embodiments, a micellar membrane is comprised of at least one polymer, such as for example a biocompatible and / or biodegradable polymer.

[0183] Nucleic acid / Polynucleotide'. As used herein, the term “nucleic acid” refers to a polymer of at least 10 nucleotides or more. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises a mixture of DNA and RNA. In some embodiments, a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises a single stranded nucleic acid. In some embodiments, a nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single and double-stranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate, phosphorodithioate, phosphoramide, phosphite-borane complexes, or 5'-N-phosphoramidite linkages and / or one or more peptide bonds, e.g., as in a “peptide nucleic acid”. In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non-natural residues. In some embodiments, a non-natural residue comprises a nucleoside analog (e.g, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo -pyrimidine, 3 -methyl adenosine, 5 -methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5 -fluorouridine, C5 -iodouridine, C5-propynyl -uridine, C5-propynyl -cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides long.

[0184] Nucleic acid particle: A “nucleic acid particle” can be used to deliver nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like). A nucleic acid particle may be formed from at least one cationic or cationically ionizable lipid or lipid-like material, at least one cationic polymer such as protamine, or a mixture thereof and nucleic acid. Nucleic acid particles include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations.

[0185] Nucleotide: As used herein, the term “nucleotide” refers to its art-recognized meaning. When a number of nucleotides is used as an indication of size, e.g., of a polynucleotide, a certain number of nucleotides refers to the number of nucleotides on a single strand, e.g., of a polynucleotide.

[0186] Parenteral: The phrases “parenteral administration” and “administered parenterally” as used herein have their art-understood meaning referring to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal, and intrastemal injection and infusion.

[0187] Partially unsaturated: As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond between ring atoms. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic (e.g., aryl or heteroaryl) moieties, as herein defined.

[0188] Patient or subject: As used herein, the term “patient” or “subject” refers to any organism to which a provided composition is or may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients or subjects include animals (e.g., mammals such as mice, rats, rabbits, non -human primates, and / or humans). In some embodiments, a patient is a human. In someembodiments, a patient or a subject is suffering from or susceptible to one or more disorders or conditions. In some embodiments, a patient or subject displays one or more symptoms of a disorder or condition. In some embodiments, a patient or subject has been diagnosed with one or more disorders or conditions. In some embodiments, a patient or a subject is receiving or has received certain therapy to diagnose and / or to treat a disease, disorder, or condition.

[0189] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in unit dose amount appropriate for administration in a therapeutic or dosing regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces. Pharmaceutically acceptable: As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0190] Pharmaceutically acceptable salt: The term “pharmaceutically acceptable salt”, as used herein, refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1- 19 (1977). Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate,nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluene sulfonate, undecanoate, valerate salts, and the like.

[0191] Further, acids which are generally considered suitable for the formation of pharmaceutically useful salts from basic pharmaceutical compounds are discussed, for example, by P. Stahl et al., Camille G. (eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use. (2002) Zurich: Wiley-VCH; S. Berge et al., Journal of Pharmaceutical Sciences 1977, 66(1), 1-19; P. Gould, International J. of Pharmaceutics 1986, 33, 201-217; Anderson et al., The Practice of Medicinal Chemistry (1996), Academic Press, New York; and in The Orange Book (Food & Drug Administration, Washington, D. C. on their website). These disclosures are incorporated herein by reference.

[0192] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N (C’i 4alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.

[0193] Physiological conditions: as used herein, the term “physiological conditions” has its art-understood meaning referencing conditions under which cells or organisms live and / or reproduce. In some embodiments, the term refers to conditions of the external or internal mileu that may occur in nature for an organism or cell system. In some embodiments, physiological conditions are those conditions present within the body of a human or non-human animal, especially those conditions present at and / or within a surgical site. Physiological conditions typically include, e.g., a temperature range of 20 - 40°C, atmospheric pressure of 1, pH of 6-8, glucose concentration of 1-20 mM, oxygen concentration at atmospheric levels, and gravity as it is encountered on earth. In some embodiments, conditions in a laboratory are manipulated and / or maintained at physiologic conditions. In some embodiments, physiological conditions are encountered in an organism.

[0194] Polycyclic: As used herein, the term “polycyclic” refers to a saturated or unsaturated ring system having two or more rings (for example, heterocyclyl rings, heteroaryl rings, cycloalkyl rings, or aryl rings), having between 7 and 20 atoms, in which one or more carbon atoms are common to two adjacent rings. For example, in some embodiments, a polycyclic ring system refers to a saturated or unsaturated ring system having three or more rings (for example, heterocyclyl rings, heteroaryl rings, cycloalkyl rings, or aryl rings), having between 14 and 20 atoms, in which one or more carbon atoms are common to two adjacent rings. The rings in a polycyclic ring system may be fused (i.e., bicyclic or tricyclic), spirocyclic, or a combination thereof. An example polycyclic ring is a steroid.

[0195] Polypeptide: The term “polypeptide” or “peptide”, as used herein, typically has its art-recognized meaning of a polymer of at least three amino acids or more. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional, biologically active, or characteristic fragments, portions or domains (e.g., fragments, portions, or domains retaining atleast one activity) of such complete polypeptides. In some embodiments, polypeptides may contain L-amino acids, D-amino acids, or both and / or may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, polypeptides may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof (e.g., may be or comprise peptidomimetics).

[0196] Prevent or prevention: As used herein, the terms “prevent” or “prevention”, when used in connection with the occurrence of a disease, disorder, and / or condition, refer to reducing the risk of developing the disease, disorder and / or condition and / or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.

[0197] Reference: As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.

[0198] Ribonucleotide: As used herein, the term “ribonucleotide” encompasses unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g., replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, and (d) intemucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. The term “ribonucleotide” also encompasses ribonucleotide triphosphates including modified and non-modified ribonucleotide triphosphates.

[0199] Ribonucleic acid (RNA): As used herein, the term “RNA” refers to a polymer of ribonucleotides. In some embodiments, an RNA is single stranded. In some embodiments, an RNA is double stranded. In some embodiments, an RNA comprises both single and double stranded portions. In some embodiments, an RNA can comprise a backbone structure as described in the definition of “Nucleic acid / Polynucleotide" above. An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc ), or a messenger RNA (mRNA). In some embodiments where an RNA is a mRNA. In some embodiments where an RNA is a mRNA, an RNA typically comprises at its 3’ end a poly(A) region. In some embodiments where an RNA is a mRNA,an RNA typically comprises at its 5’ end an art-recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation. In some embodiments, an RNA is a synthetic RNA. Synthetic RNAs include RNAs that are synthesized in vitro (e.g., by enzymatic synthesis methods and / or by chemical synthesis methods).

[0200] Substituted or optionally substituted: As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. “Substituted” applies to one or more hydrogens that are either explicit or implicit from the

[0201]

[0202] r

[0203]

[0204] 1R1, orr1). Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subj ected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes provided herein. Groups described as being “substituted” preferably have between 1 and 4 substituents, more preferably 1 or 2 substituents. Groups described as being “optionally substituted” may be unsubstituted or be “substituted” as described above. Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; –(CH2)0–4R°; –(CH2)0–4OR°: –O(CH2)0–4R°, –O–(CH2)0–4C(O)OR°; -(CH2)o-4CH(OR°)2; –(CH2)0–4SR°; –(CH2)0–4Ph. which may be substituted with R°; –(CH2)0–4O(CH2)0–1Ph which may be substituted with R°; -CH=CHPh, which may be substituted with R°; –(CH2)0–4O(CH2)0–1-pyridyl which may be substituted with R°; -NO2; -CN; -N3; –(CH2)0–4N(R°)2: –(CH2)0–4N(R°)C(O)R°; -N(R°)C(S)R°; –(CH2)0–4N(R°)C(O)NR°2; –N(R°)C(S)NR°2; -(CH2)O^N(R°)C(0)OR°; N(R°)N(R°)C(O)R°; –N(R°)N(R°)C(O)NR°2; -N(R°)N(R°)C(O)OR°; –(CH2)0–4C(O)R°; C(S)R°; –(CH2)0–4C(O)OR°; –(CH2)0–4C(O)SR°; –(CH2)0–4C(O)OSiR°3; –(CH2)0–4OC(O)R°; –OC(O)(CH2)0–4SR°: –(CH2)0–4SC(O)R°; –(CH2)0–4C(O)NR°2; –C(S)NR°2; -C(S)SR°; -SC(S)SR°, –(CH2)0–4OC(O)NR°2; -C(O)N(OR°)R°; -C(O)C(O)R°; –C(O)CH2C(O)R°; -C(NOR°)R°; –(CH2)0–4SSR°; –(CH2)0–4S(O)2R°; –(CH2)0–4S(O)2OR°: –(CH2)0–4OS(O)2R°; –S(O)2NR°2; –(CH2)0–4S(O)R°: –N(R°)S(O)2NR°2; -N(R°)S(O)2R°; -N(OR°)R°; –C(NH)NR°2; -P(O)2R°; -P(O)R°2; -OP(O)R°2; -OP(O)(OR°)2; SiR°3; –(C1–4straight or branched alkylene)O–N(R°)2; or –(C1–4straight or branched alkylene)C(O)O–N(R°)2, whereineach R° may be substituted as defined below and is independently hydrogen, C1–6aliphatic, –CH2Ph. –O(CH2)0–1Ph, -CH2-(5- to 6-membered heteroaryl ring), or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3 - to 12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0205] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, –(CH2)0–2R*, -(haloR*), -(CH2)o 2OH, –(CH2)0–2OR*, –(CH2)0–2CH(OR*)2, -O(haloR’), -CN, -N3, –(CH2)0–2C(O)R*, –(CH2)0–2C(O)OH, –(CH2)0–2C(O)OR*, –(CH2)0–2SR*, –(CH2)0–2SH, –(CH2)0–2NH2, –(CH2)0–2NHR*, –(CH2)0–2NR*2, -NO2, -SiR’s, -oSiR’s, –C(O)SR*, –(C1–4straight or branched alkylene)C(O)OR*, or -SSR* wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from Ci^ aliphatic, -CH2PI1, –O(CH2)0–1Ph, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0206] Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: =0 (“oxo”), =S, =NNR*2, =NNHC(0)R*, =NNHC(0)0R*, =NNHS(O)2R*, =NR*, =N0R*, –O(C(R*2))2–3O–, or–S(C(R*2))2–3S–, wherein each independent occurrence of R* is selected from hydrogen, C1–6aliphatic which may be substituted as defined below, or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: –O(CR*2)2–3O–, wherein each independent occurrence of R* is selected from hydrogen, C1–6aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0207] Suitable substituents on the aliphatic group of R* include halogen, -R*, -(haloR*). -OH, -OR’, -O(haloR’), -CN, -C(O)OH, -C(O)OR*, -NH2, -NHR*, -NR*2, or -NO2, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently Ci^ aliphatic, -CH2PI1, –O(CH2)0–1Ph, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0208] Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include –R†. –NR†2, –C(O)R†, –C(O)OR†, –C(O)C(O)R†, –C(O)CH2C(O)R†, –S(O)2R†, –S(O)2NR†2, –C(S)NR†2, –C(NH)NR†2, or –N(R†)S(O)2R†: wherein each R' is independently hydrogen, C1-6 aliphatic which may be substituted as defined below, unsubstituted –OPh, or an unsubstituted 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R†, taken together with theirintervening atom(s) form an unsubstituted 3 -to 12-membered saturated, partially unsaturated, or aryl mono-or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0209] Suitable substituents on the aliphatic group of R' are independently halogen, -R*, -(haloR*). -OH, -OR’, -O(haloR’), -CN, -C(O)OH, -C(O)OR’, -NH2, -NHR*, -NR or -NO2, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently Ci- 4 aliphatic, -CH2PI1, –O(CH2)0–1Ph, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0210] Suffering from. An individual who is “suffering from” a disease, disorder, and / or condition has been diagnosed with and / or displays one or more symptoms of a disease, disorder, and / or condition.

[0211] Small molecule: As used herein, the term “small molecule” means a low molecular weight organic and / or inorganic compound. In general, a “small molecule” is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, a small molecule is not a polymer.

[0212] In some embodiments, a small molecule does not include a polymeric moiety. In some embodiments, a small molecule is not and / or does not comprise a protein or polypeptide (e.g., is not an oligopeptide or peptide). In some embodiments, a small molecule is not and / or does not comprise a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, a small molecule is not and / or does not comprise a polysaccharide; for example, in some embodiments, a small molecule is not a glycoprotein, proteoglycan, glycolipid, etc.). In some embodiments, a small molecule is not a lipid.

[0213] In some embodiments, a small molecule is a modulating agent (e.g., is an inhibiting agent or an activating agent). In some embodiments, a small molecule is biologically active. In some embodiments, a small molecule is detectable (e.g., comprises at least one detectable moiety). In some embodiments, a small molecule is a therapeutic agent.

[0214] Those of ordinary skill in the art, reading the present disclosure, will appreciate that certain small molecule compounds described herein may be provided and / or utilized in any of a variety of forms such as, for example, crystal forms (e.g., polymorphs, solvates, etc), salt forms, protected forms, pro-drug forms, ester forms, isomeric forms (e.g., optical and / or structural isomers), isotopic forms, etc.

[0215] Those of ordinary skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more stereoisomeric forms. In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in the form of an individual enantiomer, diastereomer or geometric isomer, or may be in the form of a mixture of stereoisomers; in some embodiments, such a small molecule may be utilized in accordance with the present disclosure in a racemic mixture form.

[0216] Those of skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more tautomeric forms. In some embodiments, such a small molecule may be utilized inaccordance with the present disclosure in the form of an individual tautomer, or in a form that interconverts between tautomeric forms.

[0217] Those of skill in the art will appreciate that certain small molecule compounds have structures that permit isotopic substitution (e.g.,2H or3H for H;nC,13C or14C for12C;13N or15N for14N;17O or18O for16O;36C1 for35C1 or37C1;18F for19F;131I for127I; etc.). In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in one or more isotopically modified forms, or mixtures thereof. In some embodiments, reference to a particular small molecule compound may relate to a specific form of that compound. In some embodiments, a particular small molecule compound may be provided and / or utilized in a salt form (e.g., in an acid-addition or base-addition salt form, depending on the compound); in some such embodiments, the salt form may be a pharmaceutically acceptable salt form.

[0218] In some embodiments, where a small molecule compound is one that exists or is found in nature, that compound may be provided and / or utilized in accordance in the present disclosure in a form different from that in which it exists or is found in nature. Those of ordinary skill in the art will appreciate that, in some embodiments, a preparation of a particular small molecule compound that contains an absolute or relative amount of the compound, or of a particular form thereof, that is different from the absolute or relative (with respect to another component of the preparation including, for example, another form of the compound) amount of the compound or form that is present in a reference preparation of interest (e.g., in a primary sample from a source of interest such as a biological or environmental source) is distinct from the compound as it exists in the reference preparation or source. Thus, in some embodiments, for example, a preparation of a single stereoisomer of a small molecule compound may be considered to be a different form of the compound than a racemic mixture of the compound; a particular salt of a small molecule compound may be considered to be a different form from another salt form of the compound; a preparation that contains only a form of the compound that contains one conformational isomer ((Z) or (E)) of a double bond may be considered to be a different form of the compound from one that contains the other conformational isomer ((E) or (Z)) of the double bond; a preparation in which one or more atoms is a different isotope than is present in a reference preparation may be considered to be a different form; etc.

[0219] Those skilled in the art will further appreciate that, in small molecule structures, the symbol, as used herein, when drawn across a bond, refers to a point of attachment between two atoms. For example:

[0220]

[0221] Additionally or alternatively, the symbol refers to a point of attachment ring in a spirocyclic manner. Additionally or alternatively, the symbol, when drawn in place of a bond, is

[0222] intended to represent a chiral center having either (R) or (S) configuraiton. For example,

[0223]

[0224] \ is

[0225] intended to represent both:

[0226]

[0227] and.

[0228] Treat: As used herein, the terms “treat,” “treatment,” or “treating” refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduceincidence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and / or condition, for example, for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0229] Cationic or Ionizable Lipid Compounds

[0230] The present disclosure provides, among other things, cationic or ionizable lipid compounds useful for forming particles comprising nucleic acids. As described herein, in some embodiments, the present disclosure provides a compound represented by formula I:

[0231] L1— X1— T1

[0232] G-L3-N

[0233]

[0234] \2- X2- T2

[0235] I

[0236] or a pharmaceutically acceptable salt thereof, wherein:

[0237] L1and L2are each independently an optionally substituted C1-C30 aliphatic group;

[0238] L3is a bond or optionally substituted C1-C10 aliphatic group;

[0239] X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -S(O)2N(R’)-, -N(R1)S(O)2-, -S(O)-, -S(O)2-, -S(O)2C(R1)2-, -OC(S)C(R1)2-, -C(R1)2C(S)O-, -S-, -N(H)C(O)N(R1)-, -NHC(O)-, -C(O)N(R’)-, -NHS(O)2N(R1)-, and -C(O)-;

[0240] each R1is, independently, at each instance, optionally substituted C1-C20 aliphatic or H;

[0241] T1and T2are each independently an optionally substituted C3-C30 aliphatic;

[0242] G is -N(R2)C(S)N(R2)2, -OH, -N(R2)2, -N+(R3)3, -N(R5)C(O)R3, -N(R5)S(O)2R3, -N(R5)C(O)N(R3)2, –CH(N–R2), -R4, -S(O)2R3, S(O)R3, or -SR3;

[0243] each R2is, independently, at each instance, selected from the group consisting of H, optionally substituted Ci-Ce aliphatic and OR3;

[0244] R3is selected from the group consisting of H and optionally substituted C1-C10 aliphatic;

[0245] R4is optionally substituted 4- to 12-membered heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S, optionally substituted 4- to 12 membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S, optionally substituted Ce-Cn aryl, or optionally substituted C3-C12 cycloaliphatic; and each R5is independently selected from H and optionally substituted Ci-Ce aliphatic.

[0246] In some embodiments, a compound represented by formula I is not a compound selected from the table below:Name Structure

[0247] I

[0248] o BNT-008

[0249] z

[0250] BNT-009

[0251] BNT-10

[0252] ^^z

[0253] BNT-20 o I y

[0254] o o I I BNT-21

[0255] BNT-22

[0256]

[0257] Name Structure

[0258] I BNT-23 O

[0259] \ / Z——

[0260] BNT-24

[0261] 'co co—

[0262] Wo X t—

[0263] BNT-46 N'^X^XOH / z — —

[0264] 0 o X

[0265] BNT-50 0

[0266] O 'V. P

[0267] N^S / \Z\z^ / \

[0268] BNT-51 C2X2X^ N—

[0269] c / 'o

[0270] x^XX / XsBNT-52

[0271] 1 H |

[0272] 6z'b

[0273]

[0274] Name Structure

[0275] ct p

[0276] I^^NAN, OH BNT-54

[0277] 1 H |

[0278] X

[0279] O / 6'"o >w

[0280] IZ

[0281] rz. - BNT-56. Z. < - -. z. - — —

[0282] b d

[0283] b J OO / X^X

[0284] W( t / ) x

[0285] ^O; oz\c cnn 'O / - '° \ / ' O

[0286] BNT-57 ^V \ xO. o / , \O (

[0287] ; » xC«> W

[0288] O OO O ' ' > ' '' ''

[0289] ^ 'z —

[0290] ' ' z — —

[0291] BNT-59

[0292] ^ O z o I BNT-71

[0293] BNT-72

[0294]

[0295] Name Structure

[0296] BNT-73

[0297] ^^1 0

[0298] II

[0299] 0 N^^OH BNT-74

[0300] o / K >

[0301] b b d <

[0302] ^ ^;o; Oo c c wocnwn=-=

[0303] ' '°° \ \ / / '' o o — —

[0304] BNT-75

[0305] ^ ' ^ ' zzZ — — - - o o o I T BNT-76

[0306] N'^N^OHBNT-96

[0307] °, x°

[0308] BNT-115

[0309]

[0310] Name Structure

[0311] T Z

[0312] O o BNT-120

[0313] zz Z \—

[0314] z—

[0315] BNT-140 7

[0316] < OT

[0317] 7 \ / O

[0318] 77 ° \x

[0319] zz—Wo ov, / \, z..

[0320] \ 7 \;'; co« o / ( °

[0321] " —O O' \ /

[0322] ) < C w 7o \ 77 \ \ / ° ' —

[0323] / ° \ / " 77 7 o \ \ o..

[0324] W7W / z / / \ \' \ 7 \ o' o _

[0325] 7 ° \x

[0326] BNT-141W / z

[0327] / \' o

[0328] ' ' z —

[0329] ^Z

[0330] O„O

[0331] zz >wzz N^ z o IOHo — BNT-142 II J <w\ I= z — /

[0332] BNT-143

[0333] BNT-123

[0334]

[0335] Name Structure

[0336] 0 O

[0337] BNT-124 N —

[0338] 1 H Y--3 bz'b

[0339] 0 O

[0340] BNT-125 N — ^^^X^N" X 1 H L_y b'xb

[0341] 0 O

[0342] BNT-126

[0343] b / xb

[0344] 1

[0345] N OH BNT-146

[0346] o

[0347] II

[0348] 1

[0349] 1ST OH BNT-147

[0350] o

[0351] H

[0352] O1 ^ Xx

[0353] i\r OH BNT-148

[0354]

[0355] Name Structure

[0356] N — | ^ OH BNT-149 J C -lQ

[0357] ^^1 0 II

[0358] 1^ N^OHBNT-150 cP

[0359] 0 ii

[0360] \^N\^ \ \) |

[0361] 0

[0362] BNT-151 Ci®

[0363] 1 NT I — N 1 / W BNT-152

[0364] Cl®1<=1®

[0365] ^^^1 0

[0366] II

[0367] 1 / ^ A 1 —^ Ni / o BNT-153

[0368] Ci®1cP o

[0369]

[0370] Name Structure

[0371] X0

[0372] ii

[0373] °

[0374] BNT-154

[0375] Cl®1cP

[0376] BNT-155

[0377] ^ 0, jhoo cmn==

[0378] 65fZ<

[0379] ^^1 0

[0380] II BNT-156

[0381] ^ 'Z -

[0382] / -Z —.

[0383] IZ\ BNT-157

[0384] N^N^N^ BNT-158

[0385]

[0386] Name Structure

[0387] ^^1 0

[0388] ii

[0389] BNT-159

[0390] 10

[0391] II

[0392] \ \z / S II. -'^ \z \

[0393] ° / NT N BNT-160

[0394] BNT-161

[0395] o

[0396] II

[0397] N^^;;\^i::i^^QHBNT-162

[0398] 0

[0399] II

[0400] \ 'x / / S. xz \

[0401] 0 1

[0402] N — — OH BNT-163

[0403]

[0404] I Name Structure O

[0405] BNT-164

[0406] 0 \ / Z——

[0407] ii

[0408] BNT-165

[0409] WO X C—

[0410] BNT-166 00

[0411] N^$x.

[0412] BNT-167 j ^\^0H

[0413] ^^^1 0

[0414] II

[0415] N^$x.

[0416] BNT-168 ° / \ / \ / \ / \ j ^X^OH

[0417]

[0418] Name Structure

[0419] T I

[0420] O o I

[0421] 0 BNT-169

[0422] \ \ / /

[0423] \ / / z— ——

[0424] / \ z°

[0425] BNT-170 \C zW

[0426] o / r\°~

[0427] °%=,x

[0428] \ / (z°

[0429] ° / \( \Z) < /

[0430] O5 OOTw. OT^== 0U /

[0431] BNT-171 0 * '

[0432] o I BNT-172

[0433] BNT-173

[0434]

[0435] I o Name Structure

[0436] T o o I I

[0437] O BNT-174 >

[0438] ^Z.—-

[0439] ^ z \ Z— z^. z^.

[0440] / \,

[0441] / \ / z \ z / ° \ z°o°

[0442] BNT-175 \ zC \OT\C \ Z zCWM

[0443] / r\ / / / r\°°°°~ 'x s s

[0444] / \ c\ \ / \Z\ / \ \ \ / z\ (Oz zo° / °

[0445] / \ / \C / \ w

[0446] / \ °W< >' Z

[0447] / \ \' o

[0448] O \x

[0449] " > Cn z

[0450] / \' o

[0451] H / i ° '

[0452] BNT-176

[0453] ^z

[0454] /

[0455] \

[0456] BNT-177 o

[0457] I

[0458] BNT-178

[0459]

[0460] Name Structure

[0461] / / z IZ — BNT-179

[0462] > > y / ZZ. -—

[0463] \ Z \ Z——

[0464] / / \ \ z Z°°

[0465] BNT-180 \ \ zCOT

[0466] / / °°s s

[0467] o> co \ / \ / \ \z,°o

[0468] / \ c / /

[0469] ri | CF3BNT-181

[0470] JN^OH ' ' z — -

[0471] o z BNT-182

[0472] BNT-183

[0473] JN^OH

[0474] I CF3BNT-184

[0475] JN^-0H

[0476]

[0477] Name Structure

[0478] | F T o BNT-185

[0479] / Z c - - ] F BNT-186 N^^^OH v co>

[0480] OT) (f

[0481] BNT-187

[0482] i r

[0483] y

[0484] o BNT-188 N^^O IH

[0485] BNT-189

[0486] BNT-190

[0487] JN'— OH

[0488]

[0489] Name Structure

[0490] BNT-191 I O

[0491] /

[0492] \,Z- - HOU0 —N°" S" '°N

[0493] 1 H

[0494] BL-199 Z y—

[0495] ,, Z ) ( Oozc-^ \ / — P' '

[0496] 0 );> o< / O Z / '

[0497] Z y—

[0498] / <.'0z V

[0499] ° / \s

[0500] to to

[0501] / (° ' > W x

[0502] ZQ\

[0503] BL-200 > ( < O / o / xx

[0504] ' ) > <co

[0505] y z— 'ZQ Z^ \

[0506] < o / / x

[0507] ' > <co

[0508] o 0

[0509] r T

[0510] ^0

[0511] BL-201

[0512] ^0

[0513] BL-202

[0514] BL-203

[0515]

[0516] Name Structure

[0517] BL-204

[0518] / I

[0519] o o Z y—

[0520] > z—

[0521] Z y—

[0522] BL-205 Z Z y— y—

[0523] O / O O / \ / \ \x*x

[0524] >

[0525] / / (' O' °' O Z Z Z^^^

[0526] 1 H ( ( / O / O / / xx

[0527] ' ' > <cn > <tn

[0528] !! t o' 'o >' < °

[0529] BL-206

[0530] M i

[0531] o" 'o

[0532] BL-207

[0533] 1 H

[0534] N N _ _ _ _ _ _ _ _ _ N s k °z'° U BL-208

[0535] \^ OzS';oN0000

[0536]

[0537] Name Structure

[0538] I

[0539] o BL-209

[0540] \ \ \ z —

[0541] Z z z———

[0542] <<< « w co===

[0543] ZI ZI ZI BL-212

[0544] Z Z Z y— y— y—

[0545] \

[0546] «w

[0547] C )O \o / Z

[0548] ( < O \ \ '* 'OO, Zz z— ^

[0549] / C C ) ' )OZoo ^ \ \ ) <

[0550] ' '°°o \ / \ \ / / \,

[0551] < >

[0552] BL-210

[0553] I oz'o

[0554] BL-211

[0555] \^ OzS':oNOOC

[0556] BL-213

[0557]

[0558]

[0559] In some embodiments, a compound of formula I is a compound represented by formula IA:

[0560] L1-X1-T1

[0561]

[0562] \2- X2— T2

[0563] IA

[0564] or a pharmaceutically acceptable salt thereof, wherein:

[0565] L1and L2are each independently an optionally substituted C1-C30 aliphatic group;

[0566] L3is a bond or optionally substituted C1-C10 aliphatic;

[0567] X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -S(O)2N(R’)-, -N(R1)S(O)2-, -S(O)-, -S(O)2-, -S(O)2C(R1)2-, -OC(S)C(R1)2-, -C(R1)2C(S)O-, -S-, -N(H)C(O)N(R1)-, -NHC(O)-, -C(O)N(R’)-, -NHS(O)2N(R1)-, and -C(O)-;

[0568] each R1is, independently, at each instance, optionally substituted C1-C20 aliphatic or H;

[0569] T1and T2are each independently an optionally substituted C3-C30 aliphatic;

[0570] G1is -S(O)R3or -SR3; and

[0571] R3is optionally substituted C1-C10 aliphatic.

[0572] In some embodiments, a compound of formula I is a compound represented by formula IB:L1-X1-T1

[0573] G2-L3-N / /

[0574] \2- X2- T2

[0575] IB

[0576] or a pharmaceutically acceptable salt thereof, wherein:

[0577] L1and L2are each independently an optionally substituted C1-C30 aliphatic group;

[0578] L3is a bond or optionally substituted C1-C10 aliphatic;

[0579] X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -S(O)2N(R’)-, -N(R’)S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R1)2-, -OC(S)C(R1)2-, -C(R1)2C(S)O-, -S-, and -N(H)C(O)N(R1);

[0580] each R1is, independently, at each instance, optionally substituted C1-C20 aliphatic or H;

[0581] T1and T2are each independently an optionally substituted C3-C30 aliphatic;

[0582] G2is -S(O)2R3a;

[0583] R3ais -(CH2)0-6-R3bor Ci-Ce aliphatic; and

[0584] R3bis -OH, -NH(CH3), or -N(CH3)2.

[0585] In some embodiments of formula IB, when R3ais -CH3, then X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -N(H)C(O)N(R1)-, -NHC(O)-, -C(O)N(R1)-, and -NHS(O)2N(R1)-. In some embodiments of formula IB, when R3ais -CH3, then T1and T2are each branched optionally substituted C1-C20 aliphatic.

[0586] In some embodiments, a compound of formula I is a compound represented by formula IB:

[0587] L1-X1-T1

[0588] G2-L3-N

[0589] 2- X2- T2

[0590] IB

[0591] or a pharmaceutically acceptable salt thereof, wherein:

[0592] L1and L2are each independently an optionally substituted C1-C30 aliphatic group;

[0593] L3is a bond or optionally substituted C1-C10 aliphatic;

[0594] X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -S(O)2N(R’)-, -N(R’)S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R1)2-, -OC(S)C(R1)2-, -C(R1)2C(S)O-, -S-, and -N(H)C(O)N(R1);

[0595] each R1is, independently, at each instance, optionally substituted C1-C20 aliphatic or H;

[0596] T1and T2are each independently an optionally substituted C3-C30 aliphatic;

[0597] G2is -S(O)2R3a;

[0598] R3ais -(CH2)0-6-R3bor Ci-Ce aliphatic; and

[0599] R3bis -OH, -NH(CH3), or -N(CH3)2, provided that when R3ais -CH3, then T1and T2are each branched optionally substituted C1-C20 aliphatic.

[0600] In some embodiments, a compound of formula I is a compound represented by formula IB:L1-X1-T1

[0601] G2-L3-N / /

[0602] \2- X2- T2

[0603] IB

[0604] or a pharmaceutically acceptable salt thereof, wherein:

[0605] L1and L2are each independently an optionally substituted C1-C30 aliphatic group;

[0606] L3is a bond or optionally substituted C1-C10 aliphatic;

[0607] X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -S(O)2N(R’)-, -N(R’)S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R1)2-, -OC(S)C(R1)2-, -C(R1)2C(S)O-, -S-, and -N(H)C(O)N(R1);

[0608] each R1is, independently, at each instance, optionally substituted C1-C20 aliphatic or H;

[0609] T1and T2are each independently an optionally substituted C3-C30 aliphatic;

[0610] G2is -S(O)2R3a;

[0611] R3ais -(CH2)0-6-R3bor Ci-Ce aliphatic; and

[0612] R3bis -OH, -NH(CH3), or -N(CH3)2, provided that, when R3ais -CH3, then X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -N(H)C(O)N(R1)-, -NHC(O)-, -C(O)N(R1)-, and -NHS(0)2N(R’)-, and T1and T2are each branched optionally substituted C1-C20 aliphatic.

[0613] In some embodiments, a compound of formula I is a compound represented by formula IC:

[0614] L1— X1— T1

[0615] G3-L3-N / /

[0616] 2- X2- T2

[0617] IC

[0618] or a pharmaceutically acceptable salt thereof, wherein:

[0619] L1and L2are each independently an optionally substituted C1-C30 aliphatic group;

[0620] L3is a bond or optionally substituted C1-C10 aliphatic;

[0621] X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -N(H)C(O)N(R’)-, -NHC(O)-, and -C(O)N(R1)-;

[0622] each R1is, independently, at each instance, optionally substituted C1-C20 aliphatic or H;

[0623] T1and T2are each independently an optionally substituted C3-C30 aliphatic group;

[0624]

[0625] each R30is independently selected from -SH,-OH, -NH2, -NH(CH3), -N(CH3)2, -C(O)NH2, and optionally substituted C1-C6aliphatic;

[0626] provided that when G3is -OH, -NH2, -NH(CH3), or -N(CH3)2, and X1or X2is a bond, -OC(O)-, or -C(O)O-, then L3is C2-C10 alkenyl, or C2-C10 alkynyl.

[0627] In some embodiments, a compound of formula I or IB is represented by formula IIA:

[0628] O / L1—X1- T1

[0629] HO— (CH2)1-6— S-(CH2)1-6— /

[0630]

[0631] O L2- X2— T2

[0632] IIA

[0633] or a pharmaceutically acceptable salt thereof, wherein L1, L2, X1, X2, T1, and T2are as described in classes and subclasses herein, both singly and in combination.

[0634] In some embodiments, a compound of formula I or IB is represented by formula IIA-1:

[0635] / L1— X1— T1

[0636] HO^(CH2)I-6“N

[0637]

[0638] \2- X2- T2

[0639] IIA-1

[0640] or a pharmaceutically acceptable salt thereof, wherein L1, L2, X1, X2, T1, and T2are as described in classes and subclasses herein, both singly and in combination.

[0641] In some embodiments, a compound of formula I or IB is represented by formula IIA-2:

[0642] v

[0643] O / L1— 0

[0644] HO— (CH2)1-6— S-(CH2)1-6—

[0645] O L2- O

[0646]

[0647] cT2

[0648] IIA-2

[0649] or a pharmaceutically acceptable salt thereof, wherein L1, L2, T1, and T2are as described in classes and subclasses herein, both singly and in combination.In some embodiments, a compound of formula I or IB is represented by formula IIA-3

[0650] O O O L1— S-T1

[0651] HO— (CH2)1-6— S-(CH2)1-6—

[0652] O L2—S-T2

[0653]

[0654] o o

[0655] IIA-3

[0656] or a pharmaceutically acceptable salt thereof, wherein L1, L2, T1, and T2are as described in classes and subclasses herein, both singly and in combination.

[0657] In some embodiments, a compound of formula I or IB is represented by formula IIA-4:

[0658] O O O L1— S-N-T1

[0659] HO— (CH2)1-6— S-(CH2)1-6— R1

[0660] O L2— S —N— T2

[0661]

[0662] O O R1

[0663] IIA-4

[0664] or a pharmaceutically acceptable salt thereof, wherein L1, L2, T1, T2, and R1are as described in classes and subclasses herein, both singly and in combination.

[0665] In some embodiments, a compound of formula I or IA is represented by formula IIB:

[0666] O / L1— X1— T1

[0667] R3-S-(CH2)1-6—N

[0668]

[0669] L2— x2- T2

[0670] IIB

[0671] or a pharmaceutically acceptable salt thereof, wherein R3, L1, L2, X1, X2, T1, and T2are as described in classes and subclasses herein, both singly and in combination.

[0672] In some embodiments, a compound of formula I or IA is represented by formula IIB- 1:

[0673] zL1- X1- T1

[0674] R3-S-(CH2)1-6—

[0675] L2- X2- T2

[0676] IIB-1

[0677] or a pharmaceutically acceptable salt thereof, wherein R3, L1, L2, X1, X2, T1, and T2are as described in classes and subclasses herein, both singly and in combination.

[0678] In some embodiments, a compound of formula IC is represented by formula IIC:

[0679] X1— T1

[0680] I

[0681] L1

[0682]

[0683] HO^^^^N'L2-X2-T2

[0684] IIC

[0685] or a pharmaceutically acceptable salt thereof,

[0686] wherein X1and X2are each independently selected from a bond, -OC(O)-, and -C(O)O-; andwherein L1, L2, T1, and T2are as described in classes and subclasses herein, both singly and in combination. In some embodiments, a compound of formula IC is represented by formula IID:

[0687]

[0688] or a pharmaceutically acceptable salt thereof,

[0689] wherein X1and X2are each independently selected from a bond, -OC(O)-, and -C(O)O-; and wherein L1, L2, T1, and T2are as described in classes and subclasses herein, both singly and in combination. In some embodiments, a compound of formula IC is represented by formula IIE:

[0690]

[0691] or a pharmaceutically acceptable salt thereof,

[0692] wherein X1and X2are each independently selected from a bond, -OC(O)-, and -C(O)O-; and wherein L1, L2, T1, and T2are as described in classes and subclasses herein, both singly and in combination. In some embodiments, a compound of formula I or IC is represented by formula IIF

[0693] X1-T1

[0694] I

[0695] |-1

[0696] ^^2)1-6 _ X2-T2

[0697] (R30)

[0698]

[0699] ' '1-2

[0700] IIF

[0701] or a pharmaceutically acceptable salt thereof, wherein L1, L2, X1, X2, T1, T2and R30are as described in classes and subclasses herein, both singly and in combination.

[0702] In some embodiments, a compound of formula I or IC is represented by formula IIF-1:

[0703] O

[0704] O — /

[0705] I 'T1

[0706] II T

[0707] N\^-(CH2)l-6— N'L2-0

[0708] (R3^2 / W2

[0709]

[0710] o

[0711] IIF-1

[0712] or a pharmaceutically acceptable salt thereof, wherein L1, L2, T1, T2and R30are as described in classes and subclasses herein, both singly and in combination.

[0713] In some embodiments, a compound of formula I or IC is represented by formula IIF-2:0'1

[0714] OL<CH2>l-«—^'L2- / 2

[0715]

[0716] 1-2

[0717] IIF-2

[0718] or a pharmaceutically acceptable salt thereof, wherein L1, L2, T1, T2and R30are as described in classes and subclasses herein, both singly and in combination.

[0719] As described herein with respect to any of formula I, IA, IB, IC, IIA, IIA-1, IIA-2, IIA-3, IIA-4, IIB, IIB-1, IIC, IID, IIE, IIF, IIF-1 and IIF-2, L1and L2are each independently selected from an optionally substituted C1-C30 aliphatic group. In some embodiments, L1is optionally substituted C1-C30 aliphatic. In some embodiments, L1is C1-C10 aliphatic. In some embodiments, L1is optionally substituted C1-C30 alkylene. In some embodiments, L1is optionally substituted C1-C30 alkenylene. In some embodiments, L1is C1-C10 alkylene. In some embodiments, L1is -(CH2)1-10-. In some embodiments, L1is -(CH2)6-. In some embodiments, L1is -(CH2)7-. In some embodiments, L1is -(CH2)s-. In some embodiments, L1is -(CH2)9-. In some embodiments, L2is optionally substituted C1-C30 aliphatic. In some embodiments, L2is C1-C10 aliphatic. In some embodiments, L2is optionally substituted C1-C30 alkylene. In some embodiments, L2is optionally substituted C1-C30 alkenylene. In some embodiments, L2is C1-C10 alkylene. In some embodiments, L2is -(CH2)1-10-. In some embodiments, L2is -(CH2)6-. In some embodiments, L2is -(CH2)7-. In some embodiments, L2is -(CH2)s-. In some embodiments, L2is -(CH2)9-. In some embodiments, L1and L2are each C1-C30 alkylene. In some embodiments, L1and L2are each -(CH2)6-12-. In some embodiments, L1and L2are each -(CH2)6-10-. In some embodiments, L1and L2are each -(CH2)6-. In some embodiments, L1and L2are each -(CH2)7-. In some embodiments, L1and L2are each -(CH2)s-. In some embodiments, L1and L2are each -(CH2)9-.

[0720] In some embodiments, L1and L2are the same. In some embodiments, L1and L2are different.

[0721] As described herein with respect to any of formula I, IA, IB, IIA, IIA-1, IIB, IIB- 1, and IIF, X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -S(O)2N(R1)-, -N(R1)S(O)2-, -S(O)-, -S(O)2-, -S(O)2C(R1)2-, -OC(S)C(R1)2-, -C(R1)2C(S)O-, -S-, -N(H)C(O)N(R1)-, -NHC(O)-, -C(O)N(R’)-, -NHS(O)2N(R’)-, and -C(O)-. In some embodiments, X1and X2are each independently selected from -OC(O)-, -C(O)O-, -S(O)2N(R1)-, -N(R1)S(O)2-, -S(O)-, -S(O)2-, -S(O)2C(R1)2-, -OC(S)C(R1)2-, -C(R1)2C(S)O-, -S-, -N(H)C(O)N(R1)-, -NHC(O)-, -C(O)N(R’)-, -NHS(O)2N(R1)-, and -C(O)-. In some embodiments, X1and X2are each independently selected from -OC(O)-, -C(O)O-, -S(O)2N(R’)-, -N(R1)S(O)2-, -S(O)-, -S(O)2-, -S(O)2C(R1)2-, -OC(S)C(R1)2-, -C(R1)2C(S)O-, -S-, -N(H)C(O)N(R1)-, -NHC(O)-, -C(O)N(R’)-, -NHS(O)2N(R1)-, and -C(O)-. In some embodiments, X1and X2are each independently selected from -OC(O)-, -C(O)O-, -S(O)2N(R1)-, -N(R1)S(O)2-, and -S(O)2-.

[0722] As described herein with respect to any of formula I, IA, IB, IIA, IIA-1, IIB, IIB- 1, and IIF, X1is selected from the group consisting of a bond, -OC(O)-, -C(O)O-, -S(O)2N(R1)-, -N(R1)S(O)2_, -S(O)-, -S(O)2-, -S(O)2C(R1)2-, -OC(S)C(R1)2-, -C(R1)2C(S)O-, -S-, -N(H)C(O)N(R1)-, -NHC(O)-, -C(O)N(R1)-, -NHS(O)2N(R1)-, and -C(O)-. In some embodiments, X1is -C(O)O-, -OC(O)-, -S(O)2N(R1)-, -N(R1)S(O)2, or -S(O)2-. In some embodiments, X1is -OC(O)-, or -C(O)O-. In some embodiments, X1is a bond. In some embodiments, X1is -OC(O)-. In some embodiments, X1is -C(O)O-. In some embodiments, X1is -S(O)2N(R’)-. In some embodiments, X1is -N(R’)S(O)2-. In some embodiments, X1is -S(O)-. In some embodiments, X1is -S(O)2-. In some embodiments, X1is -S(O)2C(R’)2-. In some embodiments, X1is -OC(S)C(R’)2-. In some embodiments, X1is -C(R1)2C(S)O-. In some embodiments, X1is -S-. In some embodiments, X1is -N(H)C(O)N(R1)-. In some embodiments, X1is -NHC(O)-. In some embodiments, X1is -C(O)N(R’)-. In some embodiments, X1is -NHS(O)2N(R1)-. In some embodiments, X1is -C(O)-. As described herein with respect to any of formula I, IA, IB, IIA, IIA-1, IIB, IIB-1, and IIF, X2is selected from the group consisting of a bond, -OC(O)-, -C(O)O-, -S(O)2N(R1)-, -N(R1)S(O)2., -S(O)-, -S(O)2-, -S(O)2C(R1)2-, -OC(S)C(R1)2-, -C(R1)2C(S)O-, -S-, -N(H)C(O)N(R1)-, -NHC(O)-, -C(O)N(R1)-, -NHS(O)2N(R1)-, and -C(O)-. In some embodiments, X2is -C(O)O-, OC(O)-, -S(O)2N(R1)-, -N(R1)S(O)2, or -S(O)2-. In some embodiments, X2is -OC(O)-, or -C(O)O-. In some embodiments, X2is a bond. In some embodiments, X2is -OC(O)-. In some embodiments, X2is -C(O)O-. In some embodiments, X2is -S(O)2N(R’)-. In some embodiments, X2is -N(R’)S(O)2-. In some embodiments, X2is -S(O)-. In some embodiments, X2is -S(O)2-. In some embodiments, X2is -S(O)2C(R’)2-. In some embodiments, X2is -OC(S)C(R’)2-. In some embodiments, X2is -C(R1)2C(S)O-. In some embodiments, X2is -S-. In some embodiments, X2is -N(H)C(O)N(R1)-. In some embodiments, X2is -NHC(O)-. In some embodiments, X2is -C(O)N(R’)-. In some embodiments, X2is -NHS(O)2N(R1)-. In some embodiments, X2is -C(O)-. As described herein with respect to any of formula IC, IIC, IID, and IIE, X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -N(H)C(O)N(R1)-, -NHC(O)-, and -C(O)N(R’)-. In some embodiments, X1and X2are each independently selected from -OC(O)-, -C(O)O-, -N(H)C(O)N(R1)-, -NHC(O)-, and -C(O)N(R’)-. In some embodiments, X1and X2are each independently selected from -OC(O)-, -C(O)O-, -NHC(O)-, and -C(O)N(R’)-. In some embodiments, X1and X2are each independently selected from -OC(O)- and -C(O)O-. In some embodiments, X1and X2are each independently selected from -NHC(O)-, and -C(O)N(R’)-. In some embodiments, X1and X2are each -N(H)C(O)N(R1)-.

[0723] As described herein with respect to any of formula IC, IIC, IID, and IIE, X1is selected from the group consisting of a bond, -OC(O)-, -C(O)O-, -N(H)C(O)N(R1)-, -NHC(O)-, and -C(O)N(R’)-. In some embodiments, X1is selected from the group consisting of -OC(O)-, -C(O)O-, -N(H)C(O)N(R1)-, -NHC(O)-, and -C(O)N(R’)-. In some embodiments, X1is selected from the group consisting of -OC(O)-, -C(O)O-, -NHC(O)-, and -C(O)N(R’)-. In some embodiments, X1is a bond. In some embodiments, X1is -OC(O)-. In some embodiments, X1is -C(O)O-. In some embodiments, X1is -N(H)C(O)N(R1)-. In some embodiments, X1is -NHC(O)-. In some embodiments, X1is -C(O)N(R’)-.

[0724] As described herein with respect to any of formula IC, IIC, IID, and IIE, X2is selected from the group consisting of a bond, -OC(O)-, -C(O)O-, -N(H)C(O)N(R1)-, -NHC(O)-, and -C(O)N(R’)-. In some embodiments, X2is selected from the group consisting of -OC(O)-, -C(O)O-, -N(H)C(O)N(R1)-, -NHC(O)-, and -C(O)N(R’)-. In some embodiments, X2is selected from the group consisting of -OC(O)-, -C(O)O-,-NHC(O)-, and -C(O)N(R’)-. In some embodiments, X2is a bond. In some embodiments, X2is -OC(O)-. In some embodiments, X2is -C(O)O-. In some embodiments, X2is -N(H)C(O)N(R1)-. In some embodiments, X2is -NHC(O)-. In some embodiments, X2is -C(O)N(R’)-.

[0725] In some embodiments, X1and X2are each -OC(O)-. In some embodiments, X1and X2are each -C(O)O-. In some embodiments, X1and X2are each -S(O)2-. In some embodiments, X1and X2are each -S-.

[0726] In some embodiments, X1and X2are each -S(O)2N(R’)-, where each R1is independently R1is Ci-Cio aliphatic. In some embodiments, X1and X2are each -S(O)2N(R’)-, where each R1is -(CH2)3-10-CH3. In some embodiments, X1and X2are each -S(O)2N(R’)-, where each R1is -(CH2)3-CH3. In some embodiments, X1and X2are each -S(O)2N(R’)-, where each R1is -(CH2)4-CH3. In some embodiments, X1and X2are each -S(O)2N(R’)-, where each R1is -(CH2)5-CH3. In some embodiments, X1and X2are each -S(O)2N(R’)-, where each R1is -(CH2)6-CH3In some embodiments, X1and X2are each -S(O)2N(R’)-, where each R1is -(CH2)7-CH3

[0727] In some embodiments, X1and X2are the same. In some embodiments, X1and X2are different.

[0728] As described herein with respect to any of formula I, IA, IB, IC, IIA, IIA-1, IIA-2, IIA-3, IIA-4, IIB, IIB-1, IIC, IID, IIE, IIF, IIF-1, and IIF-2, T1and T2are each independently an optionally substituted C3-C30 aliphatic. In some embodiments, T1is C3-C30 aliphatic. In some embodiments, T1is optionally substituted C3-C30 alkyl. In some embodiments, T1is optionally substituted C3-C20 alkyl. In some embodiments, T1is optionally substituted C5-C20 alkyl. In some embodiments, T1is optionally substituted straight-chained C3-C20 alkyl. In some embodiments, T1is optionally substituted branched C3-C20 alkyl. In some embodiments, T1is optionally substituted C3-C20 alkenyl. In some embodiments, T1is optionally substituted C10-C20 alkenyl. In some embodiments, T2is C3-C30 aliphatic. In some embodiments, T2is optionally substituted C3-C30 alkyl. In some embodiments, T2is optionally substituted C3-C20 alkyl. In some embodiments, T2is optionally substituted C5-C20 alkyl. In some embodiments, T2is optionally substituted straight chained C3-C20 alkyl. In some embodiments, T2is optionally substituted branched C3-C20 alkyl. In some embodiments, T2is optionally substituted C3-C20 alkenyl. In some embodiments, T1is optionally substituted C10-C20 alkenyl. In some embodiments, T1and T2are the same. In some embodiments, T1and T2are different. In some embodiments, T1and T2are each independently selected from:

[0729]

[0730]

[0731] In some embodiments, moiety -L’-X’-T1is selected from the group consisting of:

[0732] OT1 O | - (CHJO.,2— O — (CH2)6.12- S- N" — (CH2)6.12- S- T1O O R16 i — (CH2)6.12-S ozR1N-T1s i? k'-'1'2^6-12 \\ | (CH2)6-12—Q

[0733]

[0734] In some embodiments, moiety -L2-X2-T2is selected from the group consisting of:

[0735] s ° zT2. 2n- (CH2)6.12— O I — (CH2)6.12-S-N i — (CH2)6.12- S- T2; ■2

[0736] O R16? - (CH2)6.12-S-T2

[0737] R1I (CH2)6. I2: - (CH2)6.12-S-T

[0738]

[0739] In some embodiments, moiety -L'-X'-T1and moiety -L2-X2-T2are each independently selected from:

[0740]

[0741] o o

[0742] and

[0743]

[0744] As described herein with respect to any of formula I, IA, IB, or IC, L3is a bond or optionally substituted C1-C10 aliphatic group. In some embodiments, L3is a bond. In some embodiments L3is an optionally substituted C1-C10 aliphatic group. In some embodiments, L3is optionally substituted C1-C10 alkylene. In some embodiments, L3is optionally substituted C1-C10 alkenylene. In some embodiments, L3is optionally substituted alkynylene. In some embodiments, LC1-C6C1-C6alkylene. In some embodiments, L3is -(CH2)I-6-. In some embodiments, L3is -(CH2)2-4-. In some embodiments, L3is -(CH2)2-. In some embodiments, L3is -(CH2)3-. In some embodiments, L3is -(CH2)4-. In some embodiments, L3is -(CH2)5- In some embodiments, L3is -(CH2)6-. In some embodiments, L3is X=Z

[0745]

[0746] In some embodiments, L3is

[0747]

[0748] In some embodiments, L3is

[0749]

[0750] . In some embodiments,

[0751]

[0752] L3is

[0753] As described herein with respect to formula -, G is - N(R2)C(S)N(R2)2, -OH, -N(R2)2, -N+(R3)s, -N(R5)C(O)R3, -N(R5)S(O)2R3, -N(R5)C(O)N(R3)2, –CH(N–R2), -R4, -S(O)2R3, S(O)R3, or -SR3. In some embodiments, G is -N(R2)C(S)N(R2)2, -OH, -N(R2)2, -N(R5)C(O)R3, -N(R5)S(O)2R3, -N(R5)C(O)N(R3)2, –CH(N–R2), -R4, or -S(O)2R3.

[0754] In some embodiments, G is -N(R2)C(S)N(R2)2. In some embodiments, G is -N(H)C(S)N(R2)2. In some embodiments, G is -N(H)C(S)N(H)(R2) In some embodiments, G is -N(CH3)C(S)N(R2)2. In some embodiments, G is -N(OH)C(S)N(R2)2.

[0755] In some embodiments, G is -N(H)C(S)N(R2)2, where each R2is selected from optionally substituted C1-C6aliphatic and OH. In some embodiments, G is -N(H)C(S)N(OH)(R2), where R2is optionally substituted C1-C6aliphatic. In some embodiments, G is -N(H)C(S)N(R2)2, where each R2is selected from optionally substituted C1-C6aliphatic. In some embodiments, G is -N(H)C(S)N(R2)2, where each R2is methyl, ethyl, propyl, or butyl. In some embodiments, G is -N(H)C(S)N(CH3)2. In some embodiments, G is -N(H)C(S)N(CH3)(OCH3). In some embodiments, G is -N(H)C(S)N(CH3)(OH). In some embodiments, G is -N(OH)C(S)N(CH3)2.In some embodiments, G is -N(H)C(S)N(R2)2, where two instances of R2come together with the atoms to which they are attached to form an optionally substituted 4- to 12-membered heterocycle ring or an optionally substituted 4- to 12-membered heteroaryl ring comprising 1 to 4 heteroatoms selected from N, O, and S. In some embodiments, G is -N(H)C(S)N(R2)2, where two instances of R2come together with the atoms to which they are attached to form an optionally substituted 4- to 12-membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S. In some embodiments, G is -N(H)C(S)N(R2)2, where two instances of R2come together with the atoms to which they are attached to form an optionally substituted azetidine, pyrrolidine, piperidine, piperazine, or azepane ring. I- some embodiments, G is -N(R2)C(S)N(H)(R2), where two instances of R2come together with the -toms to which they are attached to form an optionally substituted 4- to 12-membered heterocycle ring comprising 1 to 4 heteroatoms selected from N, O, and S. In some embodiments G is:

[0756] S S

[0757]

[0758] or

[0759] In some embodiments, G is -OH.

[0760] In some embodiments, G is -N(R2)2. In some embodiments, G is -N(R2)2, wherein each R2is independently H or C1-C6optionally substituted C1-C6aliphatic. In some embodiments, G is -N(R2)2, wherein each R2is optionally substituted C1-C6aliphatic. In some embodiments, G is -N(R2)2, wherein each R2is independently methyl, ethyl, propyl, butyl, pentyl, or hexyl. In some embodiments, G is -N(CHs)2.

[0761] In some embodiments, G is -N+(R3)2. It is understood that when G is -N+(R3)2, G is paired with a suitable counterion, such as halogen (e.g., Cl’, F’, Br, or I) to provide a chemically stable compound at neutral or physiological pH. In some embodiments, G is -N+(CH3)3.

[0762] In some embodiments, G is -N(R5)C(O)R3. In some embodiments G is -N(H)C(O)R3. In some embodiments, G is -N(H)C(O)-C1-C6aliphatic. In some embodiments, G is -N(H)C(O)-CH3.

[0763] In some embodiments, G is -N(R5)S(O)2R3. In some embodiments, G is -N(H)S(O)2R3. In some embodiments, G is -N(H)S(O)2-C1-C6aliphatic. In some embodiments, G is -N(H)S(O)2-CH3.

[0764] In some embodiments, G is -N(R5)C(O)N(R3)2. In some embodiments, G is -N(H)C(O)N(R3)2. In some embodiments, G is -N(H)C(O)N(H)(R3). G is -N(H)C(O)N(H)(CH3). G is -N(H)C(O)N(CH3)2.

[0765] In some embodiments, G is –CH(N–R2). In some embodiments, G is -CH(N-Ci-Ce aliphatic). In some N''

[0766] |

[0767] embodiments, G is -CH(N-CH3). In some embodiments. G is

[0768] In some embodiments, G is R4, and R4is optionally substituted 4- to 12-membered heterocycle. In some embodiments, G is optionally substituted 4- to 6-membered monocyclic heterocycle. In some embodiments, G is optionally substituted azetidine, pyrrolidine, piperidine, piperazine, or azepane. In some embodiments, G is 6- to 12-membered bicyclic heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S. In some embodiments, G is 4- to 12-membered heterocycle comprising 1 to 4 heteroatomsselected from N, O, and S, and optionally substituted with -(CH2)0-4N(C1-C6) or -(CH2)1-4OR°. and where R° is hydrogen or Ci-Ce aliphatic.

[0769] In some embodiments, G is R4and R4is optionally substituted 5- to 12-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S. In some embodiments, G is optionally substituted 5- to 6-membered monocyclic heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, G is optionally substituted pyrrole, imidazole, pyrazole, thiazole, oxazole, or furan. In some embodiments, G is pyrrole, imidazole, pyrazole, thiazole, oxazole, or furan optionally substituted with -(CH2)0-4OR°. In some embodiments, G is pyrrole, imidazole, pyrazole, thiazole, oxazole, or furan optionally substituted with -(CH2)0-4OR°. and where R° is hydrogen. In some embodiments, G is pyrrole, imidazole, pyrazole, thiazole, oxazole, or furan optionally substituted with -(CH2)0-4C(O)N(R°)2. In some embodiments, G is pyrrole, imidazole, pyrazole, thiazole, oxazole, or furan optionally substituted with -(CH2)0-4C(O)N(R°)2, where each R° is hydrogen. In some embodiments, G is 6- to 12-membered bicyclic heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S. In some embodiments, G is 5- to 12-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S, and optionally substituted with - (CCI-C64OR° or -(CH2)0-4SR°. and where R° is hydrogen or Ci-Ce aliphatic. In some embodiments, G is

[0770]

[0771] In some embodiments, G is R4, where R4is optionally substituted Ce-Cn aryl. In some embodiments, G is R4, and R4is Ce-Cn aryl optionally substituted with -(CH2)0-4OR°. In some embodiments, G is R4, and R4

[0772] OH

[0773]

[0774] In some embodiments, G is R4, where R4is optionally substituted C3-C12. In some embodiments, G is C3-Ce cycloaliphatic substituted with one or more of oxo, -(CH2)o 4-OR0, or -(CH2)o-4-N(R°)2. In some embodiments, G is cyclobutyl, cyclopentyl, or cyclohexyl optionally substituted with one or more of oxo, -OH, or-N(R°)2.

[0775] In some embodiments, G is -S(O)2R3. In some embodiments, G is -S(O)2R3, where R3is optionally substituted Ci-Ce aliphatic. In some embodiments, G is -S(O)2R3, where R3is C1-C10 aliphatic optionallysubstituted with -OR°, where R° is H. In some embodiments, G is -S(O)2R3, where R3is -(CH2)i-e-OH. In 0

[0776] 11 X / S. / OH V11some embodiments, G is -S(O)2R3, where R3is -CH3. In some embodiments, G is 0

[0777] 0

[0778] II

[0779]

[0780] In some embodiments, G is -S(O)R3. In some embodiments, G is -S(O)R3, where R3is optionally substituted C1-C6aliphatic. In some embodiments, G is -S(O)R3, where R3is C1-C10 aliphatic optionally substituted with -OH. In some embodiments, G is -S(O)R3, where R3is -(CH2)i-e-OH. In some embodiments, G is -

[0781] S(O)R3, where R3is -CH3 or -CH2-CH3 In some embodiments, G

[0782]

[0783] is

[0784]

[0785] In some embodiments, G is -SR3. In some embodiments, G is -SR3, where R3is optionally substituted Ci-Ce aliphatic. In some embodiments, G is -SR3, where R3is C1-C10 aliphatic optionally substituted with -OH. In some embodiments, G is -SR3, where R3is -(CH2)i-e-OH. In some embodiments, G is -SR3, where

[0786] R3is -CH3In some embodiments, G is

[0787]

[0788] O O O O O

[0789]

[0790] 15 In some embodiments, G is selected from:

[0791]

[0792] In some embodiments, a moiety -L3-G is selected from:

[0793]

[0794] As described herein with respect to formula IA, G1is -S(O)R3or -SR3. In some embodiments, G1is -S(O)RC1-C6n some embodiments, G1is -S(O)R3, where R3is optionally substituted Ci-Ce aliphatic. In some embodiments, G1is -S(O)R3, where R3is C1-C10 aliphatic optionally substituted with -OR°, where R° is H. In some embodiments, G1is -S(O)R3, where R3is -(CH2)i-e-OH. In some embodiments, G1is -

[0795] S(O)R3, where R3is -CH3 or -CH2-CH3 In some embodiments, G

[0796]

[0797] 1is

[0798]

[0799]

[0800] In some embodiments, GC1-C6-SR3. In some embodiments, G1is -SR3, where R3isoptionally substituted Ci-Ce aliphatic. In some embodiments, G1is -SR3, where R3is C1-C10 aliphatic optionally substituted with -OH. In some embodiments, G1is -SR3, where R3is -(CH2)i-e-0H. In some

[0801] embodiments, G1is -SR3, where R3is -CH3. In some embodiments, G1is

[0802]

[0803]

[0804] In some embodiments, a moiety -L3-G’ is selected from:

[0805]

[0806]

[0807] As described herein with respect to formula IB, G2is -S(O)2R3a. In some embodiments, G2is -S(O)2R3a, where R3ais optionally substituted Ci-Ce aliphatic. In some embodiments, G2is -S(O)2R3a, where R3ais optionally substituted C2-C6 aliphatic. In some embodiments, G2is -S(O)2R3a, where R3ais -CH3. In some embodiments, G2is -S(O)2R3a, where R3ais -(CH2)0-6R3bIn some embodiments, G2is -S(O)2R3a, where R3ais -(CH2)i-6-R3b. In some embodiments, G2is -S(O)2R3a, where R3ais -(CH2)1-6R3b, where R3bis -OH.

[0808] O O O o II II II II

[0809] 0 II OH

[0810]

[0811] In some embodiments, a moiety -L3-G2is selected from:

[0812]

[0813] O and As described herein with respect to formula IB, R3ais -(CH2)0-6-R3bC1-C6C1-C6aliphatic. In some embodiments, R3ais -(CH2)0-6-R3b. In some embodiments, R3ais Ci-Ce aliphatic. In some embodiments, R3ais -CH2-R3b. In some embodiments, R3ais -(CH2)2-R3b. In some embodiments, R3ais -(CH2)3-R3b. In some embodiments, R3ais -(CH2)4-R3b. In some embodiments, R3ais -(CH2)5-R3b. In some embodiments, R3ais -(CH2)6-R3b. In some embodiments, R3ais methyl, ethyl, propyl, butyl, pentyl, or hexyl. In some embodiments, R3ais -CH3.

[0814] As described herein with respect to formula IB, R3bis -OH, -NH(CH3), or -N(CH3)2. In some embodiments, R3bis -OH. In some embodiments, R3bis -NH(CH3). In some embodiments, R3bis -N(CH3)2.

[0815] As described herein with respect to formula IC, G3is selected from -OH, -NH2, -NH(CH3), -N(CHs)2,

[0816]

[0817] In some embodiments, G3is selected from

[0818] -OH, -NH2, -NH(CH3), -N(CH3)2.

[0819]

[0820] As described herein with respect to any of formula IC, IIF, IIF-1 or IIF-2, each R30is independently selected from -SH,-OH, -NH2, -NH(CHs), -N(CHs)2, -C(0)NH2, and optionally substituted Ci-Ce aliphatic. In some embodiments, R30is -C(0)NH2 In some embodiments, R30is optionally substituted Ci-Ce aliphatic. In some embodiments, R30is Ci-Ce aliphatic optionally substituted with optionally substituted with -(CH2)o 4OR0. In some embodiments, R30is Ci-Ce aliphatic optionally substituted with optionally substituted with -(CH2)0-4OR°. where R° is hydrogen. In some embodiments, R30is -CH2-OH or -CH2-CH2-OH. In some embodiments, R30is -CH2-OH. In some embodiments, R30is -CH2-CH2-OH.

[0821] As described herein with respect to formula I, each R2is, independently, at each instance, selected from the group consisting of H, optionally substituted Ci-Ce aliphatic and OR3. In some embodiments, R2is H. In some embodiments, R2is optionally substituted Ci-Ce aliphatic. In some embodiments, R2is methyl, ethyl, propyl, butyl, pentyl, or hexyl. In some embodiments, R2is -CH3.

[0822] As described herein with respect to formula I, each R3is, independently, at each instance, selected from the group consisting of H and optionally substituted C1-C10 aliphatic. In some embodiments, R3is H. In some embodiments, R3is optionally substituted C1-C10 aliphatic. In some embodiments, R3is C1-C10 aliphatic optionally substituted with one or more instances of -(CH2)0-4R°, -(CH2)0-4OR°, and -(CH2)0-4N(R°)2. In some embodiments, R3is C1-C10 aliphatic optionally substituted with -OR°, or -N(R°)2. In some embodiments, R3is C1-C10 aliphatic optionally substituted -OH, -NH, -NH(CH3), or -N(CH3)2.

[0823] As described herein with respect to any of formula IA, IIB, and IIB-1, R3is optionally substituted C1-C10 aliphatic. In some embodiments, R3is C1-C10 aliphatic optionally substituted with one or more instances of -(CH2)0-4R°, -(CH2)0-4OR°, and -(CH2)0-4N(R°)2. In some embodiments, R3is C1-C10 aliphatic optionally substituted with -OR°, or -N(R°)2. In some embodiments, R3is C1-C10 aliphatic optionally substituted -OH, -NH, -NH(CH3), or -N(CH3)2.

[0824] As described herein with respect to formula I, R4is optionally substituted 4- to 12-membered heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S, optionally substituted 4- to 12 membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S, optionally substituted Ce-Cn aryl, or optionally substituted C3-C12 cycloaliphatic. In some embodiments, R4is optionally substituted 4- to 12-membered heterocycle comprising 1 to 4 heteroatoms selected from N, O, and S. In some embodiments, R4is optionally substituted 4- to 12 membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O, and S. In some embodiments, R4is optionally substituted Ce-Cn aryl. In some embodiments, R4is optionally substituted C3-C12 cycloaliphatic.

[0825] As described herein with respect to formula I, each R5is independently selected from H and optionally substituted Ci-Ce aliphatic.

[0826] In some embodiments, a compound of formula I is a compound selected from Table 1, or a pharmaceutically acceptable salt thereof:

[0827] Table 1

[0828] Compound

[0829] Structure

[0830] No.

[0831] 9 11 011

[0832] 1 j o

[0833] BNT-47

[0834] BNT-91

[0835] O

[0836] 0 0

[0837] II11

[0838] J j o

[0839] BNT-103

[0840] 0 II

[0841] Oz xo 10

[0842] BNT-106

[0843] 0 0

[0844] II11

[0845] J j0

[0846] BNT-110

[0847] 0\

[0848]

[0849] Compound

[0850] Structure

[0851] No.

[0852] BNT-114 X

[0853] o

[0854] K c /

[0855] 7 7 ° / \ \x

[0856] ° 7 ' ( o \ / —

[0857] \ \ 7 ( \' o o — —

[0858] BNT-119 77 ° 777 ° \ ex \s \ (s. ' —

[0859] / \' o

[0860] X>'>)z° ° ( ot < Z (>

[0861] I u; ^z

[0862] BNT-121 °77 \= 7 \ ° —

[0863] ^z

[0864] OOw==

[0865] ( O ^z° tn co

[0866] °z b' ^ / Us> O b x' Q- BNT-129

[0867] O o O X I X

[0868] BNT-130

[0869] BNT-131

[0870]

[0871] Compound

[0872] Structure

[0873] No.

[0874] ZE O BNT-132

[0875] ZOT

[0876] ^ °Z

[0877] ? ° \° / \ / /

[0878] ° \ /

[0879] ° / \ Z * —

[0880] o / = / \

[0881] \° 2= o

[0882] BNT-133 \ o

[0883] \ °

[0884] 0 / ( °= Xz

[0885] O / \

[0886] J O / \ / = j 0 0 BNT-134

[0887] 01 \oz

[0888] CW

[0889] 0 F

[0890] o o I T0BNT-135

[0891] 0\

[0892] BNT-136

[0893]

[0894] Compound

[0895] Structure

[0896] No.

[0897] T BNT-138 o

[0898] ^0 < ^o

[0899] 0 ° / \ /

[0900] VC ( c° ' <zn — z.^

[0901] °o \ /

[0902] IW / s

[0903] / / xo j o'xo

[0904] w° \ / \

[0905] BNT-139 O /

[0906] 0\

[0907] ^ ^ ^zzz

[0908] BL-234

[0909] ^ O Oxx..

[0910] ^ ^cc <oo O \ / /

[0911] i C / \

[0912] 7 °

[0913] o O z T BL-235 o I

[0914] BL-236

[0915] / ^OH o' 'o J 6Z 0

[0916] BL-237

[0917] _

[0918] o'xo

[0919]

[0920] Compound

[0921] Structure

[0922] No.

[0923] X BL-238 O T o o o o o T T T T

[0924] ) ° x?p < I W / *

[0925] K C /

[0926] BL-239

[0927] ^ z^^ Z v z z— z \— z—

[0928] BL-240 ) \ZO

[0929] \C zOT

[0930] \ \ z° ) / \°^z s

[0931] \ / >

[0932] O / x )X ) ) /

[0933] / \ ° ° \ Z

[0934] x / OT°

[0935] 2 \ z \ z

[0936] w i —WWT

[0937] >^x)>^x) O O ° °

[0938] \ / ZZ

[0939] \Q <7\ > ° °- '

[0940] BL-241

[0941] -

[0942] BL-242

[0943] BL-243

[0944] os zo

[0945] \

[0946] o'xo 1

[0947] BL-244

[0948] o' 'o

[0949]

[0950]

[0951] Compound

[0952] Structure

[0953] No.

[0954] BL-252

[0955] X T X o O o O o ^zo ^ ^ ^° / wzz°ozo

[0956] '' ^ oxCW OT'

[0957] BL-253

[0958] . \^— z z \—

[0959] \ \' o z^ Z

[0960] BL-254

[0961] / \ ^°

[0962] / r\°~x

[0963] / (z° \

[0964] / / w^z

[0965] / x / / \ ° / w°

[0966] \ z \> -OTW

[0967] ¥\s / x / ° ° °~ '

[0968] \ / ZW

[0969] BL-255 \ / \ 7z^ ° 'zri

[0970] r c 7

[0971] o i BL-256

[0972] 0 ° J o“o

[0973] BL-257

[0974] BL-258

[0975] o' "o 1 o'xo

[0976] BL-259

[0977] z's^^

[0978] o'xo

[0979]

[0980] Compound

[0981] Structure

[0982] No.

[0983] X X

[0984] o BL-260 X I 0 0 o ^$ o znt ^ / °

[0985] ^ZO

[0986] BL-261; w ^ o'

[0987] / 0

[0988] VC°zOx / \ Z —

[0989] / / sW° \ z

[0990] \ \z°O

[0991] z \—

[0992] BL-262

[0993] / \ z°

[0994] \ zCOT

[0995] / / wO X

[0996] \ / \z / \ / ° ° ' Z

[0997] / \xzwOT /

[0998] \ z50—

[0999] x / O °

[1000] BL-263

[1001] )0—

[1002] h p' "

[1003] V3° \ z

[1004] zZ <> $0yo 0 X BL-264 0

[1005] X

[1006] BL-265

[1007] BL-266

[1008]

[1009] Compound

[1010] Structure

[1011] . o I I No o T I O

[1012] oz3 w

[1013] o ° \6

[1014] zz<

[1015] \

[1016] BL-267 o \z

[1017] / z >.^ z.— °

[1018] z \—

[1019] z—

[1020] \ / wz

[1021] BL-268 / \ \' o

[1022] X' Q \-z—x- / \ / O

[1023] \ '^ / 7

[1024] / \' o

[1025] \ \ / / °° '

[1026] \ / \Z / x / O °

[1027] / \) C / / / O

[1028] \ \' / O \' "

[1029] w

[1030] / Q / \>\' / ■ ' O^ °Z

[1031] ^z

[1032] BL-269

[1033] \1 \ °'

[1034] ° \ z

[1035] zcn

[1036] BL-270

[1037] F

[1038] o i BL-271

[1039] °v°

[1040]

[1041] Compound

[1042] Structure o o I X X o No.

[1043] BL-273x / z o

[1044] zOw

[1045] ° ° \ \Cz'

[1046] 2O

[1047] 77 C \k

[1048] ' — ^ z. z \—

[1049] (wz

[1050] BL-274 \ \' o

[1051] / / Ck

[1052] W\ z

[1053] \ \' o

[1054] ) \ z°

[1055] \ zCW

[1056] ) r\°~

[1057] \ / (z°x

[1058] 577 \ / s 7 °

[1059] a \ z*

[1060] BL-275 >^s) O °Zij

[1061] ^z

[1062] r7

[1063] ° \ z

[1064] \o,

[1065] ( ' / °Z (>

[1066] BL-276

[1067] o o X X BL-277

[1068] oxS zo oxX

[1069] BL-278 Os zO I

[1070]

[1071] I X Compound o X o o I Structure O No.

[1072] zw / xvx / o

[1073] o <

[1074] BL-279ZOT

[1075] o \ ° \ o \ ' 'z

[1076] / °

[1077] z^ z. z—

[1078] —

[1079] BL-280

[1080] / \ / / \ / °°

[1081] \ \

[1082] \ / \ \z° / / r\°°~x s

[1083] \ / \z> (Czo co°

[1084] \ \z° / \ / \ / / Z) <oz'

[1085] BL-281 \

[1086] / ° / \ "

[1087] 0;'

[1088] OxX zp (Dx <0

[1089] BL-282 Ox ZO I

[1090] BL-283

[1091] oz so 1 oz xo

[1092] BL-284

[1093] z'sC^^

[1094] oz0

[1095]

[1096] Compound

[1097] Structure

[1098] No.

[1099] I

[1100] o

[1101] BL-285

[1102] Z

[1103] ° \ 'OT

[1104] °°= / \ / / / \

[1105] ° ° f 7 ' « / \ / / O \ \ —K.

[1106] ' (w —z^ z

[1107] BL-286 \ \z° / ° O \ / Z (> \ / / \ / O O 2 oK

[1108] M\ z

[1109] \ \z°

[1110] / \ZO

[1111] \C zW

[1112] z / r\°~

[1113] \ / \os

[1114] / \ wz^ ^zz

[1115] ^z

[1116] BL-287

[1117] O / x

[1118] 0\zo 0° \ / xO

[1119] >z° \o,

[1120] c°' O o

[1121] o i

[1122] BL-288 OK c° I

[1123] ZO [ ° \ O /

[1124] C° IK c° o I BL-289

[1125] BL-290

[1126]

[1127] Compound

[1128] Structure

[1129] No.

[1130] I

[1131] o p C BL-291 \ C /

[1132] ^ o' BL-292 ° / \ /

[1133] ° co ( ' \ / W —

[1134] / ° co \ /

[1135] \ o

[1136] Oxx / O

[1137] *'

[1138] Ox ZO [

[1139] BL-293

[1140] )K \ C / —

[1141] / O / \ " \ z —

[1142] ii

[1143] \ ^ O /

[1144] ;s; hr ^zOCs w / / ; \ OH oz'o 1 ozCsz <>o

[1145] c°

[1146] BL-294 c° o

[1147] Oc Io I. >z° o I

[1148] o' 'o

[1149] BL-295

[1150] BL-296

[1151]

[1152] Compound

[1153] Structure

[1154] No.

[1155] o I T I I BL-297 o o OSZ OTo cczC C pCoo °W

[1156] 77 ° \ CK / / \ ^

[1157] zy z

[1158] —Z.— - ( 'M— Z^.

[1159] ° z

[1160] \ \z / \ O

[1161] / / CK

[1162] BL-298W\ z

[1163] ° \ \' / \ o o 2

[1164] 7 \ z°

[1165] \ zCW

[1166] / °s

[1167] \ / \' 7 7w OT

[1168] / \ / x 7 °O

[1169] \ 22 w w k — \ zW

[1170] —

[1171] j o ^ ' z —z

[1172] BL-299

[1173] ^ °x

[1174] XZ() / C / 1>z°! c° o O I z BL-300

[1175] BL-301

[1176] BL-302

[1177]

[1178] Compound

[1179] Structure

[1180] No.

[1181] osS zp

[1182] ^^1 J °'X° BL-303 Os ZO I

[1183] I

[1184] o p C w \ z—

[1185] BL-304

[1186] /

[1187] OW=

[1188] / \ z° Z V—

[1189] \ X

[1190] / r\°x

[1191] \ / \z / o °Z

[1192] \? / \o t°='

[1193] BNT-45 ( o \z

[1194] ? / \o=

[1195] a i •

[1196] 02

[1197] BNT-48 o r

[1198] 0

[1199] J J II BNT-101 / xP0J0

[1200] XXXXX^J^O^ —z>—

[1201] 0 II J 0' '0 1

[1202] BNT-102

[1203] o o II11BNT-109

[1204] 0\

[1205]

[1206] Compound

[1207] Structure

[1208] No.

[1209] 0 ii

[1210] BL-231

[1211] o \>= _ J 00J II BL-227

[1212] 1 0 o

[1213] BL-228

[1214] h.

[1215] _ I J II Z^x / S 0 BL-229

[1216] hh

[1217] 0' '0 J o

[1218] BL-230

[1219] X^Z'J / O'ON,'s; J

[1220] 0 0

[1221] o II II 1

[1222] BL-232 o >

[1223] II

[1224] o

[1225] 0 9 II 1

[1226] BL-2330

[1227] _ o II J 1

[1228] II

[1229] °

[1230]

[1231] Compound

[1232] Structure

[1233] No.

[1234] 0

[1235] BNT-108 0 <

[1236] >wIZ

[1237] Z 1 —

[1238] BNT-140

[1239] 7 o

[1240] 7 / \ ex

[1241] ( 'M- z

[1242] >ZJ ° o

[1243] (a>z

[1244] \ \' o

[1245] \ \ z°

[1246] > C w

[1247] / / O "

[1248] BNT-141 \ \zo ^z

[1249] )o v c'

[1250] 7o 7 \ '

[1251] ' z —

[1252] z —

[1253] /

[1254] ° r°H

[1255] ZI BL-194 o <

[1256] z — /

[1257] BL-222

[1258] Os ZO

[1259] BL-223

[1260] COOON

[1261] 6 o

[1262]

[1263] Compound

[1264] Structure

[1265] No.

[1266] Ox ZO

[1267] J BL-305 J1

[1268] d o

[1269] Oxzo

[1270] N^^N- BL-306

[1271] 0003 o' 0

[1272] 0

[1273] BNT-111 o <

[1274] o

[1275] N NH BL-224 0

[1276] N

[1277] 0

[1278] BL-226 0 r^

[1279] N

[1280] 0

[1281] N NH

[1282] 1Hj

[1283] BL-310 0 <

[1284] N

[1285] 1 H

[1286]

[1287] Compound

[1288] Structure

[1289] No.

[1290] BL-311

[1291] X

[1292] o < 2o / =0 22= \

[1293] ° ° \ / / \ \ Z —

[1294] / ° 2=

[1295] BNT-93 20= \

[1296] \ 2 o o

[1297] / zx

[1298] \(O=^z

[1299] 2 ZX < - BNT-94 o / =

[1300] o I o SH X

[1301] 0 HN--4 II N BNT-97

[1302] 0\

[1303] 8

[1304] 107 V 4>__SHL-N BNT-98

[1305] ° 1

[1306]

[1307] Compound

[1308] Structure

[1309] No.

[1310] o p=Non BNT-104

[1311] ° / \ /

[1312] 0 ° \ / N=N II L NH

[1313] ° / \

[1314] o /

[1315] < HC) BNT-105

[1316] 0\

[1317] ^z

[1318] HC>0\ I _

[1319] / ^D <x%o —^ BNT-107

[1320] 0 / N^

[1321] N^\ < NH 00J ) BNT-116

[1322] BNT-117

[1323]

[1324] Compound

[1325] Structure

[1326] No.

[1327] N=\ L NH

[1328] 1 °0j \

[1329] < HO BNT-122

[1330] o \o / =

[1331] ° / \

[1332] ° \ /

[1333] BL-312 o /

[1334] 0 /

[1335] OH

[1336] \ z —

[1337] A f / BL-313

[1338] / O — \0[ V i\= X^ / XXy^Q / XXX /

[1339] Zi^

[1340] BL-314

[1341] 0

[1342] A A~NH BL-315

[1343] 0 < ^OH

[1344] O N=\ II L NH

[1345] / XZ S ^OH BL-316

[1346] 01

[1347]

[1348] Compound

[1349] Structure

[1350] No.

[1351] O N=\

[1352] II T NH

[1353] J j V^ / OH

[1354] BL-317

[1355] o 1

[1356]

[1357] In some embodiments, a compound of formula IA is a compound selected from Table 1A, or a pharmaceutically acceptable salt thereof:

[1358] /

[1359] Table 1A Ow=

[1360] Compound Z v—

[1361] Structure z

[1362] No.

[1363] / ° o cn=

[1364] \?°=

[1365] ) ( o

[1366] BNT-45 / \o=

[1367] BNT-48

[1368] 0

[1369] ^Y o N S

[1370] BNT-101 o f °

[1371] 0

[1372] II

[1373] J o' 'o 1

[1374] BNT-102

[1375] 0 0

[1376] II11

[1377] BNT-109

[1378] ° [

[1379]

[1380] Compound

[1381] Structure

[1382] No.

[1383] 0 ii

[1384] BL-231

[1385] o) \= _ J d' O 1 || Z\X x^ 0 BL-227

[1386] / x^x / x^Xg / x / x / xz i Z —

[1387] 1 0 o

[1388] BL-228

[1389] > O W=

[1390] xzXx-~XX'\x^sx-X^X'^^

[1391] _ I J II BL-229

[1392] x'X'X^^

[1393] ph

[1394] x / CCO^s^^

[1395] O' '0 J o

[1396] BL-230

[1397] J C^ O _ J

[1398] ,'s;

[1399] 0 0

[1400] o II II 1

[1401] BL-232 o >

[1402] II

[1403] o

[1404] 0 9

[1405] 1

[1406] BL-233 0 x^

[1407] _ o II J 1

[1408] II

[1409] 0

[1410]

[1411] Compound

[1412] Structure

[1413] No.

[1414] X BNT-108 o

[1415] «)

[1416]

[1417] In some embodiments, a compound of formula IB is a compound selected from Table 1B, or a pharmaceutically acceptable salt thereof:

[1418] / °X

[1419] ( \

[1420] ) \ o —O=

[1421] \ / o / =

[1422]

[1423] Compound

[1424] Structure

[1425] No.

[1426] 0 0

[1427] II11

[1428] J J0

[1429] X BNT-110 o

[1430] 01

[1431] K c /

[1432] 7 ° / \x

[1433] ° 7 ) < )w\ /

[1434] \ \ 7 ( \' o o — —

[1435] 77 ° 777 ° \ ex \x \s(s.

[1436] BNT-114 'wsOT< — 'o / — z

[1437] / \' o

[1438] V X)7>)z° °

[1439] < Z (>

[1440] \ \\z6\ oZ

[1441] u ^z

[1442] °77 \= 7 \ ° —

[1443] ^z

[1444] BNT-119

[1445] OOw== ^ °z ^ '° cn

[1446] °z

[1447] ' / / Us> O? ocn b x' Q- BNT-121 o O I X

[1448] BNT-129

[1449] BNT-130

[1450]

[1451] Compound

[1452] Structure

[1453] No.

[1454] ZE

[1455] o BNT-131

[1456] ZOT

[1457] ^ °Z

[1458] °° / / \ / / \

[1459] < ' ° o — \ / Z * —

[1460] BNT-132

[1461] ° \ /

[1462] O /

[1463] \ °

[1464] / ( °=

[1465] 0

[1466] O / \

[1467] BNT-133 ° / \ / =

[1468] ^ ° /

[1469] zw

[1470] < O 0zcn

[1471] J j F 0 0 o I

[1472] o BNT-134 I01

[1473] 0

[1474] / xJ0BNT-135

[1475] 0\

[1476]

[1477] Compound

[1478] Structure

[1479] No.

[1480] X

[1481] o BNT-136

[1482] ° / \ /

[1483] ° / = \ / ^Z.— ■

[1484] y n ° / \

[1485] O / \

[1486] / 2°=

[1487] < °5 \W

[1488] Oo

[1489] BNT-138 \ \ \z° /

[1490] \ o

[1491] /

[1492] 0OT

[1493] z

[1494] \ / OT - ^z

[1495] ^Z

[1496] I j O' 0 BNT-139

[1497] 0\ < O \ / /

[1498] cnso

[1499] X

[1500] o o I X BL-234

[1501] BL-235

[1502] OsxZO

[1503] BL-236 > °

[1504] Os ZZ

[1505] O I

[1506]

[1507] Compound

[1508] Structure

[1509] No.

[1510] ^OH I o' "0 \ o'0I I o o o BL-237

[1511] s$ ° izocp \ xZ> < ° o

[1512] o' 'o

[1513] \ z—

[1514] ^^ z z

[1515] BL-238

[1516] y 'W\ z

[1517] \ \' o

[1518] BL-239

[1519] / x / / ° / OT w

[1520] 2n v < — \ 'OT^Z

[1521] >^j o °Z

[1522] ) \ / OT

[1523] OxxZO

[1524] Q < > °Z

[1525] ^cz>

[1526] J 6' °

[1527] BL-240 Oxzo [

[1528] 7o I

[1529] \ / OONX;s; i\r

[1530] o' 'b I 6'0OH BL-241

[1531] zsC^^

[1532] o'so

[1533] BL-242

[1534] BL-243

[1535]

[1536] Compound

[1537] Structure

[1538] No.

[1539] ox ZO o'xo 1 I - 4 o o T BL 2 4

[1540] )wx^x^ ° ° o'xo

[1541] \^ °

[1542] BL- ^ ZOz

[1543] 245

[1544] Ox / O

[1545] / x / / °OT

[1546] BL-246

[1547] x ) O °Z

[1548] h "

[1549] M BL-247

[1550] Os ZO o' 'o 1

[1551] BL-248

[1552] J

[1553] o' 'o

[1554] J J o'XO BL-249

[1555] 00J o' o BL-250

[1556]

[1557] Compound

[1558] Structure

[1559] No.

[1560] °v°

[1561] J Oz xo I BL-251 Ox / O [ o o I I o ^ / ° ^ / (° w ^ ^o w / °'' ^ oC

[1562] °'W'

[1563] ^

[1564] BL-252V('°ZOz

[1565] tw /

[1566] / / x°

[1567] z \—

[1568] \ \' o z—

[1569] BL-253

[1570] ^z

[1571] / w''Z—

[1572] / / x / w°

[1573] \ <-W

[1574] ^\x / °~

[1575] BL-254

[1576] A °s

[1577] 7 ’• ^

[1578] w /

[1579] iW

[1580] ' O ^ °

[1581] o \ NT I;s;,s; — O O IH o' 'o 1 O' 'o

[1582] BL-255

[1583] j

[1584] o' 'o

[1585] BL-256

[1586] 6' o I 6z'b

[1587] BL-257

[1588] BL-258

[1589]

[1590] Compound

[1591] Structure

[1592] No.

[1593] X X

[1594] o o o'xo 1 o' 'o

[1595] BL-259 $

[1596] w ^ / °

[1597] o' "oz^ o'

[1598] BL-260

[1599] / w z < —

[1600] z—

[1601] \ \' o

[1602] BL-261

[1603] /

[1604] / x / °OT

[1605] z' z —

[1606] \ zOT

[1607] f\x / o- °~

[1608] ^ ox

[1609] BL-262w✓

[1610] ^zo

[1611] h ”

[1612] ip:

[1613] Vs-Szo; ''N ^sf $ ° 0 'o 1 o'xo o X BL-263

[1614] \ ^xXXXXxN _ J

[1615] o'xo

[1616] BL-264

[1617] BL-265

[1618]

[1619] Compound

[1620] Structure

[1621] No.

[1622] C\x ZO < DX <0I T o o BL-266 Ox ZO \ 6 <

[1623] Z

[1624] o \x^ OW

[1625] '

[1626] w '

[1627] 7 / \ Ox. / °

[1628] ( '7*3— z Z^—

[1629] BL-267 \ \zo

[1630] / / Ox

[1631] 7W\

[1632] \ \zo

[1633] / \ / O

[1634] \x z«

[1635] / r\° '

[1636] \ / (z°

[1637] / X / \ OTOT

[1638] BL-268

[1639] ;r

[1640] ° \ /

[1641] ( cn

[1642] BL-269

[1643] x^°

[1644] o I

[1645] 0*zp 0xX

[1646] BL-270 0x z0 I

[1647] °< o'so 1

[1648] BL-271

[1649] _ J

[1650] o'xo

[1651]

[1652] Compound

[1653] Stru tur 0 O X X c e No.

[1654] x Z9^

[1655] / oOT

[1656] BL-272 ° \ zntz

[1657] 20

[1658] 77 C \k

[1659] 7 ( ' ' co — — ^ z.

[1660] BL-273 7 ■ \ \zo

[1661] / / Ck

[1662] 7 cWo \ z

[1663] \ \' o

[1664] / \ z°

[1665] \X ZW

[1666] 0*z0 OxXQ

[1667] / r\°~x

[1668] \ / (z°

[1669] 7 \

[1670] BL-274 a (\zu0 [

[1671] ^ ^zz

[1672] r7 Hi

[1673] ° \ z

[1674] '° co \o \ z z

[1675] ' / '° CO

[1676] BL-275

[1677] 0 0 0 X I X BL-276

[1678] BL-277

[1679]

[1680] X Co p u d X o m o n

[1681] S ruc ure o X t t o No.

[1682] J BL-278

[1683] z zwzn Vcv <

[1684] o \ ° \ ow

[1685] 'z

[1686] j?J 7 / \ ex

[1687] Mz \—

[1688] ( ' - z \ z z^.—

[1689] \zO >

[1690] BL-279 / / Os

[1691] <toz

[1692] \ \' o

[1693] / \ / z \ z°°

[1694] \\ z \W

[1695] /

[1696] \ / z / r\ r\°°~~

[1697] \ / \z(o°x s

[1698] / \ w / s / z°

[1699] \ zOT

[1700] BL-280 >^j o °Z^z

[1701] \ /

[1702] 0 Q s

[1703] r^ > '

[1704] \ \o,

[1705] W' / °s

[1706] / '°

[1707] BL-281

[1708] OxX / zo OxXzo ' o X J:

[1709] BL-282 Os ZO I

[1710] BL-283

[1711]

[1712] Compound

[1713] Structure X No. o zOTo \

[1714] O T O X i ' o

[1715] BL-284Z ZOOT

[1716] ° \ z0< '

[1717] o / ° \ ' '

[1718] z < —

[1719] ° 7= \ /

[1720] ( ° ' / tn \ — ^ z \ z—

[1721] ^ z

[1722] ° \ / ot \

[1723] \ o

[1724] BL-285

[1725] \ \ / \ZO / \ / °

[1726] \ \ z°

[1727] / / / / \o / "°°~s x

[1728] \ \ \ /

[1729] \ \ Z°z\z°o

[1730] / \ / c / / x / / °

[1731] / ° / \ " / \ ^z

[1732] ¥\x / ° °~Z^z

[1733] \ /

[1734] BL-286

[1735] p i0 \ °

[1736] ° \ z

[1737] / C /

[1738] 0 \o - c°

[1739] O

[1740] c° i

[1741] o I BL-287

[1742] BL-288

[1743] BL-289

[1744]

[1745] Compound

[1746] Structure

[1747] No.

[1748] X X

[1749] o o p C° \\ c C X C O / / BL-290 ^ oo / '

[1750] P < / x° / '

[1751] s z. z \——

[1752] °° / \ / / \ /

[1753] ° ° \ / / \ ^ z

[1754] BL-291 / 20=

[1755] ° / \ O o / /

[1756] \ c°

[1757] / / O 2 "OT

[1758] \ \z / o\ / °

[1759] BL-292 2 ) \C < \ w w <- — - / °w

[1760] s) ° / \ ° "Z^ ^zz

[1761] Q > < °'

[1762] ^ O / o / K

[1763] c°

[1764] BL-293 >z° o

[1765] o I X

[1766] \;s;zs; OH o' 'o 1 o'so

[1767] BL-294

[1768] _ j

[1769] z's; ^^

[1770] o' 'o

[1771] BL-295

[1772]

[1773] Compound

[1774] Structure

[1775] No.

[1776] o o I I BL-296 *> o o c ZW

[1777] ) ° / \ / O / \'.

[1778] w z v —

[1779] ( 'OTf - ' w — z Z.^

[1780] ) ° \ \z\ O

[1781] / / Ox

[1782] / Wco \ co \ z

[1783] ° \ \' / \ o

[1784] ) o

[1785] BL-297 ) \ z°

[1786] \C zW

[1787] ) / \°Z^S

[1788] \ / \z°

[1789] / \ w / \ / ' °

[1790] >^x) O °Z^ \ ^z

[1791] ^ zz —z

[1792] BL-298 ^

[1793] h P " ^^) ° Ox°x ^XZ<)z °' Coz )Z° 0 c° c° o o o I IO I I

[1794] BL-299

[1795] BL-300

[1796] BL-301

[1797]

[1798]

[1799] In some embodiments, a compound of formula IC is a compound selected from Table 1C, or a pharmaceutically acceptable salt thereof.

[1800] Table 1C

[1801] Compound

[1802] Structure

[1803] No.

[1804] 0<0H

[1805] BL-194 o <

[1806] N 1

[1807] BL-222 0 <

[1808]

[1809] Compound

[1810] Structure

[1811] No.

[1812] ° roHBL-194 o <

[1813] ° / / \

[1814] i d ( ' o —

[1815] BNT-111 \ \o=

[1816] )° /

[1817] / o

[1818] BL-224

[1819] ^z

[1820] o / z o I BL-226 o

[1821] N

[1822] 0

[1823] N NH

[1824] 1H\

[1825] BL-310 ' 0 <

[1826] 1 H

[1827] 0

[1828] 1H\

[1829] BL-311 0 <

[1830] N

[1831] 1 H

[1832]

[1833] Compound

[1834] Structure

[1835] No.

[1836] ° roHBL-194 o <

[1837] 0

[1838] ° \ / /

[1839] ° / \

[1840] ° \ /

[1841] BNT-93 o /

[1842] O 1

[1843] ^z—

[1844] BNT-94

[1845] o I SH

[1846] 0 HN--4 II j. N BNT-97

[1847] 01

[1848] ^XXXXX^ A0^XXXXXXXXXN^XX^N^SH^xA A ^-N BNT-98

[1849] ° 1

[1850]

[1851] Compound

[1852] Structure

[1853] No.

[1854] ° roHBL-194 o <

[1855] o° / \ / f=N\ / 0H° \ /

[1856] ° / \

[1857] BNT-104 \ o

[1858] o 1

[1859] 0 II ' z — N L=N NH

[1860] \ i _.

[1861] S ^ o r HC> BNT-105

[1862] ° 1

[1863] HO.

[1864] °

[1865] BNT-107 ^x > > N^= / 0 <

[1866] BNT-117

[1867] o

[1868] / X^y\ / yy^0^\ / -^^x^x^

[1869] BL-312

[1870] o / 0H

[1871]

[1872] Compound

[1873] Structure

[1874] No.

[1875] ° roHBL-194 o <

[1876] OH

[1877] \o= /

[1878] A ° / \

[1879] / 02=

[1880] BL-313 \ o

[1881] 01

[1882] x'''XX~X^^

[1883] \ z —

[1884] BL-314

[1885] / O-\- A I\=zi^ 0

[1886] / Z-NH BL-315

[1887] 0 < ^OH

[1888] 0 N=\

[1889] II j. NH ZX-X / X^o / x^X / ^^^N^xX / \^ X^\A > ADH BL-316

[1890] 0[

[1891] x'-X^X^^

[1892] 0 N=\ II L NH

[1893] J j A / OHBL-317

[1894] O y

[1895]

[1896] In some embodiments, a compound of formula I is a compound selected from Table ID, or a pharmaceutically acceptable salt thereof.

[1897]

[1898]

[1899] In some embodiments, compounds are provided and / or utilized in a salt form (e.g., a pharmaceutically acceptable salt form). Reference to a compound provided herein is understood to include reference to salts thereof, unless otherwise indicated.

[1900] Particles for Nucleic Acid Delivery

[1901] In some embodiments, particles of the present disclosure comprise a compound described herein, a nucleic acid (such as RNA (e.g., mRNA), DNA or mixtures thereof), and one or more of a polymer-conjugated lipid, a helper lipid, and a steroid. In some embodiments, particles of the present disclosure comprise a compound described herein, a nucleic acid (such as RNA (e.g., mRNA), DNA or mixtures thereof), a helper lipid, and a steroid. In some embodiments, particles of the present disclosure comprise a compound described herein, a nucleic acid (such as RNA (e.g., mRNA), DNA or mixtures thereof), a polymer- conjugated lipid, a helper lipid, and a steroid.

[1902] In some embodiments, particles described herein (e.g., nucleic acid particles, e.g., ribonucleic acid particles or deoxyribonucleic acid particles) comprise more than one type of nucleic acid molecules, where the molecular parameters of the nucleic acid molecules may be similar or different from each other, like with respect to molar mass or fundamental structural elements such as molecular architecture, capping, coding regions or other features.

[1903] In some embodiments, a nucleic acid particle described herein is a nanoparticle. As used in the present disclosure, “nanoparticle” refers to a particle having an average diameter suitable for parenteral administration and is less than 1000 nm in diameter. In some embodiments, a composition comprising nanoparticles can have an average nanoparticle size (e.g., mean diameter) of about 30 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 70 to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, a composition comprising nanoparticles can have an average nanoparticle size (e.g., mean diameter) of about 50 nm to about 100 nm. In some embodiments, a composition comprising nanoparticles can have an average nanoparticle size (e.g., mean diameter) of about 50 nm to about 150 nm. In some embodiments, a composition comprising nanoparticles can have an average nanoparticle size (e.g., mean diameter) of about 60 nm to about 120 nm. In some embodiments, a composition comprisingnanoparticles can have an average nanoparticle size (e.g., mean diameter) of 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.

[1904] A composition comprising nucleic acid particles (e.g., ribonucleic acid particles or deoxyribonucleic acid particles) described herein may exhibit a polydispersity index less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less of said nanoparticles. By way of example, a composition comprising nucleic acid particles (e.g., ribonucleic acid particles or deoxyribonucleic acid particles) described herein can exhibit a polydispersity index in a range of about 0.1 to about 0.3 or about 0.2 to about 0.3.

[1905] Nucleic acid particles (e.g., ribonucleic acid particles or deoxyribonucleic acid particles) described herein can be characterized by an “N / P ratio,” which is the molar ratio of cationic (nitrogen) groups (the “N” in N / P) in the cationic polymer to the anionic (phosphate) groups (the “P” in N / P) in RNA. It is understood that a cationic group is one that is either in cationic form (e.g., N+), or one that is ionizable to become cationic. Use of a single number in an N / P ratio (e.g., an N / P ratio of about 5) is intended to refer to that number over 1, e.g., an N / P ratio of about 4 is intended to mean about 4:1. In some embodiments, a nucleic acid particle (e.g., a ribonucleic acid particle) described herein has an N / P ratio greater than or equal to 4. In some embodiments, a nucleic acid particle (e.g., a ribonucleic acid particle) described herein has an N / P ratio that is about 4 to about 16. In some embodiments, a nucleic acid particle (e.g., a ribonucleic acid particle) described herein has an N / P ratio that is about 6 to about 12. In some embodiments, a nucleic acid particle described herein has an N / P ratio that is about 4 to about 12. In some embodiments, a nucleic acid particle (e.g., a ribonucleic acid particle) described herein has an N / P ratio that is about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, an N / P ratio for a nucleic acid particle (e.g., a ribonucleic acid particle) described herein is about 6. In some embodiments, an N / P ratio for a nucleic acid particle (e.g., a ribonucleic acid particle) described herein is about 12.

[1906] Compounds described herein are also referred to as “ionizable” or “cationic” lipids. Such lipids are intended to mean compounds that, in some embodiments, are capable of becoming cationic (i.e., becoming positively charged) at physiological pH.

[1907] The term “average diameter” or “mean diameter” refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PDI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here “average diameter,” “mean diameter,” “diameter,” or “size” for particles is used synonymously with this value of the Z-average.

[1908] The “polydispersity index” is preferably calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the “average diameter.” Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of ribonucleic acid nanoparticles (e.g., ribonucleic acid nanoparticles).

[1909] Different types of nucleic acid particles have been described previously to be suitable for delivery of nucleic acid in particulate form (e.g. Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60). For non-viral nucleicacid delivery vehicles, nanoparticle encapsulation of nucleic acid physically protects nucleic acid from degradation and, depending on the specific chemistry, can aid in cellular uptake and endosomal escape. Some embodiments described herein relate to compositions, methods and uses involving more than one, e.g., 2, 3, 4, 5, 6 or even more nucleic acid species. The nucleic acid species may be RNA and / or DNA. For example, the particles described herein may contain one species of RNA (e.g., one species of mRNA) and one species of DNA.

[1910] In a nucleic acid particle composition, it is possible that each nucleic acid species is separately formulated as an individual nucleic acid particle formulation. In that case, each individual nucleic acid particle formulation will comprise one nucleic acid species. The individual nucleic acid particle formulations may be present as separate entities, e.g., in separate containers. Such formulations are obtainable by providing each nucleic acid species separately (typically each in the form of a nucleic acid -containing solution) together with a particle -forming agent, thereby allowing the formation of particles. Respective particles will contain exclusively the specific nucleic acid species that is being provided when the particles are formed (individual particulate formulations).

[1911] In some embodiments, a composition such as a pharmaceutical composition comprises more than one individual nucleic acid particle formulation. Respective pharmaceutical compositions are referred to as “mixed particulate formulations.” Mixed particulate formulations according to the present disclosure are obtainable by forming, separately, individual nucleic acid particle formulations, as described above, followed by a step of mixing of the individual nucleic acid particle formulations. By the step of mixing, a formulation comprising a mixed population of nucleic acid-containing particles is obtainable. Individual nucleic acid particle populations may be together in one container, comprising a mixed population of individual nucleic acid particle formulations.

[1912] Alternatively, it is possible that different nucleic acid species are formulated together as a “combined particulate formulation.” Such formulations are obtainable by providing a combined formulation (typically combined solution) of different nucleic acid species together with a particle-forming agent, thereby allowing the formation of particles. As opposed to a “mixed particulate formulation,” a “combined particulate formulation” will typically comprise particles that comprise more than one nucleic acid species. In a combined particulate composition different nucleic acid species are typically present together in a single particle.

[1913] In certain embodiments, nucleic acids, when present in provided nucleic acid particles are resistant in aqueous solution to degradation with a nuclease.

[1914] In some embodiments, a nucleic acid is RNA, DNA, or mixtures thereof. In some embodiments, a nucleic acid is RNA. In some embodiments, an RNA is mRNA. In some embodiments, RNA is modRNA, circRNA, saRNA, taRNA, or uRNA.

[1915] In some embodiments, a nucleic acid is DNA. In some embodiments, DNA is linear DNA, plasmid DNA, minicircle DNA, nanoplasmid DNA, doggybone DNA, or a transposon.Lipid Nanoparticles (LNPs)

[1916] In some embodiments, a particle described herein is a lipid nanoparticle (LNP). LNPs have emerged as particularly useful vehicles for delivery of nucleic acids, for example as described in Theranostics, 2022 Oct 24;12(17):7509-7531. It is understood that a LNP is structurally distinct from other nanoparticles previously used for nucleic acid delivery, such as a liposome, or a lipoplex. LNPs, as described herein, typically do not comprise a bilayer (unilamellar), or a concentric series of multiple bilayers (multi -lamellar) separated by aqueous compartments, enclosing a central aqueous compartment. Moreover, LNPs, as described herein, typically do not comprise a central aqueous core or compartment. LNPs as described herein typically comprise nucleic acids (e.g., DNA or RNA such as mRNA) and lipids forming a disordered, non-lamellar phase. LNPs as described herein may be considered as oil-in-water emulsions in which the LNP core materials are preferably in liquid state and hence have a melting point below body temperature. See, e.g., ACS Nano 2021, 15, 11, 16982–17015; Aldosari, et al., Pharmaceutics, 2021, 13, 206.

[1917] LNPs described herein generally comprise four categories of lipids in addition to a nucleic acid agent (e.g., DNA or RNA such as mRNA): a cationic or cationically ionizable lipid (e.g., a lipid compound described herein), a polymer-conjugated lipid, a helper lipid, and a steroid. A person of skill in the art will understand that various combinations of these four categories of lipids can be used to prepare lipid nanoparticles for use in delivering nucleic acid agents. Lipid nanoparticles described herein are characterized by molar percentage (mol%) of components in the lipid nanoparticle. A mol% used in reference to a lipid component of a lipid nanoparticle is relative to the total other lipid components in the lipid nanoparticle.

[1918] (i) Helper lipids

[1919] As described herein, lipid nanoparticles of the present disclosure comprise a helper lipid. In some embodiments, a helper lipid is or comprises phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin. In some embodiments, a helper lipid is a phospholipid. In some embodiments, a helper lipid is or comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), 1 -palmitoyl -2 -oleoyl-sn-glycero-3 -phosphocholine (POPC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), sphingomyelins, N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), l,2-diacylglyceryl-3-O-4’-(N, N, N-trimethyl)-homoserine (DGTS), ceramides, and their derivatives. In some embodiments, a helper lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DSPE, and SM. In some embodiments, the helper lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the helper lipid is DSPC.

[1920] Helper lipids may be synthetic or naturally derived. Other helper lipids suitable for use in a lipid nanoparticle are described in WO 2021 / 026358, WO 2017 / 075531, and WO 2018 / 081480, the entire contents of each of which are incorporated herein by reference.

[1921] In some embodiments, a lipid nanoparticle comprises about 5 to about 15 mol% of a helper lipid. In some embodiments, a lipid nanoparticle comprises about 5 to about 15 mol% of a phospholipid. In someI l l

[1922] embodiments, a lipid nanoparticle comprises about 8 to about 12 mol% of a phospholipid. In some embodiments, a lipid nanoparticle comprises about 10 mol% of a phospholipid. In some embodiments, a lipid nanoparticle comprises about 5 to about 15 mol% of DSPC. In some embodiments, a lipid nanoparticle comprises about 8 to about 12 mol% of DSPC. In some embodiments, a lipid nanoparticle comprises about 10 mol% of DSPC.

[1923] (ii) Polymer-conjugated lipids

[1924] As described herein, LNPs of the present disclosure comprise a polymer-conjugated lipid. In some embodiments, a polymer-conjugated lipid is a lipid conjugated to polyethylene glycol (a “PEG-lipid”). In some embodiments, a polymer conjugated lipid is selected from the group consisting of a polyethylene glycol) (PEG) -conjugated lipid, a poly(sarcosine) (pSar)-conjugated lipid, a poly(aminoethoxy ethoxy acetic acid) (pAEEA) -conjugated lipid; and a poly (2 -methylaminoethoxy ethoxy acetic acid) (pMAEEA)-conjugated lipid. In some embodiments, a PEG-lipid is selected from pegylated diacylglycerol (PEG-DAG) such as 1 -(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG) (e.g., 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG)), a pegylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2’,3’-di(tetradecanoyloxy)propyl- 1 -0-(co-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000 amine), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate, and 2,3-di(tetradecanoxy)propyl-N-(co-methoxy(polyethoxy)ethyl)carbamate. In some embodiments, a PEG group that is part of a PEG-lipid has, on average in a composition comprising one or more PEG-lipid molecules, a number average molecular weight (Mn) of about 2000 g / mol.

[1925] In some embodiments, a PEG-lipid is DMG-PEG. In some embodiments, a PEG-lipid is PEG2000-DMG:

[1926]

[1927] In some embodiments, a PEG-lipid is provided in WO 2021 / 026358, WO 2017 / 075531, or WO 2018 / 081480, each of which is incorporated by reference in its entirety.

[1928] In some embodiments, a PEG-lipid is a compound of Formula PCL-I:

[1929]

[1930] PCL-I

[1931] or a pharmaceutically acceptable salt thereof, wherein, as applied to formula PGL-I, R8and R9are each independently C10-C30 aliphatic, optionally interrupted by one or more ester bonds, and w is an integer from 30 to 60.

[1932] In some embodiments, the PEG-lipid is represented by:

[1933]

[1934] wherein n has a mean value ranging from 30 to 60. In some embodiments, n is 50. In one embodiment, the PEG-conjugated lipid (pegylated lipid) is PEG2000-C-DMA which preferably refers to 3-N-[(co-methoxy poly(ethylene glycol)2000)carbamoyl]-l,2-dimyristyloxy-propylamine (MPEG-(2 kDa)-C-DMA) or methoxy-polyethylene glycol-2,3-bis(tetradecyloxy) propylcarbamate (2000).

[1935] In some embodiments, a PEG-lipid is selected from PEG-DAG, PEG-PE, PEG-S-DAG, PEG2000-DMG, PEG2000-C-DMA PEG-S-DMG, PEG-cer, and combinations thereof. In some embodiments, a PEG-lipid is PEG2000-DMG.

[1936] In some embodiments, a polymer-conjugated lipid is a polysarcosine-conjugated lipid, also referred to herein as sarcosinylated lipid or pSar-lipid. The term “sarcosinylated lipid” refers to a molecule comprising both a lipid portion and a polysarcosine (poly(N-methylglycine)) portion.

[1937] In some embodiments, a polymer-conjugated lipid is one described in WO 2024 / 028325, which is incorporated herein by reference in its entirety. In some embodiments, a polymer-conjugated lipid is represented by formula PCL-II:

[1938]

[1939] or a pharmaceutically acceptable salt thereof, wherein, as applied to formula PCL-II: X2and X1taken together are optionally substituted amide, optionally substituted thioamide, ester, or thioester; Y is -CH2-, -(CH2)2-, or -(CH2)3-; z is 2 to 24; and n is 1 to 100. In some embodiments of formula PCL-II: (i) when X1is -C(O)- then X2is -NR1-; (ii) when X1is -NR1- then X2is -C(O)-; (iii) when X1is -C(S)- then X2is -NR1-; (iv) when X1is -NR1- then X2is -C(S)-; (v) when X1is -C(O)- then X2is -O-; (vi) when X1is -O- then X2is -C(O)-; (vii) when X1is -C(S)- then X2is -O-; (viii) when X1is -O- then X2is -C(S)-; (ix) when X1is -C(O)- then X2is -S-; or (x) when X1is -S- then X2is -C(O)-; wherein R1is hydrogen or Ci-s alkyl. In some embodiments of formula PCL-II: (i) when X1is -C(O)- then X2is -NR1-; (ii) when X1is -NR1- then X2is -C(O)-; (iii) when X1is -C(S)- then X2is -NR1-; (iv) when X1is -NR1- then X2is -C(S)-; (v) when X1is -C(O)- then X2is -O-; or (vi) when X1is -O- then X2is -C(O)-; wherein R1is hydrogen or Ci-s alkyl. In some embodiments, a polymer-conjugated lipid comprises monomers of 2-(2-(2-aminoethoxy)ethoxy)acetic acid. In some embodiments, the polymer of the polymer-conjugated lipid is or comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments, a polymer-conjugated lipid comprises monomers of unit PCL-II-1:

[1940]

[1941] PCL-II-1

[1942] In some embodiments, a polymer-conjugated lipid comprises, 5 to 50, 5 to 25 or 10 to 25 monomers of PCL-II-1. In some embodiments, a polymer-conjugated lipid comprises 14 to 17 monomers of PCL-II-1. In some embodiments, a polymer-conjugated lipid comprises 8 to 14 monomers of PCL-II-1. In some embodiments, a polymer-conjugated lipid is selected from the table below:

[1943] DSPE- 0 0 ZZ' '' 0

[1944] U II » IZ11

[1945] .0.

[1946] AEEA14- ^35^17 ®: 0 J n 0 N"

[1947] H

[1948] A35C,?0 HO

[1949] C “H. 14

[1950] r

[1951] 0 o

[1952] o

[1953] VE- U H AEEA14- 1 I I xl II o

[1954] AC - 14

[1955] VE- (AEEA)8- U H

[1956] _ / L, O

[1957] AC O

[1958] 8

[1959] DMA- (AEEA)14- AC 3

[1960] 4

[1961] DMA- (AEEA)8- AC 0

[1962] 8

[1963] DMG- o

[1964] (AEEA)8- o

[1965] AC 8

[1966] o

[1967]

[1968] In some embodiments, an LNP comprises an polysarcosine-conjugated or a pAEEA / pMAEEA-conjugated lipid, as described herein. In some embodiments, nucleic acid particles (e.g., DNA or RNA particles) described herein comprise a polysarcosine-conjugated or a pAEEA / pMAEEA-conjugated lipid and are substantially free of a pegylated lipid (or do not contain a pegylated lipid).In some embodiments, a lipid nanoparticle comprises about 0.5 to about 5.0 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises about 1.0 to about 2.5 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises about 1.5 to about 2.0 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises about 1.5 to about 1.8 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises about 1.5 mol% to about 1.8 mol% (relative to the total amount of lipids in a lipid nanoparticle) of a polymer-conjugated lipid selected from the group consisting of: DSPE-AEEA14-AC; VE-AEEA14-AC; ALC-0I59 and PEG2000-DMG. In some embodiments, a lipid nanoparticle comprises about 1.5 mol% to about 1.8 mol% (relative to the total amount of lipids in a lipid nanoparticle) of a polymer-conjugated lipid selected from the group consisting of: DSPE-AEEA14-AC, VE-AEEA14-AC, and PEG2000-DMG. In some embodiments, a molar ratio of a cationic or cationically ionizable lipid to a polymer-conjugated lipid is from about 2: 1 to about 8:1.

[1969] (Hi) Steroids

[1970] As described generally herein, lipid nanoparticles further comprise a steroid. In some embodiments, a steroid is a sterol. In some embodiments, a sterol is P-sitosterol, stigmasterol, cholesterol, cholecalciferol, ergocalciferol, calcipotriol, botulin, lupeol, ursolic acid, oleanolic acid, cycloartenol, lanosterol, or a-tocopherol. In some embodiments, a sterol is cholesterol. In some embodiments, a lipid nanoparticle comprises about 39 to about 49 mol% of a steroid. In some embodiments, a lipid nanoparticle comprises about 40 to about 46 mol% of a steroid. In some embodiments, a lipid nanoparticle comprises about 40 to about 44 mol% of a steroid.

[1971] In some embodiments, a lipid nanoparticle comprises: about 30 to about 60 mol% of a cationic or ionizable lipid compound described herein; about 18.5 to about 48.5 mol% of a steroid (e.g., cholesterol); about 0 to about 30 mol% of a helper lipid (e.g., DSPC); and about 0 to about 10 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises: about 35 to about 55 mol% of a cationic or ionizable lipid compound described herein; about 30 to about 40 mol% of a steroid (e.g., cholesterol); about 5 to about 25 mol% of a helper lipid (e.g., DSPC); and about 0 to about 10 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle comprises: about 40 to about 50 mol% of a cationic or ionizable lipid compound described herein; about 30 to about 45 mol% of a steroid (e.g., cholesterol); about 5 to about 15 mol% of a helper lipid (e.g., DSPC); and about 1 to about 2.5 mol% of a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle described herein comprises: 47.5 mol% of a cationic or ionizable lipid compound described herein; 10 mol% of a helper lipid; 40.7 mol% of a steroid; and 1.8 mol% of a polymer conjugated lipid.

[1972] (iv) Manufacturing

[1973] Lipids and lipid nanoparticles comprising nucleic acids and their method of preparation are known in the art, including, e.g., as described in U. S. Patent Publication Nos. 2016 / 0009637, 2015 / 0273068, 2014 / 0200257, 2013 / 0338210, 2013 / 0245107, 2013 / 0123338, 2013 / 0017223, 2012 / 0183581, 2012 / 0027803, 2011 / 0311583, 2011 / 0216622, 2011 / 0117125, 2007 / 0042031, 2006 / 0083780,2005 / 017054, 2004 / 0142025, 2007 / 0042031, 1999 / 009076 and PCT Pub. Nos. WO 99 / 39741, WO 2018 / 081480, WO 2017 / 004143, WO 2017 / 075531, WO 2015 / 199952, WO 2013 / 086322, WO 2013 / 016058, WO 2013 / 086373, WO 2011 / 141705, WO 2022 / 016089, WO 2022 / 081752, the full disclosures of which are herein incorporated by reference in their entirety for the purposes described herein. For example, in some embodiments, cationic or ionizable lipids, helper lipids, and steroids are solubilized in an organic solvent such as ethanol, at a predetermined weight or molar ratios / percentages (e.g., ones described herein). In some embodiments, lipid nanoparticles are prepared at a total lipid to nucleic acid (e.g., RNA) weight ratio of approximately 10: 1 to 50:1. In some embodiments, such nucleic acid (e.g., RNA) can be diluted to 0.1 to 1.0 mg / mL (e.g., 0.4 mg / mL) in an acidic buffer, such as citrate or acetate having a pH of between about 4 to about 6.

[1974] In some embodiments, using an ethanol injection technique, a colloidal lipid dispersion comprising nucleic acids (e.g., RNAs) can be formed as follows: an ethanol solution comprising lipids, such as cationic lipids, helper lipids, steroids, and polymer-conjugated lipids, is combined with, e.g., injected into or continuously mixed with, an aqueous solution comprising nucleic acids.

[1975] In some embodiments, lipid and nucleic acid (e.g., RNA) solutions can be mixed at room temperature by pumping each solution (e.g., a lipid solution comprising a cationic lipid, a helper lipid, cholesterol, a conjugated lipid, and any other additives) at controlled flow rates into a mixing unit, for example, using piston pumps. In some embodiments, the flow rates of a lipid solution and a nucleic acid (e.g., RNA) solution into a mixing unit are maintained at a ratio of 1:3. Upon mixing, nucleic acid-lipid particles are formed as the ethanolic lipid solution is diluted with aqueous nucleic acids (e.g., RNAs). The lipid solubility is decreased, while cationic lipids bearing a positive charge interact with the negatively charged nucleic acid (e.g., RNA).

[1976] In some embodiments, a solution comprising nucleic acid (e.g., RNA) -encapsulated lipid nanoparticles can be processed by one or more of concentration adjustment, buffer exchange, formulation, and / or fdtration.

[1977] RNA

[1978] In some embodiments, a particle described herein comprises one or more oligosaccharide compositions and a nucleic acid. In some embodiments, a nucleic acid is RNA.

[1979] In some embodiments, an RNA amenable to technologies described herein is a single -stranded RNA. In some embodiments, an RNA as disclosed herein is a linear RNA. In some embodiments, a single -stranded RNA is a non-coding RNA in that its nucleotide sequence does not include an open reading frame (or complement thereof). In some embodiments, a single -stranded RNA has a nucleotide sequence that encodes (or is the complement of a sequence that encodes) a polypeptide or a plurality of polypeptides (e.g., epitopes) of the present disclosure.

[1980] In some embodiments, an RNA is or comprises an siRNA, a miRNA, or other non-coding RNA.

[1981] In many embodiments, a relevant RNA includes at least one open reading frame (ORF) (e.g., is an mRNA); in some embodiments, a relevant RNA includes a single ORF; in some embodiments, a relevant RNA includes more than one ORF.In some embodiments, an RNA comprises an ORF, e.g., encoding a polypeptide of interest or encoding a plurality of polypeptides of interest. In some embodiments, an RNA produced in accordance with technologies provided herein comprises a plurality of ORFs (e.g., encoding a plurality of polypeptides). In some embodiments, an RNA produced in accordance with technologies herein comprises a single ORF that encodes a plurality of polypeptides. In some such embodiments, polypeptides are or comprise antigens or epitopes thereof (e.g, relevant antigens).

[1982] In some embodiments, an ORF for use in accordance with the present disclosure encodes a polypeptide that includes a signal sequence, e.g., that is functional in mammalian cells, such as an intrinsic signal sequence or a heterologous signal sequence. In some embodiments, a signal sequence directs secretion of an encoded polypeptide, in some embodiments, a signal sequence directs transport of an encoded polypeptide into a defined cellular compartment, preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal-lysosomal compartment.

[1983] In some embodiments, an ORF encodes a polypeptide that includes a multimerization element (e.g., an intrinsic or heterologous multimerization element). In some embodiments, an ORF that encodes a surface polypeptide (e.g, that includes a signal sequence directing surface localization) includes a multimerization element.

[1984] In some embodiments, an ORF encodes a polypeptide that includes a transmembrane element or domain. In some embodiments, an ORF is codon-optimized for expression in a cells of a particular host, e.g., a mammalian host, e.g., a human.

[1985] In some embodiments, an RNA includes unmodified uridine residues; an RNA that includes only unmodified uridine residues may be referred to as a “uRNA”. In some embodiments, an RNA includes one or more modified uridine residues; in some embodiments, such an RNA (e.g., an RNA including entirely modified uridine residues) is referred to as a “modRNA”. In some embodiments, an RNA may be a selfamplifying RNA (saRNA). In some embodiments, an RNA may be a trans -amplifying RNA (taRNA) (see, for example, WO20I7 / I6246I).

[1986] In some embodiments, a relevant RNA includes a polypeptide -encoding portion or a plurality of polypeptide -encoding portions. In some particular embodiments, such a portion or portions may encode a polypeptide or polypeptides that is or comprises a biologically active polypeptide or portion thereof (e.g., an enzyme or cytokine or therapeutic protein such as a replacement protein or antibody or portion thereof). In some particular embodiments, such a portion or portions may encode a polypeptide or polypeptides that is or comprises an antigen (or an epitope thereof), a cytokine, an enzyme, etc. In some embodiments, an encoded polypeptide or polypeptides may be or include one or more neoantigens or neoepitopes associated with a tumor. In some embodiments, an encoded polypeptide or polypeptides may be or include one or more antigens (or epitopes thereof) of an infectious agent (e.g., a bacterium, fungus, virus, etc.). In certain embodiments, an encoded polypeptide may be a variant of a wild type polypeptide.

[1987] In some embodiments, a single -stranded RNA (e.g., mRNA) may comprise a secretion signal -encoding region (e.g, a secretion signal -encoding region that allows an encoded target entity or entities to be secreted upon translation by cells). In some embodiments, such a secretion signal -encoding region may be orcomprise a non-human secretion signal. In some embodiments, such a secretion signal -encoding region may be or comprise a human secretion signal.

[1988] In some embodiments, a single -stranded RNA (e.g., mRNA) may comprise at least one non-coding element (e.g., to enhance RNA stability and / or translation efficiency). Examples of non -coding elements include but are not limited to a 3’ untranslated region (UTR), a 5’ UTR, a cap structure (e.g, in some embodiments, an enzymatically-added cap; in some embodiments, a co -transcriptional cap), a poly adenine (poly A) tail (e.g., that, in some embodiments, may be or comprise 100 A residues or more, and / or in some embodiments may include one or more “interrupting” [i.e., non-A] sequence elements), and any combinations thereof. Exemplary embodiments of such non-coding elements may be found, for example, in WO2011015347, WO2017053297, US 10519189, US 10494399, W02007024708, W02007036366, W02017060314, W02016005324, W02005038030, WO2017036889, WO2017162266, and WO20I7I6246I, each of which is incorporated herein by referenced in its entirety.

[1989] Formats

[1990] At least four formats useful for RNA pharmaceutical compositions (e.g., immunogenic compositions or vaccines) have been developed, namely non-modified uridine containing mRNA (uRNA), nucleoside-modified mRNA (modRNA), self-amplifying mRNA (saRNA), and trans -amplifying RNAs.

[1991] Features of a non-modified uridine platform may include, for example, one or more of intrinsic adjuvant effect, good tolerability and safety, and strong antibody and T cell responses.

[1992] Features of modified uridine (e.g., pseudouridine) platform may include reduced adjuvant effect, blunted immune innate immune sensor activating capacity and thus augmented antigen expression, good tolerability and safety, and strong antibody and CD4-T cell responses. As noted herein, the present disclosure provides an insight that such strong antibody and CD4 T cell responses may be particularly useful for vaccination. Features of self-amplifying platform may include, for example, long duration of polypeptide (e.g., protein) expression, good tolerability and safety, higher likelihood for efficacy with very low vaccine dose.

[1993] In some embodiments, a self-amplifying platform (e.g., s / RNA) comprises a nucleic acid molecule, encoding both a replicase (e.g., a viral replicase) and a gene of interest, wherein the nucleic acid molecule is capable of being replicated by said replicase in cis (c / .s-rcplication system). In some embodiments, a transamplifying platform (e.g., toRNA) comprises two nucleic acid molecules, wherein one nucleic acid molecule encodes a replicase (e.g., a viral replicase) and the other nucleic acid molecule is capable of being replicated (e.g., a replicon) by said replicase in trans ( / ram-replication system). In some embodiments, a self / trans-amplifying platform (e.g., RNA) comprises a plurality of nucleic acid molecules, wherein said nucleic acids encode a plurality of replicase s and / or replicons.

[1994] In some embodiments, a trans -replication system comprises the presence of both nucleic acid molecules in a single host cell.

[1995] In some such embodiments, a nucleic acid encoding a replicase (e.g., a viral replicase) is not capable of self-replication in a target cell and / or target organism. In some such embodiments, a nucleic acid encodinga replicase (e.g., a viral replicase) lacks at least one conserved sequence element important for (-) strand synthesis based on a (+) strand template and / or for (+) strand synthesis based on a (-) strand template. In some embodiments, a self-amplifying RNA comprises a 5 ’-cap; in some / ram-replication systems, at least an RNA encoding a replicase is capped. Without wishing to be bound by any one theory, it has been found that a 5’-cap can be important for high level expression of a gene of interest in trans.

[1996] In some embodiments, a self / trans-amplifying platform does not require propagation of virus particles (e.g., is not associated with undesired virus -particle formation). In some embodiments, a self / trans-amplifying platform is not capable of forming virus particles.

[1997] In some embodiments, an RNA may comprise an Internal Ribosomal Entry Site (IRES) element. In some embodiments, an RNA does not comprise an IRES site; in particular, in some embodiments, an saRNA does not comprise an IRES site. In some such embodiments, translation of a gene of interest and / or replicase is not driven by an IRES element. In some embodiments, an IRES element is substituted by a 5’-cap. In some such embodiments, substitution by a 5 ’-cap does not affect the sequence of a polypeptide encoded by an RNA.

[1998] In some embodiments, a complex described herein comprises modRNA, saRNA, taRNA, or uRNA. In some embodiments, a complex comprises modRNA. In some embodiments, a complex comprises saRNA. In some embodiments, a complex comprises taRNA. In some embodiments, a complex comprises uRNA.

[1999] Methods of Use

[2000] Particles described herein are useful in the treatment and prophylaxis in a subject of diseases, disorders, and conditions described herein. In some embodiments, the present disclosure provides a method of treating a disease, disorder or condition comprising administering to a patient a composition comprising particles described herein. In some embodiments, the present disclosure provides use of a composition comprising particles described herein for the treatment of a disease, disorder, or condition. In some embodiments, a disease, disorder, or condition is an infectious disease, cancer, an autoimmune disease, or a rare disease.

[2001] In some embodiments, an infectious disease is caused by or associated with a viral pathogen. In some embodiments, a viral pathogen is of a family selected from poxviridae, rhabdoviridae, fdoviridae, paramyxoviridae, hepadnaviridae, coronaviridae, caliciviridae, picomaviridae, reoviridae, retroviridae, and orthomyxoviridae. In some embodiments, an infectious disease is caused by or associated with a virus selected from SARS-CoV-2, influenza, Crimean-Congo Hemorrhagic Fever (CCHF), Ebola virus, Lassa virus, Marburg virus, HIV, Nipah virus, and MERS-CoV.

[2002] In some embodiments, an infectious disease is caused by or associated with a bacterial pathogen. In some embodiments, a bacterial pathogen is of a species selected from Actinomyces israelii, bacillus antracis, Bacteroides fragilis, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campolobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium idphteriae, Ehrlichiacanis, Ehrlichia chaffeensis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Nocardia asteroids, Rickettsia ricektssii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Shigella dysenteriae, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, Treponema pallidum, Vibrio cholerae, and Yersinia pestis.

[2003] In some embodiments, an infectious disease is caused by or associated with a parasite. In some embodiments, a parasite is of a family selected from Plasmodium, Leishmania, Cryptosporidium, Entamoeba, Trypanosomas, Schistosomes, Ascaris, Echinococcus and Taeniidae.

[2004] In some embodiments, a disease, disorder, or condition is a cancer. In some embodiments, a cancer is selected from bladder cancer, breast cancer, colorectal cancer, kidney cancer, lung cancer, lymphoma, melanoma, oral / oropharyngeal cancer, pancreatic cancer, prostate cancer, thyroid cancer, and uterine cancer.

[2005] In some embodiments, a disease, disorder, or condition is a genetic disorder. In some embodiments, a genetic disorder is associated with a gain-of-function mutation or a loss-of-function mutation.

[2006] In some embodiments, a disease, disorder, or condition is an autoimmune disease. In some embodiments, an autoimmune disease is selected from Addison disease, celiac disease, rheumatoid arthritis, lupus, inflammatory bowel disease, dermatomyositis, multiple sclerosis, diabetes, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, pernicious anemia, graves’ disease, Hashimoto’s thyroiditis, myasthenia gravis, and vasculitis Sj drgen syndrome.

[2007] In some embodiments, a disease, disorder, or condition is a rare disease. As described herein, a rare disease refers to a life-threatening or chronically debilitating diseases which are of such low prevalence (e.g., fewer than 1 / 2000 people) that special combined efforts are needed to address them.

[2008] In some embodiments, the present disclosure provides complexes that can selectively target particular systems within a body. As used herein, reference to “targeting” a particular system refers to causing increased expression of RNA derived from cargo in the complex in the desired system. For example, in some embodiments, complexes described herein can selectively target the lungs, liver, spleen, heart, brain, lymph nodes, bladder, kidneys, and pancreas. As described herein, a complex “selectively targets” an organ when a single target expresses mRNA in an amount that is 65% or greater than expression in other organs post administration (e.g., 65% or more of mRNA throughout the body is expressed from a single organ, with the remaining 35% distributed between one or more different organs). In some embodiments, a complex described herein selectively targets the lungs. In some embodiments, a complex described herein selectively targets the liver. In some embodiments, a complex described herein selectively targets the spleen. In some embodiments, a complex described herein selectively targets the heart.Methods of Delivery

[2009] The present disclosure provides, among other things, a particle that is incorporated into a composition (e.g., a pharmaceutical composition or a pharmaceutical formulation, as referred to herein) to be administered to a subject. For example, in some embodiments, a composition comprising particles described herein is administered as a monotherapy. In some embodiments, a composition comprising particles described herein is administered as part of a combination therapy. In some embodiments, a concentration of total RNA (e.g., a total concentration of all of the one or more RNA molecules) in a composition described herein is of about 0.01 mg / mL to about 0.5 mg / mL, or about 0.05 mg / mL to about 0.1 mg / mL.

[2010] Compositions (also referred to as pharmaceutical compositions) may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.

[2011] In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by the United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[2012] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and / or perfuming agents can be present in the composition, according to the judgment of the formulator.

[2013] General considerations in the formulation and / or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

[2014] In some embodiments, pharmaceutical compositions provided herein may be formulated with one or more pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice ofPharmacy 21sted., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).Pharmaceutical complexes and compositions described herein can be administered by appropriate methods known in the art. As will be appreciated by a skilled artisan, the route and / or mode of administration may depend on a number of factors, including, e.g., but not limited to stability and / or pharmacokinetics and / or pharmacodynamics of pharmaceutical compositions described herein.

[2015] In some embodiments, pharmaceutical compositions described herein are formulated for parenteral administration, which includes modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion.

[2016] In some embodiments, pharmaceutical compositions described herein are formulated for intravenous administration. In some embodiments, pharmaceutically acceptable carriers that may be useful for intravenous administration include sterile aqueous solutions or dispersions and sterile powders for preparation of sterile injectable solutions or dispersions.

[2017] In some particular embodiments, pharmaceutical compositions described herein are formulated for subcutaneous (s.c) administration. In some particular embodiments, pharmaceutical compositions described herein are formulated for intramuscular (i.m) administration.

[2018] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, dispersion, powder (e.g., lyophilized powder), microemulsion, lipid nanoparticles, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. In some embodiments, prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[2019] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.

[2020] In some embodiments, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[2021] Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[2022] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into pharmaceutical compositions described herein. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[2023] Formulations of pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing active ingredient(s) into association with a diluent or another excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into a desired single- or multi -dose unit.

[2024] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of at least one RNA product produced using a system and / or method described herein.

[2025] In some embodiments, an active agent that may be included in a pharmaceutical composition described herein is or comprises a therapeutic agent administered in a combination therapy described herein. Pharmaceutical compositions described herein can be administered in combination therapy, i.e., combined with other agents. In some embodiments, such therapeutic agents may include agents leading to depletion or functional inactivation of regulatory T cells. For example, in some embodiments, a combination therapy can include a provided pharmaceutical composition with at least one immune checkpoint inhibitor.

[2026] In some embodiments, pharmaceutical composition described herein may be administered in conjunction with radiotherapy and / or autologous peripheral stem cell or bone marrow transplantation.

[2027] In some embodiments, a pharmaceutical composition described herein can be frozen to allow long-term storage.

[2028] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation.Exemplary Embodiments

[2029] The following numbered embodiments, while non-limiting, are exemplary of certain aspects of the present disclosure:

[2030] 1. A compound represented by formula IB:

[2031] L1-X1-T1

[2032] G2-L3-N / /

[2033] \2- X2— T2

[2034] IB

[2035] or a pharmaceutically acceptable salt thereof, wherein:

[2036] L1and L2are each independently an optionally substituted C1-C30 aliphatic group;

[2037] L3is a optionally substituted C1-C10 aliphatic or a bond;

[2038] X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -S(O)2N(R’)-, -N(R’)S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R1)2-, -OC(S)C(R1)2-, -C(R1)2C(S)O-, -S-, and -N(H)C(O)N(R1);

[2039] each R1is, independently, at each instance, optionally substituted C1-C20aliphatic or H;

[2040] T1and T2are each independently an optionally substituted C3-C30 aliphatic;

[2041] G2is -S(O)2R3a;

[2042] R3ais -(CH2)0-6-R3bor Ci-Ce aliphatic;

[2043] R3bis -OH, -NH(CH3), or -N(CH3)2.

[2044] 2. The compound of Embodiment 1, wherein R3ais -(CH2)0-6-R3b.

[2045] 3. The compound of Embodiments 1 or 2, wherein R3bis -OH.

[2046] 4. The compound of Embodiment 1, wherein R3ais C1-C10aliphatic.

[2047] 0 ii X / S. / OH11The compound of Embodiment 1, wherein G2is 0

[2048]

[2049]

[2050] 6. The compound of any one of Embodiments 1-5, wherein, when R3ais -CH3, then X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -N(H)C(O)N(R’)-, -NHC(O)-, -C(O)N(R’)-, and -NHS(O)2N(R1)-.

[2051] 7. A compound represented by formula IA:L1-X1-T1

[2052] G1-L3—

[2053] \2- X2— T2

[2054] IA

[2055] or a pharmaceutically acceptable salt thereof, wherein:

[2056] L1and L2are each independently an optionally substituted C1-C30 aliphatic group;

[2057] L3is a bond or optionally substituted C1-C10 aliphatic;

[2058] X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -S(O)2N(R’)-, -N(R1)S(O)2-, -S(O)-, -S(O)2-, -S(O)2C(R1)2-, -OC(S)C(R1)2-, -C(R1)2C(S)O-, -S-, -N(H)C(O)N(R1)-, -NHC(O)-, -C(O)N(R’)-, -NHS(O)2N(R1)-, and -C(O)-;

[2059] each R1is, independently, at each instance, optionally substituted C1-C20 aliphatic or H;

[2060] T1and T2are each independently an optionally substituted C3-C30 aliphatic;

[2061] G1is -S(O)R3or -SR3; and

[2062] R3is optionally substituted C1-C10 aliphatic.

[2063] 8. The compound of Embodiment 7, wherein G1is -S(O)R3.

[2064] 9. The compound of Embodiments 7 or 8, wherein R3is optionally substituted Ci-Ce aliphatic.

[2065] 10. The compound of Embodiment 9, wherein R3is -(CH2)i-e-OH.

[2066] 11. The compound of Embodiment 9, wherein R3is -CH, or -CH2-CH3.

[2067] 12. The compound of any one of Embodiments 7-11, wherein G1is

[2068]

[2069]

[2070] 13. The compound of Embodiment 7, wherein G1is -SR3.

[2071] 14. The compound of Embodiment 13, wherein R3is optionally substituted Ci-Ce aliphatic.

[2072] 15. The compound of Embodiment 14, wherein R3is -(CH2)I-6-OH.The compound of any one of Embodiments 13-15, wherein G1is

[2073]

[2074] 17. A compound represented by formula IC:

[2075] L1-X1-T1G3-L3-N / /

[2076] \2- X2- T2

[2077]

[2078] or a pharmaceutically acceptable salt thereof, wherein:

[2079] L1and L2are each independently an optionally substituted C1-C30 aliphatic group;

[2080] L3is a bond or optionally substituted C1-C10 aliphatic;

[2081] X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -N(H)C(O)N(R’)-, -NHC(O)-, and -C(O)N(R1)-;

[2082] each R1is, independently, at each instance, optionally substituted C1-C20 aliphatic or H;

[2083] T1and T2are each independently an optionally substituted C3-C30 aliphatic group;

[2084]

[2085] each R30is independently selected from -SH,-OH, -NH2, -NH(CH3), -N(CHs)2, -C(0)NH2, and optionally substituted Ci-Ce aliphatic;

[2086] provided that when G3is -OH, -NH2, -NH(CH3), or -N(CH3)2, and X1or X2is a bond, -OC(O)-, or -C(O)O-, then L3is C2-C10 alkenyl, or C2-C10 alkynyl.

[2087] 18. The compound of Embodiment 17, wherein G3is selected from -OH, -NH2, -NH(CH3), -N(CHs)2,

[2088]

[2089] The compound of Embodiment 1, wherein the compound is represented by formula IIA:

[2090] O / L1—X1- T1

[2091] HO— (CH2)I.6— S-(CH2)^—

[2092]

[2093] O L2- X2- T2

[2094] IIA

[2095] or a pharmaceutically acceptable salt thereof.

[2096] The compound of Embodiment 7, wherein the compound is represented by formula IIB:

[2097] O / L1— X1— T1

[2098]

[2099] R3— S- (CH2)I_6— N

[2100] 2- X2- T2

[2101] IIB

[2102] or a pharmaceutically acceptable salt thereof.

[2103] The compound of Embodiment 17, wherein the compound is represented by formula IIC:

[2104] X1— T1

[2105] I

[2106] L

[2107] i1

[2108]

[2109] HO L2— X2T2

[2110] IIC

[2111] or a pharmaceutically acceptable salt thereof,

[2112] wherein X1and X2are each independently selected from a bond, -OC(O)-, and -C(O)O-.

[2113] The compound of Embodiment 17, wherein the compound is represented by formula IID:

[2114] X1— T1

[2115]

[2116] or a pharmaceutically acceptable salt thereof,

[2117] wherein X1and X2are each independently selected from a bond, -OC(O)- and -C(O)O-.

[2118] The compound of Embodiment 17, wherein the compound is represented by formula IIE:L1

[2119]

[2120] N'L2-X2-T2

[2121] IIE

[2122] or a pharmaceutically acceptable salt thereof,

[2123] wherein X1and X2are each independently selected from a bond, -OC(O)-, and -C(O)O-.

[2124] 24. The compound of Embodiment 17, wherein the compound is represented by formula IIF

[2125] L

[2126] i1

[2127]

[2128] or a pharmaceutically acceptable salt thereof.

[2129] 25. The compound of any one of Embodiments 1-24, wherein L1and L2are each Ci-Cio alkylene.

[2130] 26. The compound of any one of Embodiments 1-25, wherein L1and L2are each independently -(CH2)6-10-.

[2131] 27. The compound of any one of Embodiments 1-16, 19-20, or 25-26, wherein X1and X2are each independently selected from -OC(O)-, -C(O)O-, -S(O)2N(R1)-, -N(R1)S(O)2., -S(O)-, -S(O)2-, -S(O)2C(R1)2-, -OC(S)C(R1)2-, -C(R1)2C(S)O-, -S-, -N(H)C(O)N(R1)-, -NHC(O)-, -C(O)N(R1)-, -NHS(O)2N(R1)-, and -C(O)-.

[2132] 28. The compound of any one of Embodiments 1-16, 19-20, or 25-27, wherein X1and X2are each independently selected from -OC(O)-, -C(O)O-, -S(O)2N(R1)-, -N(R1)S(O)2-, and -S(O)2-.

[2133] 29. The compound ofany one of Embodiments 17, 18, or 24, wherein X1and X2are each independently selected from -OC(O)-, -C(O)O-, -N(H)C(O)N(R1)-, -NHC(O)-, and -C(O)N(R1)-.

[2134] 30. The compound of any one of Embodiments 17, 18, or 24, wherein -OC(O)-, -C(O)O-, -NHC(O)-, and -C(O)N(R1)-.

[2135] 31. The compound of any one of Embodiments 1 -30, wherein T1and T2are each independently selected from optionally substituted C2-C2o alkyl.32. The compound of any one of Embodiments 1-31, wherein T1and T2are each independently selected from:

[2136]

[2137] 33. The compound of any one of Embodiments 1-32, wherein amoiety -L'-X'-T1is selected from the group consisting of:

[2138] oT- O (CH2)6. I2O - (CH2)6.12- s- NZ— (CH _2)6.12-S-T O -11o R1o - (CH2)6.12- s- T1o^T’

[2139] ! (CH2)6. I2- (CH2)6.12— S— T1

[2140]

[2141] 34. The compound of any one of Embodiments 1-33, wherein a moiety -L2-X2-T2is selected from the group consisting of:

[2142] O -p O: (CH2)6. I2- (CH2)6.12- s- NZ— (CH2)6.12-S-T2O _ -1 o R1o — (CH2)6.12-s-T2

[2143] R1N-T2- (CH2)6.12— S— T2

[2144]

[2145] o35. The compound of any one of Embodiments 1-34, wherein a moiety -L'-X'-T1and moiety -L2-X2-T2are each independently selected from:

[2146]

[2147] 36. The compound of Embodiment 1, wherein the compound is selected from Table IB.

[2148] 37. The compound of Embodiment 7, wherein the compound is selected from Table 1A.

[2149] 38. The compound of Embodiment 17, wherein the compound is selected from Table 1C.

[2150] 39. The compound of Embodiment 1, wherein the compound is selected from Table ID.

[2151] 40. A particle comprising a compound of any one of Embodiments 1-39, and a nucleic acid.41. The particle of Embodiment 40, wherein the nucleic acid is RNA, DNA, or mixtures thereof.

[2152] 42. The particle of Embodiment 41, wherein the RNA is mRNA.

[2153] 43. The particle of Embodiment 42, wherein the RNA is modRNA, circRNA, saRNA, taRNA, or uRNA.

[2154] 44. The particle of Embodiment 40, wherein the DNA is linear DNA, plasmid DNA, minicircle DNA, nanoplasmid DNA, doggybone DNA, or a transposon.

[2155] 45. The particle of any one of Embodiments 40-44, wherein the particle further comprises one or more of a helper lipid, a polymer-conjugated lipid, or a sterol.

[2156] 46. The particle of Embodiment 45, wherein the helper lipid is a phospholipid.

[2157] 47. The particle of Embodiments 45 or 46, wherein the helper lipid is or comprises l,2-distearoyl-5«-glycero-3-phosphocholine (DSPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), 1 -palmitoyl -2 -oleoyl-sn-glycero-3 -phosphocholine (POPC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), sphingomyelins, N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), l,2-diacylglyceryl-3-O-4’-(N, N, N-trimethyl)-homoserine (DGTS), ceramides, and their derivatives.

[2158] 48. The particle of any one of Embodiments 45-47, wherein the polymer-conjugated lipid is selected from the group consisting of a poly(ethylene glycol) (PEG) -conjugated lipid, a poly(sarcosine) (pSar)-conjugated lipid, a poly(aminoethoxy ethoxy acetic acid) (pAEEA) -conjugated lipid; and a poly(2-methylaminoethoxy ethoxy acetic acid) (pMAEEA) -conjugated lipid.

[2159] 49. The particle of any one of Embodiments 45-47, wherein the polymer-conjugated lipid is a polymer conjugated lipid is selected from the group consisting of a polyethylene glycol) (PEG) -conjugated lipid, a poly(sarcosine) (pSar)-conjugated lipid, a poly(aminoethoxy ethoxy acetic acid) (pAEEA) -conjugated lipid; and a poly (2 -methylaminoethoxy ethoxy acetic acid) (pMAEEA)-conjugated lipid. In some embodiments, a PEG-lipid is selected from pegylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG) (e.g., 1,2-dimyristoyl-rac-glycero -3 -methoxypolyethylene glycol-2000 (PEG2000-DMG)), a pegylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2’,3’-di(tetradecanoyloxy)propyl-l-0-(co-methoxy(polyethoxy)ethyl)bntane-dioate (PEG-S-DMG), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino-(polyethylene glycol)-2000] (DSPE-PEG2000 amine), a pegylatedceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate, and 2,3 -di(tetradecanoxy)propyl-N-(co-methoxy(polyethoxy)ethyl)carbamate.

[2160] 50. The particle of any one of Embodiments 45-49, wherein the sterol is selected from P-sitosterol, stigmasterol, cholesterol, cholecalciferol, ergocalciferol, calcipotriol, botulin, lupeol, ursolic acid, oleanolic acid, cycloartenol, lanosterol, or a-tocopherol.

[2161] 51. The particle of any one of Embodiments 45-50, wherein the particle is characterized by an N / P ratio that is about 4 to about 16.

[2162] 52. The particle of any one of Embodiments 45-51, wherein the particle comprises:

[2163] about 30 to about 60 mol% of the compound;

[2164] about 20 to about 60 mol% of the steroid;

[2165] about 1 to about 2 mol% of the polymer-conjugated lipid; and

[2166] about 5 to about 15 mol% of the neutral lipid.

[2167] 53. A suspension comprising a dispersed phase and an aqueous phase, and wherein the dispersed phase comprises one or more particles of any one of Embodiments 45-52.

[2168] 54. A method of increasing or causing increased expression of RNA in a target in a subject, the method comprising administering to the subject a composition comprising particles of any one of Embodiments 45-52, or the suspension of Embodiment 50.

[2169] 55. The method of Embodiment 54, wherein the target is selected from the lungs, liver, spleen, heart, brain, lymph nodes, bladder, kidneys, and pancreas.

[2170] 56. A method of treating a disease, disorder, or condition in a subject comprising administering to the subject a composition comprising the particles of any one of Embodiments 42-49, or a suspension of Embodiment 53.

[2171] 57. The method of Embodiment 56, wherein the disease, disorder, or condition is an infectious disease, cancer, a genetic disorder, an autoimmune disease, or a rare disease.

[2172] 58. The method of any one of Embodiments 54-57, wherein the particle or the suspension is administered parenterally or intranasally.59. The method of Embodiment 58, wherein the particle or the suspension is administered intramuscularly, subcutaneously, intradermally, or intravenously

[2173] EXAMPLES

[2174] The Examples provided herein document and support certain aspects of the present disclosure but are not intended to limit the scope of any claim. The following non -limiting examples are provided to further illustrate certain teachings provided by the present disclosure. Those of skill in the art, in light of the present application, will appreciate that various changes can be made in the specific embodiments that are illustrated in the present Examples without departing from the spirit and scope of the present teachings. The following abbreviations may be used in the Examples below: aq. (aqueous); ACN (acetonitrile); BLI (Bioluminescent Imaging); BM (benchmark); CHOL (cholesterol); d (day or days); Da / kDa (Daltons / kiloDaltons); DCM (dichloromethane); DLS (dynamic light scattering); DMF (N, N-dimethylformamide); DIPEA (N, N-diisopropylethylamine); DMAP (4-dimethylaminopyridine); DODMA (l,2-dioleyloxy-3-dimethylaminopropane), DOPE (l,2-dioleoyl-sn-glycero-3-phosphoethanolamine); DMSO (dimethyl sulfoxide); DSPC (distearoylphosphatidylcholine); EA (ethyl acetate); ee (enantiomeric excess); equiv. (equivalent); ELSD (evaporative light scattering detector) EtOH (Ethanol); h or hr (hour or hours); Hex (hexanes); HPLC (high-performance liquid chromatography); i.m (intramuscular); i.v (intravenous); KHMDS (potassium bis(trimethylsilyl)amide); LAH (lithium aluminum hydride); LCMS (liquid chromatography-mass spectrometry); LDA (lithium diisopropylamide); LiHMDS (lithium bis(trimethylsilyl)amide); LNP (lipid nanoparticle); MeOH (methanol); min (minute or minutes); NMR (nuclear magnetic resonance); PBS (phosphate buffered saline); Pd / C (palladium on carbon); PEG-DMG (l,2-dimyristoyl-rac-glycero-3 -methoxypolyethylene glycol-2000); rb (round-bottomed); Rf (retention factor); rt or RT (room temperature); s.c (subcutaneous); SM (starting material); TEA (triethylamine); THF (tetrahydrofuran); THP (tetrahydropyran); TLC (thin layer chromatography); TsOH (p-toluenesulfonic acid or tosylic acid); and UV (ultraviolet).

[2175] Preparation of ionizable lipids

[2176] Example 1: Synthesis of BNT-102, BNT-106, BNT-114, BNT-116, and BNT-140

[2177] General Scheme 10

[2178] ^II O n

[2179] o ox. TEA II

[2180]

[2181] DCM, 0°C to rt„ 2 h

[2182] 2-butyloctan-1-ol 2-butyloctyl methanesulfonate

[2183] DMF, 85°C, 12 h

[2184] O

[2185] NaOH

[2186]

[2187] EtOH, 40°C, 2 h

[2188] 2-butyloctane-1 -thiol S-(2-butyloctyl) ethanethioate

[2189] OH

[2190] 9-bromononan-1-ol NaH MeOH, 0°C to 65°C 3 h Na2O4.2H2O H2O2 / ACOH 9-((2-butyloctyl)thio)nonan-1 -ol 2 h, 50°C 9-((2-butyloctyl)sulfonyl)nonan-1-ol

[2191] BNT-102, R = CH2-CH2-CH2-CH2-SO-CH3BNT-106, R = CH2-CH2-CH2-CH2-SO2-CH3BNT-114, R = CH2-CH2-CH2-CH2-SO2-CH2-CH2-OH BNT-140, R = CH2-CH2-CH2-CH2-NH-C=S-N(CH3)2BNT-116,

[2192]

[2193] OH Intermediate 1: 2 -Butyloctyl methane sulfonate

[2194]

[2195] A solution of2-butyloctan-l-ol (10.0 g, 53.6 mmol, 1.00 equiv.), and triethylamine (16.2 g, 160 mmol, 3.00 equiv.) in 100 ml DCM was stirred under argon at 0°C. Methanesulfonic anhydride (14.0 g, 80.4 mmol, 1.5 equiv.) was added in portions, the reaction mixture was warmed and stirred for 2 h at rt. The progress of the reaction was monitored with TLC by using solvent mixture (n-hexane / EtOAc; 4:1). The reaction mixture was neutralized with (200 ml) NaHCOs, the organic layer was separated, and the aq. Layer was further extracted with (2 X 100 ml) DCM. The combined organic phase was washed with (2 X 100 ml) brine, dried over Na2SC>4, and the solvent was evaporated under reduced pressure. The product was used in the next step without any further purification.

[2196] Intermediate 2: 2-Butyloctyl ethanethioacetate

[2197] O

[2198]

[2199] A solution of 2-butyloctyl methanesulfonate (5.00 g, 20.5 mmol, 1.00 equiv.) in 10 ml DMF was stirred under argon at rt., potassium thioacetate (7.00 g, 61.5 mmol, 3.00 equiv.) was added in portions, the reaction mixture was stirred under argon at 85°C for 12 h. The resulting reaction mixture was subsequently diluted with (200 ml) EtOAc and washed with (100 ml) 1 M HCL, (100 ml) NaHCOs, (100 ml) H2O, and (2 X 100 ml) brine. The organic phase was collected, dried over Na2SO4, and the solvent was evaporated under reduced pressure. The product was purified with column chromatography (SiCE; w-hcxanc / EtOAc = 95 / 5).

[2200] Intermediate 3: 2-butyloctane-l-thiol

[2201]

[2202] A solution of NaOH (2.00 g, 50.0 mmol, 2.00 equiv.) in 2.5 ml H2O was added dropwise to a solution of 2-butyloctyl methanethioacetate (5.00 g, 24.7 mmol, 1.00 equiv.) in 20 ml EtOH. The reaction mixture was stirred at 40°C for 2 h. The progress of the reaction was monitored with TLC by using solvent mixture (n-hexane). The reaction mixture was neutralized with 2 M HC1 at 0°C, the mixture was diluted with (100 ml) diethyl ether, washed with (2 X 100 ml) H2O, and (2 X 100 ml) brine. The organic layer was collected, dried over Na2SO4, and the solvent was evaporated under reduced pressure. The product was purified with column chromatography (SiCE; w-hcxanc).

[2203] Intermediate 4: 9-((2-Butyloctyl)thio)nonan-l-ol

[2204]

[2205] In a 3-neck round botom flask, 60 ml MeOH was added under argon, NaH 60% (780 mg, 19.7 mmol, 1.00 equiv.) was added in portions under argon at 0°C for 10 min, then 2 -butyloctane- 1 -thiol (4.00 g, 19.7 mmol, 1.00 equiv.) was added under argon at rt., and the reaction mixture was stirred for 10 min, after that 9-bromonona-l-ol (4.40 g, 19.7 mmol, 1.00 equiv.) was added under argon, the reaction mixture was stirred for 3 h at 65°C. The solvent was evaporated under reduced pressure, dilute the mixture was (200 ml) n-hexane, NaH was filtered, the filtrate was collected, washed (2 X 100 ml) brine. The organic layer was collected, dried over Na2SC>4, and the solvent was evaporated under reduced pressure. The product was purified with column chromatography (SiCh; w-hcxanc / EtOAc: 4 / 1).

[2206] Intermediate 5: 9-((2-Butyloctyl)sulfonyl)nonan-l-ol

[2207]

[2208] A solution of 9-((2-butyioctayl)thio)nonan-l-ol (4.00 g, 11.5 mmol, 1.00 equiv.) in 10 ml glacial AcOH was stirred at rt. H2O230% (2.00 ml, 92.0 mmol, 8.00 equiv.) was added dropwise to the reaction followed by the addition of Na2WC>4.2H2O as (5%, as a catalyst), and the reaction mixture was stirred for 2 h at 50°C. The reaction mixture was neutralized with (10 ml) NaHCOs at 0°C, the mixture was diluted (100 ml) EtOAc, washed with (2 X 50 ml) H2O, then with (2 X 50 ml) brine. The organic layer was collected, dried over Na2SC>4, and the solvent was evaporated under reduced pressure. The product was purified with column chromatography (SiCh; w-hcxanc / EtOAc: 1 / 1).

[2209] Intermediate 6: 9-((2-Butyloctyl)sulfonyl)nonanal

[2210]

[2211] A solution of 9-((2-butyloctyl)sulfonyl)nonan-l-ol (3.00 g, 8.00 mmol, 1.00 equiv.) in 10 ml DCM was stirred at rt., DMP (5.00 g, 12.0 mmol, 1.50 equiv.) was added to the reaction mixture, then H2O (159 pl, 8.80 mmol, 1.10 equiv.) was added dropwise over 30 min. The reaction was quenched with 10 ml H2O, diluted with (100 ml) DCM, washed with (2 X 100 ml) NaHCOs, (2 X 100 ml) brine. The organic layer was collected, dried over Na2SC>4, and the solvent was evaporated under reduced pressure. The product was purified with column chromatography (SiCh; w-hcxanc / EtOAc: 4 / 1).

[2212] Synthesis of BNT-102, BNT-106, BNT-114, BNT-116, and BNT-140BNT-102, R = CH2-CH2-CH2-CH2-SO-CH3

[2213] BNT-106, R = CH2-CH2-CH2-CH2-SO2-CH3 BNT-114, R = CH2-CH2-CH2-CH2-SO2-CH2-CH2-OH BNT-140, R = CH2-CH2-CH2-CH2-NH-C=S-N(CH3)2 BNT-116, R N NH

[2214]

[2215] OH A solution of 9-((2-butyloctyl)sulfonyl)nonanal (2.00 equiv.), NH2-R ( 1.00 equiv.) in 5 ml DCM was stirred under argon for 30 min. at rt., then NaBH(OAc)s (4.00 equiv.) was added to the reaction mixture under argon and the reaction mixture was stirred for extra 12 h at rt. The reaction was diluted with (50 ml) DCM, washed with (3 X 50 ml) NaHCCE. (2 X 50 ml) brine. The organic layer was collected, dried over Na2SC>4, and the solvent was evaporated under reduced pressure. The product was purified with column chromatography (SiCE; from w-hcxanc / EtOAc / TEA: 4 / 1 / 1% to w-hcxanc / EtOAc / TEA: 1 / 1 / 1% for BNT-102 & 106). The product was purified with column chromatography (SiCh; from w-hcxanc / EtOAc / TEA: 1 / 1 / 1 %to EtOAc / TEA; 99% / l % for BNT-114, -116 & -140) to afford a colorless oil compound.

[2216] For example, for preparation of BNT-116: the starting material is 4-(2-aminoethyl)-1H-imidazol-5-yl)methanol dihydrochloride, which should be neutralized with (2.00 equiv.) DIEA in DCM before addition to the 9-((2-butyloctyl)sulfonyl)nonanal.

[2217] Example 2: Synthesis of BNT-47, 48, 93, 94, 97, 98, 101, 103, 104, 105, 107, 108, 109, 110, 111, 117, 129, 130, 131, 132, 133, 134, 135, 136, 138, 139 and BL-222

[2218] General Scheme 2O SOCI2

[2219] DMF, DCM

[2220] BNT-47, R = CH2-CH2-SO-CH3

[2221] BNT-48, R = CH2-CH2-SO-CH2-CH3

[2222] BNT-101, R = CH2-CH2-CH2-SO-CH3SH

[2223] BNT-103, R = CH2-CH2-CH2-CH2-SO2-CH3HN\,

[2224] BNT-108, R = CH2-CH2-CH2-S-CH2-CH2-OH BNT-97, R = BNT-117, R =

[2225] BNT-109, R = CH2-CH2-CH2-SO-CH2-CH2-OH

[2226] BNT-110, R = CH2-CH2-CH2-CH2-SO2-CH2-CH2-OH

[2227] BNT-111, R = CH2-C≡C-CH2-OH

[2228] BNT-129, R = CH2-CH2-CH2-SO2-CH2-CH2-OH

[2229] BNT-130, R = CH2-CH2-CH2-CH2-CH2-SO2-CH2-CH2-OH

[2230] BNT-131, R = CH2-CH2-CH2-CH2-CH2-SO2-CH2-CH2-CH2-OH

[2231] BNT-132, R = CH2-CH2-CH2-CH2-SO2-CH2-CH2-CH2-CH2-CH2-OH

[2232] BNT-133, R = CH2-CH2-CH2-CH2-SO2-CH2-CH2-CH2-CH2-OH

[2233] BNT-134, R = CH2-CH2-CH2-CH2-CH2-SO2-CH2-CH2-CH2-OH

[2234] BNT-135, R = CH2-CH2-CH2-SO2-CH3

[2235] BNT-136, R = CH2-CH2-CH2-CH2-CH2-CH2-SO2-CH2-CH2-CH2-CH2-OH

[2236] BNT-138, R = CH2-CH2-CH2-CH2-SO2-CH2-CH2-CH2-OH

[2237] BNT-139, R = CH2-CH2-CH2-CH2-CH2-SO2-CH2-CH2-CH2-CH2-OH

[2238]

[2239] BL-222, R = CH2-CH=CH-CH2-N(CH3)2 (E-configuration) Intermediate 1: 2-Butyloctanoyl chloride

[2240] O

[2241]

[2242] Thionyl chloride (1.00 equiv.) was added dropwise to 20 ml DCM at 0°C. After 10 min. 2-butyloctanoic acid (1.00 equiv.) was added to the reaction mixture, later, few drops of DMF were added, then the reaction mixture was stirred under argon for 2 h at 40 °C. The progress of the reaction was monitored by using the TLC with solvent mixture (w-hexane / EtOAc; 9 / 1). The solvent was evaporated under reduced pressure and the compound was dried under vacuum for 2 h. The product was directly used for the next step without any further purification.

[2243] Intermediate 2: 9-Bromononyl-2-butyloctanoateo

[2244]

[2245] In a one neck round botom flask, 2-butyloctanoyl chloride and 9-bromononan-l-ol were stirred under reduced pressure 50 mbar at 50 °C for 12 h. The product was purified with column chromatography (SiO2; from w-hexane / EtOAc; 4 / 1) to afford a colorless oil compound.

[2246] Intermediate 3: 9-Oxononyl-2-butyloctanoate

[2247] O

[2248]

[2249] A solution of 9-bromononyl-2-butyloctanoate (1.00 equiv.) and trimethylamine N-oxide (5.00 equiv.) in DMSO (1.50 equiv.) was stirred under argon at 0 °C for 3 h. The progress of the reaction was monitored by using the TLC with solvent mixture (w-hexane / EtOAc; 4 / 1). The reaction was quenched with NaHCOs (50 ml). The product was extracted with chloroform (3X 50 ml), The organic layer was collected, dried over Na2SC>4, and the solvent was evaporated under reduced pressure. The product was purified with column chromatography (SiCE; from w-hcxanc / EtOAc: 4 / 1) to afford a colorless oil compound.

[2250] Synthesis of BNT-47, 48, 101, 103, 108, 109, 110, 111, 129, 130, 131, 132, 133, 134, 135, 136, 138, 139 and BL-222

[2251]

[2252] BNT-47, R = CH2-CH2-SO-CH3

[2253] BNT-48, R = CH2-CH2-SO-CH2-CH3

[2254] BNT-101, R = CH2-CH2-CH2-SO-CH3

[2255] BNT-103, R = CH2-CH2-CH2-CH2-SO2-CH3

[2256] BNT-108, R = CH2-CH2-CH2-S-CH2-CH2-OH

[2257] BNT-109, R = CH2-CH2-CH2-SO-CH2-CH2-OH

[2258] BNT-110, R = CH2-CH2-CH2-CH2-SO2-CH2-CH2-OH

[2259] BNT-111, R = CH2-C=C-CH2-OH

[2260] BNT-129, R = CH2-CH2-CH2-SO2-CH2-CH2-OH

[2261] BNT-130, R = CH2-CH2-CH2-CH2-CH2-SO2-CH2-CH2-OH

[2262] BNT-131, R = CH2-CH2-CH2-CH2-CH2-SO2-CH2-CH2-CH2-OH

[2263] BNT-132, R = CH2-CH2-CH2-CH2-SO2-CH2-CH2-CH2-CH2-CH2-OH BNT-133, R = CH2-CH2-CH2-CH2-SO2-CH2-CH2-CH2-CH2-OH

[2264] BNT-134, R = CH2-CH2-CH2-CH2-CH2-SO2-CH2-CH2-CH2-OH

[2265] BNT-135, R = CH2-CH2-CH2-SO2-CH3

[2266] BNT-136, R = CH2-CH2-CH2-CH2-CH2-CH2-SO2-CH2-CH2-CH2-CH2-OH BNT-138, R = CH2-CH2-CH2-CH2-SO2-CH2-CH2-CH2-OH

[2267] BNT-139, R = CH2-CH2-CH2-CH2-CH2-SO2-CH2-CH2-CH2-CH2-OH BL-222, R = CH2-CH=CH-CH2-N(CH3)2(E-configuration)A solution of 9-oxononyl-2 -butyloctanoate (2.00 equiv.), NH2-R (1.00 equiv.) in 5 ml DCM was stirred under argon for 30 min. at rt., then NaBH(OAc)s (4.00 equiv.) was added to the reaction mixture under argon and the reaction mixture was stirred for extra 12 h at rt. The reaction was diluted with (50 ml) DCM, washed with (3 X 50 ml) NaHCOs, (2 X 50 ml) brine. The organic layer was collected, dried overNa2SC>4, and the solvent was evaporated under reduced pressure. The product was purified with column chromatography (SiCE; from w-hcxanc / EtOAc / TEA: 4 / 1 / 1% to w-hcxanc / EtOAc / TEA: 1 / 1 / 1% for BNT-47, 48, 101, 103, 106 & 135). The product was purified with column chromatography (SiCh; from n-hexane / EtOAc / TEA; l / l / l%to EtOAc / TEA; 99% / l% for BNT-108, 109, 110, 111, 129, 130, 131, 132, 133, 134, 136, 138, 139 & BL-222) to afford a colorless oil compound.

[2268] Synthesis of BNT-93, 9497, 98, 104, 105, 107, and 117

[2269] BNT-104, R =

[2270]

[2271] A solution of 9-oxononyl-2 -butyloctanoate (2.00 equiv.), NH2-R (1.00 equiv.) and DIEA (2.00 equiv.) in 5 ml DCM was stirred under argon for 30 min. at rt., then NaBH(OAc)3 (4.00 equiv.) was added to the reaction mixture under argon and the reaction mixture was stirred for extra 12 h at rt. The reaction was diluted with (50 ml) DCM, washed with (3 X 50 ml) NaHCOs, (2 X 50 ml) brine. The organic layer was collected, dried over Na2SC>4, and the solvent was evaporated under reduced pressure. The product was purified with column chromatography (SiCE; from w-hcxanc / EtOAc / TEA: 4 / 1 / 1% to w-hcxanc / EtOAc / TEA: 1 / 1 / 1% for BNT-92, 93, 97, 98 & 117). The product was purified with column chromatography (SiCE; from n-hexane / EtOAc / TEA; l / l / l%to EtOAc / TEA; 99% / l% for BNT-104, 105 & 107) to afford a colorless oil compound.The following compounds were prepared using the general schemes and procedures described herein. Table 2.1

[2272] Cpd. No Calculated Mass Measured Mass [M+H]+

[2273] BNT-47 771.64 772.27

[2274] BNT-48 769.66 770.30

[2275] BNT-101 769.66 770.30

[2276]

[2277] Example 3: Synthesis of BNT-45 and BNT-91

[2278] General Scheme 3

[2279] H2N-R DCM, 12 h, rt NaBH(OAc)3

[2280] BNT-45, R = CH2-CH2-SO-CH3 BNT-91, R = CH2-CH2-SO2-CH3

[2281] Intermediate 1: 2-Hexyldecanoyl chloride

[2282]

[2283] Thionyl chloride (1.00 equiv.) was added dropwise to 20 ml DCM at 0°C. After 10 min. 2-hexyloctanoic acid (1.00 equiv.) was added to the reaction mixture, later, a few drops of DMF were added, then the reaction mixture was stirred under argon for 2 h at 40 °C. The progress of the reaction was monitored by using the TLC with solvent mixture (w-hcxanc / EtOAc: 9 / 1). The solvent was evaporated under reducedpressure and the compound was dried under vacuum for 2 h. The product was directly used for the next step without any further purification.

[2284] Intermediate 2: 6-Bromohexyl-2-hexyldecanoate

[2285] O

[2286]

[2287] In a one neck round bottom flask, 2-hexyldecanoyl chloride and 6-bromohexane-l-ol were stirred under reduced pressure 50 mbar at 50 °C for 12 h. The product was purified with column chromatography (SiO2; from n-hexane / EtOAc; 4 / 1) to afford a colorless oil compound.

[2288] Intermediate 3: 6-Oxohexyl-2-hexyldecanoate

[2289] O

[2290]

[2291] A solution of 6-bromohexyl-2-hexyldecanoate (1.00 equiv.) and trimethylamine N-oxide (5.00 equiv.) in DMSO (1.50 equiv.) was stirred under argon at 0 °C for 3 h. The progress of the reaction was monitored by using the TLC with solvent mixture (n-hexane / EtOAc: 4 / 1). The reaction was quenched with NaHCO3(50 ml). The product was extracted with chloroform (3X 50 ml), The organic layer was collected, dried over Na2SC>4, and the solvent was evaporated under reduced pressure. The product was purified with column chromatography (SiO2; from n-hexane / EtOAc: 4 / 1) to afford a colorless oil compound.

[2292] Synthesis of BNT-45 and BNT-91

[2293] H2N-R NaBH(OAc)36-oxohexyl 2-hexyldecanoate DCM 12 h rt

[2294]

[2295] BNT-45, R = CH2-CH2-SO-CH3 BNT-91, R = CH2-CH2-SO2-CH3 A solution of 6-oxohexyl-2 -hexyldecanoate (2.00 equiv.), NH2-R (1.00 equiv.) in 5 ml DCM was stirred under argon for 30 min. at rt., then NaBH(OAc)3 (4.00 equiv.) was added to the reaction mixture under argon and the reaction mixture was stirred for extra 12 h at rt. The reaction was diluted with (50 ml) DCM, washed with (3 X 50 ml) NaHCOs, (2 X 50 ml) brine. The organic layer was collected, dried over Na2SO4, and the solvent was evaporated under reduced pressure. The product was purified with column chromatography (SiO2; from n-hexane / EtOAc / TEA: 4 / 1 / 1% to n-hexane / EtOAc / TEA: 1 / 1 / 1%) to afford a colorless oil compound.The following compounds were prepared using the general schemes and procedures described herein. Table 3.1

[2296] Cpd. No Calculated Mass Measured Mass [M+H]+

[2297] BNT-45 783.68 784.32

[2298] BNT-91 799.67 800.32

[2299]

[2300] Example 4: Synthesis of BNT-119, 121, 122 and 141

[2301] General Scheme 4

[2302] O O

[2303] S'O'Sx.TEA

[2304] 0 0 DCM, 0°C to rt., 2 h

[2305] 2-hexyldecan-1-ol

[2306] O

[2307] NaOH

[2308]

[2309] EtOH, 40°C, 2 h

[2310] 2-hexyldecane-1 -thiol

[2311] S-(2-hexyldecyl)

[2312] ethanethioate

[2313]

[2314] 7-bromoheptan-1 -ol

[2315] NaH MeOH, 0°C to 65°C

[2316] 3 hNa2O4.2H2O

[2317] H2O2 / ACOH

[2318]

[2319] 6'0

[2320] 7-((2-hexyldecyl)thio)heptan-1-ol 2 h, 50°C 7-((2-hexyldecyl)sulfonyl)heptan-1-ol

[2321] DMP, H2O DCM, 1 h, rt. 73 ll ll

[2322] 6'6 7-((2-hexyldecyl)sulfonyl)heptanal

[2323] BNT-119, CH2-CH2-CH2-CH2-SO-CH3BNT-121, CH2-CH2-CH2-CH2-SO2-CH2-CH2-OH BNT-141, R = CH2-CH2-CH2-CH2-NH-CO-N(CH3)2BNT-122,

[2324]

[2325] Intermediate 1: 2-Hexyldecyl methanesulfonate

[2326] O

[2327] 11

[2328]

[2329] A solution of 2 -hexyldecane -l-ol (10.0 g, 53.6 mmol, 1.00 equiv.), and triethylamine (16.2 g, 160 mmol, 3.00 equiv.) in 100 ml DCM was stirred und argon at 0°C. Methane sulfonic anhydride (14.0 g, 80.4 mmol, 1.5 equiv.) was added in portions, the reaction mixture was warmed and stirred for 2 h at rt. The progress of the reaction was monitored with TLC by using solvent mixture (w-hexane / EtOAc; 4:1). The reaction mixture was neutralized with (200 ml) NaHCOs, the organic layer was separated, and the aq. Layer was further extracted with (2 X 100 ml) DCM. The combined organic phase was washed with (2 X 100 ml) brine, dried over Na2SO4, and the solvent was evaporated under reduced pressure. The product was used in the next step without any further purification.

[2330] Intermediate 2: 2-Hexyldecane methanethioacetate

[2331] O

[2332]

[2333] A solution of 2-hexyldecyl methanesulfonate (5.00 g, 20.5 mmol, 1.00 equiv.) in 10 ml DMF was stirred under argon at rt., potassium thioacetate (7.00 g, 61.5 mmol, 3.00 equiv.) was added in portions, the reaction mixture was stirred under argon at 85°C for 12 h. The resulting reaction mixture was subsequently diluted with (200 ml) EtOAc and washed with (100 ml) 1 M HCL, (100 ml) NaHCOs, (100 ml) H2O, and (2 X 100ml) brine. The organic phase was collected, dried over Na2SC>4, and the solvent was evaporated under reduced pressure. The product was purified with column chromatography (SiCE; w-hcxanc / EtOAc = 95 / 5).

[2334] Intermediate 3: 2-Hexyldecane-l-thiol

[2335]

[2336] A solution of NaOH (2.00 g, 50.0 mmol, 2.00 equiv.) in 2.5 ml H2O was added dropwise to a solution of 2 -hexyldecane methanethioacetate (5.00 g, 24.7 mmol, 1.00 equiv.) in 20 ml EtOH. The reaction mixture was stirred at 40°C for 2 h. The progress of the reaction was monitored with TLC by using solvent mixture (n-hexane). The reaction mixture was neutralized with 2 M HCl at 0°C, the mixture was diluted with (100 ml) diethyl ether, washed with (2 X 100 ml) H2O, and (2 X 100 ml) brine. The organic layer was collected, dried over Na2SC>4, and the solvent was evaporated under reduced pressure. The product was purified with column chromatography (SiCE; w-hcxanc).

[2337] Intermediate 4: 7-((2-Hexyldecyl)thio)heptan-l-ol

[2338]

[2339] In a 3-neck round bottom flask, 60 ml MeOH was added under argon, NaH 60% (780 mg, 19.7 mmol, 1.00 equiv.) was added in portions under argon at 0°C for 10 min, then 2-hexyldecane-l-thiol (4.00 g, 19.7 mmol, 1.00 equiv.) was added under argon at rt., and the reaction mixture was stirred for 10 min, after that 7-bromoheptan-l-ol (4.40 g, 19.7 mmol, 1.00 equiv.) was added under argon, the reaction mixture was stirred for 3 h at 65°C. The solvent was evaporated under reduced pressure, dilute the mixture was (200 ml) w-hcxanc. NaH was filtered, the filtrate was collected, washed (2 X 100 ml) brine. The organic layer was collected, dried over Na2SC>4, and the solvent was evaporated under reduced pressure. The product was purified with column chromatography (SiCE; w-hcxanc / EtOAc: 4 / 1).

[2340] Intermediate 5: 7-((2-Hexyldecyl)sulfonyl)heptan-l-ol

[2341]

[2342] A solution of 7-((2-hexyldeccyl)thio)heptan-l-ol (4.00 g, 11.5 mmol, 1.00 equiv.) in 10 ml glacial AcOH was stirred at rt. H2O230% (2.00 ml, 92.0 mmol, 8.00 equiv.) was added dropwise to the reaction followed by the addition of Na2WC>4.2H2O as (5%, as a catalyst), and the reaction mixture was stirred for 2 h at 50°C. The reaction mixture was neutralized with (10 ml) NaHCOs at 0°C, the mixture was diluted (100 ml) EtOAc, washed with (2 X 50 ml) H2O, then with (2 X 50 ml) brine. The organic layer was collected, dried over Na2SC>4, and the solvent was evaporated under reduced pressure. The product was purified with column chromatography (SiCE; w-hcxanc / EtOAc: 1 / 1).Intermediate 6: 7-((2-Hexyldecyl)sulfonyl)heptanal

[2343] O

[2344]

[2345] d / S<b

[2346] A solution of 7-((2-hexyldecyl)sulfonyl)heptan-l-ol (3.00 g, 8.00 mmol, 1.00 equiv.) in 10 ml DCM was stirred at rt., DMP (5.00 g, 12.0 mmol, 1.50 equiv.) was added to the reaction mixture, then H2O (159 pl, 8.80 mmol, 1.10 equiv.) was added dropwise over 30 min. The reaction was quenched with 10 ml H2O, diluted with (100 ml) DCM, washed with (2 X 100 ml) NaHCOs, (2 X 100 ml) brine. The organic layer was collected, dried over Na2SC>4, and the solvent was evaporated under reduced pressure. The product was purified with column chromatography (SiCE; w-hcxanc / EtOAc: 4 / 1).

[2347] Synthesis of BNT-119, 121, 122 & 141

[2348] H2N-R NaBH(OAc)3DCM, 12 h, rt.

[2349] 7-((2-hexyldecyl)sulfonyl)heptanal BNT-119, R = CH2-CH2-CH2-CH2-SO-CH3 BNT-121, R = CH2-CH2-CH2-CH2-SO2-CH2-CH2-OH BNT-141, R = CH2-CH2-CH2-CH2-NH-CO-N(CH3)2 BNT-122, R =

[2350]

[2351] OH A solution of 7-((2-hexyldecyl)sulfonyl)heptanal (2.00 equiv.), NH2-R (1.00 equiv.) in 5 ml DCM was stirred under argon for 30 min. at rt., then NaBH(OAc)3 (4.00 equiv.) was added to the reaction mixture under argon and the reaction mixture was stirred for extra 12 h at rt. The reaction was diluted with (50 ml) DCM, washed with (3 X 50 ml) NaHCOs, (2 X 50 ml) brine. The organic layer was collected, dried over Na2SC>4, and the solvent was evaporated under reduced pressure. The product was purified with column chromatography (SiCE; from w-hcxanc / EtOAc / TEA: 4 / 1 / 1% to w-hcxanc / EtOAc / TEA: 1 / 1 / 1% for BNT- 119). The product was purified with column chromatography (Si O2I from w-hcxanc / EtOAc / TEA: 1 / 1 / 1 %to EtOAc / TEA; 99% / l% for BNT-121, 122 & 141) to afford a colorless oil compound.

[2352] For BNT-122: the starting material is 4-(2 -aminoethyl)- l / / -imidazol-5-yl)mcthanol dihydrochloride, which should be neutralized with (2.00 equiv.) DIEA in DCM before addition to the 9-((2-butyloctyl)sulfonyl)nonanal.

[2353] Characterization of Ionizable Lipids

[2354] Characterization Methods

[2355] Particle Size MeasurementAnalysis of particle size was performed by dynamic light scattering (DLS) using a DynaPro Plate Reader II (Wyatt, Dernbach, Germany). LNP formulations were diluted to 0.005 mg / mL in IX PBS and the 120 pL of diluted sample were measured in triplicate in a 96- well plate. From the measurements, size (Zaverage), and polydispersity indices (PDI) were calculated from the cumulant analysis using Dynamics 7.8.1.3 software.

[2356] Measurement of Zeta Potential (electrophoretic mobility)

[2357] Zeta potential of particles was determined by photon correlation spectroscopy using particles sizer (zeta potential / particle sizer, NicompTM 380 ZLS, Santa Barbara, CA, USA) with an E-field strength of 4 V / cm and an electrode spacing of 0.4 cm in a plastic cuvette. LNP formulations were diluted 1:26 in 0.1XPBS for zeta potential measurement. The electrostatic mobility was converted to the zeta potential using the Helmholtz-Smoluchowski equation. All measurements were carried out at a temperature of 23 °C.

[2358] mRNA Accessibility Method

[2359] RNA accessibility and total RNA concentration in the formulations was measured by a modified Quant-iT RiboGreen RNA assay (Invitrogen, Carlsbad, CA). LNP samples were diluted in lx TE buffer pH 7.4 to a mRNA concentration between 2 and 5 ng / pL. Accessible mRNA was measured by diluting the sample in lx TE, and the total RNA amount was quantified by diluting the sample in 2% Triton X-100 (VWR International GmbH, Darmstadt, Germany). Ribogreen reagent was added to each sample, and the fluorescent signal was quantified in an Infinite F200PRO microplate reader (Tecan, Mannedorf, Switzerland). For the determination of total RNA concentration, a standard curve of Ribogreen fluorescence versus mRNA was used between 0 - 2.5 pg / mL mRNA in 1% Triton X-100 (pH 7.4).

[2360] Agarose Gel Electrophoresis

[2361] Agarose gel electrophoresis (AGE) was performed to evaluate free RNA. The gel was poured by using 1 g agarose dissolved in 100 mL of lx TAE Buffer pH 7.4 (Tris-acetate-EDTA) (Rotiphorese® 50X TAE, Carl Roth, Karlsruhe, Germany), 1 mL of 5% Sodium hypochlorite, and 10 pL of GelRed Nucleic Acid Gel Stain (Biotium, Hayward, CA, USA). The gel was allowed to set for at least 25 min at room temperature. The gel was then placed in a gel electrophoresis tank and lx TAE running buffer (pH 7.4) was used. Before loading, the samples were incubated at 40 °C with or without 2% of Triton X- 100, for total and free RNA, respectively. The gel was run at 80 V for 40 minutes. Gel images were taken on a Chemidoc XRS imaging system (Bio-Rad, Berkeley, CA, USA).

[2362] Capillary Electrophoresis

[2363] Fragment Analyzer (mRNA integrity)

[2364] mRNA integrity is determined by capillary electrophoresis using Agilent Fragment analyzer. 10 pl of LNPs (at 0.05 mg / ml mRNA concentration) were mixed with 20 pl of 20% Triton-X-100 in 30% ethanol and incubated for 20 min at 30°C on an orbital shaker at 600 rpm. 2 pl of the solution was mixed with 18 pl of the Agilent’s high-sensitivity RNA diluent marker solution in a PCR plate and sealed with adhesive seal.The plate is incubated for 2 min at room temperature and subsequently heated to 70°C for 2 min on an orbital shaker (600 rpm). After cooling to room temperature, the plate was inserted to the Fragment Analyzer for the measurement. Data analysis has been carried out by ProSize software.

[2365] Luciferase Assay

[2366] Luciferase assays are a reliable and sensitive method to detect the expression of luciferase in live cells. In this assay, firefly luciferase catalyze the mono -oxygenation of beetle luciferin via ATP and are Mg2+-dependent. This results in emission of light in the range of 550 to 620 nm. For cell transfections, HepG2, C2C12, and RAW 264.7 cells were seeded at respective cell number of 20000 cells / well, 5 000 cells / well and 40 000 cells / well in Nunc white flat-bottom 96-well plate (Merck KGaA, Darmstadt, Germany) and centrifuged at 500 x g for 5 min. After 18-24h, the media was replaced by fresh media at 90 pl / well. DPBS and only medium were used as negative and blank control, respectively. Formulation samples were tested at mRNA assay concentration between 50 to 125 ng per well. Samples and controls were added at 10 pl / well. After 24 h, luciferase expression was determined by Bright-Glo™ Luciferase assay (Promega, Madison, WI, USA) according to manufacturer’s protocol. Briefly, the reagent was added to the cells in medium in 1: 1 (v / v) ratio, followed by an incubation of 5 min in the dark to allow for complete cell lysis. Viability was measured by CellTiter-Glo® (Promega, Madison, WI, USA) according to manufacturer’s protocol. Briefly, the reagent was added to the cells in medium in 1: 1 (v / v) ratio, followed by an incubation of 10 min in shaker followed by 20 min to allow for stabilization the signal. Alternatively, luciferase and viability was determined by ONE-Glo™ + Tox Luciferase Reporter and Cell Viability Assay (Promega GmbH, Madison, WI, USA) according to manual instructions. Briefly, 20 pl of 5X CellTiter-Fluor™ Reagent was added to the wells, mix by orbital shaking (300-500rpm for ~30 sec). After incubation for 30 min at 37°C, the fluorescence was measured with excitation wavelength at 400 nm and emission at 505 nm (viability). Then, ONE-Glo™ Reagent was added at 100 pl / well. After incubation for three minutes, the bioluminescence was measured (luciferase expression). Bioluminescence signals (photons per second [p / s] ) and fluorescence were measured using a microplate luminescence reader Infinite M200 (Tecan, Mannedorf, Switzerland). Relative luminescence was calculated by subtracting the signal of DPBS control from the sample control. Relative viability was calculated by:

[2367] Viability (%) = (RLUsample−RLUblank) / (RLUPBS−RLUblank) × 100.

[2368]

[2369] (RLUPBS−RLUblank)

[2370] Hemolysis Assay

[2371] A hemolysis assay was performed to study the hemolytic properties of the formulations. Human blood diluted in UPBS to a 20% vol / vol blood solution. In a 96-well round bottom plate, 100 pL LNPs diluted at an equivalent concentration of 0.05 mg mRNA / mL were added to 100 pL blood solution in PBS and incubated at 37 °C for 1 h (triplicates). After incubation, the plate was centrifuged at 23 °C at 500 x g for 5 min. And then, the supernatant was transferred into a clear 96-well plate and UV absorption was read at540 nm using Tecan Pro200 Plate Reader. Positive and negative controls were carried out with 0.2% Triton-X and buffer alone, respectively.

[2372] Complement Activation Assay

[2373] SC5b-9 levels were determined in vitro by Microvue SC5b-9 Plus ELISA kit (Quidel Co., San Diego, CA, USA). Briefly, samples and positive control (Cremophor El, Merck, Darmstadt, Germany; Cobra venom factor; Quidel Co., San Diego, CA, USA) and negative control (lx DPBS and Intralipid) were incubated with Normal Human Serum Complement (Innovative Research, Michigan, USA) at the ratio of 20:80 (specimen: serum) for 1 h at 37 °C. LNP formulations were incubated with human serum at a final mRNA concentration of 0.01 mg / ml, the theoretical concentration (corresponding to a dose of 0.49 mg / kg). SC5b-9 EIA kit (Quidel, San Diego, USA) was executed according to manufactures protocol.

[2374] Example 5: Preparation of Lipid Nanoparticle (LNP) Formulations

[2375] Lipid nanoparticles were prepared by mixing an aqueous phase (0.15 mg / mL mRNA diluted in 0.05 M acetate buffer, pH 4.0, or in 0.05 M citrate buffer, pH 4.0) and an organic phase (a lipid mixture of ionizable lipid:phospholipid:cholesterol: PEGylated-lipid dissolved in ethanol at a various molar fractions, with N / P=6 and a total concentration of 17.05 mM) at a 3: 1 volume ratio and 12 mL / min, using a microfluidic instrument (NanoAssemblr® Benchtop, Precision NanoSystems, Vancouver, Canada). The mixture was dialyzed against lx DPBS (GIBCO, pH 7.4) for 2-3 h in a Slide-A-Lyser 10K MWCO dialysis cassette (Thermo Fisher Scientific, Waltham, MA, USA.) In these experiments, residual ethanol was regularly controlled by osmolality measurements. The physicochemical characterization (size, polydispersity, zeta potential, RNA accessibility and total RNA concentration) was performed on the day of preparation. After complete characterization, formulations were stored at 4°C for not more than 1 day. Lipid nanoparticles were diluted in PBS to the desired RNA concentration prior to in vitro testing.

[2376] Example 6: LNP formulations comprising ionizable lipid compounds: BL-194, BNT-47, or BNT -91 The present example describes process for preparing lipid nanoparticles (LNPs) using particular compounds (e.g., BL-194, BNT-47, or BNT -91) described herein, as well as in vitro characterization and biological effects. LNPs of the present example have been prepared by the microfluidic mixing procedure described in Example 5. The structures of various LNP components are listed in Table 6.1 below. A summary of LNP formulations used in this example are presented in Table 6.2. Each of the below formulations was prepared using 100mM CB, pH 4.0 as RNA buffer, and 1X PBS as dialysis buffer.

[2377] Table 6.1. List of LNP componentsComponent Lipid Chemical Structure

[2378] z —— \ ° r°HIonizable Lipid BL- 194 _CLko °

[2379] '''''''' 0 <

[2380] / b °'

[2381] O OOT==

[2382] o V

[2383] O \o X=—'

[2384] y ° / <= Z—

[2385] Ionizable Lipid BNT-47

[2386] / °

[2387] Ionizable Lipid BNT-91

[2388] o=\

[2389] ° \ /

[2390] ° r i PEGylated- A, ^o- XX. / DMG-PEG2000 ( o= /

[2391] lipid o \

[2392] o

[2393] z— \ / —

[2394] oow== Phospholipid DSPC /

[2395] Sterol Cholesterol

[2396] I fHjT /

[2397] JT TH Y A

[2398]

[2399] Table 6.2. Example 6 LNP Formulations

[2400] Ionizable PEGylated

[2401] Ionizable Sterol Phospholipid RNA Final cone. LNP No. Lipid Lipid N / P

[2402] Lipid (mol%) (mol%) construct (mg / mL)

[2403] (mol%) (mol%)

[2404] LNP#01 BL- 194 47.5 40.7 10 1.8 6 mod. 0.05 LNP #02 BNT-47 47.5 40.7 10 1.8 6 Luciferase 0.05 LNP #03 BNT-91 47.5 40.7 10 1.8 6 mRNA 0.05

[2405]

[2406] Size Distribution of Example 6 LNPsThe sizes of the LNPs have been analyzed by DLS as described above. The hydrodynamic diameters for all formulations have been found to be below 100 nm (Figure 1). The formulations displayed narrow size distribution (PDI < 0.3) except BNT-91 LNPs. The ionizable lipids with identical methyl sulfonyl head groups (BNT-47 and BNT-91) displayed significantly different sizes and polydispersity indices due to differences in the tail group. BNT-47 LNPs, LNP #02, showed larger size with lower PDI compared to BNT-91 LNPs, LNP #03, indicating that the position of ester branching effect the hydrodynamic diameter and PDI.

[2407] Zeta Potential

[2408] The zeta (Q potential values of the formulation have been analyzed by electrophoretic light scattering (Figure 2). The formulations showed neutral to negative zeta potential values.

[2409] RNA Accessibility

[2410] The accessibility of RNA (also referred to as mRNA encapsulation efficiency) and the amount of free RNA in the LNP formulations was measured by Ribogreen Assay (Figure 3A) and Agarose gel electrophoresis (Figure 3B) methods, respectively. BL- 194 showed very high encapsulation efficiency (>85%) whereas LNPs #02 and #03 showed high accessibility (Figure 3A). Agarose gel electropherogram has also confirmed that there is significant amount of free mRNA with LNP #02 and LNP #03 formulations. (Figure 3B). In contrast, LNP #01 formulation has not showed any free mRNA as in line with Ribogreen results.

[2411] RNA Integrity

[2412] mRNA integrity of the formulations has been determined by capillary electrophoresis, fragment analyzer (Figure 4). As seen in Figure 4, negligible amount of mRNA fragmentation has been observed for formulations except LNP #02 formulations. For LNP #02 formulations, percent mRNA integrity was found to be significantly lower (-50%)

[2413] In vitro transfection of Example 6 LNPs

[2414] To investigate Example 6 ionizable lipid LNP functionality, cytotoxicity, and transfection efficiency, mRNA expression was evaluated in vitro using a skeletal muscle cell line (C2C12), a hepatocarcinoma cell line (HepG2), a human embryonic kidney cell line (HEK293T), and a murine macrophage cell line (RAW 264.7). For this purpose, the LNP formulations comprising firefly luciferase -encoding mRNA were incubated with the cells at three dosages, 12.5, 25 and 50 ng per well and luciferase expression was assessed 24 h post-incubation. In general, the formulations have not showed significant cytotoxicity at the tested conditions (Figures 5A-5D). BNT-47 and BNT-91 formulations, LNP #02 and LNP #03, respectively, showed low transfection efficiency (Figures 6A-6D) most likely due to unfavorable quality characteristics of these formulations (very high mRNA accessibility, significant amount of free mRNA, low mRNA integrity). However LNP #01 showed high transfection efficiency in dose-dependent manner.Example 7: LNP formulations comprising ionizable lipid compounds BNT-101, BNT-102, or BNT-103

[2415] The present example describes process for preparing lipid nanoparticles (LNPs) using particular lipid / compounds (e.g., BNT-101, BNT-102, or BNT-103) described herein, as well as in vitr / oQ~ characterization \ °

[2416] and biological effects. LNPs of the present example have been prepared by the microfluidic mixing Zoo

[2417] procedure described in Example 5. The structures of various LNP components a / )reo=listed in Table 7.1 below. A summary of LNP formulations used in this example are presented in Table 7.2. Each of the below formulations was prepared using 100mM CB, pH 4.0 as RNA buffer, and 1X PBS as dialysis buffer.

[2418] ( /

[2419] Table 7.1. List of LNP components

[2420] Component Lipid Chemical Struct / )ure

[2421] o

[2422] J J II

[2423] Ionizable Lipid BNT-101 o J °

[2424] 0

[2425] Ionizable Lipid BNT-102

[2426] 0 0

[2427] II11Ionizable Lipid BNT-103 J 0 < J0

[2428] o

[2429] PEGylated- DMG-PEG2000

[2430] lipid 44

[2431] o

[2432] 0

[2433] OvI Phospholipid DSPC

[2434] Hx ^° °-10

[2435] Phospholipid DOPE

[2436] 3 ~ i' ' / ' NH, D

[2437]

[2438] Component Lipid Chemical Structure

[2439] Sterol Cholesterol

[2440] i fHjT /

[2441] f TH Y A

[2442]

[2443] Table 7.2. Example 7 LNP Formulations

[2444] Ionizable PEGylated Final LNP Ionizable Sterol Phospholipid RNA

[2445] Lipid Phospholipid Lipid N / P cone. No. Lipid (mol%) (mol%) construct (mol%) (mol%) (mg / mL) LNP#04 BNT-101 47.5 40.5 DSPC 10 2 6 0.05

[2446] mod.

[2447] LNP #05 BNT-102 47.5 40.5 DSPC 10 2 6 0.05

[2448] Luciferase

[2449] LNP #06 BNT-102 47.5 40.5 DOPE 10 2 6 0.05

[2450] mRNA

[2451] LNP #07 BNT-103 47.5 40.5 DSPC 10 2 6 0.05

[2452]

[2453] Size Distribution of Example 7 LNPs

[2454] The sizes of the LNPs have been analyzed by DLS as described above and shown in Figure 7. The hydrodynamic diameters for BNT-101 (LNP #04 and #05) formulations (ionizable lipid with sulfone tail and methyl sulfoxide head) was found to be above 100 nm (large size). LNP #06 formulation with DOPE phospholipid has led to smaller size compared to its DSPC counterpart, LNP #05. PDI values was found to be < 0.3 indicating a narrow size distribution. BNT-101 and BNT-103, LNP #04 and #07, respectively, formulations have led to smaller size (109 and 66 nm, respectively). The significantly smaller size of BNT-103 formulation, LNP #07, has been attributed to the ionizable lipid’s head group, since BNT-101 and BNT-103 ionizable lipids consist of identical lipid tail.

[2455] Zeta Potential

[2456] The zeta (Q potential values of the formulation have been analyzed by electrophoretic light scattering (Figure 8). The formulations showed similar neutral to negative zeta potential values.

[2457] RNA Accessibility

[2458] The accessibility of RNA (also referred to as mRNA encapsulation efficiency) and the amount of free RNA in the LNP formulations was measured by Ribogreen Assay (Figure 9A) and Agarose gel electrophoresis (Figure 9B) methods, respectively. The formulations were able to effectively encapsulate the mRNA (Figure 9A; encapsulation efficiency > 84%). No free mRNA / negligible free mRNA has been seen as shown by AGE (Figure 9B).RNA Integrity

[2459] mRNA integrity of the formulations has been determined by capillary electrophoresis, fragment analyzer (Figure 10). As seen in Figure 10, slightly lower mRNA integrity has been observed with BNT-101 formulation, LNP #04.

[2460] In vitro transfection of Example 7 LNPs

[2461] To investigate Example 7 ionizable lipid LNP functionality, cytotoxicity, and transfection efficiency, mRNA expression was evaluated in vitro using a skeletal muscle cell line (C2C12), a hepatocarcinoma cell line (HepG2), and a murine macrophage cell line (RAW 264.7). For this purpose, the LNP formulations comprising firefly luciferase-encoding mRNA were incubated with the cells at three dosages, 12.5, 25 and 50 ng per well and luciferase expression was assessed 24 h post-incubation. In general, the formulations have not showed significant cytotoxicity at the tested conditions (Figures 11A-11C). Formulations were effectively able to transfect the cell lines that have been tested. In general, dose dependent increase in the transfection efficiency has been observed (Figures 12A-12C). BNT-101, LNP #04, showed the highest transfection efficiency in C2C12 and HepG2 cell lines. Depending on the cell line, BNT-102 DOPE, LNP #06, showed either comparable or higher transfection efficiency compared to its DSPC counterpart, LNP# 05.

[2462] Hemolysis Analysis and Complement Activation

[2463] The hemolytic effect and complement activation of BNT-101 LNPs was determined as described herein. The in vitro hemolytic effect of the BNT-101, LNP #04, has been investigated at the mRNA dose of 16 mg / kg (the mRNA dose equals to 16 mg / kg in in vivo matrix) (Figure 13A). Even at very high mRNA dosage, LNP #04 formulations showed negligible hemolysis indicating that the formulations do not affect the integrity of cell membranes of red blood cells (PBS served as negative control, Triton (0.2%) served as positive control). In addition, the effect of LNP# 04 formulations on complement activation has been determined (Figure 13B). The final complement complex concentration (SC5b-9) was found to be similar to the negative control, PBS, showing that the formulations do not lead to the complement activation (Cobra venom factor (CVF) served as positive control; untreated SO (only serum sample), untreated SI (only serum sample) and PBS as negative controls).

[2464] Example 8: LNP formulations comprising ionizable lipid compound BNT-106

[2465] The present example describes process for preparing lipid nanoparticles (LNPs) using particular lipid compounds (e.g., BNT-106) described herein, as well as in vitro characterization and biological effects. LNPs of the present example have been prepared by the microfluidic mixing procedure described in Example 5. The effect of helper lipid and N / P ratio on BNT-106 formulations have been analyzed herein. Formulations at two different N / P ratio (6 or 12) with two different helper lipids (DOPE or DSPC) were prepared by microfluidic mixing of the lipid mixture in ethanol and RNA solution in aqueous, acidic buffer. The structures of various LNP components are listed in Table 8.1 below. A summary of LNP formulationsused in this example are presented in Table 8.2. Each of the below formulations was prepared using 100mM CB, pH 4.0 as RNA buffer, and 1X PBS as dialysis buffer.

[2466] Table 8.1. List of LNP components

[2467] Component Lipid Chemical Structure

[2468] >z.— ^ D i

[2469] D

[2470] Ionizable Lipid BNT-106

[2471] / \ z°

[2472] (C w

[2473] \ / O "

[2474] / \ \zo —

[2475] o

[2476] / o

[2477] PEGylated- DMG-PEG2000

[2478] lipid 44

[2479] o

[2480] o

[2481] OxI Phospholipid DSPC

[2482] H<0o- 1 0

[2483] Phospholipid DOPE C<

[2484] 3 „ o oj

[2485] i' ^NH, D

[2486] Sterol Cholesterol

[2487] I fHX 7

[2488] f T A T A

[2489] HO*^ —

[2490]

[2491] Table 8.2. Example 8 LNP Formulations

[2492] Ionizable PEGylated Final LNP Ionizable Sterol Phospholipid RNA

[2493] Lipid Phospholipid Lipid N / P cone. No. Lipid (mol%) (mol%) construct (mol%) (mol%) (mg / mL) LNP#08 BNT-106 47.5 40.5 DOPE 10 2 6 0.05 mod.

[2494] LNP #09 BNT-106 47.5 40.5 DSPC 10 2 6 0.05 Luciferase LNP #10 BNT-106 47.5 40.5 DOPE 10 2 12 0.05 mRNA

[2495] LNP #11 BNT-106 47.5 40.5 DSPC 10 2 12 0.05

[2496]

[2497] Size Distribution of Example 8 LNPs

[2498] The sizes of the LNPs have been analyzed by DLS as described above and shown in Figure 14. The hydrodynamic diameters for all formulations have been found to be 95-122 nm. PDI values was found to be <0.2 indicating a narrow size distribution.

[2499] Zeta Potential

[2500] The zeta (Q potential values of the formulation have been analyzed by electrophoretic light scattering (Figure 15). The formulations showed similar neutral zeta potential values.

[2501] RNA Accessibility

[2502] The accessibility of RNA (also referred to as mRNA encapsulation efficiency) a in the LNP formulations was measured by Ribogreen Assay method (Figure 16). The formulations were able to effectively encapsulate the mRNA (Figure 16; encapsulation efficiency > 60%). LNP #08 formulation was found to have slightly more accessible mRNA compared to other formulations.

[2503] RNA Integrity

[2504] mRNA integrity of the formulations has been determined by capillary electrophoresis, fragment analyzer (Figure 17). As seen in Figure 17, negligible amount of mRNA fragmentation has been observed for all formulations.

[2505] In vitro transfection of Example 8 LNPs

[2506] To investigate Example 8 ionizable lipid LNP functionality, cytotoxicity, and transfection efficiency, mRNA expression was evaluated in vitro using a skeletal muscle cell line (C2C12), a hepatocarcinoma cell line (HepG2), and a murine macrophage cell line (RAW 264.7). For this purpose, the LNP formulations comprising firefly luciferase-encoding mRNA were incubated with the cells at three dosages, 12.5, 25 and 50 ng per well and luciferase expression was assessed 24 h post-incubation. In general, the formulations did not show significant cytotoxicity at the tested conditions (Figures 18A-18C). LNP #10 showed slight decrease in viability in the HepG2 and Raw 264.7 cells at 50 ng of mRNA dosage. Formulations were effectively able to transfect the cell lines that have been tested (Figures 19A-19C). LNP #08 showed the highest transfection efficiency. At both N / P ratios (6 or 12), formulations with DOPE helper lipid showed profoundly higher transfection efficiency compared to formulations with DSPC helper lipid. For DOPE formulations, increasing N / P ratio from 6 to 12 has not provided any significant increase in the luciferase expression, however for DSPC formulation transfection efficiency has increased with the increase in N / P ratio.

[2507] In vivo Bioluminescence Imaging

[2508] Based on the in vitro results, LNP #08 formulation was selected and tested in vivo. For the formulation, 6 mice (Balb / c) were injected with 2 pg / mouse RNA dose (mod. luciferase mRNA). After 6 h ventral in vivobioluminescence imaging was performed for 6 animals and 3 animals were sacrificed. Organs (heart, lung, liver, spleen, kidneys and lymph nodes (popliteal and inguinal)) were collected and ex vivo imaging of organs was performed. After 24 h, the same in vivo and ex vivo imaging were performed with the remaining 3 animals. The details of the workflow have been presented in Table 8.3.

[2509] Table 8.3 Workflow of BLI

[2510] LNP Ionizable RNA Size Timepoints

[2511] Dosage Phospholipid N / P

[2512] No. Lipid construct (n=X) Groups OH 6H 24H mod. la

[2513] LNP#08 BNT-106 Luciferase 2 μg 6 DOPE 6

[2514] lb

[2515] mRNA

[2516]

[2517]

[2518] wherein

[2519] Administration (i.v.), volume 100 μL

[2520] In vivo Imaging

[2521] Ex vivo Imaging

[2522] End of Experiment

[2523]

[2524] BLI results (Figures 20A-20D) showed that BNT-106 has showed strong signal in the liver-spleen region after 6 h (Figure 20A). The signal has decreased after 24 h as expected (Figure 20B). The highest expression has been observed in the liver and followed by spleen (Figure 20C). Approximately 80% of the signal has been found to be coming from the liver after 6 h (Figure 20D). Biodistribution has not changed significantly after 24 h (Figure 20D).

[2525] Example 9: LNP formulations comprising ionizable lipid compounds BNT-129, BNT-110, BNT-130 or BNT-131

[2526] The present example describes process for preparing lipid nanoparticles (LNPs) using particular lipid compounds (e.g., BNT-129, BNT-110, BNT-130 or BNT-131) described herein, as well as in vitro characterization and biological effects. LNPs of the present example have been prepared by the microfluidic mixing procedure described in Example 5. The effect of varying the number of methylene units between tertiary amine and sulfone group is analyzed herein. Ionizable lipids with head groups having from 3 to 6 methylene group in between the tertiary amine and sulfone group were formulated by microfluidic mixing of the lipid mixture in ethanol and RNA solution in aqueous, acidic buffer. The structures of various LNP components are listed in Table 9.1 below. A summary of LNP formulations used in this example are presented in Table 9.2. Each of the below formulations was prepared using 100mM CB, pH 4.0 as RNA buffer, and 1X PBS as dialysis buffer.

[2527] Table 9.1. List of LNP componentsComponent Lipid Chemical Structure I o \ z ——

[2528] Ionizable Lipid BNT-129,or 1°

[2529] b °'

[2530] z—

[2531] Yo- \o x=

[2532] 0 O / <= o

[2533] N / \Z''\XSX / XQHj o Ionizable Lipid BNT-110

[2534] o f

[2535] 0

[2536] J J O-b Ionizable Lipid BNT-130 o <

[2537] V

[2538] Ionizable Lipid BNT-131 Q X

[2539] ^

[2540] o r i PEGylated- DMG-PEG2000

[2541] lipid

[2542] o

[2543] Phospholipid DSPC

[2544] Sterol Cholesterol

[2545] XHXL /

[2546] f THJ

[2547] H0*AXX^X>

[2548]

[2549] Table 9.2. Example 9 LNP FormulationsIonizable PEGylated Final

[2550] LNP Ionizable Sterol Phospholipid RNA

[2551] Lipid Lipid N / P cone.

[2552] No. Lipid (mol%) (mol%) construct

[2553] (mol%) (mol%) (mg / mL) LNP BNT-129 47.5 40.5 10 2 6 0.05 #12

[2554] LNP BNT-110 47.5 40.5 10 2 6 mod. 0.05 #13

[2555] Luciferase

[2556] LNP BNT-130 47.5 40.5 10 2 6 mRNA 0.05 #14

[2557] LNP BNT-131 47.5 40.5 10 2 6 0.05 #15

[2558]

[2559] Size Distribution of Example 9 LNPs

[2560] The sizes of the LNPs have been analyzed by DLS as described above and shown in Figure 21. The hydrodynamic diameters for all LNPs have been found to be <80 nm. The size of the LNPs were found to be similar for all formulations indicating that the number of methylene unit has no significant effect on the size of the formulations. PDI values was found to be approximately <0.3 indicating a narrow size distribution.

[2561] Zeta Potential

[2562] The zeta (Q potential values of the formulation have been analyzed by electrophoretic light scattering (Figure 22). The formulations showed similar neutral to slightly negative zeta potential values.

[2563] RNA Accessibility

[2564] The accessibility of RNA (also referred to as mRNA encapsulation efficiency) a in the LNP formulations was measured by Ribogreen Assay method (Figure 23). The formulations were able to effectively encapsulate the mRNA (Figure 23; encapsulation efficiency <90%).

[2565] RNA Integrity

[2566] mRNA integrity of the formulations has been determined by capillary electrophoresis, fragment analyzer (Figure 24). As seen in Figure 24, negligible amount of mRNA fragmentation has been observed for all formulations.

[2567] In vitro transfection of Example 9 LNPs

[2568] To investigate Example 9 ionizable lipid LNP functionality, cytotoxicity, and transfection efficiency, mRNA expression was evaluated in vitro using a skeletal muscle cell line (C2C12), a hepatocarcinoma cell line (HepG2), a human embryonic kidney cell line (HEK293T) and a murine macrophage cell line (RAW264.7). For this purpose, the LNP formulations comprising firefly luciferase -encoding mRNA were incubated with the cells at three dosages, 12.5, 25 and 50 ng per well and luciferase expression was assessed 24 h post-incubation. In general, the formulations did not show significant cytotoxicity at the tested conditions (Figures 25A-25D). Formulations were effectively able to transfect the cell lines that have been tested (Figures 26A-26D). BNT-131 (with 6 methylene groups) formulation, LNP #15, showed the lowest transfection efficiency. This is attributed to the increased distance between the sulfone group and tertiary amine in the ionizable lipid head. BNT-129, BNT-110 and BNT-130 LNPs, LNP #12, LNP #13, and LNP #14, respectively, showed similar transfection efficiency (methylene groups number 3 to 5). BNT-130, LNP #14, showed slightly higher efficiency (with 5 methylene groups) indicating that 5 methylene group might be the optimum number to reach the highest transfection efficiency.

[2569] Example 10: LNP formulations comprising ionizable lipid compounds BNT-108, BNT-109, BNT-111, BNT-119, BNT-48 or BNT-121

[2570] The present example describes process for preparing lipid nanoparticles (LNPs) using particular lipid compounds (e.g., BNT-108, BNT-109, BNT-111, BNT-119, BNT-48 or BNT-121) described herein, as well as in vitro characterization and biological effects. LNPs of the present example have been prepared by the microfluidic mixing procedure described in Example 5. Based on previous findings, BNT-119 and BNT-121 LNPs were formulated with DOPE and prepared using 50 mM CB, pH 4.0 as RNA buffer, and IX PBS as dialysis buffer. Ionizable lipids with sulfone tails showed higher transfection efficiency when formulated with DOPE instead of DSPC. Other LNP components have been kept the same for all formulations. LNPs were formulated by microfluidic mixing of the lipid mixture in ethanol and RNA solution in aqueous, acidic buffer. The structures of various LNP components are listed in Table 10.1 below. A summary of LNP formulations used in this example are presented in Table 10.2. Each of the below formulations was prepared using 100mM CB, pH 4.0 as RNA buffer, and 1X PBS as dialysis buffer unless otherwise denoted above.

[2571] Table 10.1. List of LNP components

[2572] Component Lipid Chemical Structure

[2573] O

[2574] Ionizable Lipid BNT-108 0 <

[2575]

[2576] Component Lipid Chemical Structure

[2577] o o II

[2578] X- / X^OH z Ionizable Lipid BNT-109 / °~

[2579] \ °

[2580] O [

[2581] / o o

[2582] / )o=

[2583] 0

[2584] ^s^0HO'% Ionizable Lipid BNT-111 0 <

[2585] ( /

[2586] ) \

[2587] Ionizable Lipid BNT-119 ° °

[2588] o o

[2589] IIH

[2590] Ionizable Lipid BNT-48 0 <

[2591] %<° ''''~x'x''~x'''''~x''''^^ 'xxX^0H ° ° J

[2592] Ionizable Lipid BNT-121

[2593] ° \ z " u> ^ / b ° r

[2594] PEGylated- DMG-PEG2000

[2595] lipid 44

[2596] o

[2597] o

[2598] X / X. I / Phospholipid DSPC 1 0

[2599] Phospholipid DOPE

[2600] 3 ^+NH3 D

[2601]

[2602] Component Lipid Chemical Structure

[2603] Sterol Cholesterol

[2604] i fHjT /

[2605] f TH Y A

[2606]

[2607] Table 10.2. Example 10 LNP Formulations

[2608] Ionizable PEGylated Final LNP Ionizable Sterol Phospholipid RNA

[2609] Lipid Phospholipid Lipid N / P cone. No. Lipid (mol%) (mol%) construct (mol%) (mol%) (mg / mL) LNP BNT-108 47.5 40.5 DSPC 10 2 6 0.05 #16

[2610] LNP BNT-109 47.5 40.5 DSPC 10 2 6 0.05 #17

[2611] LNP BNT-111 47.5 40.5 DSPC 10 2 6 mod. 0.05 #18

[2612] Luciferase LNP BNT-119 47.5 40.5 DOPE 10 2 6 mRNA 0.05 #19

[2613] LNP 0.05 BNT-48 47.5 40.5 DSPC 10 2 6

[2614] #20

[2615] LNP 0.05 BNT-121 47.5 40.5 DOPE 10 2 6

[2616] #21

[2617]

[2618] Size Distribution of Example 10 LNPs

[2619] The sizes of the LNPs have been analyzed by DLS as described above and shown in Figure 27. The hydrodynamic diameters for all LNPs have been found to be <100 nm, with a PDI of approx. < 0.3.

[2620] Zeta Potential

[2621] The zeta (Q potential values of the formulation have been analyzed by electrophoretic light scattering (Figure 28). The formulations showed similar neutral to slightly negative zeta potential values.

[2622] RNA Accessibility

[2623] The accessibility of RNA (also referred to as mRNA encapsulation efficiency) in the LNP formulations was measured by Ribogreen Assay method (Figure 29). All formulations showed high mRNA encapsulation efficiency (mRNA encapsulation efficiency > 80%), except BNT-108 and BNT-111 formulations, LNP #16 and LNP #18, respectively.RNA Integrity

[2624] mRNA integrity of the formulations has been determined by capillary electrophoresis, fragment analyzer (Figure 30). As seen in Figure 30, mRNA integrity was found to be slightly lower for BNT-108 and BNT- 111 formulations, LNP #16 and LNP #18, respectively, most likely due to high mRNA accessibility for these formulations. All other formulations have showed high mRNA integrity.

[2625] In vitro transfection of Example 10 LNPs

[2626] To investigate Example 10 ionizable lipid LNP functionality, cytotoxicity, and transfection efficiency, mRNA expression was evaluated in vitro using a skeletal muscle cell line (C2C12), a hepatocarcinoma cell line (HepG2), and a murine macrophage cell line (RAW 264.7). For this purpose, the LNP formulations comprising firefly luciferase-encoding mRNA were incubated with the cells at three dosages, 12.5, 25 and 50 ng per well and luciferase expression was assessed 24 h post-incubation. In general, the formulations did not show significant cytotoxicity at the tested conditions (Figures 31A-31C). BNT-111, LNP #18, showed low transfection efficiency due to high mRNA accessibility / free mRNA as expected (Figures 32A-32C). BNT-119 and BNT-121 formulations, LNP #19 and LNP #21, respectively, showed the highest transfection efficiency indicating the potential benefit of sulfone groups in the lipid head and tail. Other formulations have showed moderate transfection efficiency.

[2627] Example 11: LNP formulations comprising ionizable lipid compounds BNT-110 or BNT-114 The present example describes process for preparing lipid nanoparticles (LNPs) using particular lipid compounds (e.g., BNT-110 or BNT-114) described herein, as well as in vitro characterization and biological effects. LNPs of the present example have been prepared by the microfluidic mixing procedure described in Example 5. BNT-114 has additionally been formulated with DOPE to investigate the effect of helper lipid on the BNT-114 formulation. LNPs were formulated by microfluidic mixing of the lipid mixture in ethanol and RNA solution in aqueous, acidic buffer. The structures of various LNP components are listed in Table 11.1 below. A summary of LNP formulations used in this example are presented in Table 11.2. Each of the below formulations was prepared using 100mM CB, pH 4.0 as RNA buffer, and 1X PBS as dialysis buffer.

[2628] Table 11.1. List of LNP components

[2629] Component Lipid Chemical Structure

[2630] O O II11

[2631] J J0Ionizable Lipid BNT-110

[2632] ° 1

[2633]

[2634] Component Lipid Chemical Structure

[2635] o ii

[2636] O O 1 z o

[2637] / o— Ionizable Lipid BNT-114

[2638] \ °

[2639] / o o

[2640] / )o=

[2641] o

[2642] PEGylated- '\ / O'

[2643] DMG-PEG2000

[2644] lipid 44

[2645] ( / o

[2646] o

[2647] / ) OvI Phospholipid DSPC _ _P'

[2648] °-10

[2649] Phospholipid DOPE

[2650] 3 „ i' ^NH, D

[2651] Sterol Cholesterol

[2652] i £hL 7

[2653] f TA J A

[2654]

[2655] Table 11.2. Example 11 LNP Formulations

[2656] Ionizable PEGylated Final LNP Ionizable Sterol Phospholipid RNA

[2657] Lipid Phospholipid Lipid N / P cone. No. Lipid (mol%) (mol%) construct (mol%) (mol%) (mg / mL) LNP BNT-114 47.5 40.5 DSPC 10 2 6 0.05 #22

[2658] mod.

[2659] LNP BNT-114 47.5 40.5 DOPE 10 2 6 Luciferase 0.05 #23

[2660] mRNA

[2661] LNP BNT-IIO 47.5 40.5 DSPC 10 2 6 0.05 #24

[2662]

[2663] Size Distribution of Example 11 LNPsThe sizes of the LNPs have been analyzed by DLS as described above and shown in Figure 33. The hydrodynamic diameters for BNT-114 and BNT-110 formulations with DSPC, LNP #22 and LNP #24, respectively, were found to be 130 and 76 nm, respectively, indicating that sulfone tail is leading to larger size in the LNP formulations. DOPE formulation of BNT-114, LNP #23, showed smaller size compared to DSPC formulation, LNP #22, (119 and 130 nm, respectively). PDI values was found to be approximately <0.2 indicating a narrow size distribution.

[2664] Zeta Potential

[2665] The zeta (Q potential values of the formulation have been analyzed by electrophoretic light scattering (Figure 34). The formulations showed similar neutral zeta potential values.

[2666] RNA Accessibility

[2667] The accessibility of RNA (also referred to as mRNA encapsulation efficiency) a in the LNP formulations was measured by Ribogreen Assay method (Figure 35). All formulation was able to effectively encapsulate the mRNA. BNT-114 formulated with DOPE, LNP# 23, showed slightly lower encapsulation efficiency compared to other formulations (68%).

[2668] RNA Integrity

[2669] mRNA integrity of the formulations has been determined by capillary electrophoresis, fragment analyzer (Figure 36). As seen in Figure 36, negligible amount of mRNA fragmentation has been observed for all formulations.

[2670] In vitro transfection of Example 11 LNPs

[2671] To investigate Example 11 ionizable lipid LNP functionality, cytotoxicity, and transfection efficiency, mRNA expression was evaluated in vitro using a skeletal muscle cell line (C2C12), a hepatocarcinoma cell line (HepG2), a human embryonic kidney cell line (HEK293T) and a murine macrophage cell line (RAW 264.7). For this purpose, the LNP formulations comprising firefly luciferase -encoding mRNA were incubated with the cells at three dosages, 12.5, 25 and 50 ng per well and luciferase expression was assessed 24 h post-incubation. In general, the formulations did not show significant cytotoxicity at the tested conditions (Figures 37A-37D). Formulations were effectively able to transfect the cell lines that have been tested (Figures 38A-38D). BNT-114 formulated with DOPE, LNP #23, showed higher transfection efficiency compared to BNT-114 formulated with DSPC, LNP #22. Without being bound to any particular theory or conclusion, this result also indicates that sulfone -branched lipids show higher transfection efficiency when formulated with DOPE as a helper lipid (instead of DSPC). BNT-110 formulated with DSPC, LNP #24, showed slightly higher transfection efficiency than BNT-114 formulated with DOPE, LNP #23, on HepG2, Raw 264.7 and Hek 293 cell lines; however for C2C12 cells, BNT-114 formulated with DOPE, LNP #23, showed the highest transfection efficiency. Without being bound to any particulartheory or conclusion, this may be attributed to the different affinity of the ester and sulfone tails to the cell lines. Sulfone groups might potentially lead to differed targeting / cell-receptor interactions.

[2672] Example 12: LNP formulations comprising ionizable lipid compounds BNT-132, BNT-133, BNT-134, BNT-135, BNT-136, BNT-138, BNT-139, BNT-140, or BNT-141

[2673] The present example describes process for preparing lipid nanoparticles (LNPs) using particular lipid compounds (e.g., BNT-132, BNT-133, BNT-134, BNT-135, BNT-136, BNT-138, BNT-139, BNT-140, or BNT-141) described herein, as well as in vitro characterization and biological effects. The effect of varying the position of the sulfone group is analyzed herein. LNPs of the present example have been prepared by the microfluidic mixing procedure described in Example 5. LNPs were formulated by microfluidic mixing of the lipid mixture in ethanol and RNA solution in aqueous, acidic buffer. The structures of various LNP components are listed in Table 12.1 below. A summary of LNP formulations used in this example are presented in Table 12.2. Each of the below formulations was prepared using 50mM AB, pH 4.0 as RNA buffer, and IX PBS as dialysis buffer. 07= / \

[2674] ° / \

[2675] / 2°=

[2676] Table 12.1. List of LNP components o 2

[2677] Component Lipid Chemical Structure

[2678] A V

[2679] Ionizable Lipid BNT-132

[2680] 01

[2681] J U)

[2682] 6^

[2683] A V

[2684] o Ionizable Lipid BNT-133 I o [

[2685] Ionizable Lipid BNT-134

[2686]

[2687] Component Lipid Chemical Structure / z — TZ

[2688] Ionizable Lipid BNT-135

[2689] ^ Z

[2690] \ \ / °

[2691] /

[2692] \z CO°

[2693] > \C w —

[2694] Ionizable Lipid BNT-136 / ° / \ '

[2695] ° \ / /

[2696] ° 7= ° / \ \ /

[2697] Ionizable Lipid BNT-138 ° \ /

[2698] \o= / o \ o \ /

[2699] / o° 2=

[2700] \ o

[2701] z z

[2702] z

[2703] Ionizable Lipid BNT-139

[2704] ' z —

[2705] 3 ^'o CO

[2706] H \ 1zo

[2707] o o T 1 00 0I s o I nizable Lipid BN - 4 o I I _ _

[2708] BNT-141 Ionizable Lipid

[2709] H'.>OHH I \ PEGylated- C16-Ceramide-Ho '45PEG2000 lipid

[2710] 0

[2711]

[2712] Component Lipid Chemical Structure

[2713] 0

[2714] oxI Phospholipid DSPC

[2715] H> T0O_ 1 0

[2716] Sterol Cholesterol

[2717] I fHX /

[2718] f TAT A

[2719] HO*^ —

[2720]

[2721] Table 12.2. Example 12 LNP Formulations

[2722] Ionizable PEGylated Final LNP Ionizable Sterol Phospholipid RNA

[2723] Lipid Phospholipid Lipid N / P cone. No. Lipid (mol%) (mol%) construct (mol%) (mol%) (mg / mL) BM Benchmark

[2724] 47.5 40.5 DSPC 10 2 6 0.05 LNP (BM)

[2725] LNP BNT-132 47.5 40.5 DSPC 10 2 6 0.05 #25

[2726] LNP BNT-133 47.5 40.5 DSPC 10 2 6 0.05 #26

[2727] LNP BNT-134 47.5 40.5 DSPC 10 2 6 0.05 #27

[2728] LNP BNT-135 47.5 40.5 DSPC 10 2 6 mod. 0.05 #28

[2729] Luciferase LNP BNT-136 47.5 40.5 DSPC 10 2 6 mRNA 0.05 #29

[2730] LNP BNT-138 47.5 40.5 DSPC 10 2 6 0.05 #30

[2731] LNP BNT-139 47.5 40.5 DSPC 10 2 6 0.05 #31

[2732] LNP BNT-140 47.5 40.5 DSPC 10 2 6 0.05 #32

[2733] LNP BNT-141 47.5 40.5 DSPC 10 2 6 0.05 #33

[2734]

[2735] Size Distribution of Example 12 LNPsThe sizes of the LNPs have been analyzed by DLS as described above and shown in Figure 39. The hydrodynamic diameter of BNT-134 formulation, LNP #27, was found to be 130 nm and BNT-139 formulation, LNP #31, was found to be 117 nm. Whereas all other formulations showed a small size range from 70 to 96 nm, which is a little bit bigger than the benchmark (BM) formulation, BM LNP (59 nm). The formulations have showed PDI values range from 0.10 to 0.26, except BNT-136 formulation, LNP #29, which has a PDI valve 0.34 indicating a larger size distribution for BNT-136 formulation, LNP #29.

[2736] Zeta Potential

[2737] The zeta (Q potential values of the formulation have been analyzed by electrophoretic light scattering (Figure 40). The formulations showed similar neutral to slightly negative zeta potential values.

[2738] RNA Accessibility

[2739] The accessibility of RNA (also referred to as mRNA encapsulation efficiency) in the LNP formulations was measured by Ribogreen Assay method (Figure 41). Some formulations such as LNP #27 and LNP #31 (BNT-134 and BNT-139, respectively) displayed significant mRNA accessibility; on the other hand, some formulations have displayed more than 80% encapsulation efficiency, LNP #26, #29, and #32. Moreover, some formulations such as LNP #25, #28, #31, and #33 (BNT-132, BNT-135, BNT-139, and BNT-141, respectively) have about 50% encapsulation efficiency.

[2740] Free RNA

[2741] The amount of free RNA in the LNP formulations was measured by agarose gel electrophoresis method (Figure 42). As shown in Figure 42, a small amount of free mRNA has been detected for BNT-135, BNT- 138, and BNT-140 formulations, LNP #28, #30, and #32, respectively, whereas no free mRNA has been detected for the others LNPs.

[2742] RNA Integrity

[2743] mRNA integrity of the formulations has been determined by capillary electrophoresis, fragment analyzer (Figure 43). As seen in Figure 36, all formulations showed mRNA integrity more than 75%. Reduced mRNA integrity was seen for BNT-138 formulation, LNP #30.

[2744] In vitro transfection of Example 12 LNPs

[2745] To investigate Example 12 ionizable lipid LNP functionality, cytotoxicity, and transfection efficiency, mRNA expression was evaluated in vitro using a hepatocarcinoma cell line (HepG2) and a human embryonic kidney cell line (HEK293T). For this purpose, the LNP formulations comprising firefly luciferase -encoding mRNA were incubated with the cells at three dosages, 12.5, 25 and 50 ng per well and luciferase expression was assessed 24 h post-incubation. In general, the formulations did not show significant cytotoxicity at the tested conditions (Figures 44A and 44B). Formulations were effectively ableX o to transfect the tested cell lines, except BNT-140 and BNT-141 formulations, LNP #30 and #31, which \ showed slight O z —— \

[2746] ly lower transfection efficiency as compared to other formulations (Figures 45A and 45B).

[2747] 11

[2748] ^ % o o J

[2749] o Example 13: LNP formulations comprising ionizable lipid compounds BNT-222 or BNz

[2750] T-223 b °'

[2751] The present example describes process for preparing lipid nanoparticles (LNPs) 1 _ _ 1 using particular lipid o V o

[2752] compounds (e.g., BNT-222 or BNT-223) described herein, as well as in \ \o v x= z—itro characterization and o / <=

[2753] biological effects. LNPs of the present example have been prepared by the microfluidic mixing procedure described in Example 5. LNPs were formulated by microfluidic mixing o / \= h of t°he lipid mixture in ethanol and RNA solution in aqueous, acidic buffer. The structures of various LNP components are listed in Table 13.1 below. A summary of LNP formulations used in this example \° axre presented in Table 13.2. Each of the below formulations was prepared using 50mM AB, pH 4.0 as RNA buffer, and HEPES, pH 6.0, as dialysis buffer.

[2754] Table 13.1. List of LNP components

[2755] Component Lipid Chemical Structure

[2756] A V° Ionizable Lipid BNT-222

[2757] 01

[2758] Ionizable Lipid BNT-223

[2759] PEGylated- DMG-PEG2000

[2760] lipid

[2761] Phospholipid DSPC

[2762] Sterol Cholesterol

[2763] I fHjT /

[2764] f TH Y A

[2765]

[2766] Table 13.2. Example 13 LNP Formulations

[2767] Ionizable PEGylated Final

[2768] LNP Ionizable Sterol Phospholipid RNA

[2769] Lipid Lipid N / P cone.

[2770] No. Lipid (mol%) (mol%) construct

[2771] (mol%) (mol%) (mg / mL) BM1 Benchmark

[2772] 47.5 40.5 10 2 6 0.05

[2773] LNP 1 (BM1)

[2774] BM2 Benchmark

[2775] 47.5 40.5 10 2 6 mod. 0.05

[2776] LNP 2 (BM2)

[2777] Luciferase

[2778] LNP BNT-222 47.5 40.5 10 2 6 mRNA 0.05 #34

[2779] LNP BNT-223 47.5 40.5 10 2 6 0.05 #35

[2780]

[2781] Size Distribution of Example 13 LNPs

[2782] The sizes of the LNPs have been analyzed by DLS as described above and shown in Figure 46. The hydrodynamic diameter of BL-222 formulation, LNP #34, was found to be 85 nm and BL-223 formulation, LNP #35, was found to be 63 nm. The formulations showed PDI values of 0.21 and 0.28, respectively.

[2783] Zeta Potential

[2784] The zeta (Q potential values of the formulation have been analyzed by electrophoretic light scattering (Figure 47). The formulations showed similar neutral zeta potential values.

[2785] RNA Accessibility

[2786] The accessibility of RNA (also referred to as mRNA encapsulation efficiency) in the LNP formulations was measured by Ribogreen Assay method. Both formulations displayed significant mRNA encapsulation efficiency (Figure 48).

[2787] Free RNA

[2788] The amount of free RNA in the LNP formulations was measured by agarose gel electrophoresis method (Figure 49). As shown in Figure 49, no free mRNA was detected for either formulation.

[2789] RNA Integrity

[2790] mRNA integrity of the formulations has been determined by capillary electrophoresis, fragment analyzer (Figure 50). As seen in Figure 50, both formulations showed mRNA integrity more than 90%.

[2791] In vitro transfection of Example 13 LNPsTo investigate Example 13 ionizable lipid LNP functionality, cytotoxicity, and transfection efficiency, mRNA expression was evaluated in vitro using a hepatocarcinoma cell line (HepG2) and a human embryonic kidney cell line (HEK293T). For this purpose, the LNP formulations comprising firefly luciferase-encoding mRNA were incubated with the cells at three dosages, 12.5, 25 and 50 ng per well and luciferase expression was assessed 24 h post-incubation. In general, the formulations did not show significant cytotoxicity at the tested conditions (Figures 51A and 51B). Formulations were also effectively able to transfect the tested cell lines (Figures 52A and 52B).

[2792] Example 14: LNP formulations comprising ionizable lipid compounds BNT-93 or BNT-94

[2793] The present example describes process for preparing lipid nanoparticles (LNPs) using particular lipid compounds (e.g., BNT-93 or BNT-94) described herein, as well as in vitro characterization and biological effects. LNPs of the present example have been prepared by the microfluidic mixing procedure described in Example 5. LNPs were formulated by microfluidic mixing of the lipid mixture in ethanol and RNA solution in aqueous, acidic buffer. The structures of various LNP components are listed in Table 14.1 below. A summary of LNP formulations used in this example are presented in Table 14.2. Each of the below formulations was prepared using 100mM CB, pH 4.0 as RNA buffer, and 1X PBS as dialysis buffer.

[2794] Table 14.1. List of LNP components

[2795] Component Lipid Chemical Structure

[2796] 0

[2797] Ionizable Lipid BNT-93

[2798] 01

[2799] 0 r^jr^OH

[2800] Ionizable Lipid BNT-94

[2801] O \

[2802] o

[2803] PEGylated- DMG-PEG2000

[2804] lipid

[2805] o

[2806]

[2807] Component Lipid Chemical Structure

[2808] 0

[2809] oxI Phospholipid DSPC. P.

[2810] H> T0O_ 1 0

[2811] Sterol Cholesterol

[2812] I fHX /

[2813] f TAT A

[2814]

[2815] Table 14.2. Example 14 LNP Formulations

[2816] Ionizable PEGylated Final

[2817] LNP Ionizable Sterol Phospholipid RNA

[2818] Lipid Lipid N / P cone.

[2819] No. Lipid (mol%) (mol%) construct

[2820] (mol%) (mol%) (mg / mL) LNP BNT-93 47.5 40.7 10 1.8 6 mod. 0.05 #36

[2821] Luciferase

[2822] LNP BNT-94 47.5 40.7 10 1.8 6 mRNA 0.05 #37

[2823]

[2824] Size Distribution of Example 14 LNPs

[2825] The sizes of the LNPs have been analyzed by DLS as described above and shown in Figure 53. The hydrodynamic diameters for all formulations have been found to be below 70 nm. The formulations have displayed narrow size distribution (PDI < 0.22).

[2826] Zeta Potential

[2827] The zeta (Q potential values of the formulation have been analyzed by electrophoretic light scattering (Figure 54). The formulations showed similar slightly negative zeta potential values.

[2828] RNA Accessibility

[2829] The accessibility of RNA (also referred to as mRNA encapsulation efficiency) in the LNP formulations was measured by Ribogreen Assay method. Both formulations showed relatively high encapsulation efficiency (>74%) (Figure 55).

[2830] Free RNA

[2831] The amount of free RNA in the LNP formulations was measured by agarose gel electrophoresis method (Figure 56). As shown in Figure 57, negligible amount of mRNA was detected for either formulation.RNA Integrity

[2832] mRNA integrity of the formulations has been determined by capillary electrophoresis, fragment analyzer (Figure 57). As seen in Figure 58, no significant mRNA fragmentation was observed.

[2833] In vitro transfection of Example 14 LNPs

[2834] To investigate example ionizable lipid LNP functionality, cytotoxicity, and transfection efficiency, mRNA expression was evaluated in vitro using a skeletal muscle cell line (C2C12), a hepatocarcinoma cell line (HepG2), a human embryonic kidney cell line (HEK293T) and a murine macrophage cell line (RAW 264.7). For this purpose, the LNP formulations comprising firefly luciferase -encoding mRNA were incubated with the cells at three dosages, 12.5, 25 and 50 ng per well and luciferase expression was assessed 24 h post-incubation. In general, the formulations did not show significant cytotoxicity at the tested conditions (Figures 58A-58D). Both formulations showed dose-dependent transfection efficiency (Figures 59A-59D). In most of the tested cell lines, BNT-94 formulation, LNP #37, showed higher transfection efficiency.

[2835] Example 15: LNP formulations comprising ionizable lipid compounds BNT-97 or BNT-98

[2836] The present example describes process for preparing lipid nanoparticles (LNPs) using particular lipid compounds (e.g., BNT-97 or BNT-98) described herein, as well as in vitro characterization and biological effects. LNPs of the present example have been prepared by the microfluidic mixing procedure described in Example 5. LNPs were formulated by microfluidic mixing of the lipid mixture in ethanol and RNA solution in aqueous, acidic buffer. The structures of various LNP components are listed in Table 15.1 below. A summary of LNP formulations used in this example are presented in Table 15.2. Each of the below formulations was prepared using 100mM CB, pH 4.0 as RNA buffer, and 1X PBS as dialysis buffer.

[2837] Table 15.1. List of LNP components

[2838] Component Lipid Chemical Structure

[2839] SH

[2840] 0 HN-^ II N

[2841] Ionizable Lipid BNT-97

[2842] ° 1

[2843]

[2844] Component Lipid Chemical Structure

[2845] \ z ——

[2846] 107 lo V X"SHIonizable Lipid BNT-98 b °'

[2847] O 1

[2848] o V

[2849] O \o x=—>

[2850] o / <=

[2851] o

[2852] PEGylated- DMG-PEG2000

[2853] lipid 44

[2854] o

[2855] Phospholipid DSPC

[2856] Sterol Cholesterol

[2857] i fhX /

[2858] f TA J A

[2859]

[2860] Table 15.2. Example 15 LNP Formulations

[2861] Ionizable PEGylated Final

[2862] LNP Ionizable Sterol Phospholipid RNA

[2863] Lipid Lipid N / P cone.

[2864] No. Lipid (mol%) (mol%) construct

[2865] (mol%) (mol%) (mg / mL) LNP BNT-97 47.5 40.5 10 2.0 6 mod. 0.05 #38

[2866] Luciferase

[2867] LNP BNT-98 47.5 40.5 10 2.0 6 mRNA 0.05 #39

[2868]

[2869] Size Distribution of Example 15 LNPs

[2870] The sizes of the LNPs have been analyzed by DLS as described above and shown in Figure 60. The hydrodynamic diameters for both formulations were found to be below 65 nm. PDI values were found to be < 0.3 indicating a narrow size distribution.

[2871] Zeta Potential

[2872] The zeta (Q potential values of the formulation have been analyzed by electrophoretic light scattering (Figure 61). BNT-97 formulation, LNP #38, showed negative zeta potential whereas BNT-98 formulation, LNP #39, displayed neutral to slightly negative zeta potential.RNA Accessibility

[2873] The accessibility of RNA (also referred to as mRNA encapsulation efficiency) in the LNP formulations was measured by Ribogreen Assay method. While BNT-98 formulation, LNP #39, was able to effectively encapsulate the mRNA (mRNA inaccessibility > 76%); BNT-97 formulation, LNP #38, showed high RNA accessibility (Figure 62).

[2874] Free RNA

[2875] The amount of free RNA in the LNP formulations was measured by agarose gel electrophoresis method (Figure 63). As shown in Figure 64, the electropherogram showed significant amount of free mRNA for LNP #38.

[2876] RNA Integrity

[2877] mRNA integrity of the formulations has been determined by capillary electrophoresis, fragment analyzer. Although BNT-97 formulation, LNP #38, showed slightly lower mRNA integrity, both formulations showed high mRNA integrity (mRNA integrity > 80%) (Figure 64).

[2878] In vitro transfection of Example 15 LNPs

[2879] To investigate Example 15 ionizable lipid LNP functionality, cytotoxicity, and transfection efficiency, mRNA expression was evaluated in vitro using a skeletal muscle cell line (C2C12), a hepatocarcinoma cell line (HepG2), and a murine macrophage cell line (RAW 264.7). For this purpose, the LNP formulations comprising firefly luciferase-encoding mRNA were incubated with the cells at three dosages, 12.5, 25 and 50 ng per well and luciferase expression was assessed 24 h post-incubation. In general, the formulations did not show significant cytotoxicity at the tested conditions (Figures 65A-65C). Both formulations showed dose -dependent transfection efficiency (Figures 66A-66C). For all cell lines, BNT-97 formulation, LNP #38, showed lower transfection efficiency most likely due to lower mRNA encapsulation efficiency.

[2880] Example 16: LNP formulations comprising ionizable lipid compounds BNT-104, BNT-105, BNT-107 or BNT-122

[2881] The present example describes process for preparing lipid nanoparticles (LNPs) using particular lipid compounds (e.g., BNT-104, BNT-105, BNT-107 or BNT-122) described herein, as well as in vitro characterization and biological effects. LNPs of the present example have been prepared by the microfluidic mixing procedure described in Example 5. LNPs comprising BNT-122 were formulated using DOPE as helper lipid and were prepared using 50mM CB, pH 4.0 as RNA buffer, and IX PBS as dialysis buffer. LNPs were formulated by microfluidic mixing of the lipid mixture in ethanol and RNA solution in aqueous, acidic buffer. The structures of various LNP components are listed in Table 16.1 below. A summary of LNP formulations used in this example are presented in Table 16.2. Each of the below formulations wasprepared using 100mM CB, pH 4.0 as RNA buffer, and 1X PBS as dialysis buffer unless otherwise denoted above.

[2882] Table 16.1. List of LNP components

[2883] Component Lipid Chemical Structure

[2884] O / 0H

[2885] Ionizable Lipid BNT-104

[2886] ° [

[2887] 0 N=\ II j. NH

[2888] < H(J Ionizable Lipid BNT-105

[2889] HOX

[2890] °

[2891] Ionizable Lipid BNT-107 J > N== /

[2892] N=\ L NH

[2893] 1 °0j \

[2894] / H0 / Ionizable Lipid BNT-122

[2895] ° r 1 PEGylated- DMG-PEG2000

[2896] lipid

[2897] o

[2898] o

[2899] osI Phospholipid DSPC

[2900] H*T^° O_ 1 0

[2901]

[2902] <>*

[2903] T

[2904] Component Lipid Chemical Structure + z

[2905] o

[2906] / OO0- 1=- Phospholipid DOPE / o—

[2907] \ °

[2908] / o o

[2909] / )o=

[2910] Sterol Cholesterol

[2911] i fHL /

[2912] f TAJ A ( /

[2913]

[2914] ) \ l

[2915] Table 16.2. Example 16 LNP Formulations

[2916] Ionizable PEGylated Final LNP Ionizable Sterol Phospholipid RNA

[2917] Lipid Phospholipid Lipid N / P cone. No. Lipid (mol%) (mol%) construct (mol%) (mol%) (mg / mL) LNP BNT-104 47.5 40.5 DSPC 10 2.0 6 0.05 #40

[2918] LNP BNT-105 47.5 40.5 DSPC 10 2.0 6 mod. 0.05 #41

[2919] Luciferase

[2920] LNP BNT-107 47.5 40.5 DSPC 10 2.0 6 mRNA 0.05 #42

[2921] LNP BNT-122 47.5 40.5 DOPE 10 2.0 6 0.05 #43

[2922]

[2923] Size Distribution of Example 16 LNPs

[2924] The sizes of the LNPs have been analyzed by DLS as described above and shown in Figure 67. The hydrodynamic diameters for all formulations were found to be below 60 nm. PDI values were found to be < 0.25 for BNT-104, BNT-105 and BNT-107 formulations (LNP #40, LNP #41, and LNP #42, respectively), indicating a narrow size distribution. For BNT-122 formulation (LNP #43), PDI has been found to be slightly larger (0.36).

[2925] Zeta Potential

[2926] The zeta (Q potential values of the formulation have been analyzed by electrophoretic light scattering (Figure 68). All formulations displayed neutral to slightly negative zeta potential.

[2927] RNA AccessibilityThe accessibility of RNA (also referred to as mRNA encapsulation efficiency) in the LNP formulations was measured by Ribogreen Assay method. Tested formulations have showed high mRNA encapsulation efficiency (mRNA inaccessibility > 80%) (Figure 69).

[2928] RNA Integrity

[2929] mRNA integrity of the formulations has been determined by capillary electrophoresis, fragment analyzer. All formulations have showed high mRNA integrity (mRNA integrity > 88%). (Figure 70).

[2930] In vitro transfection of Example 16 LNPs

[2931] To investigate Example 16 ionizable lipid LNP functionality, cytotoxicity, and transfection efficiency, mRNA expression was evaluated in vitro using a skeletal muscle cell line (C2C12), a hepatocarcinoma cell line (HepG2), and a murine macrophage cell line (RAW 264.7). For this purpose, the LNP formulations comprising firefly luciferase-encoding mRNA were incubated with the cells at three dosages, 12.5, 25 and 50 ng per well and luciferase expression was assessed 24 h post-incubation. In general, the formulations did not show significant cytotoxicity at the tested conditions (Figures 71A-71C). The viability of C2C12 and HEPG2 cells have slightly decreased at 50 ng of mRNA dosage of BNT-107 formulation, LNP #42. For all cell lines, LNP #42 (BNT-107) showed lowest transfection efficiency while LNP #41 (BNT-105) showed highest transfection efficiency (Figures 72A-72C). Without being bound to any theory or conclusion in particular, the distance between imidazole group and tertiary amine in the lipid head plays a crucial role in transfection efficiency as LNP #41 comprising BNT-105 (lipid having two methylene group in between imidazole group and central tertiary amine group) showed profoundly higher transfection efficiency than LNP #42 comprising BNT-107 (lipid having one methylene group in between imidazole group and central tertiary amine group). Moreover, the orientation of imidazole group (type of ring atom (C or N) where the imidazole binds the rest of the lipid) is an important design criteria as LNP #41 comprising BNT-105 showed higher transfection efficiency than LNP #40 comprising BNT-104 - these lipids, BNT-104 and BNT-105, have identical chemical formulae, but the imidazole rings are bound to the lipid via different atoms (N for BNT-104 and C for BNT-105). The use of an ester branching group led to higher transfection efficiency in this case compared to sulfone branching group (LNP #41 vs LNP #43).

[2932] Hemolysis Analysis and Complement Activation (BNT-105)

[2933] The hemolytic effect and complement activation of BNT-105 LNPs was determined as described herein. The in vitro hemolytic effect of the BNT-105, LNP #41, has been investigated at the mRNA dose of 16 mg / kg (the mRNA dose equals to 16 mg / kg in in vivo matrix) (Figure 73A). Even at very high mRNA dosage, LNP #42 formulations showed negligible hemolysis indicating that the formulations do not affect the integrity of cell membranes of red blood cells (PBS served as negative control, Triton (0.2%) served as positive control). In addition, the effect of LNP #42 formulations on complement activation has been determined (Figure 73B). The final complement complex concentration (SC5b-9) was found to be similar to the negative control, PBS, showing that the formulations do not lead to the complement activation (Cobravenom factor (CVF) served as positive control; untreated SO (only serum sample), untreated SI (only serum sample) and PBS as negative controls).

[2934] In vivo Bioluminescence Imaging (BNT-105)

[2935] Based on the in vitro results, LNP #41 formulation was selected and tested in vivo. For the formulation, 3 mice (Balb / c) were injected with 16 pg / mouse mRNA dose (1 pg of mod. luciferase mRNA + 15 ig of BNT mRNA). Ventral in vivo bioluminescence imaging was performed at 6 h and 24 h timepoints. Following the 24h timepoint, blood and organs (heart, lung, liver, spleen, kidneys and lymph nodes (popliteal and inguinal)) were collected and ex vivo imaging of organs was performed. The details of the workflow have been presented in Table 12.3.

[2936] Size Timepoints Ionizable RNA N /

[2937] LNP No. Dosage (n=X

[2938] Lipid construct P OH 6H 24H

[2939] )

[2940] 0.9%NaCl

[2941] - - - 1 - (Control)

[2942] mod.

[2943] 16 pg (1 pg of mod. luciferase

[2944] LNP #42 BNT-105 Luciferase 3 6

[2945] mRNA + 15 pg of BNT mRNA)

[2946] mRNA

[2947]

[2948] wherein

[2949] Administration (i.v.), volume 100 μL

[2950] In vivo Imaging

[2951] Ex vivo Imaging

[2952] Blood collection (final) + sera generation

[2953]

[2954] BLI results (Figures 74A-74E) showed that LNP #42 (BNT-105) showed strong signal in the liver-spleen region after 6 h (Figure 75A). The signal has decreased after 24 h as expected (Figure 75A and 75B). The highest expression has been observed in the liver and spleen followed by inguinal lymph node (Figure 75C). Other organs showed similar expression levels to the control group (Figure 75D) indicating no off targeting. Approximately 50% of the signal was found to be coming from the liver while 50% of the signal was found to be coming from the spleen after 24 h (Figure 75E).

[2955] Example 17: LNP formulations comprising ionizable lipid compounds BNT-116 or BNT-117 The present example describes process for preparing lipid nanoparticles (LNPs) using particular lipid compounds (e.g., BNT-116 or BNT-117) described herein, as well as in vitro characterization and biological effects. LNPs of the present example have been prepared by the microfluidic mixing procedure described in Example 5. LNPs were formulated by microfluidic mixing of the lipid mixture in ethanol and RNA solution in aqueous, acidic buffer. The structures of various LNP components are listed in Table 17.1below. A summary of LNP formulations used in this example are presented in Table 17.2. BNT-116

[2956] \ formulations was prepared using lOOmM CB, pH 4.0 as RNA buffer, and IX PBS as dialysis buf zf ——er while BNT-117 formulations was prepared using 50mM CB, pH 4.0 as RNA buffer, and IX PBS as dialysis ^ 46 z

[2957] buffer.

[2958] \ b ° °'

[2959] Yo / o o- Table 17.1. List of LNP components / o ) \o x=°— / =

[2960] o / / =

[2961] Component Lipid Chemical Structure

[2962] N^\

[2963] L NH

[2964] J d' o i )

[2965] ( / S HO

[2966] Ionizable Lipid BNT-116

[2967] ) \ I

[2968] °= / \ z

[2969] ° \ /

[2970] Ionizable Lipid BNT-117 ° \ z

[2971] \ o

[2972] O PEGylated- \-Z—. -\ / O'

[2973] DMG-PEG2000

[2974] lipid

[2975] o \ X _ _

[2976] o

[2977] Phospholipid DSPC

[2978] Phospholipid DOPE

[2979] ^NH3

[2980] Sterol Cholesterol

[2981] I XHjT?

[2982] f TH Y A

[2983]

[2984] Table 17.2. Example 13 LNP FormulationsIonizable PEGylated Final LNP Ionizable Sterol Phospholipid RNA

[2985] Lipid Phospholipid Lipid N / P cone. No. Lipid (mol%) (mol%) construct (mol%) (mol%) (mg / mL) LNP BNT-116 47.5 40.5 DOPE 10 2.0 6 mod. 0.05 #44

[2986] Luciferase

[2987] LNP BNT-117 47.5 40.5 DSPC 10 2.0 6 mRNA 0.05 #45

[2988]

[2989] Size Distribution of Example 17 LNPs

[2990] The sizes of the LNPs have been analyzed by DLS as described above and shown in Figure 75. The hydrodynamic diameter of LNP #44 (BNT-116) was found to be 167 nm whereas LNP #45 (BNT-117) showed smaller size (56 nm). The formulations showed PDI values of 0.26 and 0.43, respectively indicating a larger size distribution for BNT-117 formulation, LNP #45.

[2991] Zeta Potential

[2992] The zeta (Q potential values of the formulation have been analyzed by electrophoretic light scattering (Figure 76). BNT-116 formulation, LNP #44, showed negative zeta potential whereas BNT-117 formulation, LNP #45, displayed neutral to slightly negative zeta potential.

[2993] RNA Accessibility

[2994] The accessibility of RNA (also referred to as mRNA encapsulation efficiency) in the LNP formulations was measured by Ribogreen Assay method. Both formulations displayed significant mRNA accessibility (Figure 77).

[2995] Free RNA

[2996] The amount of free RNA in the LNP formulations was measured by agarose gel electrophoresis method (Figure 78). As shown in Figure 79, the electropherogram showed significant amount of free mRNA for LNP #44 (BNT-116) whereas no free mRNA was detected for LNP #45 (BNT-117).

[2997] RNA Integrity

[2998] mRNA integrity of the formulations has been determined by capillary electrophoresis, fragment analyzer. Reduced mRNA integrity was seen for BNT-116 formulation, LNP #44 (Figure 79).

[2999] In vitro transfection of Example 17 LNPs

[3000] To investigate Example 17 ionizable lipid LNP functionality, cytotoxicity, and transfection efficiency, mRNA expression was evaluated in vitro using a skeletal muscle cell line (C2C12), a hepatocarcinoma cell line (HepG2), a human embryonic kidney cell line (HEK293T), and a murine macrophage cell line (RAW 264.7). For this purpose, the LNP formulations comprising firefly luciferase -encoding mRNA wereincubated with the cells at three dosages, 12.5, 25 and 50 ng per well and luciferase expression was assessed 24 h post-incubation. In general, the formulations did not show significant cytotoxicity at the tested conditions (Figures 80A-80D). LNP # 44 (BNT-116) showed notable transfection efficiency. In general, LNP #45 (BNT-117) has shown higher transfection efficiency (Figures 81A-81C).

[3001] The embodiments of the disclosure described above are intended to be merely exemplary, numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention as defined in any appended claims.

Claims

CLAIMS1. A compound represented by formula IB:L1-X1-T1G2-L3-N / / \2- x2- T2IBor a pharmaceutically acceptable salt thereof, wherein:L1and L2are each independently an optionally substituted C1-C30 aliphatic group;L3is a optionally substituted C1-C10 aliphatic or a bond;X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -S(O)2N(R’)-, -N(R’)S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R1)2-, -OC(S)C(R1)2-, -C(R1)2C(S)O-, -S-, and -N(H)C(O)N(R1);each R1is, independently, at each instance, optionally substituted C1-C20aliphatic or H;T1and T2are each independently an optionally substituted C3-C30 aliphatic;G2is -S(O)2R3a;R3ais -(CH2)0-6-R3bor Ci-Ce aliphatic;R3bis -OH, -NH(CH3), or -N(CH3)2.

2. The compound of claim 1, wherein R3ais -(CH2)0-6-R3b.

3. The compound of claims 1 or 2, wherein R3bis -OH.

4. The compound of claim 1, wherein R3ais C1-C10aliphatic.

6. The compound of any one of claims 1-5, wherein, when R3ais -CH3, then X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -N(H)C(O)N(R’)-, -NHC(O)-, -C(O)N(R’)-, and -NHS(O)2N(R1)-.

7. A compound represented by formula IA:L1-X1-T1G1-L3—\2- X2— T2IAor a pharmaceutically acceptable salt thereof, wherein:L1and L2are each independently an optionally substituted C1-C30 aliphatic group;L3is a bond or optionally substituted C1-C10 aliphatic;X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -S(O)2N(R’)-, -N(R1)S(O)2-, -S(O)-, -S(O)2-, -S(O)2C(R1)2-, -OC(S)C(R1)2-, -C(R1)2C(S)O-, -S-, -N(H)C(O)N(R1)-, -NHC(O)-, -C(O)N(R’)-, -NHS(O)2N(R1)-, and -C(O)-;each R1is, independently, at each instance, optionally substituted C1-C20 aliphatic or H;T1and T2are each independently an optionally substituted C3-C30 aliphatic;G1is -S(O)R3or -SR3; andR3is optionally substituted C1-C10 aliphatic.

8. The compound of claim 7, wherein G1is -S(O)R3.

9. The compound of claims 7 or 8, wherein R3is optionally substituted Ci-Ce aliphatic.

10. The compound of claim 9, wherein R3is –(CH2)1-6-OH.

11. The compound of claim 9, wherein R3is -CH3or -CH2-CH3.

12. The compound of any one of claims 7-11, wherein G113. The compound of claim 7, wherein G1is -SR3.

14. The compound of claim 13, wherein R3is optionally substituted Ci-Ce aliphatic.

15. The compound of claim 14, wherein R3is –(CH2)1-6-OH.The compound of any one of claims 13-15, wherein G1is17. A compound represented by formula IC:L1-X1-T1G3-L3-N / / \2- X2- T2or a pharmaceutically acceptable salt thereof, wherein:L1and L2are each independently an optionally substituted C1-C30 aliphatic group;L3is a bond or optionally substituted C1-C10 aliphatic;X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -N(H)C(O)N(R’)-, -NHC(O)-, and -C(O)N(R1)-;each R1is, independently, at each instance, optionally substituted C1-C20 aliphatic or H;T1and T2are each independently an optionally substituted C3-C30 aliphatic group;each R30is independently selected from -SH,-OH, -NH2, -NH(CH3), -N(CHs)2, -C(0)NH2, and optionally substituted Ci-Ce aliphatic;provided that when G3is -OH, -NH2, -NH(CH3), or -N(CH3)2, and X1or X2is a bond, -OC(O)-, or -C(O)O-, then L3is C2-C10 alkenyl, or C2-C10 alkynyl.

18. The compound of claim 17, wherein G3is selected from -OH, -NH2, -NH(CH3), -N(CHs)2,S19. The compound of claim 1, wherein the compound is represented by formula IIA:O / L1—X1- T1HO— (CH2)I.6— S-(CH2)^—O L2- X2- T2IIAor a pharmaceutically acceptable salt thereof.

20. The compound of claim 7, wherein the compound is represented by formula IIB:O / L1— X1— T1R3— S- (CH2)I_6— N2- X2- T2IIBor a pharmaceutically acceptable salt thereof.

21. The compound of claim 17, wherein the compound is represented by formula IIC:X1— T1IHO L2— X2T2IICor a pharmaceutically acceptable salt thereof,wherein X1and X2are each independently selected from a bond, -OC(O)-, and -C(O)O-.

22. The compound of claim 17, wherein the compound is represented by formula IID:X1— T1or a pharmaceutically acceptable salt thereof,wherein X1and X2are each independently selected from a bond, -OC(O)- and -C(O)O-.

23. The compound of claim 17, wherein the compound is represented by formula IIE:or a pharmaceutically acceptable salt thereof,wherein X1and X2are each independently selected from a bond, -OC(O)-, and -C(O)O-.

24. The compound of claim 17, wherein the compound is represented by formula IIFX1-T1I7^L1N, J-ICH,),.6-N,L2-X2. T2R30IIFor a pharmaceutically acceptable salt thereof.

25. The compound of any one of claims 1-24, wherein L1and L2are each C1-C10 alkylene.

26. The compound of any one of claims 1-25, wherein L1and L2are each independently -(CH2)6-10-.

27. The compound of any one of claims 1-16, 19-20, or 25-26, wherein X1and X2are each independently selected from -OC(O)-, -C(O)O-, -S(O)2N(R1)-, -N(R1)S(O)2., -S(O)-, -S(O)2-, -S(O)2C(R1)2-, -OC(S)C(R1)2-, -C(R1)2C(S)O-, -S-, -N(H)C(O)N(R1)-, -NHC(O)-, -C(O)N(R1)-, -NHS(O)2N(R1)-, and -C(O)-.

28. The compound of any one of claims 1-16, 19-20, or 25-27, wherein X1and X2are each independently selected from -OC(O)-, -C(O)O-, -S(O)2N(R1)-, -N(R1)S(O)2-, and -S(O)2-.

29. The compound of any one of claims 17, 18, or 24, wherein X1and X2are each independently selected from -OC(O)-, -C(O)O-, -N(H)C(O)N(R1)-, -NHC(O)-, and -C(O)N(R1)-.

30. The compound of any one of claims 17, 18, or 24, wherein -OC(O)-, -C(O)O-, -NHC(O)-, and -C(O)N(R’)-.

31. The compound of any one of claims 1 -30, wherein T1and T2are each independently selected from optionally substituted C3-C20 alkyl.

32. The compound of any one of claims 1-31, wherein T1and T2are each independently selected from:

33. The compound of any one of claims 1-32, wherein a moiety -L’-X’-T1is selected from the group consisting of:OT- O - (CH2)6.12- S- N o '" 4 — (CH2)6.12- S- T1O R1o — (CH2)6.12-s-T1R1N-T1S. Il - (CH2)6-12—4 I - (CH2)6.12— S— T1 XQ34. The compound of any one of claims 1-33, wherein a moiety -L2-X2-T2is selected from the group consisting of:S 9 zT2°n- (CH2)6.12— O I - (CH2)6.12-S-N I - (CH2)6.12- S- T2fl O R16 I - (CH2)6.12-S-T2g R1N-T2s, - (CH2)6.12^ I - (CH2)6.12— S— T2 XQ35. The compound of any one of claims 1-34, wherein a moiety -L'-X'-T1and moiety -L2-X2-T2are each independently selected from:

36. The compound of claim 1, wherein the compound is selected from Table IB.

37. The compound of claim 7, wherein the compound is selected from Table 1A.

38. The compound of claim 17, wherein the compound is selected from Table 1C.

39. The compound of claim 1, wherein the compound is selected from Table ID.

40. A particle comprising a compound of any one of claims 1-39, and a nucleic acid.

41. The particle of claim 40, wherein the nucleic acid is RNA, DNA, or mixtures thereof.

42. The particle of claim 41, wherein the RNA is mRNA.

43. The particle of claim 42, wherein the RNA is modRNA, circRNA, saRNA, taRNA, or uRNA.

44. The particle of claim 40, wherein the DNA is linear DNA, plasmid DNA, minicircle DNA, nanoplasmid DNA, doggybone DNA, or a transposon.

45. The particle of any one of claims 40-44, wherein the particle further comprises one or more of a helper lipid, a polymer-conjugated lipid, or a sterol.

46. The particle of claim 45, wherein the helper lipid is a phospholipid.

47. The particle of claims 45 or 46, wherein the helper lipid is or comprises 1,2-distearoyl-5«-glycero-3-phosphocholine (DSPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), 1 -palmitoyl -2 -oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), sphingomyelins, N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), l,2-diacylglyceryl-3-O-4’-(N, N, N-trimethyl)-homoserine (DGTS), ceramides, and their derivatives.

48. The particle of any one of claims 45-47, wherein the polymer-conjugated lipid is selected from the group consisting of a polyethylene glycol) (PEG) -conjugated lipid, a poly(sarcosine) (pSar)-conjugated lipid, a poly(aminoethoxy ethoxy acetic acid) (pAEEA)-conjugated lipid; and a poly(2 -methylaminoethoxy ethoxy acetic acid) (pMAEEA) -conjugated lipid.

49. The particle of any one of claims 45-47, wherein the polymer-conjugated lipid is a polymer conjugated lipid is selected from the group consisting of a polyethylene glycol) (PEG) -conjugated lipid, a poly(sarcosine) (pSar)-conjugated lipid, a poly(aminoethoxy ethoxy acetic acid) (pAEEA) -conjugated lipid; and a poly (2 -methylaminoethoxy ethoxy acetic acid) (pMAEEA)-conjugated lipid. In some embodiments, a PEG-lipid is selected from pegylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG) (e.g., 1,2-dimyristoyl-rac-glycero -3 -methoxypolyethylene glycol-2000 (PEG2000-DMG)), a pegylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2’,3’-di(tetradecanoyloxy)propyl-l-0-(co-methoxy(polyethoxy)ethyl)bntane-dioate (PEG-S-DMG), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino-(polyethylene glycol)-2000] (DSPE-PEG2000 amine), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate, and 2,3 -di(tetradecanoxy)propyl-N-(co-methoxy(polyethoxy)ethyl)carbamate.

50. The particle of any one of claims 45-49, wherein the sterol is selected from P-sitosterol, stigmasterol, cholesterol, cholecalciferol, ergocalciferol, calcipotriol, botulin, lupeol, ursolic acid, oleanolic acid, cycloartenol, lanosterol, or a-tocopherol.

51. The particle of any one of claims 45-50, wherein the particle is characterized by an N / P ratio that is about 4 to about 16.

52. The particle of any one of claims 45-51, wherein the particle comprises:about 30 to about 60 mol% of the compound;about 20 to about 60 mol% of the steroid;about 1 to about 2 mol% of the polymer-conjugated lipid; andabout 5 to about 15 mol% of the neutral lipid.

53. A suspension comprising a dispersed phase and an aqueous phase, and wherein the dispersed phase comprises one or more particles of any one of claims 45-52.

54. A method of increasing or causing increased expression of RNA in a target in a subject, the method comprising administering to the subject a composition comprising particles of any one of claims 45-52, or the suspension of claim 50.

55. The method of claim 54, wherein the target is selected from the lungs, liver, spleen, heart, brain, lymph nodes, bladder, kidneys, and pancreas.

56. A method of treating a disease, disorder, or condition in a subject comprising administering to the subject a composition comprising the particles of any one of claims 42-49, or a suspension of claim 53.

57. The method of claim 56, wherein the disease, disorder, or condition is an infectious disease, cancer, a genetic disorder, an autoimmune disease, or a rare disease.

58. The method of any one of claims 54-57, wherein the particle or the suspension is administered parenterally or intranasally.

59. The method of claim 58, wherein the particle or the suspension is administered intramuscularly, subcutaneously, intradermally, or intravenously.

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