Lipidoid Compounds and Related Compositions and Uses

Abstract

Compositions comprising lipidoid compounds, methods for preparing such compositions, and the use of these compositions in gene delivery applications are disclosed.

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 480,821, filed January 20, 2023, U.S. Provisional Application No. 63 / 515,987, filed July 27, 2023, and U.S. Provisional Application No. 63 / 600,961, filed November 20, 2023. The contents of each of the foregoing patent applications are incorporated herein by reference in their entirety.

[0002] Sequence Listing The Sequence Listing XML associated with this application has been provided electronically in XML format and is incorporated herein by reference. The XML file containing the Sequence Listing XML is named "POTH-077_WO_SeqList.xml." The XML file is 34,029 bytes in size, was created on January 19, 2024, and has been electronically submitted by the USPTO Patent Center.

[0003] Field The present invention relates generally to lipidoid compounds, compositions containing such compounds, methods for preparing these compounds, and the use of these compositions in gene delivery. [Background technology]

[0004] background There has been a long-felt but unmet need in the art for compositions and methods for delivering nucleic acids to cells and for genetically modifying cells in vivo, ex vivo, and in vitro. Widely accepted gene delivery and genetic modification techniques, such as the use of viral vectors, including AAV, can cause acute toxicity and adverse side effects in patients. The present disclosure provides improved compositions, methods, and kits for delivering nucleic acids to various types of cells, including hepatocytes, in vivo, ex vivo, and in vitro. More specifically, the present disclosure provides improved lipid nanoparticle compositions and methods for using the same. These lipid nanoparticle compositions and methods enable the delivery of nucleic acids to cells with high efficiency and low toxicity. Thus, the compositions and methods of the present disclosure are widely applicable to many diverse fields, including gene therapy. Summary of the Invention

[0005] overview In some embodiments, novel compounds are provided. In one embodiment, the novel compounds are compounds of formula (I): TIFF2026503550000002.tif12170 formula (I) or a salt thereof, wherein A, TIFF2026503550000003.tif70170H Each B independently: TIFF2026503550000004.tif19170Here, * indicates a bond to A, ** indicates a bond to C, Each C, independently, TIFF2026503550000005.tif17170n is an integer in the range of 2 to 6; a is an integer ranging from 1 to 5; b is an integer ranging from 1 to 5; each y is independently an integer ranging from 1 to 10; Each R1 independently represents one or more of C3 to C 12 Unbranched C1-C optionally substituted with cycloalkyl18 is alkyl; Each R' is independently an unbranched C1-C 18 is alkylene; R3 is a C1-C optionally substituted with one or more hydroxyl 10 alkyl or -NH-(C=O)-(C1-C6 alkyl), or a salt thereof.

[0006] In another aspect, the novel compounds are compounds of formula (II): TIFF2026503550000006.tif11170 formula (II) or a salt thereof, wherein A, TIFF2026503550000007.tif90170Each B is independent, TIFF2026503550000008.tif17170where * indicates a bond to A, ** indicates a bond to C, Each C, independently, TIFF2026503550000009.tif17170 or C1~C 18 is alkyl; n is an integer ranging from 2 to 6; a is an integer ranging from 1 to 5; b is an integer ranging from 1 to 5; Each R1 is independently C1 to C 18 Alkyl or C2-C 18 Alkenyl, C1-C 18 Alkyl or C2-C 18 One or more C3-C alkenyl groups 12 optionally substituted with cycloalkyl; Each R' is independently an unbranched C1-C 18 is alkylene; R3 is (i) a C1-C optionally substituted with one or more hydroxyl 10 alkyl, -NH-(C=O)-(C1-C6 alkyl) or phenyl, or (ii) cyclohexyl or -(C1-C6 alkylene)-hydroxyl optionally substituted with one or more hydroxyl; Each Y is independently TIFF2026503550000010.tif13170where *** indicates binding to R1; each p is independently an integer ranging from 0 to 3; each q is independently 0 or 1; Each z is independently 0 or 1, or a salt thereof.

[0007] In some embodiments, novel lipid nanoparticles ("LNPs") are provided that include novel compounds. In one embodiment, the novel compound is a compound of formula (I). In another embodiment, the novel compound is a compound of formula (II).

[0008] In some embodiments, a pharmaceutical composition is provided comprising a composition of the present disclosure and at least one pharmaceutically acceptable excipient or diluent.

[0009] In some embodiments, methods are provided for delivering at least one nucleic acid to at least one cell, comprising contacting the at least one cell with at least one composition of the present disclosure.

[0010] In some embodiments, methods of genetically modifying at least one cell are provided, comprising contacting the at least one cell with at least one composition of the present disclosure.

[0011] In some embodiments, methods are provided for treating at least one disease or disorder in a subject in need thereof, comprising administering to the subject at least one therapeutically effective amount of at least one composition of the present disclosure.

[0012] In some embodiments, methods are provided for delivering at least one nucleic acid to at least one cell, comprising contacting the at least one cell with at least one composition of the present disclosure.

[0013] In some aspects, cells modified by the methods of the present disclosure are provided.

[0014] Any of the aspects and / or embodiments described herein may be combined with any other aspect and / or embodiment herein.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein, unless the context clearly dictates otherwise, the singular includes the plural; by way of example, the terms "a," "an," and "the" are either singular or plural, and the term "or" is understood to be inclusive. By way of example, "an element" means one or more elements. Throughout this specification, the word "comprising," or variations such as "comprises" or "comprising," are understood to mean the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps. About can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about."

[0016] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. References cited herein are not admitted to be prior art to the claimed invention. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the present disclosure will become apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0017] [Figure 1] 10 shows that in the presence or absence of recombinant ApoE4, LNP compositions of the present disclosure demonstrated an increase in the percentage of GFP-positive HepG2 cells with tannic acid concentration.

[0018] [Figure 2A-2B] Shown are whole body luminescence imaging (BLI) measurements 48 hours post-dose (FIG. 2A) and body weight loss (BWL) measurements 24 hours post-dose (FIG. 2B) for mice treated with LNP compositions of the present disclosure with or without tannic acid.

[0019] [Figure 3A-3C] Shown are whole body luminescence imaging (BLI) measurements 48 hours after administration (FIG. 3A), body weight loss (BWL) measurements 24 hours after administration (FIG. 3B), and cytokine levels (FIG. 3C) of mice treated with LNP compositions of the present disclosure with or without tannic acid.

[0020] [Figure 4A-4B] Shown are whole body luminescence imaging (BLI) measurements of mice treated with LNP compositions of the present disclosure with or without tannic acid at 48 hours (FIG. 4A) or 1 week (FIG. 4B) post-administration.

[0021] [Figure 5A-5B] FIG. 5B shows that in the presence (FIG. 5A) or absence (FIG. 5B) of recombinant ApoE4, the LNP compositions of the present disclosure demonstrated higher luciferase expression in HepG2 cells with the addition of proanthocyanidins.

[0022] [Figures 6A-6B] FIG. 6B shows that in the presence (FIG. 6A) or absence (FIG. 6B) of recombinant ApoE4, the LNP compositions of the present disclosure demonstrated higher luciferase expression in HepG2 cells with the addition of proanthocyanidins.

[0023] [Figures 7A-7B] 7A and 7B show that the LNP compositions of the present disclosure demonstrated higher luciferase expression in HepG2 cells with the addition of ellagic acid or punicalagin in the presence (FIG. 7B) or absence (FIG. 7A) of recombinant ApoE4.

[0024] [Figure 8] 1 shows whole body luminescence imaging (BLI) measurements at 48 hours after administration of mice treated with LNP compositions of the present disclosure with or without proanthocyanidins, ellagic acid, or punicalagins.

[0025] [Figure 9] 10 shows that the LNP composition of the present disclosure demonstrated higher PCCA-HA expression in mice supplemented with tannic acid. DETAILED DESCRIPTION OF THE INVENTION

[0026] Detailed Description The present disclosure provides novel lipidoid compositions, novel lipid nanoparticle compositions (LNPs) comprising novel lipidoid compositions, methods for preparing LNPs, and methods for using them.In a non-limiting example, the compositions and methods of the present disclosure can be used for gene delivery.In a non-limiting example, the compositions and methods of the present disclosure can be widely used to deliver nucleic acids to liver cells in vivo, ex vivo, or in vitro for the treatment of certain diseases and disorders, including but not limited to liver damage.In a non-limiting example, the compositions and methods of the present disclosure can be widely used to deliver nucleic acids and induce the expression of secreted therapeutic proteins.

[0027] Compositions of the present disclosure In one aspect, the present invention provides a compound of formula (I): TIFF2026503550000011.tif12170 formula (I) or a salt thereof, wherein A, TIFF2026503550000012.tif70170Each B is independent, TIFF2026503550000013.tif19170Here, * indicates a bond to A, ** indicates a bond to C, Each C, independently, TIFF2026503550000014.tif17170n is an integer in the range of 2 to 6; a is an integer ranging from 1 to 5; b is an integer ranging from 1 to 5; each y is independently an integer ranging from 1 to 10; Each R1 independently represents one or more of C3 to C 12 Unbranched C1-C optionally substituted with cycloalkyl 18 is alkyl; Each R' is independently an unbranched C1-C 18 is alkylene; R3 is a C1-C optionally substituted with one or more hydroxyl 10alkyl or -NH-(C=O)-(C1-C6 alkyl), or a salt thereof.

[0028] In some embodiments, TIFF2026503550000015.tif19170

[0029] In some embodiments, each C is TIFF2026503550000016.tif11170 In some embodiments, each C In some embodiments, each R is C alkyl. In some embodiments, each R is TIFF2026503550000018.tif7170 In some embodiments, y is 1. In some embodiments, a is 1 and b is 1. In some embodiments, a is 2 and b is 2.

[0030] In some embodiments, each C is TIFF2026503550000019.tif17170In some embodiments, each R1' is a C1 alkylene. In some embodiments, each R1' is a C2 alkylene. In some embodiments, each R1' is a C4 alkylene. In some embodiments, y is 7.

[0031] In some embodiments, In some embodiments, each R is C alkyl. In some embodiments, each R is TIFF2026503550000021.tif7170 In some embodiments, y is 1. In some embodiments, a is 1 and b is 1. In some embodiments, a is 2 and b is 2.

[0032] In some embodiments, TIFF2026503550000022.tif18170 In some embodiments, each R' is a C1 alkylene. In some embodiments, each R' is a C2 alkylene. In some embodiments, each R' is a C4 alkylene. In some embodiments, each R' is a C1 alkylene and y is 7. In some embodiments, each R' is a C2 alkylene and y is 7. In some embodiments, each R' is a C4 alkylene and y is 1.

[0033] In some embodiments, each R1 is C4 alkyl.

[0034] In some embodiments, each R1' is a C1 alkylene.

[0035] In some embodiments, each R1' is a C2 alkylene.

[0036] In some embodiments, each R1' is a C4 alkylene.

[0037] In some embodiments, R3 is CH3.

[0038] In some embodiments, R3 is a C1-C substituted with one or more hydroxyl. 10 In some embodiments, TIFF2026503550000023.tif17170 In some embodiments, TIFF2026503550000024.tif24170 In some embodiments, TIFF2026503550000025.tif27170

[0039] In some embodiments, TIFF2026503550000026.tif37170

[0040] In some aspects, each R is C alkyl and y is 1. In some embodiments, a is 1 and b is 1. In some embodiments, a is 2 and b is 2.

[0041] In some aspects, each R1 is a C1 alkylene or a C2 alkylene and y is 7. In some embodiments, a is 2 and b is 2.

[0042] In some aspects, each R is a C alkylene and y is 1. In some embodiments, a is 1 and b is 1. In some embodiments, a is 2 and b is 2.

[0043] In some embodiments, a is 1.

[0044] In some embodiments, b is 1.

[0045] In some embodiments, a is 1 and b is 1.

[0046] In some embodiments, a is 2.

[0047] In some embodiments, b is 2.

[0048] In some embodiments, a is 2 and b is 2.

[0049] In some embodiments, n is 4.

[0050] In some embodiments, y is 1.

[0051] In some embodiments, y is 7.

[0052] In some embodiments, the compound of formula (I) is: A compound selected from TIFF2026503550000027.tif253170TIFF2026503550000028.tif69170.

[0053] In one aspect, the present disclosure provides a compound of formula (II): TIFF2026503550000029.tif12170 formula (II) or a salt thereof, wherein A, TIFF2026503550000030.tif90170Each B is independent, TIFF2026503550000031.tif17170Here, * indicates a bond to A, ** indicates a bond to C, Each C, independently, TIFF2026503550000032.tif17170 or C1~C 18 is alkyl; n is an integer ranging from 2 to 6; a is an integer ranging from 1 to 5; b is an integer ranging from 1 to 5; Each R1 is independently C1 to C 18 Alkyl or C2-C 18 Alkenyl, C1-C 18 Alkyl or C2-C 18 One or more C3-C alkenyl groups 12 optionally substituted with cycloalkyl; Each R' is independently an unbranched C1-C 18 is alkylene; R3 is (i) a C1-C optionally substituted with one or more hydroxyl 10 alkyl, -NH-(C=O)-(C1-C6 alkyl) or phenyl, or (ii) -cyclohexyl or -(C1-C6 alkylene)-hydroxyl optionally substituted with one or more hydroxyl; Each Y is independently TIFF2026503550000033.tif13170where *** indicates binding to R1; each p is independently an integer ranging from 0 to 3; each q is independently 0 or 1; Each z is independently 0 or 1, or a salt thereof.

[0054] In some embodiments, TIFF2026503550000034.tif19170

[0055] In some embodiments, TIFF2026503550000035.tif19170

[0056] In some embodiments, TIFF2026503550000036.tif19170

[0057] In some embodiments, each B is TIFF2026503550000037.tif17170Here, * indicates a bond to A and ** indicates a bond to C.

[0058] In some embodiments, each B is TIFF2026503550000038.tif16170Here, * indicates a bond to A and ** indicates a bond to C.

[0059] In some embodiments, each C is TIFF2026503550000039.tif14170 In some embodiments, each C TIFF2026503550000040.tif14170 In some embodiments, each C TIFF2026503550000041.tif14170

[0060] In some embodiments, each C is TIFF2026503550000042.tif11170 In some embodiments, each C TIFF2026503550000043.tif11170 In some embodiments, each C TIFF2026503550000044.tif11170

[0061] In some embodiments, each C is TIFF2026503550000045.tif17170

[0062] In some embodiments, each C is TIFF2026503550000046.tif17170

[0063] In some embodiments, each C is TIFF2026503550000047.tif11170 or C1~C 18 It is alkyl.

[0064] In some embodiments, each Y is TIFF2026503550000048.tif12170Here, *** indicates binding to R1.

[0065] In some embodiments, each Y is TIFF2026503550000049.tif14170Here, *** indicates binding to R1.

[0066] In some embodiments, each Y is TIFF2026503550000050.tif7170

[0067] In some embodiments, a is 2.

[0068] In some embodiments, b is 2.

[0069] In some embodiments, a is 2 and b is 2.

[0070] In some embodiments, each R is C 18 In some embodiments, each R is alkyl. TIFF2026503550000051.tif12170 In some embodiments, each R1 is TIFF2026503550000052.tif7170 In some embodiments, each R1 is The file is TIFF2026503550000053.tif6170.

[0071] In some embodiments, TIFF2026503550000054.tif12170Here, *** indicates a bond to R1, and each R1 is C1 to C 18 In some embodiments, each R is alkyl. TIFF2026503550000055.tif12170 In some embodiments, each R1 is TIFF2026503550000056.tif7170 In some embodiments, each R1 is TIFF2026503550000057.tif6170

[0072] In some embodiments, each R is C 18 In some embodiments, each R is alkenyl. TIFF2026503550000058.tif7170

[0073] In some embodiments, TIFF2026503550000059.tif12170Here, *** indicates a bond to R1, and each R1 is C2 to C 18 In some embodiments, each R is alkenyl. TIFF2026503550000060.tif7170.

[0074] In some embodiments, each R is C 18 In some embodiments, each R is alkyl. TIFF2026503550000061.tif11170 In some embodiments, each R1 is TIFF2026503550000062.tif6170 In some embodiments, each R1 is one or more of C3 to C 12 Cycloalkyl-substituted C1-C 18 In some embodiments, each R is alkyl. TIFF2026503550000063.tif11170

[0075] In some embodiments, TIFF2026503550000064.tif12170 Each R1 is C1~C 18 In some embodiments, each R is alkyl. TIFF2026503550000065.tif11170 In some embodiments, each R1 is TIFF2026503550000066.tif6170 In some embodiments, each R1 is one or more of C3 to C 12 Cycloalkyl-substituted C1-C 18 In some embodiments, each R is alkyl. TIFF2026503550000067.tif11170

[0076] In some embodiments, TIFF2026503550000068.tif12170 Each R1 is C1~C 18 In some embodiments, each R is alkyl. TIFF2026503550000069.tif11170 In some embodiments, each R1 is TIFF2026503550000070.tif9170

[0077] In some embodiments, TIFF2026503550000071.tif13170Here, *** indicates a bond to R1, and each R1 is C1 to C 18 In some embodiments, each R is alkyl. TIFF2026503550000072.tif8170

[0078] In some embodiments, z is 1.

[0079] In some embodiments, TIFF2026503550000073.tif19170z is 1.

[0080] In some embodiments, z is 0.

[0081] In some embodiments, TIFF2026503550000074.tif19170z is 0.

[0082] In some embodiments, q is 0.

[0083] In some embodiments, q is 1.

[0084] In some embodiments, p is 0.

[0085] In some embodiments, TIFF2026503550000075.tif19170p is 1.

[0086] In some embodiments, p is 1.

[0087] In some embodiments, p is 2.

[0088] In some embodiments, p is 3.

[0089] In some embodiments, n is 4.

[0090] In some embodiments, R3 is CH3.

[0091] In some embodiments, R3 is CH2CH2CH3.

[0092] In some embodiments, R3 is a C1-C substituted with one or more hydroxyl. 10 In some embodiments, TIFF2026503550000076.tif16170 In some embodiments, TIFF2026503550000077.tif24170 In some embodiments, TIFF2026503550000078.tif27170 In some embodiments, TIFF2026503550000079.tif27170 In some embodiments, TIFF2026503550000080.tif30170 In some embodiments, TIFF2026503550000081.tif19170 In some embodiments, TIFF2026503550000082.tif15170 In some embodiments, TIFF2026503550000083.tif15170 In some embodiments, TIFF2026503550000084.tif15170 In some embodiments, TIFF2026503550000085.tif19170 In some embodiments, TIFF2026503550000086.tif19170 In some embodiments, TIFF2026503550000087.tif19170 In some embodiments, TIFF2026503550000088.tif10170

[0093] In some embodiments, R3 is a C1-C substituted with one or more phenyl. 10 It is alkyl.

[0094] In some embodiments, R3 is -CH2-phenyl.

[0095] In some aspects, R3 is cyclohexyl substituted with one or more hydroxyl or -(C1-C6 alkylene)-hydroxyl. TIFF2026503550000089.tif24170 In some embodiments, TIFF2026503550000090.tif27170

[0096] In some embodiments, TIFF2026503550000091.tif37170

[0097] In some embodiments, the compound of formula (II) is TIFF2026503550000092.tif234170TIFF2026503550000093.tif239170TIFF2026503550000094.tif244170TIF F2026503550000095.tif242170TIFF2026503550000096.tif196170TIFF2026503550000097.tif242170TIFF20 26503550000098.tif252170TIFF2026503550000099.tif243170TIFF2026503550000100.tif233170TIFF2026503550000101.tif218170TIFF2026503550000102.tif232170TIFF2026503550000103.tif95170.

[0098] It will be understood that the compounds of any one of the formulas disclosed herein, and any pharmaceutically acceptable salts thereof, include stereoisomers, mixtures of stereoisomers, and polymorphs of all isomeric forms of said compounds.

[0099] However, it will be understood that the compounds disclosed herein may be presented without a specified configuration (e.g., without a specified stereochemistry). Such presentation is intended to encompass all available isomers, tautomers, positional isomers, and stereoisomers of the compound. In some embodiments, the presentation of a compound herein without a specific configuration is intended to refer to each of the available isomers, tautomers, positional isomers, and stereoisomers of the compound, or any mixture thereof.

[0100] It should be understood that the compounds of any formula described herein include the compounds themselves, as well as their salts, and optionally their solvates.Salts can be formed, for example, between an anion on the substituted compounds disclosed herein and a positively charged group (e.g., amino).Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetic acid, glutamic acid, glucuronic acid, glutaric acid, malate, maleic acid, succinic acid, fumaric acid, tartaric acid, tosylate, salicylic acid, lactate, naphthalenesulfonate, acetate (e.g., trifluoroacetic acid), etc.

[0101] In any of the formulas described herein, when a "-" is used to indicate a link between two variables (e.g., A to B), it is understood that the linkage can be one or more covalent bonds.

[0102] General Methods for the Preparation of Compounds of Formula (I) or Formula (II) of the Present Disclosure Compounds of Formula (I) or Formula (II) may be prepared using the reagents, intermediates, precursors, methods and schemes disclosed herein, or using other commercially available reagents and methods known to those skilled in the art.

[0103] General procedure for the synthesis of lipidoid (A) TIFF2026503550000104.tif143170 Intermediate 2

[0104] Trans-cyclohexyl 1,4-di-methanol 1 (23 g, 0.16 mol) was dissolved in 200 ml of dry THF, and dihydropyran (13.4 g, 1.0 equiv.) was added. After cooling the reaction mixture in an ice bath, pyridinium p-toluenesulfonate (PPTS) (2 g, 0.05 equiv.) was added. The resulting reaction mixture was stirred at room temperature for 24 h. The reaction mixture was quenched with saturated NaHCO3 (200 ml), extracted sequentially with EtOAc (150 ml x 3), washed with brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified on a silica gel column (150 g, gradient elution from 10% to 30% EtOAc in hexane) to give the desired intermediate 2 (Cy-THP) (23.7 g, 65% yield). 1 H NMR(500MHz,CDCl3):δ4.56-4.54(m,1H),3.87-3.82(m,1H),3.56-3.52 dd,J=9.8Hz,9.8Hz),3.51-3.46(m,1H),3.46(d,1H,J=6Hz),3.21-3.218(dd,1H,J=3hz, 6Hz),1.92-1.76(m,5H),1.72-1.67(m,1H)1.60-1.38(m,7H),0.98-0.93(m,4H).MS:m / z 251.2(M+Na).

[0105] Intermediate 3

[0106] THP-Cy 2 (4 g, 17.54 mmol) was dissolved in dry DCM (100 mL) and dry pyridine (9 mL) at room temperature. The mixture was cooled to -78 °C in an acetone-dry ice bath, followed by the dropwise addition of anhydrous triflic acid (4.7 mL, 1.6 equiv.) at -78 °C over 30 min. The reaction mixture was stirred from -78 to -30 °C for 3 h, at which point TLC showed that most of the starting material had been consumed. The reaction mixture was diluted with 100 mL of DCM, then quenched with 1 N HCl (150 mL), extracted with DCM (100 mL x 2), washed with NaHCO3, brine, and dried over NaSO4 to give crude triflate 3.

[0107] Intermediate 5

[0108] Alkyne 4 (17.54 mmol) was placed in 50 mL of dry THF and cooled to -78 °C. 2.5 M n-BuLi (8.4 mL, 1.2 equiv.) in hexane was added dropwise over 30 min. The resulting mixture was stirred at -78 °C for 2 h, followed by the addition of HMPA (hexamethylphosphoramide) (6.3 g, 2 equiv.) at the same temperature. The mixture was stirred at -78 °C for an additional 1.0 h. Crude triflate 3 in THF (10 mL) was added to the addition funnel over 30 min at -78 °C. The reaction mixture was stirred for an additional 2–3 h until the temperature reached -30 °C and monitored by TLC until the disappearance of the triflate. The reaction mixture was quenched with saturated NH4Cl (150 mL), extracted with EtOAc (100 mL × 3), washed with NaHCO3, brine, and dried over Na2SO4. The crude product was purified by silica gel column to give pure intermediate 5 in 90% yield.

[0109] 5a 1 H NMR (500MHz, CDCl3): δ4.57-4.55(m,1H),3.86-3.83(m,1H),3.56-3.52(dd,1H,J =9.8Hz,9.8.0Hz),3.49-3.48(m,1H),3.26(dd,1H,J=4.8Hz,9.8Hz),2.53-2.51( m,1H),2.06-2.04(m,2H),1.88-1.79(m,5H),1.75-1.67(m,1H),1.62-1.49(m,5H ),1.42-1.33(m,1H),1.14-1.12((dd,6H,6H,J=9.8Hz),1.04-0.96(m,4H).MS:m / z 301.1(M+Na).

[0110] 5b 1H NMR (500MHz, CDCl3): δ4.56(T,1H,J=4.8Hz),3.86-3.83(m,1H),3.56-3.52(dd ,1H,J=9.8Hz,8.0Hz),3.49-3.48(m,1H),3.21-3.18(dd,1H,J=4.8Hz,9.6Hz),2 .07-2.03(m,4H),1.88-1.75(m,7H),1.73-1.59(m,4H),1.58-1.45(m,5H),1.4 4-1.33(m,2H),1.31-1.15(m,2H),1.12-1.05(m,1H),1.04-0.93(m,6H).MS:m / z 355.1(M+Na).

[0111] Intermediate 6

[0112] Intermediate 5 was dissolved in a mixed solvent of EtOAc / MeOH (2 / 1) and 10 wt% Pd / C was added at room temperature. The reaction mixture was treated with H2 in a balloon overnight and filtered through Celite to give intermediate 6 in quantitative yield without further purification.

[0113] Intermediate 7

[0114] Intermediate 6 was dissolved in MeOH (1 g / 10 MeOH), followed by the addition of CSA (0.05 equiv.). The resulting mixture was stirred at room temperature and neutralized with TEA to give the desired deprotected intermediate 7. After concentration and purification on a silica gel column, intermediate 7 was obtained in 85% yield.

[0115] 7a 1 H NMR (500MHz, CDCl3): δ3.45(d,2H,J=4.8Hz),1.79-1.77(m,4H),1.54-1.30(m,4H),1. 28-1.23(m,3H),1.17-1.12(m,4H),0.95-0.88(m,2H),087-0.86(d,6H,J=3Hz).MS:m / z 221.1(M+Na).

[0116] 7b 1H NMR (500MHz, CDCl3): δ3.45(d,2H,J=4.8Hz),1.78-1.76(m,4H),1.69-1.64(m ,5H),1.56-1.42(m,2H),1.26-1.09(m,12H),0.97-0.81(m,6H).MS(APCI):m / z 275.2(M+Na).

[0117] Intermediate 8

[0118] Intermediate 7 was mixed with dry DCM (15 ml / g) and 1.5 equivalents of DIEA, followed by the addition of acryloyl chloride (1.6 equivalents) in an ice bath. The resulting reaction mixture was stirred at room temperature for 2-3 hours until the starting material was consumed. The reaction mixture was quenched with cold 1N HCl (2 equivalents), extracted twice with DCM, then washed with saturated NaHCO3, brine, and dried over Na2SO4. After purification on a silica gel column, intermediate 8 was obtained in 85% yield.

[0119] Products 10a and 10b

[0120] Acrylate 7 and amine 404 (0.225 equiv. per acrylate) were mixed neat and heated at 90 °C for 48 h until the major product was the desired product, as monitored by LCMS. The reaction mixture was cooled to room temperature, diluted with DCM, and then purified on a silica gel column using DCM / EtOAC to give the desired product 10 in 40–50% yield.

[0121] 10a 1 H NMR (500MHz, CDCl3): δ3.87(d,8H,J=9.8Hz),2.77(t,8H,J=9.8Hz),2.43(t,12H,J=4.8Hz),2.28(m,4H),2.18(s,3H),1.78- 1.74(m,14H),1.58-1.49(m,20H),1.31-1.22(m,16H),1.16-1.11(m,14H),0.97-0.88(m,8H),0.86(d,24H,J=9.8Hz).MS:m / z 1155.2(M+H).

[0122] 10b1 H NMR (500MHz, CDCl3): δ3.87(d,8H,J=6Hz),2.77(t,8H,J=6Hz),2.43(t,12H,J=4.8Hz),2.19(m,4H), 2.18(s,3H),1.77-1.61(m,30H),1.25-1.19(m,20H),1.18-1.12(m,32H),0.98-0.82(m,24H).MS:m / z 1371.2(M+H).

[0123] General procedure for the synthesis of lipidoids (B) TIFF2026503550000105.tif254170 intermediate 12a

[0124] Starting material 11 (1.0 g, 6.62 mmol) was dissolved in dry THF (30 ml), and 1,3-dibromopropane (2.94 g, 2.2 equivalents) and DIEA (3.37 ml, 3.0 equivalents) were added. The mixture was heated to 55° C. for 24 hours and cooled to room temperature. The mixture was diluted with EOTAc (100 ml) and then washed with saturated NaHCO and brine to obtain the crude product. After purification on a silica gel column, 910 mg of intermediate 12a was obtained as a pale yellow oil (35% yield). 1 H NMR (500MHz, CDCl3): δ7.37-7.26(m,5H),4.52(s,2H),2.68-2.60(m,6H),2.05-1.95(m,4H).MS:m / z 392.0(M+H).

[0125] Intermediate 14

[0126] Pyridine (0.29 mL, 5.0 equiv.) and alcohol 7a (300 mg, 2.1 equiv.) were dissolved in dry DCM (10 mL), followed by the dropwise addition of malon chloride 13 (100 mg, 0.714 mmol) in an ice bath. The resulting reaction mixture was stirred at room temperature for 2-3 h until the starting material was consumed. The reaction mixture was quenched with saturated NaHCO3 and extracted with DCM. The crude product was dried, purified on silica gel, and concentrated to give the desired intermediate 14 (225 mg, 68% yield).

[0127] Intermediate 16a

[0128] Intermediate 14 (100 mg, 0.215 mmol) was dissolved in dry DMF (5 mL) and NaH (60%, 9.46 mg, 1.1 equiv.) was added in an ice bath. The resulting mixture was stirred at room temperature for 30 min, and then intermediate 12a (38 mg, 0.45 equiv.) was added at room temperature. The mixture was warmed to 40 °C for 2–3 h until the reaction was complete. The reaction mixture was quenched with saturated NH4Cl (20 mL) and extracted with EtOAc (20 mL × 3), followed by washing with NaHCO3 and brine and drying over anhydrous sodium sulfate to give the crude product, which was purified to give the desired intermediate 16a (57 mg, 51% yield).

[0129] Product 17a

[0130] A solution of intermediate 14 (360 mg, 0.78 mmol) and NaH (1 mmol, 40 mg) in dry DMF (1 mL) was stirred at room temperature for 30 min, followed by the addition of N-(2-(benzyloxy)ethyl)-3-bromo-N-(3-bromopropyl)propan-1-amine (0.44 mmol, 134 mg). The reaction was maintained at 40 °C for 12 h and then quenched with diluted ice-cold HCl solution (1 N). The mixture was washed with EtOAc (3 × 100 mL) and brine (3 × 100 mL). The combined organic layers were dried over NaSO and concentrated in vacuo. The crude material was purified by column chromatography (DCM:MeOH = 10:1 to 5:1, v / v), followed by reverse-phase Biotage to give the desired intermediate compound in 10% yield.

[0131] The intermediate compound was then treated with 10% Pd / C (80 mg) under H gas, and after 12 h the reaction was filtered. The crude material was purified by reverse phase Biotage to give the desired compound 17a in 75% yield. 1H NMR(500MHz,CDCl3)δ4.05-3.83(m,9H),3.38(t,J=7.1Hz,2H),3.22-2.99(m,5H),1.91(dd,J=10.4,5.3Hz,4H),1.89-1.66(m,19H), 1.54(ddq,J=26.6,13.3,6.7Hz,9H),1.39-1.22(m,10H),1.24-1.08(m,19H),1.01-0.88(m,12H),0.86(d,J=6.6Hz,26H).MS(ESI):C 66 H 119 Calculated value for NO9 [M+H] + 1070.9, measured value 1071.1.

[0132] Intermediate 15

[0133] Intermediate 15 was prepared according to the procedure for preparing intermediate 16a.

[0134] Intermediate 18

[0135] Intermediate 15 was dissolved in dry THF and cooled with ice water, followed by the dropwise addition of 1M LiAlH (1.5 equivalents). The resulting reaction mixture was stirred overnight at room temperature and then quenched under argon with water, 15% aqueous NaOH, and water, successively. After filtration through Celite, the filtrate was extracted with EtOAc and saturated NaHCO. The organic layers were combined and dried over NaSO. The crude product was used in the next step without separation.

[0136] Product 17b

[0137] Intermediate 18 and cyclohexyl acid (4.4 equiv.) were mixed in dry DMF, followed by the addition of EDCl (6.0 equiv.) and DMAP (8.0 equiv.) at room temperature. The resulting reaction mixture was stirred at room temperature for 24 h and purified by silica gel column (Hex / EtOAc) to give the desired product 17b.

[0138] product 17c

[0139] The Bn-protected precursor (100 mg) was prepared according to the procedure for preparing intermediate 16a. This precursor was then dissolved in MeOH (1 mL) and treated with Pd / C (10 wt%) under a H balloon for 12 h. After filtration and concentration, product 17c was obtained. The crude was purified by reverse-phase Biotage™ to give the desired compound in 75% yield. 1 H NMR(500MHz,CDCl3)δ4.12-3.91(m,13H),3.89(d,J=6.5Hz,6H),3.43-3.33(m,2H),3.13(d,J=16.1Hz,6H),2.29(t,J=7.6H) z,8H),1.96-1.71(m,22H),1.68-1.47(m,19H),1.46-1.24(m,22H),1.06-0.94(m,11H),0.89(t,J=6.8Hz,12H).MS(ESI):C 70 H 119 NO 17 Calculated value for [M+H] + 1246.8, measured value 1247.0.

[0140] Intermediate 35

[0141] To a solution of alcohol 34 (4.5 g, 18.5 mmol) and malonyl dichloride (1.06 g, 7.6 mmol) in dry DCM (70 mL) was added TEA (66 mmol, 7 g) at -70 °C. The reaction was maintained at -70 °C to -30 °C for 6 h and then quenched with 1 N HCl (200 mL). The mixture was washed with EtOAc (3 × 200 mL), saturated NaHCO (2 × 200 mL), and brine (3 × 200 mL). The combined organic layers were dried over NaSO and concentrated in vacuo. The crude product was purified by column chromatography (Hex:EA = 10:1, v / v) to give the desired intermediate 35 as a colorless oil (2.9 g) in 70% yield. 1H NMR(500MHz,CDCl3)δ4.06(d,J=7.2Hz,1H),3.97(t,J=7.1Hz,3H),3.88(d,J=6.5Hz,2H),3.37(s,2H),2.29(t,J =7.6Hz,4H),1.95-1.75(m,6H),1.71-1.47(m,9H),1.45-1.23(m,11H),1.05-0.96(m,5H),0.88(t,J=6.9Hz,6H).

[0142] 3-Bromo-N-(3-bromopropyl)-N-methylpropan-1-amine (12b)

[0143] To a solution of 3,3'-(methylazanediyl)bis(propan-1-ol) (10 mmol) in dry DCM at 0 °C was added PBr (50 mmol); the mixture was heated at 37 °C for 12 h and then quenched with saturated NaHCO in a bath. The mixture was washed with DCM (3 × 200 mL) and brine (3 × 200 mL). The combined organic layers were dried over NaSO and concentrated in vacuo. The crude product was purified by column chromatography (DCM:MeOH = 5:1, v / v) to give the desired crude product. The crude product was then purified by reverse-phase Biotage to give 12b in 35% yield. 1 H NMR(500MHz,CDCl3)δ3.52(ddd,J=7.6,5.0,2.4Hz,2H),3.28(tt,J=11.3,4.5Hz,1H),3.20-3.10( m,1H),2.82(d,J=4.6Hz,2H),2.66-2.52(m,1H),2.44(tdd,J=14.2,7.3,5.2Hz,1H).MS(ESI):C7H 15 Calculated value for Br2N [M+H] + 272.0, measured value 271.9.

[0144] product 17d

[0145] Product 17d was prepared according to the procedure for preparing intermediate 16a. 11H NMR (500 MHz, CDCl3) δ 4.13 - 3.92 (m, 11H), 3.89 (d, J = 6.5 Hz, 6H), 3.43 - 3.33 (m, 3H), 3.14 (s, 3H), 2.94 (s, 2H), 2.76 (s, 3H), 2.29 (t, J = 7.6 Hz, 8H), 1.97 - 1.73 (m, 23H), 1.67 - 1.57 (m, 14H), 1.53 (tq, J = 8.3, 4.4 Hz, 6H), 1.41 (q, J = 7.3 Hz, 7H), 1.37 - 1.25 (m, 17H), 1.06 - 0.94 (m, 12H), 0.93 - 0.85 (m, 12H). MS (ESI): C 69 H 117 NO 16 Calculated value for [M + H] related to + 1216.8, measured value 1216.6.

[0146] Product 17e

[0147] Product 17e was prepared according to the preparation procedure of intermediate 16a. 1 1H NMR (500 MHz, CDCl3) δ 3.99 - 3.86 (m, 6H), 3.35 (t, J = 7.6 Hz, 2H), 2.38 - 2.29 (m, 4H), 2.15 (s, 3H), 1.88 (q, J = 7.6 Hz, 4H), 1.81 - 1.69 (m, 11H), 1.64 - 1.43 (m, 29H), 1.26 (t, J = 5.2 Hz, 8H), 1.21 - 1.08 (m, 14H), 1.02 - 0.88 (m, 8H), 0.86 (d, J = 6.6 Hz, 18H). MS (ESI): C 65 H 117 Calculated value for [M + H] related to NO8 + 1040.9, measured value 1040.9.

[0148] <0To a solution of 4-cyclohexylbutan-1-ol (2.26 g, 14 mmol) and malonyl dichloride (0.85 g, 6 mmol) in dry DCM (50 mL) was added TEA (40 mmol, 5 g) at -70 °C. The reaction was maintained at -70 °C to -30 °C for 6 h and then quenched with 1 N HCl (200 mL). The mixture was washed with EtOAc (3 × 200 mL), saturated NaHCO (2 × 200 mL), and brine (3 × 200 mL). The combined organic layers were dried over NaSO and concentrated in vacuo. The crude material was purified by column chromatography (Hex:EA = 10:1, v / v) to give the desired product 100 as a colorless oil (1.7 g) in 75% yield. 1 H NMR(500MHz,CDCl3)δ4.13(t,J=6.8Hz,4H),3.36(s,2H),1.65(dddd,J=30.4,14 .9,8.2,4.7Hz,14H),1.41-1.28(m,4H),1.26-1.10(m,12H),0.94-0.76(m,5H).

[0149] To a solution of bis(4-cyclohexylbutyl)malonate (570 mg, 1.4 mmol) and CsCO (550 mg, 1.68 mmol) in dry THF (6 mL) was added 1,3-diiodopropane (498 mg, 1.68 mmol), and the mixture was kept at room temperature for 6 h. The mixture was concentrated in vacuo and directly purified by column chromatography (Hex:(EA:DCM = 1:6, v / v) = 50:1, v / v) to give the desired product 101 as a colorless oil (260 mg) in 35% yield.

[0150] To a solution of 101 (150 mg, 0.27 mmol) and 3-amino-1-propanol (8.3 mg, 0.11 mmol) in THF / MeCN (1:1, v / v) was added DIEA (116 mg, 0.9 mmol). The mixture was heated at 60 °C for 5 days. The mixture was purified by reverse-phase column chromatography (mobile phase A: 0.1% TFA in HO and mobile phase B: MeCN:IPA = 1:1, v / v) to give product 102. 1H NMR(500MHz,CDCl3)δ4.12(qt,J=10.8,6.8Hz,4H),3.08(d,J=9.5Hz,2H),1.91(q,J=7.3Hz,11H),1.79(d,J=7.1Hz ,2H),1.73-1.55(m,14H),1.33(tdd,J=9.6,7.1,5.3Hz,4H),1.28-1.07(m,12H),0.86(q,J=11.3Hz,4H).MS(ESI):C 55 H 97 Calculated value for NO9 [M+H] + 916.7, measured value 917.0.

[0151] To a solution of 103 (1.656 g, 3 mmol) and CsCO (1174 mg, 3.6 mmol) in dry THF (10 mL) was added 1,3-diiodopropane (1066 mg, 3.6 mmol), and the mixture was kept at room temperature for 6 h. The mixture was concentrated in vacuo and directly purified by column chromatography (Hex:(EA:DCM = 1:6, v / v) = 50:1, v / v) to give the desired product 104 as a colorless oil (496 mg) in 30% yield. 1 H NMR(500MHz,CDCl3)δ4.06(dd,J=11.7,7.2Hz,1H),4.00-3.93(m,3H),3.89(d,J=6.5Hz ,3H),3.36(t,J=7.4Hz,1H),3.18(t,J=6.8Hz,2H),2.30(t,J=7.5Hz,4H),2.05-1.98(m, 2H),1.91-1.75(m,9H),1.62(p,J=7.5Hz,7H),1.52(d,J=5.3Hz,1H),1.41(d,J=10.1Hz ,3H),1.37-1.24(m,12H),1.00(td,J=8.9,2.8Hz,6H),0.90(t,J=7.0Hz,8H).MS(ESI):C 34 H 57 Calculated value for IO8 [M+Na] + 743.3, measured value 743.2.

[0152] To a solution of 104 (160 mg, 0.22 mmol) and 3-amino-1-propanol (7.5 mg, 0.1 mmol) in THF / MeCN (1:1, v / v) was added DIEA (116 mg, 0.9 mmol), and the mixture was heated at 60 °C for 5 days. The mixture was purified by reverse-phase column chromatography (mobile phase A: 0.1% TFA in HO and mobile phase B: MeCN:IPA = 1:1, v / v) to give product 105.

[0153] General procedure for the synthesis of lipidoids (C) TIFF2026503550000108.tif162170Synthesis of acrylates General Protocol In a round-bottom flask, cyclohexyl-based carboxylic acid (1.0 equiv.), 4-dimethylaminopyridine (DMAP) (0.4 equiv.), and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) (1.5 equiv.) were dissolved in dichloromethane. The resulting solution was stirred at room temperature for 20 min, after which 2-hydroxyethyl acrylate (1.5 equiv.) was added dropwise. The resulting solution was stirred at room temperature for 20 h. Water was added and extracted with dichloromethane (3x). The combined organic extracts were washed with brine, dried over Na2SO4, filtered, and evaporated. The crude material was purified by silica gel flash column chromatography using a 3-5% EtOAc / hexane eluent to give the acrylate as a colorless oil.

[0154] Intermediate 5C

[0155] In a 100 mL round-bottom flask, trans-4-pentylcyclohexanecarboxylic acid (2.5 g, 1.0 equiv.), 4-dimethylaminopyridine (DMAP) (0.62 g, 0.4 equiv.), and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) (3.62 g, 1.5 equiv.) were dissolved in dichloromethane (30 mL). The resulting solution was stirred at room temperature for 20 minutes, after which 2-hydroxyethyl acrylate (2.2 mL, 1.5 equiv.) was added dropwise. The resulting solution was stirred at room temperature for 20 hours. Water (50 mL) was added, and the mixture was extracted with dichloromethane (3 × 50 mL). The combined organic extracts were washed with brine (20 mL), dried over NaSO, filtered, and evaporated. The crude material was purified by silica gel flash column chromatography using a 5% EtOAc / hexane eluent to give 5C as a colorless oil.

[0156] Colorless oil, 3.08 g; yield: 82%; 1 H NMR(499MHz,CDCl3)δ6.42(dd,J=17.4,1.4Hz,1H),6.13(dd,J=17.3,10.5Hz,1H),5.86(dd,J=10.5,1.4Hz,1H),4.38-4.28(m,4 H),2.24(tt,J=12.3,3.6Hz,1H),2.00-1.91(m,2H),1.84-1.76(m,2H),1.46-1.34(m,2H),1.34-1.12(m,9H),0.95-0.83(m,5H).

[0157] Intermediate C4 TIFF2026503550000109.tif16170

[0158] In a 100 mL round-bottom flask, cyclohexanepentanoic acid (1.0 g, 1.0 equiv.), 4-dimethylaminopyridine (DMAP) (0.26 g, 0.4 equiv.), and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) (1.25 g, 1.5 equiv.) were dissolved in dichloromethane (10 mL). The resulting solution was stirred at room temperature for 20 minutes, after which 2-hydroxyethyl acrylate (0.76 mL, 1.5 equiv.) was added dropwise. The resulting solution was stirred at room temperature for 20 hours. Water (50 mL) was added and extracted with dichloromethane (3 × 50 mL). The combined organic extracts were washed with brine (20 mL), dried over NaSO, filtered, and evaporated. The crude material was purified by silica gel flash column chromatography using a 5% EtOAc / hexane eluent to give C4 as a colorless oil.

[0159] Colorless oil, 0.86 g; yield: 59%; 1 H NMR(499MHz,CDCl3)δ6.43(dd,J=17.3,1.4Hz,1H),6.14(dd,J=17.3,10.5Hz,1H),5.86(dd,J=10.5,1.4Hz,1H),4. 38-4.29(m,4H),2.32(t,J=7.5Hz,2H),1.70-1.57(m,7H),1.35-1.27(m,2H),1.25-1.09(m,6H),0.90-0.79(m,2H).

[0160] Lipidoid synthesis General Protocol Polyamine (404 or 405, as shown in General Scheme C) (1.0 equiv.) and acrylate 5C, 3C, or C4 (5.0 equiv.) were combined in a scintillation glass vial. The vial was capped and the reaction mixture was stirred at 85 °C for 3 days. The cooled crude reaction was purified by silica gel flash column chromatography using 5-10% MeOH / CHCl eluent to afford the title compound as a light brown oil.

[0161] Product 5C-404

[0162] Amine 404 (116 mg, 1.0 equiv.) and 5C (1.21 g, 5.0 equiv.) were combined in a 20 mL scintillation glass vial. The capped vial was stirred at 85 °C for 3 days. The cooled reaction was purified by silica gel flash column chromatography using 4% MeOH / CHCl.

[0163] Light brown oil, 0.91 g; yield: 85%; 1 H NMR(499MHz,CDCl3)δ4.25(s,16H),2.76(t,J=7.2Hz,8H),2.46(t,J=7.3Hz,11H),2.35-2.12(m,10H),1.98-1.92(m,8 Mass:

[0164] Product 5C-405

[0165] Amine 405 (107 mg, 1.0 equiv.) and 5C (1.38 g, 5.0 equiv.) were combined in a 20 mL scintillation glass vial. The capped vial was stirred at 85 °C for 3 days. The cooled reaction was purified by silica gel flash column chromatography using 4% MeOH / CHCl.

[0166] Light brown oil, 0.80 g; yield: 67%; 1 H NMR(499MHz,CDCl3)δ4.25(s,16H),2.79(t,J=7.2Hz,8H),2.59-2.38(m,15H),2.30-2.15(m,7H),1.99 -1.91(m,8H),1.84-1.77(m,8H),1.40(qd,J=13.1,3.5Hz,8H),1.34-1.12(m,37H),0.97-0.82(m,20H).

[0167] Product C4-404 TIFF2026503550000110.tif35170

[0168] Amine 404 (29 mg, 1.0 equiv.) and C4 (0.3 g, 5.0 equiv.) were combined in a 4 mL scintillation glass vial. The capped vial was stirred at 85 °C for 3 days. The cooled reaction was purified by silica gel flash column chromatography using 4% MeOH / CHCl.

[0169] Light brown oil, 0.136 mg; Yield: 60%; 1H NMR(499MHz,CDCl3)δ4.26(s,16H),2.77(t,J=7.2Hz,8H),2.50-2.40(m,12H),2.36-2.25(m,12 H),2.18(s,3H),1.71-1.55(m,33H),1.35-1.28(m,8H),1.23-1.12(m,23H),0.89-0.80(m,8H).

[0170] Product C4-405 TIFF2026503550000111.tif32170

[0171] Amine 405 (25 mg, 1.0 equiv.) and C4 (0.3 g, 5.0 equiv.) were combined in a 4 mL scintillation glass vial. The capped vial was stirred at 85 °C for 3 days. The cooled reaction was purified by silica gel flash column chromatography using 4% MeOH / CHCl.

[0172] Light brown oil, 0.101 mg; Yield: 45%; 1H NMR(499MHz,CDCl3)δ4.26(s,16H),2.79(t,J=7.2Hz,8H),2.58-2.40(m,15H),2.32(t, J=7.5Hz,8H),2.22(s,3H),1.71-1.56(m,29H),1.34-1.10(m,32H),0.89-0.80(m,8H).

[0173] General procedure for the synthesis of lipidoids (D) TIFF2026503550000112.tif95170 intermediate 21a

[0174] To diol 20a (2 g, 13.7 mmol) in DCM (200 mL) at 0 °C was added TEA (1.38 g, 13.7 mmol) and 19 (1.08 g, 11.9 mmol) dropwise. The resulting mixture was stirred at 0 °C to room temperature overnight and washed with 1 N HCl, saturated NaHCO3, and brine. The combined organic phases were dried over Na2SO4 and concentrated. Purification by column chromatography (ethyl acetate / hexane) afforded intermediate 21a (861 mg, 36%) as a clear oil. 1 H NMR(500MHz,CDCl3)δ6.39(dd,J=17.4,1.5Hz,1H),6.11(dd,J=17.4,10.4Hz,1H),5.81(dd,J=10.4,1.5Hz, 1H),4.14(t,J=6.7Hz,2H),3.63(t,J=6.6Hz,2H),1.71-1.62(m,2H),1.61-1.52(m,2H),1.42-1.29(m,8H).

[0175] Intermediate 21b

[0176] Intermediate 21b was synthesized according to the procedure for 21a. 1 H NMR(500MHz,CDCl3)δ6.40(dd,J=17.4,1.4Hz,1H),6.12(ddd,J=17.4,10.4,0.8Hz,1H),5.82(dd,J=10 .5,1.4Hz,1H),4.09(d,J=7.2Hz,0.5H),3.99(d,J=6.5Hz,1.5H),3.55(dd,J=6.9,5.2Hz,0.5H),3.46(t ,J=5.7Hz,1.5H),1.88-1.80(m,3H),1.67(dddt,J=17.9,11.3,7.8,3.9Hz,0.5H),1.60-1.49(m,1H),1. 46(ddt,J=10.2,7.1,3.8Hz,1H),1.45-1.37(m,0.5H),1.30(dt,J=11.7,5.5Hz,1H),1.10-0.92(m,3H).

[0177] Intermediate 21c

[0178] Intermediate 21c was synthesized according to the procedure for 21a.

[0179] Intermediate 23a

[0180] To 21a (292 mg, 1.46 mmol) in DCM (20 mL) were added 22a (251 mg, 1.6 mmol, 1.1 equiv), EDCI (307 mg, 1.6 mmol, 1.1 equiv), and DMAP (196 mg, 1.6 mmol, 1.1 equiv). The resulting mixture was stirred at room temperature overnight and washed with 1N HCl, saturated NaHCO3, and brine. The combined organic phases were dried over Na2SO4 and concentrated. Purification by column chromatography (ethyl acetate / hexane) afforded intermediate 23a (332 mg, 67%) as a clear oil. 1 H NMR(500MHz,CDCl3)δ6.39(dd,J=17.4,1.5Hz,1H),6.12(dd,J=17.3,10.4Hz,1H),5.81(dd,J=10.4,1.5Hz,1H),4.15(t,J=6.7Hz,2H),4 .05(t,J=6.7Hz,2H),2.33-2.27(m,2H),1.74-1.52(m,10H),1.55-1.47(m,2H),1.41-1.29(m,7H),1.28-1.07(m,4H),0.94-0.83(m,2H).

[0181] Intermediate 23b

[0182] Intermediate 23b was synthesized according to the procedure for 23a. 1 H NMR(500MHz,CDCl3)δ6.40(dd,J=17.3,1.5Hz,1H),6.12(dd,J=17.3,10.4Hz,1H),5.81(dd,J=10.5,1.5Hz,1H),4.15(t,J=6.7Hz,2H),4.05(t,J=6.7 Hz,2H),2.17(d,J=7.0Hz,2H),1.83-1.57(m,10H),1.39-1.30(m,8H),1.2 6(qt,J=12.2,3.5Hz,2H),1.21-1.08(m,1H),0.96(qd,J=12.7,3.8Hz,2H).

[0183] Intermediate 23c

[0184] Intermediate 23c was synthesized according to the procedure for 23a. H NMR (499 MHz, CDCl) δ 6.40 (dd, J = 17.4, 1.4 Hz, 1H), 6.12 (dd, J = 17.3, 10.4 Hz, 1H), 5.82 (dd, J = 10.4, 1.4 Hz, 1H), 4.08 (d, J = 7.1 Hz, 0.5H), 3.99 (dd, J = 6.8, 4.0 Hz, 2H), 3.90 (d ,J=6.5Hz,1.5H),2.33-2.26(m,2H),1.83-1.78(m,3H),1.69-1.58(m,4H),1.57-1.51( m,1H),1.50-1.39(m,1H),1.38-1.23(m,4H),1.09-0.95(m,3H),0.89(t,J=6.9Hz,3H).

[0185] Intermediate 23d

[0186] Intermediate 23d was synthesized according to the procedure for 23a. H NMR (500 MHz, CDCl) δ 6.40 (dd, J = 17.3, 1.5 Hz, 1H), 6.12 (ddd, J = 17.4, 10.4, 0.8 Hz, 1H), 5.82 (dd, J = 10.4, 1.5 Hz, 1H), 4.08 (d, J = 7.2 Hz, 0.5H), 3.99 (dd, J = 6.9, 4.8 Hz, 2H), 3.90 (d, J =6.5Hz,1.5H),2.28(td,J=7.5,1.1Hz,2H),1.89-1.78(m,3H),1.68-1.58(m,3H),1.58-1. 51(m,3H),1.48-1.41(m,1H),1.23-1.15(m,2H),1.10-0.95(m,3H),0.88(d,J=6.6Hz,6H).

[0187] Intermediate 23e

[0188] Intermediate 23e was synthesized according to the procedure for 23a. 1H NMR(500MHz,CDCl3)δ6.40(dd,J=17.3,1.5Hz,1H),6.12(ddd,J=17.4,10.4,0.9Hz, 1H),5.82(dd,J=10.5,1.4Hz,1H),4.08(d,J=7.2Hz,0.5H),3.99(dd,J=6.9,3.3Hz, 2H),3.90(d,J=6.5Hz,1.5H),2.33-2.26(m,2H),1.89-1.78(m,3H),1.69-1.50(m,4 H),1.50-1.38(m,1H),1.34-1.22(m,11H),1.10-0.95(m,3H),0.88(t,J=6.9Hz,3H).

[0189] Intermediate 23f

[0190] Intermediate 23f was synthesized according to the procedure of 23a. 1 H NMR(500MHz,CDCl3)δ6.41(dd,J=17.3,1.5Hz,1H),6.13(ddd,J=17.4,10.4,0.9Hz,1 H),5.83(dd,J=10.5,1.4Hz,1H),5.59-5.30(m,2H),3.99(dd,J=6.9,3.3Hz,2H),3.90 (d,J=6.5Hz,3H),2.33-2.28(m,2H),2.20-2.17(m,1H),2.08-2.03(m,2H),1.85-1.8 0(m,5H),1.67-1.62(m,7H),1.38-1.25(m,14H),1.05-0.97(m,4H),0.89-0.87(m,3H)

[0191] Product 24a

[0192] A mixture of N1-(3-aminopropyl)-N1-methylpropane-1,3-diamine (11.8 mg, 0.081 mmol) and 23a (116 mg, 0.34 mmol) was heated at 90 °C for 72 h. The reaction mixture was cooled to room temperature and loaded onto a column (MeOH / DCM). The product 24a (37 mg, 30%) was purified as a clear gel.

[0193] 1H NMR(499MHz,CDCl3)δ4.05(td,J=6.8,1.6Hz,16H),2.77(t,J=7.3Hz,8H),2.43(td,J=7.0,2.2Hz,12H),2.29(q,J=7.0Hz,12H),2.1 8(s,3H),1.74-1.63(m,16H),1.61-1.54(m,32H),1.52(dt,J=8.7,7.0Hz,8H),1.32(m,24H),0.94-0.83(m,8H).MS actual value 1498.6[M+H] + , calculated value for [C87H155N3O16=1498.1]

[0194] Product 24b

[0195] Product 24b was synthesized according to the procedure for 24a. 1 H NMR(500MHz,CDCl3)δ4.05(td,J=6.8,2.1Hz,16H),2.76(t,J=7.3Hz,8H),2. 43(td,J=7.3,3.0Hz,12H),2.28(dd,J=8.9,5.8Hz,4H),2.17(d,J=7.0Hz,12 H),1.80-1.73(m,3H),1.73-1.65(m,16H),1.64-1.55(m,24H),1.32(s,32H) ,1.37-1.20(m,8H),1.20-1.08(m,4H),1.02-0.90(m,8H).MS actual value 1442.5[M+H] +, Calculated value for [C83H147N3O16=1442.08]

[0196] product 24c

[0197] Product 24c was synthesized according to the procedure of 24a. 1H NMR(500MHz,CDCl3)δ3.98(d,J=7.2Hz,5H),3.89(dd,J=6.6,1.8Hz,11H),2.77(t,J= 7.3Hz,8H),2.43(t,J=7.3Hz,12H),2.29(q,J=7.1Hz,12H),2.17(s,3H),1.85-1.75( m,14H),1.61(h,J=7.6Hz,18H),1.58-1.49(m,4H),1.45-1.37(m,4H),1.31(ddt,J=1 1.2,8.0,5.3Hz,16H),1.06-0.93(m,12H),0.89(t,J=6.9Hz,12H).MS actual value 1330.5[M+H] + , calculated value for [C75H131N3O16=1329.95]

[0198] product 24d

[0199] Product 24d was synthesized according to the procedure of 24a. 1 H NMR(499MHz,CDCl3)δ3.98(d,J=7.2Hz,5H),3.89(dd,J=6.6,2.2Hz,11H),2.77(t,J=7.4Hz,8H),2.43(t,J=7.3Hz,12H),2.28(t,J=7.5Hz,12H),2.17 (s,3H),1.85-1.78(m,2H),1.67-1.49(m,34H),1.46-1.36(m,6H),1.23-1. 15(m,10H),1.06-0.94(m,12H),0.88(d,J=6.6Hz,24H).MS actual value 1386.8[M+H] + , calculated value for [C79H139N3O16=1386.02]

[0200] product 24e

[0201] Product 24e was synthesized according to the procedure of 24a. 1H NMR(500MHz,CDCl3)δ3.98(d,J=7.2Hz,5H),3.89(dd,J=6.5,1.8Hz,11H),2.76(t,J =7.3Hz,8H),2.43(t,J=7.2Hz,12H),2.30(q,J=9.8Hz,12H),1.94(s,3H),1.83-1.7 6(m,12H),1.64-1.56(m,18H),1.59-1.50(m,5H,1.45-1.37(m,5H),1.34-1 .22(m,40H),1.06-0.93(m,12H),0.88(t,J=6.9Hz,12H).MS actual value 1498.6[M+H] + , calculated value for [C87H155N3O16=1498.14]

[0202] product 24f

[0203] Product 24f was synthesized according to the procedure of 24a. 1 H NMR (500MHz, CDCl3): δ5.27-5.32(m,8H),3.88(d,16H,J=9.8Hz),2.77(t,8H,J=12Hz),2.43(t,8H,J=9.8Hz),2.23(t,8H,J=9.8Hz),2.18(s,3H) ,2.06-2.02(dd,8H,J=3Hz,9.8Hz),1.80-1.82(m,16H),1.62-1.60(m,34 H),1.36-1.26(m,56H),1.01-0.97(m,14H),0.89(d,12H,J=3Hz).MS:m / z 1988.1(M+H).

[0204] General procedure for the synthesis of lipidoids (E) General formula E.1

[0205] The general structure of E.1 compounds is shown below. TIFF2026503550000113.tif47170

[0206] The synthetic route to E.1 compounds is shown in general Scheme E.1 below. This two-step sequence begins with the esterification reaction of trans-4-pentylcyclohexanecarboxylic acid with hydroxy-substituted alkyl bromides of different lengths (C3, C5, and C7) catalyzed by N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl) and N,N-dimethylpyridin-4-amine (DMAP). The corresponding esters, bearing bromide as a functional group handle, can be synthesized by the esterification of hydroxy-substituted amines (H n , where n=2, 3, or 4) to give the target compound. TIFF2026503550000114.tif98170

[0207] Experimental Protocol General Esterification Protocol In a 100 mL round-bottom flask, trans-4-pentylcyclohexanecarboxylic acid 5C (1.0 equiv.), EDC-HCl (1.5 equiv.), and DMAP (0.4 equiv.) were dissolved in dichloromethane (DCM). This solution was then stirred at room temperature for 20 min, after which hydroxy-substituted alkyl bromide 25 (1.5 equiv.) was added, and the resulting reaction was stirred at room temperature for 20 h. Brine solution was added, and the reaction was extracted with DCM (4x). The combined organic extracts were dried over Na2SO4, filtered, and evaporated. The crude material was purified by silica gel flash automated chromatography using a 5-10% EtOAc / hexane eluent to afford the bromo-substituted ester.

[0208] General Protocol for Amine Alkylation The amine (2-aminoethanol H2, 3-aminopropanol H3, or 4-aminobutanol H4) and bromo-substrate were weighed into a 1-dram scintillation vial. The solvent tetrahydrofuran (THF) and acetonitrile (CH3CN) (1:1) were poured into the vial, followed by N,N-diisopropylethylamine (DIPEA, 3.0 equiv.). The reaction vial was capped and stirred at 70 °C for 3 days. The reaction was then cooled, the solvent evaporated, and water (20 mL) was added. The resulting mixture was extracted with DCM (4 × 20 mL), and the combined DCM extracts were washed with brine (20 mL). The resulting extracts were dried over Na2SO4, filtered, and evaporated. The crude material was purified by flash silica gel chromatography using 2–10% MeOH / DCM.

[0209] 5CC3 TIFF2026503550000115.tif27170

[0210] Following the general protocol for esterification, trans-4-pentylcyclohexanecarboxylic acid (1.07 g) was combined with EDC (1.21 g) and DMAP (0.26 g) in 10 mL of DCM. 3-Bromopropanol (0.6 mL) was added after 15 minutes of stirring. The crude product after workup was purified using 6% EtOAc / hexane. Colorless oil, 1.26 g (73%); 1 H NMR(499MHz,CDCl3)δ4.20(q,J=6.2Hz,2H),3.53(dt,J=74.6,6.5Hz,2H),2.26-2.05(m,3H),2 .00-1.91(m,2H),1.85-1.76(m,2H),1.46-1.35(m,2H),1.32-1.15(m,9H),0.94-0.85(m,5H).

[0211] 5CC5 TIFF2026503550000116.tif24170

[0212] Following the general protocol for esterification, trans-4-pentylcyclohexanecarboxylic acid (5.0 g) was combined with EDC (5.8 g) and DMAP (1.23 g) in 50 mL of DCM. 7-Bromoheptanol (3.8 mL) was added after 15 minutes of stirring. The crude product after workup was purified using 4% EtOAc / hexane. Colorless oil, 5.3 g (62%); 1 H NMR(499MHz,CDCl3)δ4.06(t,J=6.5Hz,2H),3.47(dt,J=64.6,6.7Hz,2H),2.21(tt,J=12.2,3.6Hz,1H),1.98-1.92(m,2H),1.91-1.85 (m,1H),1.84-1.75(m,3H),1.68-1.61(m,2H),1.55-1.46(m,2H),1.40(qd,J=13.1,3.5Hz,2H),1.32-1.13(m,9H),0.94-0.86(m,5H).

[0213] 5CC7 TIFF2026503550000117.tif24170

[0214] Following the general protocol for esterification, trans-4-pentylcyclohexanecarboxylic acid (5.0 g, 1.0 equiv.), EDC-HCl (7.25 g, 1.5 equiv.), and DMAP (1.23 g, 0.4 equiv.) were dissolved in DCM (50 mL) in a 250 mL round-bottom flask. The solution was stirred at room temperature for 20 minutes, after which 1-bromoheptanol (5.8 mL, 1.5 equiv.) was added. After the reaction and workup were complete, the crude material was purified by silica gel flash automated chromatography using a 3% EtOAc / hexane eluent to give 5CC7 as a colorless oil. Colorless oil, 6.8 g; yield: 71%. 1 H NMR(499MHz,CDCl3)δ4.04(t,J=6.6Hz,2H),3.47(dt,J=62.7,6.8Hz,2H),2.20(tt,J=12.3,3.6Hz,1H ),1.99-1.91(m,2H),1.89-1.74(m,4H),1.62(p,J=6.7Hz,2H),1.48-1.13(m,17H),0.95-0.85(m,5H).

[0215] General formula E.2 The general structure of E.2 compounds is shown below. TIFF2026503550000118.tif44170

[0216] The synthetic route to E.2 compounds is shown below in general Scheme E.2. TIFF2026503550000119.tif96170

[0217] 5CC2

[0218] In a 200 mL round-bottom flask, trans-4-pentylcyclohexanecarboxylic acid (5.0 g, 1.0 equiv.) was combined with EDC (5.8 g, 1.2 equiv.) and DMAP (1.23 g, 0.4 equiv.) in DCM (50 mL). The suspension was then stirred at room temperature for 15 minutes, after which ethylene glycol (4.3 mL, 3.0 equiv.) was added, and the resulting mixture was stirred at room temperature for 20 hours. Water (20 mL) was added, and the reaction was extracted with DCM (4 × 50 mL). The combined DCM extracts were washed with brine (20 mL), dried over Na2SO4, filtered, and evaporated to dryness. The crude residue was purified by flash chromatography using a 10–15% EtOAc / hexane eluent. Colorless oil, 2.8 g (65%); 1 H NMR(499MHz,CDCl3)δ4.22-4.16(m,2H),3.84-3.78(m,2H),2.26(tt,J=12.2,3.6Hz,1H),2. 05-1.93(m,3H),1.86-1.76(m,2H),1.48-1.35(m,2H),1.32-1.15(m,9H),0.95-0.84(m,5H).

[0219] 5CC2C6

[0220] In a 200 mL round-bottom flask, 6-bromohexanoic acid (2.0 g, 1.0 equiv.) was combined with EDC (2.7 g, 1.2 equiv.) and DMAP (0.51 g, 0.4 equiv.) in DCM (30 mL). The suspension was then stirred at room temperature for 15 minutes, after which 5CC2 (2.7 g, 1.1 equiv.) was added, and the resulting mixture was stirred at room temperature for 20 hours. Water (20 mL) was added, and the reaction was extracted with DCM (4 × 50 mL). The combined DCM extracts were washed with brine (20 mL), dried over Na2SO4, filtered, and evaporated to dryness. The crude residue was purified by flash chromatography using a 6-8% EtOAc / hexane eluent. Colorless oil, 2.8 g (65%); 1 H NMR(499MHz,CDCl3)δ4.31-4.22(m,4H),3.47(dt,J=64.5,6.7Hz,2H),2.35(t,J=7.4Hz,2H),2.24(tt,J=12. 2,3.6Hz,1H),1.99-1.75(m,6H),1.70-1.61(m,2H),1.52-1.35(m,4H),1.34-1.14(m,9H),0.95-0.85(m,5H).

[0221] H2C6C25C

[0222] Following the general protocol for amine alkylation described in General Scheme E.1, amine H2 (17 mg) was combined with C6C25C (300 mg) and DIPEA (200 μL) in THF / CH3CN (1:1, 1.0 mL). After the reaction, the crude product was purified with a 4% MeOH / DCM eluent.

[0223] General formula E.3

[0224] The general structure of E.3 compounds is shown below. TIFF2026503550000120.tif49170

[0225] The synthetic route to E.3 compounds is shown below in general scheme E.3. TIFF2026503550000121.tif98170

[0226] CyO 6,10

[0227] In a 200 mL round-bottom flask, 2-hexyldecanoic acid 33 (5.0 g, 1.0 equiv.) was combined with EDC (4.56 g, 1.2 equiv.) and DMAP (0.95 g, 0.4 equiv.) in DCM (100 mL). The suspension was then stirred at room temperature for 15 min, after which trans-1,4-cyclohexanediol 32 (3.4 g, 1.5 equiv.) was added, and the resulting mixture was stirred at room temperature for 20 h. Water (20 mL) was added, and the reaction was extracted with DCM (4 × 50 mL). The combined DCM extracts were washed with brine (20 mL), dried over Na2SO4, filtered, and evaporated to dryness. The crude residue was purified by flash chromatography using a 10–15% EtOAc / hexane eluent. Colorless oil, 1.9 g (26%); 1 H NMR(499MHz,CDCl3)δ4.88-4.69(m,1H),3.85-3.63(m,1H),2.36-2.18(m,1H) ,2.03-1.91(m,4H),1.57-1.38(m,8H),1.32-1.19(m,20H),0.90-0.84(m,6H).

[0228] C6CyO 6.10

[0229] In a 200 mL round-bottom flask, 6-bromohexanoic acid (1.2 g, 1.0 equiv.) was combined with EDC (1.4 g, 1.2 equiv.) and DMAP (0.3 g, 0.4 equiv.) in DCM (20 mL). The suspension was then stirred at room temperature for 15 min before being added to CyO. 6,10 (2.5 g, 1.1 equiv.) was added and the resulting mixture was stirred at room temperature for 20 h. Water (20 mL) was added and the reaction was extracted with DCM (4 x 50 mL). The combined DCM extracts were washed with brine (20 mL); dried over Na2SO4; filtered and evaporated to dryness. The crude residue was purified by flash chromatography using a 4-5% EtOAc / hexane eluent. Colorless oil, 1.4 g (43%); 1H NMR(499MHz,CDCl3)δ4.92-4.75(m,2H),3.47(dt,J=64.2,6.7Hz,2H),2.36-2.24(m,3H) ,2.01-1.75(m,6H),1.68-1.40(m,12H),1.31-1.20(m,20H),0.87(td,J=7.0,1.6Hz,6H).

[0230] H2C6CyO 6.10

[0231] Following the general protocol for amine alkylation described in General Scheme E.1, amine H2 (19 mg) was reacted with C6CyO in THF / CH3CN (1:1, 1.0 mL). 6,10 (470 mg) and DIPEA (200 μL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent.

[0232] General formula E.4

[0233] The general structure of E.4 compounds is shown below. TIFF2026503550000122.tif52170

[0234] The synthetic route to E.4 compounds is shown below in general Scheme E.4. TIFF2026503550000123.tif90170

[0235] CyO 8,9

[0236] In a 200 mL round-bottom flask, trans-1,4-cyclohexanedicarboxylic acid 29 (1.0 g, 1.0 equiv.) was combined with EDC (0.56 g, 0.5 equiv.) and DMAP (0.15 g, 0.2 equiv.) in DCM (50 mL). The suspension was then stirred at room temperature for 15 min, after which 9-heptadecanol 30 (0.74 g, 0.5 equiv.) was added, and the resulting mixture was stirred at room temperature for 20 h. Water (20 mL) was added, and the reaction was extracted with DCM (4 × 50 mL). The combined DCM extracts were washed with brine (20 mL), dried over Na2SO4, filtered, and evaporated to dryness. The crude residue was purified by flash chromatography using a 10–15% EtOAc / hexane eluent. Colorless oil, 1.45 g (61%); 1 H NMR(499MHz,CDCl3)δ4.86(p,J=6.3Hz,1H),2.37-2.21(m,2H),2.14-2.01(m,4H),1.55-1.41(m,8H),1.31-1.21(m,24H),0.88(t,J=6.9Hz,6H).

[0237] C5CyO 8.9

[0238] In a 200 mL round-bottom flask, add CyO 8,9 (1.45 g, 1.0 equiv.) was combined with EDC (0.81 g, 1.2 equiv.) and DMAP (0.2 g, 0.4 equiv.) in DCM (20 mL). The solution was then stirred at room temperature for 15 min, after which 5-bromopentanol 31 (0.6 mL, 1.2 equiv.) was added, and the resulting mixture was stirred at room temperature for 20 h. Water (20 mL) was added, and the reaction was extracted with DCM (4 × 50 mL). The combined DCM extracts were washed with brine (20 mL), dried over Na2SO4, filtered, and evaporated to dryness. The crude residue was purified by flash chromatography using a 5-10% EtOAc / hexane eluent. Colorless oil, 1.1 g (57%); 1H NMR(499MHz,CDCl3)δ4.85(p,J=6.3Hz,1H),4.07(t,J=6.5Hz,2H),3.41(t,J=6.7Hz,2H),2.36-2.18(m,2H),2.12- 1.96(m,4H),1.94-1.81(m,2H),1.71-1.61(m,2H),1.56-1.39(m,10H),1.30-1.21(m,24H),0.87(t,J=6.9Hz,6H).

[0239] H2C5CyO 8.9

[0240] Following the general protocol for amine alkylation described in General Scheme E.1, amine H2 (11 mg) was reacted with C5Cy in THF / CH3CN (1:1, 0.8 mL). 8,9 (289 mg) and DIPEA (150 μL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent.

[0241] General formula E.5

[0242] A synthetic route to E.5 compounds is shown below in General Scheme E.5. TIFF2026503550000124.tif101170

[0243] Intermediate H2NHC75C

[0244] 5CC7 (1.0 g, 1.0 equiv.) was combined with H2 (4.83 mL, 30.0 equiv.) in ethanol (1.0 mL) in a 20 mL scintillation vial. The vial was then capped and heated to 60 °C for 20 h. The cooled reaction was then evaporated to dryness, and the resulting residue was purified by flash silica gel chromatography using 50-60% MeOH / DCM. Colorless oil, 0.34 g (36%); 1H NMR(499MHz,CDCl3)δ4.04(t,J=6.6Hz,2H),3.67-3.60(m,2H),2.80-2.74(m,2H),2.64-2.58(m,2H),2.20(tt,J= 12.2,3.6Hz,1H),2.02-1.86(m,4H),1.84-1.76(m,2H),1.65-1.57(m,2H),1.53-1.12(m,20H),0.95-0.84(m,5H).

[0245] General formula E.6

[0246] The general structure of E.6 compounds is shown below. TIFF2026503550000125.tif80170

[0247] The synthetic route to E.6 compounds is shown below in general scheme E.6. TIFF2026503550000126.tif102170

[0248] B5C

[0249] In a 200 mL round-bottom tube, trans-4-pentylcyclohexanecarboxylic acid 5C (3.95 g, 2.1 equiv.) was combined with EDC (3.91 g, 2.1 equiv.) and DMAP (1.2 g, 1.0 equiv.) in DCM (100 mL). The solution was stirred at room temperature for 20 min, after which 2-hydroxymethyl-1,3-propanediol 26 (1.02 g, 1.0 equiv.) was added. The resulting suspension was stirred at room temperature for 20 h. The hydroxy starting material slowly dissolves as the reaction proceeds. 100 mL of brine solution was added, and the reaction was stirred at ambient temperature for 20 min. The layers were separated, and the aqueous layer was extracted with DCM (4 × 50 mL). The combined extracts were dried over Na2SO4, filtered, and evaporated to dryness. The crude material was purified by silica gel flash chromatography using a 15–20% EtOAc / hexane eluent. Colorless oil, 1.51 g (34%). 1H NMR(499MHz,CDCl3)δ4.24-4.09(m,4H),3.59(t,J=6.0Hz,2H),2.29-2.14(m,4H),1.95(dt,J=12.2,3.6Hz ,4H),1.80(dt,J=15.3,3.2Hz,4H),1.40(qd,J=13.1,3.5Hz,4H),1.34-1.13(m,18H),0.96-0.83(m,10H).

[0250] 65C BC

[0251] In a 200 mL round-bottom flask, 6-bromohexanoic acid 27 (0.4 g, 1.0 equiv.) was combined with EDC (0.5 g, 1.2 equiv.) and DMAP (0.12 g, 0.5 equiv.) in DCM (20 mL). The solution was stirred at room temperature for 20 minutes, after which B5C (1.05 g, 1.1 equiv.) dissolved in 10 mL of DCM was added. The resulting solution was stirred at room temperature for 20 hours. Brine (50 mL) was added, and the reaction was stirred at room temperature for 20 hours. The layers were separated, and the aqueous layer was extracted with DCM (3 × 50 mL). The combined extracts were dried over Na2SO4, filtered, and evaporated to dryness. The crude product was purified by silica gel flash chromatography using a 6% EtOAc / hexane eluent. Colorless oil, 0.95 g (73%); 1 H NMR(499MHz,CDCl3)δ4.12(t,J=6.4Hz,6H),3.47(dt,J=64.3,6.7Hz,2H),2.40(h,J=6.0Hz,1H),2.34(t,J=7.5Hz,2H),2.22(tt,J=12.2,3.6 Hz,2H),1.99-1.76(m,10H),1.65(p,J=7.5Hz,2H),1.52-1.44(m,2H),1.39(qd,J=13.1,3.5Hz,4H),1.32-1.14(m,18H),0.94-0.84(m,10H).

[0252] HBC265

[0253] Following the general protocol for amine alkylation described in General Scheme E.1, amine H2 (12 mg) was combined with BC6B5C (292 mg) and DIPEA (200 μL) in THF / CH3CN (1:1, 0.8 mL). After the reaction, the crude product was purified with a 6% MeOH / DCM eluent.

[0254] H5-65C

[0255] In a 20 mL scintillation glass vial, 5-amino-1-pentanol (19 mg, 1.0 equiv.), BC6B5C (272 mg, 2.2 equiv.), K2CO3 (165 mg, 4.4 equiv.), and KI (62 mg, 1.0 equiv.) were combined and poured into CH3CN / THF (1:1, 3 mL). 4 Å molecular sieves were added to the suspension, and the capped vial was stirred at 75 °C for 3 days. The cooled reaction was filtered, deionized water (20 mL) was added, and the filtrate was extracted with DCM (4 × 40 mL). The combined extracts were washed with brine (20 mL), dried over Na2SO4, filtered, and evaporated. The crude material was purified by silica gel flash chromatography, and the desired product was eluted with a 4-5% MeOH / DCM eluent. TIFF2026503550000127.tif71170

[0256] Intermediate 106

[0257] In a 20 mL scintillation glass vial, 3-aminopropan-1-ol (4.0 g, 20.0 equiv.) and 5CC7 (1.0 g, 1.0 equiv.) were combined in EtOH (1.0 mL). The vial was capped and stirred at 70 °C for 20 h. The cooled reaction mixture was transferred to a 100 mL round-bottom flask, and the solvent was evaporated to dryness. The residue was dissolved in 100 mL of DCM and washed with 5% aqueous sodium bicarbonate (2 × 100 mL) followed by brine (50 mL); dried over NaSO; filtered, and evaporated. The crude material was purified by silica gel flash chromatography, and the desired product 106 eluted with a 40% MeOH / DCM eluent. Colorless oil, 301 mg (%); 1H NMR(499MHz,CDCl3)δ4.03(t,J=6.6Hz,2H),3.81(t,J=5.0Hz,2H),2.87(t,J =5.0Hz,2H),2.59(t,J=7.1Hz,2H),2.20(tt,J=12.3,3.6Hz,1H),1.99-1.90 (m,2H),1.83-1.76(m,2H),1.72-1.65(m,2H),1.64-1.56(m,2H),1.50-1.13 (m,20H),0.95-0.83(m,5H);LC-MS:Rt7.6min, m / z calculated value [M+H]:370.33,actual value 370.2.

[0258] H3-765C

[0259] In a 20 mL scintillation glass vial, 106 (120 mg, 1.0 equiv.), BC6B5C (229 mg, 1.1 equiv.), K2CO3 (161 mg, 2.2 equiv.), and KI (84 mg, 1.0 equiv.) were combined and poured into CH3CN / THF (1:1, 3 mL). 4 Å molecular sieves were added to the suspension, and the capped vial was stirred at 75 °C for 3 days. The cooled reaction was filtered, deionized water (20 mL) was added, and the filtrate was extracted with DCM (4 × 40 mL). The combined extracts were washed with brine (20 mL), dried over Na2SO4, filtered, and evaporated. The crude material was purified by silica gel flash chromatography, and the desired product was eluted with a 4-5% MeOH / DCM eluent. TIFF2026503550000128.tif53170

[0260] OH-C4

[0261] In a 200 mL round-bottom flask, cyclohexanepentanoic acid (2.2 g, 1.0 equiv.) was combined with EDC (2.8 g, 1.2 equiv.) and DMAP (0.8 g, 0.5 equiv.) in DCM (25 mL). The solution was then stirred at room temperature for 20 minutes, after which 2-hydroxymethyl-1,3-propanediol (0.86 g, 0.68 equiv.) in N,N-dimethylformamide (5 mL) was added. The resulting solution was stirred at room temperature for 20 hours. Brine solution (100 mL) was poured into the mixture and stirred at ambient temperature for 20 minutes. The layers were separated, and the aqueous layer was extracted with DCM (4 × 50 mL). The combined extracts were dried over Na2SO4, filtered, and evaporated to dryness. The crude product was purified by silica gel flash chromatography using a 15–20% EtOAc / hexane eluent. Colorless oil, 1.8 g (35%); 1 H NMR(499MHz,CDCl3)δ4.23-4.11(m,4H),3.61(t,J=6.0Hz,2H),2.32(t,J=7.5Hz,4H),2.25- 2.14(m,2H),1.70-1.56(m,14H),1.35-1.27(m,4H),1.25-1.08(m,12H),0.92-0.77(m,4H).

[0262] 6C4

[0263] In a 200 mL round-bottom flask, 6-bromohexanoic acid (0.46 g, 1.0 equiv.) was combined with EDC (0.6 g, 1.2 equiv.) and DMAP (0.2 g, 0.5 equiv.) in DCM (15 mL). The solution was then stirred at room temperature for 20 minutes, after which OH-C4 (2.2 g, 1.0 equiv.) in DCM (5 mL) was added. The resulting solution was stirred at room temperature for 20 hours. Brine solution (100 mL) was poured into the mixture and stirred at ambient temperature for 20 minutes. The layers were separated, and the aqueous layer was extracted with DCM (4 × 50 mL). The combined extracts were dried over Na2SO4, filtered, and evaporated to dryness. The crude product was purified by silica gel flash chromatography using a 5-10% EtOAc / hexane eluent. Colorless oil, 1.2 g (83%); 1H NMR(499MHz,CDCl3)δ6.09(dd,J=6.0,3.1Hz,6H),5.43(dt,J=64.4,6.7Hz,2H),4.40-4.23(m,7H),3.88-3. 71(m,2H),3.66-3.52(m,16H),3.48-3.40(m,2H),3.31-3.23(m,4H),3.23-3.03(m,12H),2.87-2.75(m,4H).

[0264] H2-6C4

[0265] In a 20 mL scintillation glass vial, H2 (17 mg, 1.0 equiv.), 6C4 (351 mg, 2.2 equiv.), K2CO3 (180 mg, 4.4 equiv.), and KI (49 mg, 1.0 equiv.) were combined and poured into CH3CN / THF (1:1, 3 mL). 4 Å molecular sieves were added to the suspension, and the capped vial was stirred at 75 °C for 3 days. The cooled reaction was filtered, deionized water (20 mL) was added, and the filtrate was extracted with DCM (4 × 40 mL). The combined extracts were washed with brine (20 mL), dried over Na2SO4, filtered, and evaporated. The crude material was purified by silica gel flash chromatography, and the desired product was eluted with a 4-5% MeOH / DCM eluent. TIFF2026503550000129.tif66170

[0266] Intermediate 107

[0267] In a 1000 mL round-bottom flask, 6-bromohexanoic acid (25 mg, 1.0 equiv.), EDC (29.5 g, 1.2 equiv.), and DMAP (7.9 g, 0.6 equiv.) were combined in DCM (250 mL) and stirred at room temperature for 30 minutes. A solution of (2,2-dimethyl-1,3-dioxan-5-yl)methanol (20.6 g, 1.1 equiv.) in DCM (15 mL) was added, and the resulting solution was stirred at room temperature for 20 hours. DI water (150 mL) was added, and the solution was extracted with DCM (4 × 150 mL). The combined DCM extracts were washed with brine (50 mL), dried over Na2SO4, filtered, and evaporated. The crude product was dissolved in a mixture of methanol (200 mL) and 1 N HCl (200 mL). The resulting solution was stirred at room temperature for 2 hours. The methanol was evaporated on a rotavapor and extracted with EtOAc (4 x 200 mL). The combined extracts were washed with brine (100 mL), dried over Na2SO4, filtered, and evaporated. The crude was carried on to the next step without purification.

[0268] In a 500 mL round-bottom flask, trans-4-n-pentylcyclohexanecarboxylic acid (10.0 g, 1.0 equiv.), EDC (11.7 g, 1.2 equiv.), and DMAP (3.1 g, 0.5 equiv.) were combined in DCM (100 mL) and stirred at room temperature for 30 min. A solution of the above crude material (17.0 g, 1.2 equiv.) in DCM (50 mL) was added, and the resulting solution was stirred at room temperature for 20 h. DI water (100 mL) was added, and the solution was extracted with DCM (4 × 100 mL). The combined extracts were washed with brine (50 mL), dried over Na2SO4, filtered, and evaporated. The crude material was purified by silica gel flash chromatography using 15–20% EtOAc / hexanes to give 107 as a colorless oil (7.8 g, 34%).

[0269] 6C125C

[0270] In a 100 mL round-bottom flask, dodecanoic acid (490 mg, 1.0 equiv.), EDC (0.62 g, 1.2 equiv.), and DMAP (0.2 g, 0.6 equiv.) were combined in DCM (10 mL) and stirred at room temperature for 15 minutes. A solution of 107 in DCM (50 mL) was added, and the resulting solution was stirred at room temperature for 20 hours. DI water (50 mL) was added, and the solution was extracted with DCM (4 x 50 mL). The combined DCM extracts were washed with brine (50 mL), dried over Na2SO4, filtered, and evaporated. The crude material was purified by flash chromatography using 5-10% EtOAc / hexanes. Colorless oil, 1.13 g (83%); 1 H NMR(499MHz,CDCl3)δ4.14-4.10(m,6H),3.47(dt,J=64.3,6.7Hz,2H),2.39(p,J=6.0Hz,1H),2.36-2.27(m,4H),2.22(tt,J=12.3 ,3.6Hz,1H),1.99-1.91(m,2H),1.89-1.75(m,4H),1.66-1.58(m,4H),1.51-1.35(m,4H),1.32-1.16(m,25H),0.95-0.83(m,8H).

[0271] H2-6C125C

[0272] In a 20 mL scintillation glass vial, H2 (11 mg, 1.0 equiv.), 6C125C (250 mg, 2.2 equiv.), K2CO3 (162 mg, 4.4 equiv.), and KI (85 mg, 1.0 equiv.) were combined and poured into CH3CN / THF (1:1, 3 mL). 4 Å molecular sieves were added to the suspension, and the capped vial was stirred at 75 °C for 3 days. The cooled reaction was filtered, deionized water (20 mL) was added, and the filtrate was extracted with DCM (4 × 40 mL). The combined extracts were washed with brine (20 mL), dried over Na2SO4, filtered, and evaporated. The crude material was purified by silica gel flash chromatography, and the desired product was eluted with a 4-5% MeOH / DCM eluent. TIFF2026503550000130.tif51170

[0273] In a 200 mL round-bottom flask, 6-bromopentanoic acid (1.17 g, 1.0 equiv.) was combined with EDC (1.4 g, 1.2 equiv.) and DMAP (0.4 g, 0.5 equiv.) in DCM (20 mL). The solution was then stirred at room temperature for 20 minutes, after which OH-5C (3.2 g, 1.0 equiv.) in DCM (10 mL) was added. The resulting solution was stirred at room temperature for 20 hours. Brine solution (100 mL) was added, and the solution was stirred at room temperature for 20 minutes. The layers were separated, and the aqueous layer was extracted with DCM (4 × 50 mL). The combined extracts were dried over Na2SO4, filtered, and evaporated to dryness. The crude product was purified by silica gel flash chromatography using a 5-10% EtOAc / hexane eluent. Colorless oil, 1.8 g (45%); 1 H NMR(499MHz,CDCl3)δ4.17-4.06(m,6H),3.47(dt,J=67.5,6.4Hz,2H),2.43-2.32(m,3H),2.22(tt,J=12.2,3.5Hz ,2H),1.98-1.85(m,5H),1.84-1.74(m,7H),1.39(qd,J=13.1,3.5Hz,4H),1.33-1.13(m,18H),0.96-0.83(m,10H).

[0274] H2-55C

[0275] In a 20 mL scintillation glass vial, H2 (14.6 mg, 1.0 equiv.), 55C (322 mg, 2.2 equiv.), K2CO3 (170 mg, 4.4 equiv.), and KI (41 mg, 1.0 equiv.) were combined and poured into CH3CN / THF (1:1, 3 mL). 4 Å molecular sieves were added to the suspension, and the capped vial was stirred at 75 °C for 3 days. The cooled reaction was filtered, deionized water (20 mL) was added, and the filtrate was extracted with DCM (4 × 40 mL). The combined extracts were washed with brine (20 mL), dried over Na2SO4, filtered, and evaporated. The crude material was purified by silica gel flash chromatography, and the desired product was eluted with a 4-5% MeOH / DCM eluent. TIFF2026503550000131.tif45170

[0276] In a 200 mL round-bottom flask, 6-bromooctanoic acid (1.4 g, 1.0 equiv.) was combined with EDC (1.5 g, 1.2 equiv.) and DMAP (0.4 g, 0.5 equiv.) in DCM (20 mL). The solution was then stirred at room temperature for 20 minutes, after which OH-5C (3.1 g, 1.0 equiv.) in DCM (10 mL) was added. The resulting solution was stirred at room temperature for 20 hours. Brine solution (100 mL) was added, and the solution was stirred at room temperature for 20 minutes. The layers were separated, and the aqueous layer was extracted with DCM (4 × 50 mL). The combined extracts were dried over Na2SO4, filtered, and evaporated to dryness. The crude product was purified by silica gel flash chromatography using a 5-10% EtOAc / hexane eluent. Colorless oil, 2.2 g (55%); 1 H NMR(499MHz,CDCl3)δ4.11(t,J=5.5Hz,6H),3.46(dt,J=62.5,6.7Hz,2H),2.42-2.35(m,1H),2.30(t,J=7.5Hz,2H),2.22( tt,J=12.2,3.6Hz,2H),1.98-1.90(m,4H),1.88-1.72(m,6H),1.65-1.58(m,2H),1.47-1.12(m,28H),0.94-0.84(m,10H).

[0277] H2-85C

[0278] In a 20 mL scintillation glass vial, H2 (11.4 mg, 1.0 equiv.), 85C (282 mg, 2.2 equiv.), K2CO3 (136 mg, 4.4 equiv.), and KI (37 mg, 1.0 equiv.) were combined and poured into CH3CN / THF (1:1, 3 mL). 4 Å molecular sieves were added to the suspension, and the capped vial was stirred at 75 °C for 3 days. The cooled reaction was filtered, deionized water (20 mL) was added, and the filtrate was extracted with DCM (4 × 40 mL). The combined extracts were washed with brine (20 mL), dried over Na2SO4, filtered, and evaporated. The crude material was purified by silica gel flash chromatography, and the desired product was eluted with a 4-5% MeOH / DCM eluent.

[0279] Lipid nanoparticles of the present disclosure The present disclosure provides lipid nanoparticles (LNPs) comprising one or more compounds of Formula (I) and / or Formula (II). In addition to one or more compounds of Formula (I) and / or Formula (II), the LNPs of the present disclosure may include one or more additional LNP components described below.

[0280] In some embodiments, LNPs of the disclosure comprise at least about 2.5%, or at least about 5%, or at least about 7.5%, or at least about 10%, or at least about 12.5%, or at least about 15%, or at least about 17.5%, or at least about 20%, or at least about 22.5%, or at least about 25%, or at least about 27.5%, or at least about 30%, or at least about 32.5%, or at least about 35%, or at least about 37.5%, or at least about 40%, or at least about 42.5%, or at least about 45%, or at least about 47.5%, or at least about 50%, or at least about 52.5%, or at least about 55%, or at least about 57.5%, or at least about 60%, or at least about 62.5%, or at least about 65%, or at least about 67.5%, or at least about 70% by molar ratio of at least one compound of the disclosure. In some embodiments, the at least one compound is at least one compound of Formula (I) or Formula (II) described herein. In some embodiments, the at least one compound of the present disclosure is a mixture of two or more compounds of Formula (I) or Formula (II).

[0281] structural lipids

[0282] In some embodiments, the LNP may further comprise at least about 2.5%, or at least about 5%, or at least about 7.5%, or at least about 10%, or at least about 12.5%, or at least about 15%, or at least about 17.5%, or at least about 20%, or at least about 22.5%, or at least about 25%, or at least about 27.5%, or at least about 30%, or at least about 32.5%, or at least about 35%, or at least about 37.5%, or at least about 40%, or at least about 42.5%, or at least about 45%, or at least about 47.5%, or at least about 50%, or at least about 52.5%, or at least about 55%, or at least about 57.5%, or at least about 60%, or at least about 62.5%, or at least about 65%, or at least about 67.5%, or at least about 70% by molar ratio of at least one structured lipid.

[0283] In some embodiments, the structured lipid may be a steroid. In some embodiments, the structured lipid may be a sterol. In some embodiments, the structured lipid may comprise cholesterol. In some embodiments, the structured lipid may comprise ergosterol. In some embodiments, the structured lipid may be a phytosterol.

[0284] In some embodiments, the at least one structured lipid is a mixture of two structured lipids.

[0285] phospholipids

[0286] In some embodiments, the LNP may further comprise at least about 2.5%, or at least about 5%, or at least about 7.5%, or at least about 10%, or at least about 12.5%, or at least about 15%, or at least about 17.5%, or at least about 20%, or at least about 22.5%, or at least about 25%, or at least about 27.5%, or at least about 30%, or at least about 32.5%, or at least about 35%, or at least about 37.5%, or at least about 40%, or at least about 42.5%, or at least about 45%, or at least about 47.5%, or at least about 50%, or at least about 52.5%, or at least about 55%, or at least about 57.5%, or at least about 60%, or at least about 62.5%, or at least about 65%, or at least about 67.5%, or at least about 70% by molar ratio of at least one phospholipid.

[0287] As used herein, the term "phospholipid" is used in the broadest sense to refer to any amphipathic molecule containing a polar (hydrophilic) head group containing phosphate and two hydrophobic fatty acid chains. In some embodiments, the phospholipid may comprise dioleoylphosphatidylethanolamine (DOPE). In some embodiments, the phospholipid may comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments, the phospholipid may comprise 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC). In some embodiments, the phospholipid may comprise DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine). In some embodiments, the phospholipid is selected from the group consisting of DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPA-NA (1,2-dierucoyl-sn-glycero-3-phosphate (sodium salt)), DEPC (1,2-dierucoyl-sn-glycero-3-phosphocholine), DEPE (1,2-dierucoyl-sn-glycero-3-phosphoethanolamine), DEPG-NA (1,2-dierucoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium salt)), DLOPC (1,2-dilinoleoyl-sn-glycero-3-phosphocholine), DLPA-NA (1,2-dilauroyl-sn-glycero-3-phosphate (sodium salt)), DLPC (1,2-dilauroyl-sn-glycero-3-phospho Choline), DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine), DLPG-NA (1,2-dilauroyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium salt)), DLPG-NH4 (1,2-dilauroyl-sn-glycero-3[phospho-rac-(1-glycerol)(ammonium salt)), DLPS-NA (1,2-dilauroyl-sn-glycero-3-phosphoserine(sodium salt)), DMPA-NA (1,2-dimyristoyl-sn-glycero-3-phosphate(sodium salt)), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DMPG-NA (1,2-Dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium salt)), DMPG-NH4 (1,2-Dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol)(ammonium salt)), DMPG-NH4 / NA (1,2-Dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium / ammonium salt)), DMPS-NA (1,2-Dimyristoyl-sn-glycero-3-phosphoserine(sodium salt)), DOPA-NA(1,2 -dioleoyl-sn-glycero-3-phosphate (sodium salt)), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOPG-NA (1,2-dioleoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium salt)), DOPS-NA (1,2-dioleoyl-sn-glycero-3-phosphoserine (sodium salt)), DPPA-NA (1,2-dipalmitoyl-sn-glycero-3 -phosphate (sodium salt)), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DPPG-NA (1,2-dipalmitoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium salt)), DPPG-NH4 (1,2-dipalmitoyl-sn-glycero-3[phospho-rac-(1-glycerol)(ammonium salt)), DPPS-NA (1,2-dipalmitoyl-sn-glycero-3-phosphoserine (sodium salt)), DSPA-NA (1,2-disulfonyl- stearoyl-sn-glycero-3-phosphate (sodium salt)), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DSPG-NA (1,2-distearoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium salt)), DSPG-NH4 (1,2-distearoyl-sn-glycero-3[phospho-rac-(1-glycerol)(ammonium salt)), DSPS-NA (1,2-distearoyl-sn-glycero-3-phosphoserine (sodium salt)), EPC (Egg-PC), HEPC (Hydrogenated Egg PC), HSPC (Hydrogenated Soybean PC), LYSOPC MYRISTIC (1-Myristoyl-sn-glycero-3-phosphocholine), LYSOPC PALMITIC (1-Palmitoyl-sn-glycero-3-phosphocholine), LYSOPC STEARIC (1-stearoyl-sn-glycero-3-phosphocholine), milk sphingomyelin (MPPC; 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine), MSPC (1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine), PMPC (1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol). amine), POPG-NA (1-palmitoyl-2-oleoyl-sn-glycero-3[phospho-rac-(1-glycerol)](sodium salt)), PSPC (1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine), SMPC (1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine), SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine), SPPC (1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine), or any combination thereof.

[0288] PEGylated lipids

[0289] In some embodiments, the LNPs may further comprise at least about 0.25%, or at least about 0.5%, or at least about 0.75%, or at least about 1.0%, or at least about 2.5%, or at least about 5%, or at least about 7.5%, or at least about 10% PEGylated lipid by molar ratio.

[0290] As used herein, the term "PEGylated lipid" refers to any lipid modified (e.g., covalently linked) to at least one polyethylene glycol molecule. In some embodiments, the PEGylated lipid may comprise 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (hereinafter referred to as DMG-PEG2000 or PEG-DMG).

[0291] In some embodiments, the at least one PEGylated lipid is a mixture of two PEGylated lipids.

[0292] Exemplary LNP Compositions

[0293] The following are exemplary LNP compositions of the present disclosure that include at least one compound of Formula (I) and / or Formula (II), at least one structured lipid, at least one PEGylated lipid, and at least one phospholipid.

[0294] In some embodiments, lipid nanoparticles containing at least one nucleic acid may comprise about 40.75% by molar ratio of at least one compound of Formula (I), about 51.75% by molar ratio of at least one structural lipid, about 5% by molar ratio of at least one phospholipid, and about 2.5% by molar ratio of at least one PEGylated lipid. In some embodiments, lipid nanoparticles containing at least one nucleic acid may comprise about 30.75% to about 50.75% by molar ratio of at least one compound of Formula (I), about 41.75% to about 61.75% by molar ratio of at least one structural lipid, about 0.1% to about 15% by molar ratio of at least one phospholipid, and about 0.1% to about 12.5% ​​by molar ratio of at least one PEGylated lipid. In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise from about 35.75% to about 45.75% by molar ratio of at least one compound of Formula (I), from about 46.75% to about 56.75% by molar ratio of at least one structural lipid, from about 1% to about 10% by molar ratio of at least one phospholipid, and from about 1% to about 7.5% by molar ratio of at least one PEGylated lipid.

[0295] In some embodiments, lipid nanoparticles containing at least one nucleic acid may comprise about 40.75% by molar ratio of at least one compound of formula (II), about 51.75% by molar ratio of at least one structural lipid, about 5% by molar ratio of at least one phospholipid, and about 2.5% by molar ratio of at least one PEGylated lipid. In some embodiments, lipid nanoparticles containing at least one nucleic acid may comprise about 30.75% to about 50.75% by molar ratio of at least one compound of formula (II), about 41.75% to about 61.75% by molar ratio of at least one structural lipid, about 0.1% to about 15% by molar ratio of at least one phospholipid, and about 0.1% to about 12.5% ​​by molar ratio of at least one PEGylated lipid. In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise about 35.75% to about 45.75% by molar ratio of at least one compound of Formula (II), about 46.75% to about 56.75% by molar ratio of at least one structural lipid, about 1% to about 10% by molar ratio of at least one phospholipid, and about 1% to about 7.5% by molar ratio of at least one PEGylated lipid.

[0296] In some embodiments, lipid nanoparticles containing at least one nucleic acid may comprise about 43.17% by molar ratio of at least one compound of formula (II), about 43.17% by molar ratio of at least one structural lipid, about 11.96% by molar ratio of at least one phospholipid, and about 1.7% by molar ratio of at least one PEGylated lipid. In some embodiments, lipid nanoparticles containing at least one nucleic acid may comprise about 33.17% to about 53.17% by molar ratio of at least one compound of formula (II), about 33.17% to about 53.17% by molar ratio of at least one structural lipid, about 1.96% to about 21.96% by molar ratio of at least one phospholipid, and about 0.1% to about 11.7% by molar ratio of at least one PEGylated lipid. In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise from about 38.17% to about 48.17% by molar ratio of at least one compound of Formula (II), from about 38.17% to about 48.17% by molar ratio of at least one structural lipid, from about 6.96% to about 16.96% by molar ratio of at least one phospholipid, and from about 1% to about 6.7% by molar ratio of at least one PEGylated lipid.

[0297] In some embodiments, lipid nanoparticles containing at least one nucleic acid may comprise about 54% by molar of at least one compound of formula (II), about 35% by molar of at least one structural lipid, about 10% by molar of at least one phospholipid, and about 1% by molar of at least one PEGylated lipid. In some embodiments, lipid nanoparticles containing at least one nucleic acid may comprise about 44% to about 64% by molar of at least one compound of formula (II), about 25% to about 45% by molar of at least one structural lipid, about 0.1% to about 20% by molar of at least one phospholipid, and about 0.1% to about 10% by molar of at least one PEGylated lipid. In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise about 49% to about 59% by molar ratio of at least one compound of Formula (II), about 30% to about 40% by molar ratio of at least one structural lipid, about 5% to about 15% by molar ratio of at least one phospholipid, and about 0.5% to about 5% by molar ratio of at least one PEGylated lipid.

[0298] Table 1A shows further exemplary LNP compositions of the present disclosure.

[0299] [Table 1A] TIFF2026503550000133.tif255170TIFF2026503550000134.tif255170TIFF2026503550000135.tif252170TIFF2026503550000136.tif247170

[0300] In some embodiments, including the LNP compositions described in Tables 1A-1C, the compound of Formula (I) or Formula (II) is one of Compound Nos. 1-76.

[0301] In some embodiments, including the LNP compositions described in Tables 1A-1C, the structural lipid can be cholesterol.

[0302] In some embodiments, including the LNP compositions described in Tables 1A-1C, the phospholipid is DOPE.

[0303] In some embodiments, including the LNP compositions described in Tables 1A-1C, the phospholipid is DSPC.

[0304] In some embodiments, including the LNP compositions described in Tables 1A-1C, the phospholipid is DOPC.

[0305] In some embodiments, including the LNP compositions described in Tables 1A-1C, the phospholipid is DPPC.

[0306] In some embodiments, including the LNP compositions described in Tables 1A-1C, the phospholipid is a mixture of DSPC and DOPC. In some embodiments, the mixture of DSPC and DOPC can include a 1:1 ratio of DSPC to DOPC (e.g., an LNP containing 10% phospholipid can include 5% DOPC and 5% DSPC).

[0307] In some embodiments of the preceding LNPs, including the LNP compositions set forth in Table 1, the PEGylated lipid can be DMG-PEG2000.

[0308] In some embodiments, including the LNP compositions described in Tables 1A-1C, the phospholipid can be DOPE and the PEGylated lipid can be DMG-PEG2000.

[0309] In some embodiments, including the LNP compositions described in Tables 1A-1C, the phospholipid can be DOPC and the PEGylated lipid can be DMG-PEG2000.

[0310] In some embodiments, including the LNP compositions described in Tables 1A-1C, the phospholipid can be DSPC and the PEGylated lipid can be DMG-PEG2000.

[0311] In some embodiments, including the LNP compositions described in Tables 1A-1C, the phospholipid can be DPPC and the PEGylated lipid can be DMG-PEG2000.

[0312] In some embodiments, including the LNP compositions described in Tables 1A-1C, the phospholipid can be a mixture of DSPC and DOPC, and the PEGylated lipid can be DMG-PEG2000. In some embodiments, the mixture of DSPC and DOPC can include DSPC and DOPC in a 1:1 ratio (e.g., an LNP containing 10% phospholipid can include 5% DOPC and 5% DSPC).

[0313] Targeting Ligands

[0314] In some embodiments, LNPs, including those described in Table 1A, can further comprise at least one targeting ligand.

[0315] In some embodiments, the LNPs of the disclosure have a molar concentration of at least about 0.05%, or at least about 0.1%, or at least about 0.15%, or at least about 0.2%, or at least about 0.25%, or at least about 0.3%, or at least about 0.35%, or at least about 0.4%, or at least about 0.45%, or at least about 0.5%, or at least about 0.55%, or at least about 0.6%, or at least about 0.65%, or at least about 0.7%, or at least about 0.75%. %, or at least about 0.8%, or at least about 0.85%, or at least about 0.9%, or at least about 0.95%, or at least about 1.0%, or at least about 1.1%, or at least about 1.2%, or at least about 1.3%, or at least about 1.4%, or at least about 1.5%, or at least about 1.6%, or at least about 1.7%, or at least about 1.8%, or at least about 1.9%, or at least about 2.0% of at least one targeting ligand.

[0316] A targeting ligand can be any ligand that confers enhanced affinity for a selected target, e.g., a molecule, cell or cell type, e.g., a cell or organ compartment, tissue, organ or region of the body, compared to, e.g., a species lacking such a ligand.

[0317] In some embodiments, compositions comprising a targeting lipid are well tolerated and provide an appropriate therapeutic index such that treatment of a patient with an effective amount of the composition is associated with an improved toxicity and / or risk profile to the patient compared to treatment of a patient with an effective amount of the composition without a targeting ligand.

[0318] In some embodiments, the targeting ligand provides enhanced affinity to the liver or liver cells (such as hepatocytes). A non-limiting example of a targeting ligand that provides enhanced affinity to the liver or liver cells is GalNac (n-acetyl-galactosamine). Thus, in some embodiments, the present invention provides LNP compositions comprising a targeting ligand that includes GalNac.

[0319] In some embodiments, the targeting ligand comprising GalNac can be a PEGylated GalNac molecule. In some embodiments, the PEGylated GalNac molecule can be Tri-GalNac-PEG2000-DESPE (referred to herein as "GalNac-PEG"), the structure of which is shown below. TIFF2026503550000137.tif36170Accordingly, in some aspects, the present disclosure provides an LNPS comprising GalNac-PEG.

[0320] In some embodiments, the targeting ligand may also include a targeting group, such as a tissue targeting agent group. A non-limiting example of a targeting group may be a multivalent GalNac molecule. Thus, in some embodiments, the present invention provides an LNP composition comprising a targeting ligand that includes a multivalent GalNac. A non-limiting example of a multivalent GalNac molecule is GalNac-PEG.

[0321] Table 1B shows exemplary LNP compositions of the present disclosure comprising at least one compound of Formula (I) and / or Formula (II), at least one structured lipid, at least one PEGylated lipid, and at least one phospholipid, and at least one targeting ligand comprising GalNac.

[0322] [Table 1B] TIFF2026503550000139.tif248170TIFF2026503550000140.tif203170

[0323] In some embodiments comprising an LNPS of Table 1B, the targeting ligand comprising GalNac is GalNac-PEG.

[0324] In some aspects, the targeting ligand can include DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine). Thus, in some embodiments, the present invention provides LNP compositions comprising a targeting ligand that includes DSPE. In some aspects, the DSPE can be PEGylated. In some aspects, the targeting ligand that includes DSPE can be 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], also referred to herein as DSPE-PEG2000 or DSPE-PEG, the structure of which is shown below: TIFF2026503550000141.tif22170

[0325] Table 1C shows exemplary LNP compositions of the present disclosure comprising at least one compound of Formula (I) and / or Formula (II), at least one structured lipid, at least one PEGylated lipid, and at least one phospholipid, and at least one targeting ligand comprising DSPE.

[0326] [Table 1C]

[0327] nucleic acid molecule

[0328] In some embodiments, the lipid nanoparticles of the present disclosure, including those listed in Tables 1A-1C, may further comprise at least one nucleic acid. In some embodiments, the lipid nanoparticles may comprise multiple nucleic acid molecules. In some embodiments, at least one nucleic acid or multiple nucleic acid molecules may be formulated in the lipid nanoparticles.

[0329] Thus, the lipid nanoparticles can comprise at least one nucleic acid, at least one compound of the present disclosure, at least one structural lipid, at least one phospholipid, and at least one PEGylated lipid. In some embodiments, the lipid nanoparticles can further comprise at least one targeting ligand.

[0330] In some embodiments, at least one nucleic acid is a DNA molecule. In one embodiment, at least one DNA molecule is a DoggyBone DNA molecule. In some embodiments, at least one DNA molecule is a DNA plasmid.

[0331] At least one nucleic acid is an RNA molecule. In some embodiments, the RNA molecule is an mRNA molecule. In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, all cytidine residues in the mRNA molecule can be 5-methylcytidine.

[0332] In some embodiments, the at least one RNA molecule is a guide RNA (gRNA) molecule.

[0333] In some embodiments, at least one nucleic acid may comprise both an mRNA molecule and a guide RNA (gRNA) molecule. That is, the LNPs of the present disclosure may comprise both an mRNA molecule and a gRNA molecule. In some embodiments in which the LNP comprises both an mRNA molecule and a gRNA molecule, the mRNA molecule comprises at least one nucleic acid sequence encoding a fusion protein, the fusion protein comprising: (i) an inactivated Cas9 (dCas9) protein or its inactivated nuclease domain; and (ii) a Clo051 protein or its nuclease domain, and the gRNA molecule encodes a guide RNA sequence that targets one or more specific genomic loci. In some embodiments, the fusion protein may be a Cas-CLOVER protein. In some embodiments, the gRNA molecule may target the psk9 gene.

[0334] In some embodiments in which the LNP comprises both mRNA and gRNA molecules, the ratio of mRNA:gRNA can be about 1:2, or about 1:3, or about 1:4, or about 1:5, or about 1:6, or about 1:7, or about 1:8, or about 1:9, or about 1:10, or about 1:1, or about 2:1, or about 3:1, or about 4:1, or about 5:1, or about 6:1, or about 7:1, or about 8:1, or about 9:1, or about 10:1.

[0335] In some embodiments, the at least one nucleic acid molecule comprises at least one RNA molecule and at least one DNA molecule, i.e., LNPs of the present disclosure can comprise both RNA and DNA molecules.

[0336] In some embodiments, the LNPs of the present disclosure may comprise both RNA and DNA molecules, where the RNA molecule comprises at least one nucleic acid sequence encoding a transposase, and the DNA molecule comprises at least one nucleic acid sequence comprising a transposon. In some embodiments, the transposase may be any of the transposases described herein. In some embodiments, the transposon may comprise at least one nucleic acid sequence encoding a FVIII polypeptide. In some embodiments, the transposon may comprise at least one nucleic acid sequence encoding a human propionyl-CoA carboxylase subunit alpha (PCCA) polypeptide.

[0337] In some embodiments in which the LNPs of the present disclosure comprise both RNA (e.g., mRNA) and DNA, the ratio of RNA to DNA (RNA:DNA) in the LNP can be about 1:2, or about 1:3, or about 1:4, or about 1:1, or about 2:1, or about 3:1, or about 4:1, or about 5:1, or about 6:1, or about 7:1, or about 8:1, or about 9:1, or about 10:1.

[0338] In some embodiments, the lipid nanoparticles may comprise lipids and nucleic acids in a specific ratio (weight / weight).

[0339] In some embodiments, lipid nanoparticles comprising at least one nucleic acid have a molecular weight ratio of about 5:1 to about 15:1, or about 10:1 to about 20:1, or about 15:1 to about 25:1, or about 20:1 to about 30:1, or about 25:1 to about 35:1, or about 30:1 to about 40:1, or about 35:1 to about 45:1, or about 40:1 to about 50:1, or about 45:1 to about 55:1, or about 50:1 to about 60:1, or about 55:1 to about 65:1, or about 60:1 to about 70:1, or about 65:1 to about 75:1, or about 70:1 to about 80:1, or about 75:1 to about The lipids and nucleic acids may be present in a ratio of 85:1, or from about 80:1 to about 90:1, or from about 85:1 to about 95:1, or from about 90:1 to about 100:1, or from about 95:1 to about 105:1, or from about 100:1 to about 110:1, or from about 105:1 to about 115:1, or from about 110:1 to about 120:1, or from about 115:1 to about 125:1, or from about 120:1 to about 130:1, or from about 125:1 to about 135:1, or from about 130:1 to about 140:1, or from about 135:1 to about 145:1, or from about 140:1 to about 150:1 (lipid:nucleic acid (weight / weight)).

[0340] In some embodiments, lipid nanoparticles comprising at least one nucleic acid have a lipid to lipid ratio of about 5:1, or about 10:1, or about 15:1, or about 20:1, or about 25:1, or about 30:1, or about 35:1, or about 40:1, or about 45:1, or about 50:1, or about 55:1, or about 60:1, or about 65:1, or about 70:1, or about 75:1, or about 8 The lipids may be present in a ratio of 0:1, or about 85:1, or about 90:1, or about 95:1, or about 100:1, or about 105:1, or about 110:1, or about 115:1, or about 120:1, or about 125:1, or about 130:1, or about 135:1, or about 140:1, or about 145:1, or about 150:1 (lipid:nucleic acid (wt / wt)).

[0341] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise lipid to nucleic acid in a ratio of about 10:1, or about 25:1, or about 40:1 (lipid:nucleic acid (wt / wt)). In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise lipid to nucleic acid in a ratio of about 20:1, or about 40:1, or about 60:1, or about 80:1, or about 120:1 (lipid:nucleic acid (wt / wt)).

[0342] In some embodiments, including LNPs described in Tables 1A-1C, the lipid to nucleic acid ratio in the nanoparticles can be about 30:1 to about 50:1 (w / w), or about 35:1 to about 45:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 40:1 (w / w).

[0343] In some embodiments, including LNPs described in Tables 1A-1C, the lipid to nucleic acid ratio in the nanoparticles can be about 40:1 to about 60:1 (w / w), or about 45:1 to about 55:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 50:1 (w / w).

[0344] In some embodiments, including LNPs described in Tables 1A-1C, the lipid to nucleic acid ratio in the nanoparticles can be about 50:1 to about 70:1 (w / w), or about 55:1 to about 65:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 60:1 (w / w).

[0345] In some embodiments, including LNPs described in Tables 1A-1C, the lipid to nucleic acid ratio in the nanoparticles can be about 70:1 to about 90:1 (w / w), or about 75:1 to about 85:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 80:1 (w / w).

[0346] In some embodiments, including LNPs described in Tables 1A-1C, the lipid to nucleic acid ratio in the nanoparticles can be about 90:1 to about 110:1 (w / w), or about 95:1 to about 105:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 100:1 (w / w).

[0347] In some embodiments, including LNPs described in Tables 1A-1C, the lipid to nucleic acid ratio in the nanoparticles can be about 110:1 to about 130:1 (w / w), or about 115:1 to about 125:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 120:1 (w / w).

[0348] Further features of the nucleic acid molecules of the present disclosure are provided herein.

[0349] Polyphenol Additives

[0350] In some embodiments, lipid nanoparticles of the present disclosure, including those set forth in Tables 1A-1C, may further comprise at least one polyphenol (also referred to herein as a "polyphenol additive").

[0351] As used herein, the term "polyphenol" refers to any compound that has at least two phenolic subunits, and phenol is an aromatic ring that has at least one hydroxyl substituent as defined herein.Polyphenols include compounds that have at least two phenolic subunits, such as flavonoids, catechins, anthocyanins, stilbenes and ellagic acid.Polyphenols also include compounds that have at least three phenolic subunits, such as proanthocyanins, tannins and punicalagins.

[0352] Therefore, lipid nanoparticles can comprise at least one compound of the present disclosure, at least one structured lipid, at least one phospholipid, at least one PEGylated lipid, and at least one polyphenol.In some embodiments, lipid nanoparticles can further comprise at least one nucleic acid, at least one targeting ligand, or any combination thereof.

[0353] A non-limiting example of a polyphenol is tannic acid. Accordingly, the present disclosure provides LNPs that include tannic acid.

[0354] Another non-limiting example of a polyphenol is proanthocyanidin. Accordingly, the present disclosure provides LNPs that include proanthocyanidins.

[0355] Another non-limiting example of a polyphenol is punicalagin. Accordingly, the present disclosure provides LNPs comprising punicalagin.

[0356] Another non-limiting example of a polyphenol is ellagic acid. Accordingly, the present disclosure provides LNPs comprising ellagic acid.

[0357] In some embodiments, the lipid nanoparticles may contain polyphenols and nucleic acids in a specific ratio (weight / weight).

[0358] In some embodiments, lipid nanoparticles comprising a polyphenol and at least one nucleic acid are in a ratio of about 0.1:1, or about 0.15:1, or about 0.2:1, or about 0.25:1, or about 0.3:1, or about 0.35:1, or about 0.4:1, or about 0.45:1, or about 0.5:1, or about 1:1, or about 1.5:1, or about 2:1, or about 2.5:1, or about 3:1, or about 3.5:1, or about 4:1, or about 4.5:1, or about 5:1, or about 5.5:1, or about 6:1, or about 6.5:1, or about 7:1, or about 7.5:1, or about 8:1, or or about 8.5:1, or about 9:1, or about 9.5:1, or about 10:1, or about 10.5:1, or about 11:1, or about 11.5:1, or about 12:1, or about 12.5:1, or about 13:1, or about 13.5:1, or about 14:1, or about 14.5:1, or about 15:1, or about 15.5:1, or about 16:1, or about 16.5:1, or about 17:1, or about 17.5:1, or about 18:1, or about 18.5:1, or about 19:1, or about 19.5:1, or about 20:1 (polyphenol:nucleic acid).

[0359] In some embodiments, lipid nanoparticles comprising tannic acid and at least one nucleic acid may comprise tannic acid and nucleic acid at a ratio of about 0.15:1, or about 0.2:1, or about 5:1, or about 7:1, or about 7.5:1, or about 10:1, or about 12.5:1, or about 15:1 (tannic acid:nucleic acid (wt / wt)). In some embodiments, the at least one nucleic acid may comprise DNA.

[0360] In some embodiments, lipid nanoparticles comprising proanthocyanidins and at least one nucleic acid may comprise proanthocyanidins and nucleic acids at a ratio of about 2.5: 1, or about 5: 1, or about 7.5: 1, or about 10: 1, or about 12.5: 1, or about 15: 1, or about 20: 1 (proanthocyanidins: nucleic acids (weight / weight)). In some embodiments, the at least one nucleic acid may comprise DNA.

[0361] In some embodiments, the lipid nanoparticles comprising ellagic acid and at least one nucleic acid may comprise ellagic acid and nucleic acid at a ratio of about 2.5:1, about 5:1, or about 10:1 (ellagic acid:nucleic acid (wt / wt)). In some embodiments, the at least one nucleic acid may comprise DNA.

[0362] In some embodiments, lipid nanoparticles comprising punicalagin and at least one DNA molecule may comprise punicalagin and DNA in a ratio of about 2.5:1, about 5:1, or about 10:1 (punicalagin:DNA (wt / wt)). In some embodiments, the at least one nucleic acid may comprise DNA.

[0363] In some embodiments, the lipid nanoparticles may contain polyphenols and nucleic acids in a specific ratio (weight / weight).

[0364] In some embodiments, the lipid nanoparticles may comprise polyphenols and lipids in a ratio of about 0.08:1, 0.1:1, or about 0.17:1, or about 0.2:1, or about 0.25:1, or about 0.3:1 (polyphenols:lipids (wt / wt)).

[0365] Exemplary LNPs of the Disclosure

[0366] The following are exemplary LNPs of the present disclosure:

[0367] In some embodiments, lipid nanoparticles are provided comprising about 40.75% by molar ratio of at least one compound of Formula (I), about 51.75% by molar ratio of at least one structural lipid, about 5% by molar ratio of at least one phospholipid, and about 2.5% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 30.75% to about 50.75% by molar ratio of at least one compound of Formula (I), about 41.75% to about 61.75% by molar ratio of at least one structural lipid, about 0.1% to about 15% by molar ratio of at least one phospholipid, and about 0.1% to about 12.5% ​​by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 35.75% to about 45.75% by molar ratio of at least one compound of Formula (I), about 46.75% to about 56.75% by molar ratio of at least one structured lipid, about 1% to about 10% by molar ratio of at least one phospholipid, and about 1% to about 7.5% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 110:1 to about 130:1 (w / w), or about 115:1 to about 125:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 120:1 (w / w). In some embodiments, the ratio of lipid to nucleic acid in the nanoparticles can be about 30:1 (w / w) to about 50:1 (w / w), or about 35:1 (w / w) to about 45:1 (w / w), hi some embodiments, the ratio of lipid to nucleic acid in the nanoparticles can be about 40:1 (w / w).

[0368] In some embodiments, lipid nanoparticles are provided comprising about 40.8% to about 45.9% by molar ratio of at least one compound of Formula (I), about 45.9% to about 53.8% by molar ratio of at least one structural lipid, about 0% to about 6.2% by molar ratio of at least one phospholipid, and about 2% to about 2.5% by molar ratio of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 30.8% to about 55.9% by molar ratio of at least one compound of Formula (I), about 35.9% to about 63.8% by molar ratio of at least one structured lipid, about 0% to about 16.2% by molar ratio of at least one phospholipid, and about 0.1% to about 12.5% ​​by molar ratio of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 35.8% to about 50.9% by molar ratio of at least one compound of Formula (I), about 40.9% to about 58.8% by molar ratio of at least one structured lipid, about 0% to about 11.2% by molar ratio of at least one phospholipid, and about 1% to about 7.5% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 30:1 to about 60:1 (w / w), or about 35:1 to about 55:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 40:1 to about 50:1 (w / w). In some embodiments, the ratio of lipid to nucleic acid in the nanoparticles can be about 40:1 (w / w). In some embodiments, the ratio of lipid to nucleic acid in the nanoparticles can be about 50:1 (w / w).

[0369] In some embodiments, lipid nanoparticles are provided comprising about 54.2% to about 60% by molar ratio of at least one compound of Formula (I), about 38% to about 39.5% by molar ratio of at least one structural lipid, about 0% to about 3.9% by molar ratio of at least one phospholipid, and about 2% to about 2.4% by molar ratio of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 44.2% to about 70% by molar ratio of at least one compound of Formula (I), about 28% to about 49.5% by molar ratio of at least one structured lipid, about 0% to about 13.9% by molar ratio of at least one phospholipid, and about 0.1% to about 12.4% by molar ratio of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 49.2% to about 65% by molar ratio of at least one compound of Formula (I), about 33% to about 44.5% by molar ratio of at least one structured lipid, about 1% to about 8.9% by molar ratio of at least one phospholipid, and about 1% to about 7.4% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 40:1 to about 60:1 (w / w), or about 45:1 to about 55:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 50:1 (w / w).

[0370] In some embodiments, lipid nanoparticles are provided comprising about 40.75% by molar ratio of at least one compound of formula (II), about 51.75% by molar ratio of at least one structural lipid, about 5% by molar ratio of at least one phospholipid, and about 2.5% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 30.75% to about 50.75% by molar ratio of at least one compound of formula (II), about 41.75% to about 61.75% by molar ratio of at least one structural lipid, about 0.1% to about 15% by molar ratio of at least one phospholipid, and about 0.1% to about 12.5% ​​by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 35.75% to about 45.75% by molar ratio of at least one compound of Formula (II), about 46.75% to about 56.75% by molar ratio of at least one structured lipid, about 1% to about 10% by molar ratio of at least one phospholipid, and about 1% to about 7.5% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 90:1 to about 110:1 (w / w), or about 95:1 to about 105:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 100:1 (w / w).

[0371] In some embodiments, lipid nanoparticles are provided comprising about 43.17% by molar of at least one compound of formula (II), about 43.17% by molar of at least one structural lipid, about 11.96% by molar of at least one phospholipid, and about 1.7% by molar of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 33.17% to about 53.17% by molar ratio of at least one compound of Formula (II), about 33.17% to about 53.17% by molar ratio of at least one structural lipid, about 1.96% to about 21.96% by molar ratio of at least one phospholipid, and about 0.1% to about 11.7% by molar ratio of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 38.17% to about 48.17% by molar ratio of at least one compound of Formula (II), about 38.17% to about 48.17% by molar ratio of at least one structured lipid, about 6.96% to about 16.96% by molar ratio of at least one phospholipid, and about 1% to about 6.7% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 90:1 to about 110:1 (w / w), or about 95:1 to about 105:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 100:1 (w / w).

[0372] In some embodiments, lipid nanoparticles are provided comprising about 50% by molar ratio of at least one compound of formula (II), about 38.5% by molar ratio of at least one structural lipid, about 10% by molar ratio of at least one phospholipid, and about 1.5% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 40% to about 60% by molar ratio of at least one compound of formula (II), about 28.5% to about 48.5% by molar ratio of at least one structural lipid, about 0.1% to about 20% by molar ratio of at least one phospholipid, and about 0.1% to about 11.5% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 45% to about 55% by molar ratio of at least one compound of Formula (II), about 33.5% to about 43.5% by molar ratio of at least one structured lipid, about 5% to about 15% by molar ratio of at least one phospholipid, and about 0.5% to about 6.5% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 30:1 to about 90:1 (w / w), about 70:1 to about 90:1 (w / w), about 75:1 to about 85:1 (w / w), or about 35:1 to about 85:1 (w / w). In some embodiments, the ratio of lipid to nucleic acid in the nanoparticles can be from about 40:1 (w / w) to about 50:1 (w / w), or from about 60:1 (w / w) to about 80:1 (w / w).

[0373] In some embodiments, lipid nanoparticles are provided comprising about 50% by molar ratio of at least one compound of formula (II), about 38% by molar ratio of at least one structured lipid, about 10% by molar ratio of at least one phospholipid, and about 2% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 40% to about 60% by molar ratio of at least one compound of formula (II), about 28% to about 48% by molar ratio of at least one structured lipid, about 0.1% to about 20% by molar ratio of at least one phospholipid, and about 0.1% to about 12% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 45% to about 55% by molar ratio of at least one compound of Formula (II), about 33% to about 43% by molar ratio of at least one structural lipid, about 5% to about 15% by molar ratio of at least one phospholipid, and about 0.5% to about 7% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 30:1 to about 60:1 (w / w), or about 35:1 to about 55:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 40:1 (w / w) or about 50:1 (w / w).

[0374] In some embodiments, lipid nanoparticles are provided comprising about 54% by molar ratio of at least one compound of formula (II), about 35% by molar ratio of at least one structural lipid, about 10% by molar ratio of at least one phospholipid, and about 1% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 44% to about 64% by molar ratio of at least one compound of formula (II), about 25% to about 45% by molar ratio of at least one structural lipid, about 0.1% to about 20% by molar ratio of at least one phospholipid, and about 0.1% to about 10% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 49% to about 59% by molar ratio of at least one compound of Formula (II), about 30% to about 40% by molar ratio of at least one structured lipid, about 5% to about 15% by molar ratio of at least one phospholipid, and about 0.5% to about 5% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 90:1 to about 110:1 (w / w), or about 95:1 to about 105:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 100:1 (w / w).

[0375] In some embodiments, lipid nanoparticles are provided comprising about 40.8% to about 54% by molar ratio of at least one compound of Formula (II), about 35% to about 51.8% by molar ratio of at least one structural lipid, about 5% to about 12% by molar ratio of at least one phospholipid, and about 1% to about 2.5% by molar ratio of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 30.8% to about 64% by molar ratio of at least one compound of Formula (II), about 25% to about 61.8% by molar ratio of at least one structural lipid, about 0.1% to about 22% by molar ratio of at least one phospholipid, and about 0.1% to about 12.5% ​​by molar ratio of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 35.8% to about 59% by molar ratio of at least one compound of Formula (II), about 30% to about 56.8% by molar ratio of at least one structured lipid, about 1% to about 17% by molar ratio of at least one phospholipid, and about 0.5% to about 7.5% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 90:1 to about 110:1 (w / w), or about 95:1 to about 105:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 100:1 (w / w).

[0376] In some embodiments, lipid nanoparticles are provided, which comprise at least one compound of formula (II) at a molar ratio of about 50%, at least one structural lipid at a molar ratio of about 38.5%, at least one phospholipid at a molar ratio of about 10%, and at least one PEGylated lipid at a molar ratio of about 1.5%, and comprise at least one nucleic acid, and at least one nucleic acid comprises at least one RNA molecule (for example, mRNA molecule).In some embodiments, lipid nanoparticles are provided, which comprise at least one compound of formula (II) at a molar ratio of about 50%, at least one structural lipid at a molar ratio of about 42.5%, at least one phospholipid at a molar ratio of about 5%, and at least one PEGylated lipid at a molar ratio of about 2.5%, and comprise at least one nucleic acid, and at least one nucleic acid comprises at least one RNA molecule (for example, mRNA molecule). In some embodiments, lipid nanoparticles are provided, which comprise at least one compound of formula (II) at a molar ratio of about 50%, at least one structural lipid at a molar ratio of about 37.5%, at least one phospholipid at a molar ratio of about 10%, and at least one PEGylated lipid at a molar ratio of about 2.5%, and comprise at least one nucleic acid, and at least one nucleic acid comprises at least one RNA molecule (for example, mRNA molecule).In some embodiments, lipid nanoparticles are provided, which comprise at least one compound of formula (II) at a molar ratio of about 50%, at least one structural lipid at a molar ratio of about 41%, at least one phospholipid at a molar ratio of about 7.5%, and at least one PEGylated lipid at a molar ratio of about 1.5%, and comprise at least one nucleic acid, and at least one nucleic acid comprises at least one RNA molecule (for example, mRNA molecule). In some embodiments, lipid nanoparticles are provided comprising about 50% by molar of at least one compound of formula (II), about 43% by molar of at least one structural lipid, about 5% by molar of at least one phospholipid, and about 2% by molar of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule).In some embodiments, lipid nanoparticles are provided, which comprise at least one compound of formula (II) at a molar ratio of about 45%, at least one structural lipid at a molar ratio of about 48.5%, at least one phospholipid at a molar ratio of about 5%, and at least one PEGylated lipid at a molar ratio of about 1.5%, wherein said lipid nanoparticles comprise at least one nucleic acid, and said at least one nucleic acid comprises at least one RNA molecule (such as mRNA molecule).In some embodiments, lipid nanoparticles are provided, which comprise at least one compound of formula (II) at a molar ratio of about 45.5%, at least one structural lipid at a molar ratio of about 7.5%, and at least one phospholipid at a molar ratio of about 2%, wherein said lipid nanoparticles comprise at least one nucleic acid, and said at least one nucleic acid comprises at least one RNA molecule (such as mRNA molecule). In some embodiments, lipid nanoparticles are provided, comprising at least one compound of formula (II) at a molar ratio of about 40%, at least one structural lipid at a molar ratio of about 52.5%, at least one phospholipid at a molar ratio of about 5%, and at least one PEGylated lipid at a molar ratio of about 2.5%, wherein said lipid nanoparticles comprise at least one nucleic acid, and said at least one nucleic acid comprises at least one RNA molecule (such as mRNA molecule).In some embodiments, lipid nanoparticles are provided, comprising at least one compound of formula (II) at a molar ratio of about 40%, at least one structural lipid at a molar ratio of about 50%, at least one phospholipid at a molar ratio of about 7.5%, and at least one PEGylated lipid at a molar ratio of about 2.5%, wherein said lipid nanoparticles comprise at least one nucleic acid, and said at least one nucleic acid comprises at least one RNA molecule (such as mRNA molecule). In some embodiments, lipid nanoparticles are provided comprising about 40% by molar of at least one compound of formula (II), about 48% by molar of at least one structural lipid, about 10% by molar of at least one phospholipid, and about 2% by molar of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule).In some embodiments, lipid nanoparticles are provided, which comprise at least one compound of formula (II) at a molar ratio of about 40%, at least one structural lipid at a molar ratio of about 53.5%, at least one phospholipid at a molar ratio of about 5%, and at least one PEGylated lipid at a molar ratio of about 1.5%, and comprise at least one nucleic acid, and at least one nucleic acid comprises at least one RNA molecule (for example, mRNA molecule).In some embodiments, lipid nanoparticles are provided, which comprise at least one compound of formula (II) at a molar ratio of about 40%, at least one structural lipid at a molar ratio of about 48.5%, at least one phospholipid at a molar ratio of about 10%, and at least one PEGylated lipid at a molar ratio of about 1.5%, and comprise at least one nucleic acid, and at least one nucleic acid comprises at least one RNA molecule (for example, mRNA molecule).

[0377] In some embodiments, lipid nanoparticles are provided comprising about 50% by molar ratio of at least one compound of formula (II), about 38.5% by molar ratio of at least one structural lipid, about 10% by molar ratio of at least one phospholipid, about 1.25% by molar ratio of at least one PEGylated lipid, and about 0.25% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 40% to about 60% by molar ratio of at least one compound of Formula (II), about 28.5% to about 48.5% by molar ratio of at least one structured lipid, about 0.1% to about 20% by molar ratio of at least one phospholipid, about 0.1% to about 11.25% by molar ratio of at least one PEGylated lipid, and about 0.1% to about 10.25% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 45% to about 55% by molar ratio of at least one compound of Formula (II), about 33.5% to about 43.5% by molar ratio of at least one structured lipid, about 5% to about 15% by molar ratio of at least one phospholipid, about 0.5% to about 6.25% by molar ratio of at least one PEGylated lipid, and about 0.1% to about 5.25% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 70:1 to about 90:1 (w / w), or about 75:1 to about 85:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 80:1 (w / w).

[0378] In some embodiments, lipid nanoparticles are provided comprising about 50% by molar ratio of at least one compound of formula (II), about 38.5% by molar ratio of at least one structural lipid, about 10% by molar ratio of at least one phospholipid, about 1.2% by molar ratio of at least one PEGylated lipid, and about 0.3% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 40% to about 60% by molar ratio of at least one compound of Formula (II), about 28.5% to about 48.5% by molar ratio of at least one structured lipid, about 0.1% to about 20% by molar ratio of at least one phospholipid, about 0.1% to about 11.2% by molar ratio of at least one PEGylated lipid, and about 0.1% to about 10.3% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 45% to about 55% by molar ratio of at least one compound of Formula (II), about 33.5% to about 43.5% by molar ratio of at least one structured lipid, about 5% to about 15% by molar ratio of at least one phospholipid, about 0.5% to about 6.2% by molar ratio of at least one PEGylated lipid, and about 0.1% to about 5.3% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 40:1 to about 60:1 (w / w), or about 45:1 to about 55:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 50:1 (w / w).

[0379] In some embodiments, lipid nanoparticles are provided comprising about 50% by molar ratio of at least one compound of formula (II), about 38.5% by molar ratio of at least one structural lipid, about 10% by molar ratio of at least one phospholipid, about 1% by molar ratio of at least one PEGylated lipid, and about 0.5% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 40% to about 60% by molar ratio of at least one compound of Formula (II), about 28.5% to about 48.5% by molar ratio of at least one structured lipid, about 0.1% to about 20% by molar ratio of at least one phospholipid, about 0.1% to about 11% by molar ratio of at least one PEGylated lipid, and about 0.1% to about 10.5% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 45% to about 55% by molar ratio of at least one compound of Formula (II), about 33.5% to about 43.5% by molar ratio of at least one structural lipid, about 5% to about 15% by molar ratio of at least one phospholipid, about 0.5% to about 6% by molar ratio of at least one PEGylated lipid, and about 0.1% to about 5.5% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 70:1 to about 90:1 (w / w), or about 75:1 to about 85:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 80:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 40:1 to about 60:1 (w / w), or about 45:1 to about 55:1 (w / w), hi some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 50:1 (w / w).

[0380] In some embodiments, lipid nanoparticles are provided comprising about 45% by molar ratio of at least one compound of formula (II), about 42.5% by molar ratio of at least one structural lipid, about 10% by molar ratio of at least one phospholipid, and about 2.5% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 35% to about 55% by molar ratio of at least one compound of formula (II), about 32.5% to about 52.5% by molar ratio of at least one structural lipid, about 0.1% to about 20% by molar ratio of at least one phospholipid, and about 0.1% to about 12.5% ​​by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 40% to about 50% by molar ratio of at least one compound of Formula (II), about 37.5% to about 47.5% by molar ratio of at least one structural lipid, about 5% to about 15% by molar ratio of at least one phospholipid, and about 0.5% to about 7.5% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 50:1 to about 70:1 (w / w), or about 55:1 to about 65:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 60:1 (w / w).

[0381] In some embodiments, lipid nanoparticles are provided comprising about 45% by molar ratio of at least one compound of formula (II), about 42.5% by molar ratio of at least one structural lipid, about 10% by molar ratio of at least one phospholipid, about 2.25% by molar ratio of at least one PEGylated lipid, and about 0.25% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 35% to about 55% by molar ratio of at least one compound of Formula (II), about 32.5% to about 52.5% by molar ratio of at least one structured lipid, about 0.1% to about 20% by molar ratio of at least one phospholipid, about 0.1% to about 12.25% by molar ratio of at least one PEGylated lipid, and about 0.1% to about 10.25% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 40% to about 50% by molar ratio of at least one compound of Formula (II), about 37.5% to about 47.5% by molar ratio of at least one structured lipid, about 5% to about 15% by molar ratio of at least one phospholipid, about 0.5% to about 7.25% by molar ratio of at least one PEGylated lipid, and about 0.1% to about 5.25% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 50:1 to about 70:1 (w / w), or about 55:1 to about 65:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 60:1 (w / w).

[0382] In some embodiments, lipid nanoparticles are provided comprising about 45% by molar ratio of at least one compound of formula (II), about 42.5% by molar ratio of at least one structural lipid, about 10% by molar ratio of at least one phospholipid, about 2% by molar ratio of at least one PEGylated lipid, and about 0.5% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 35% to about 55% by molar ratio of at least one compound of Formula (II), about 32.5% to about 52.5% by molar ratio of at least one structural lipid, about 0.1% to about 20% by molar ratio of at least one phospholipid, about 0.1% to about 12% by molar ratio of at least one PEGylated lipid, and about 0.1% to about 10.5% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 40% to about 50% by molar ratio of at least one compound of Formula (II), about 37.5% to about 47.5% by molar ratio of at least one structured lipid, about 5% to about 15% by molar ratio of at least one phospholipid, about 0.5% to about 7% by molar ratio of at least one PEGylated lipid, and about 0.1% to about 5.5% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 50:1 to about 70:1 (w / w), or about 55:1 to about 65:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 60:1 (w / w).

[0383] In some embodiments, lipid nanoparticles are provided comprising about 50% by molar ratio of at least one compound of formula (II), about 38% by molar ratio of at least one structural lipid, about 10% by molar ratio of at least one phospholipid, about 1.7% by molar ratio of at least one PEGylated lipid, and about 0.3% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 40% to about 60% by molar ratio of at least one compound of Formula (II), about 28% to about 48% by molar ratio of at least one structural lipid, about 0.1% to about 20% by molar ratio of at least one phospholipid, about 0.1% to about 11.7% by molar ratio of at least one PEGylated lipid, and about 0.1% to about 10.3% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 45% to about 55% by molar ratio of at least one compound of Formula (II), about 33% to about 43% by molar ratio of at least one structured lipid, about 5% to about 15% by molar ratio of at least one phospholipid, about 0.5% to about 6.7% by molar ratio of at least one PEGylated lipid, and about 0.1% to about 5.3% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 40:1 to about 60:1 (w / w), or about 45:1 to about 55:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 50:1 (w / w).

[0384] In some embodiments, lipid nanoparticles are provided comprising about 50% by molar ratio of at least one compound of formula (II), about 38% by molar ratio of at least one structural lipid, about 10% by molar ratio of at least one phospholipid, about 1.5% by molar ratio of at least one PEGylated lipid, and about 0.5% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 40% to about 60% by molar ratio of at least one compound of Formula (II), about 28% to about 48% by molar ratio of at least one structured lipid, about 0.1% to about 20% by molar ratio of at least one phospholipid, about 0.1% to about 11.5% by molar ratio of at least one PEGylated lipid, and about 0.1% to about 10.5% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 45% to about 55% by molar ratio of at least one compound of Formula (II), about 33% to about 43% by molar ratio of at least one structural lipid, about 5% to about 15% by molar ratio of at least one phospholipid, about 0.5% to about 6.5% by molar ratio of at least one PEGylated lipid, and about 0.1% to about 5.5% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 40:1 to about 60:1 (w / w), or about 45:1 to about 55:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 50:1 (w / w).

[0385] In some embodiments, the nucleic acid molecule is a DNA molecule. Thus, the present disclosure provides lipid nanoparticles comprising about 40.75% by molar ratio of at least one compound of Formula (I), about 51.75% by molar ratio of at least one structural lipid, about 5% by molar ratio of at least one phospholipid, and about 2.5% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 30.75% to about 50.75% by molar ratio of at least one compound of Formula (I), about 41.75% to about 61.75% by molar ratio of at least one structural lipid, about 0.1% to about 15% by molar ratio of at least one phospholipid, and about 0.1% to about 12.5% ​​by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 35.75% to about 45.75% by molar ratio of at least one compound of Formula (I), about 46.75% to about 56.75% by molar ratio of at least one structured lipid, about 1% to about 10% by molar ratio of at least one phospholipid, and about 1% to about 7.5% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one DNA molecule. In one embodiment, the at least one DNA molecule is a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule is a DNA plasmid. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 110:1 to about 130:1 (w / w), or about 115:1 to about 125:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 120:1 (w / w).

[0386] In some embodiments, the nucleic acid molecule is a DNA molecule. Thus, the present disclosure provides lipid nanoparticles comprising about 40.75% by molar ratio of at least one compound of formula (II), about 51.75% by molar ratio of at least one structural lipid, about 5% by molar ratio of at least one phospholipid, and about 2.5% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, and the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 30.75% to about 50.75% by molar ratio of at least one compound of formula (II), about 41.75% to about 61.75% by molar ratio of at least one structural lipid, about 0.1% to about 15% by molar ratio of at least one phospholipid, and about 0.1% to about 12.5% ​​by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, and the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 35.75% to about 45.75% by molar ratio of at least one compound of Formula (II), about 46.75% to about 56.75% by molar ratio of at least one structured lipid, about 1% to about 10% by molar ratio of at least one phospholipid, and about 1% to about 7.5% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one DNA molecule. In one embodiment, the at least one DNA molecule is a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule is a DNA plasmid. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 110:1 to about 130:1 (w / w), or about 115:1 to about 125:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 120:1 (w / w).

[0387] In some embodiments, lipid nanoparticles are provided comprising about 40% to about 46% by molar ratio of at least one compound of Formula (I), about 45.9% to about 51.8% by molar ratio of at least one structured lipid, about 4.9% to about 7% by molar ratio of at least one phospholipid, and about 2% to about 3% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 30% to about 56% by molar ratio of at least one compound of Formula (I), about 35.9% to about 61.8% by molar ratio of at least one structured lipid, about 0.1% to about 17% by molar ratio of at least one phospholipid, and about 0.1% to about 13% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 35% to about 51% by molar ratio of at least one compound of Formula (I), about 40.9% to about 56.8% by molar ratio of at least one structured lipid, about 1% to about 12% by molar ratio of at least one phospholipid, and about 1% to about 8% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one DNA molecule. In one embodiment, the at least one DNA molecule is a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule is a DNA plasmid. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 70:1 to about 130:1 (w / w), or about 75:1 to about 125:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 80:1 (w / w) to about 120:1 (w / w). In some embodiments, the ratio of lipid to nucleic acid in the nanoparticles can be about 80:1 (w / w). In some embodiments, the ratio of lipid to nucleic acid in the nanoparticles can be about 120:1 (w / w).

[0388] In some embodiments, lipid nanoparticles are provided comprising about 54% to about 60% by molar ratio of at least one compound of Formula (I), about 30% to about 36% by molar ratio of at least one structured lipid, about 2.8% to about 7% by molar ratio of at least one phospholipid, and about 3% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 44% to about 70% by molar ratio of at least one compound of Formula (I), about 20% to about 46% by molar ratio of at least one structured lipid, about 0.1% to about 17% by molar ratio of at least one phospholipid, and about 0.1% to about 13% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 49% to about 65% by molar ratio of at least one compound of Formula (I), about 25% to about 41% by molar ratio of at least one structured lipid, about 0.1% to about 12% by molar ratio of at least one phospholipid, and about 0.1% to about 8% by molar ratio of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one DNA molecule. In one embodiment, the at least one DNA molecule is a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule is a DNA plasmid. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 50:1 to about 110:1 (w / w), or about 55:1 to about 105:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 60:1 (w / w) to about 100:1 (w / w). In some embodiments, the ratio of lipid to nucleic acid in the nanoparticles can be about 60:1 (w / w). In some embodiments, the ratio of lipid to nucleic acid in the nanoparticles can be about 100:1 (w / w).

[0389] In some embodiments, lipid nanoparticles are provided, which comprise at least one compound of formula (II) at a molar ratio of about 50%, at least one structural lipid at a molar ratio of about 38.5%, at least one phospholipid at a molar ratio of about 10% and at least one PEGylated lipid at a molar ratio of about 1.5%, and comprise at least one nucleic acid, wherein said at least one nucleic acid comprises at least one DNA molecule and at least one RNA molecule.In some embodiments, the ratio of lipid to nucleic acid in nanoparticles can be about 50:1 (w / w).

[0390] In some embodiments, lipid nanoparticles are provided, which comprise at least one compound of formula (II) at a molar ratio of about 50%, at least one structural lipid at a molar ratio of about 38.5%, at least one phospholipid at a molar ratio of about 10%, at least one PEGylated lipid at a molar ratio of about 1%, and at least one targeting ligand, which comprises GalNac at a molar ratio of about 0.5%, and which comprise at least one nucleic acid, and said at least one nucleic acid comprises at least one DNA molecule and at least one RNA molecule.In some embodiments, the ratio of lipid to nucleic acid in nanoparticles can be about 50:1 (w / w).

[0391] In some embodiments, lipid nanoparticles are provided, which comprise at least one compound of formula (II) at a molar ratio of about 50%, at least one structural lipid at a molar ratio of about 38%, at least one phospholipid at a molar ratio of about 10% and at least one PEGylated lipid at a molar ratio of about 2%, and comprise at least one nucleic acid, wherein at least one nucleic acid comprises at least one DNA molecule and at least one RNA molecule.In some embodiments, the ratio of lipid to nucleic acid in nanoparticles can be about 50:1 (w / w).

[0392] In some embodiments, lipid nanoparticles are provided, which comprise at least one compound of formula (II) at a molar ratio of about 50%, at least one structural lipid at a molar ratio of about 38.5%, at least one phospholipid at a molar ratio of about 10% and at least one PEGylated lipid at a molar ratio of about 1.5%, and comprise at least one nucleic acid, wherein said at least one nucleic acid comprises at least one DNA molecule and at least one RNA molecule.In some embodiments, the ratio of lipid to nucleic acid in nanoparticles can be about 80:1 (w / w).

[0393] In some embodiments, lipid nanoparticles are provided, which comprise at least one compound of formula (II) at a molar ratio of about 45%, at least one structural lipid at a molar ratio of about 42.5%, at least one phospholipid at a molar ratio of about 10%, at least one PEGylated lipid at a molar ratio of about 2% and at least one targeting ligand, which comprises GalNac at a molar ratio of about 0.5%, and comprise at least one nucleic acid, wherein said at least one nucleic acid comprises at least one DNA molecule and at least one RNA molecule.In some embodiments, the ratio of lipid to nucleic acid in nanoparticles can be about 60:1 (w / w).

[0394] In some embodiments, lipid nanoparticles are provided, which comprise at least one compound of formula (II) at a molar ratio of about 50%, at least one structural lipid at a molar ratio of about 41%, at least one phospholipid at a molar ratio of about 7.5%, at least one PEGylated lipid at a molar ratio of about 1% and at least one targeting ligand, which comprises GalNac at a molar ratio of about 0.5%, and which comprise at least one nucleic acid, and said at least one nucleic acid comprises at least one DNA molecule and at least one RNA molecule.In some embodiments, the ratio of lipid to nucleic acid in nanoparticles can be about 60:1 (w / w).

[0395] In some embodiments, lipid nanoparticles are provided, which comprise at least one compound of formula (II) at a molar ratio of about 45%, at least one structural lipid at a molar ratio of about 45.75%, at least one phospholipid at a molar ratio of about 7.5%, at least one PEGylated lipid at a molar ratio of about 1.5% and at least one targeting ligand, which comprises GalNac at a molar ratio of about 0.25%, and which comprise at least one nucleic acid, and said at least one nucleic acid comprises at least one DNA molecule and at least one RNA molecule.In some embodiments, the ratio of lipid to nucleic acid in nanoparticles can be about 50:1 (w / w).

[0396] In some embodiments, lipid nanoparticles are provided, which comprise at least one compound of formula (II) at a molar ratio of about 40%, at least one structural lipid at a molar ratio of about 53.5%, at least one phospholipid at a molar ratio of about 5%, at least one PEGylated lipid at a molar ratio of about 1% and at least one targeting ligand, which comprises GalNac at a molar ratio of about 0.5%, and which comprise at least one nucleic acid, and said at least one nucleic acid comprises at least one DNA molecule and at least one RNA molecule.In some embodiments, the ratio of lipid to nucleic acid in nanoparticles can be about 50:1 (w / w).

[0397] In some embodiments, lipid nanoparticles are provided, which comprise at least one compound of formula (II) at a molar ratio of about 40%, at least one structural lipid at a molar ratio of about 48%, at least one phospholipid at a molar ratio of about 10%, at least one PEGylated lipid at a molar ratio of about 1.5% and at least one targeting ligand, which comprises GalNac at a molar ratio of about 0.5%, and which comprise at least one nucleic acid, and said at least one nucleic acid comprises at least one DNA molecule and at least one RNA molecule.In some embodiments, the ratio of lipid to nucleic acid in nanoparticles can be about 40:1 (w / w).

[0398] In some embodiments, lipid nanoparticles are provided, which comprise at least one compound of formula (II) at a molar ratio of about 40%, at least one structural lipid at a molar ratio of about 52.75%, at least one phospholipid at a molar ratio of about 5%, at least one PEGylated lipid at a molar ratio of about 2% and at least one targeting ligand, which comprises GalNac at a molar ratio of about 0.25%, and comprise at least one nucleic acid, wherein said at least one nucleic acid comprises at least one DNA molecule and at least one RNA molecule.In some embodiments, the ratio of lipid to nucleic acid in nanoparticles can be about 60:1 (w / w).

[0399] In some embodiments, lipid nanoparticles are provided, which comprise at least one compound of formula (II) at a molar ratio of about 45%, at least one structural lipid at a molar ratio of about 42.5%, at least one phospholipid at a molar ratio of about 10%, at least one PEGylated lipid at a molar ratio of about 2% and at least one targeting ligand, which comprises GalNac at a molar ratio of about 0.5%, and comprise at least one nucleic acid, wherein said at least one nucleic acid comprises at least one DNA molecule and at least one RNA molecule.In some embodiments, the ratio of lipid to nucleic acid in nanoparticles can be about 60:1 (w / w). In some embodiments, lipid nanoparticles are provided, which comprise at least one compound of formula (II) at a molar ratio of about 50%, at least one structural lipid at a molar ratio of about 38%, at least one phospholipid at a molar ratio of about 10%, at least one PEGylated lipid at a molar ratio of about 1.7% and at least one targeting ligand, which comprises GalNac at a molar ratio of about 0.3%, and which comprise at least one nucleic acid, and said at least one nucleic acid comprises at least one DNA molecule and at least one RNA molecule.In some embodiments, the ratio of lipid to nucleic acid in nanoparticles can be about 50:1 (w / w).

[0400] In some embodiments, lipid nanoparticles are provided comprising about 50% by molar ratio of at least one compound of formula (II), about 38.5% by molar ratio of at least one structural lipid, about 10% by molar ratio of at least one phospholipid, and about 1.5% by molar ratio of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 40% to about 60% by molar ratio of at least one compound of formula (II), about 28.5% to about 48.5% by molar ratio of at least one structural lipid, about 0.1% to about 20% by molar ratio of at least one phospholipid, and about 0.1% to about 11.5% by molar ratio of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 45% to about 55% by molar ratio of at least one compound of Formula (II), about 33.5% to about 43.5% by molar ratio of at least one structural lipid, about 5% to about 15% by molar ratio of at least one phospholipid, and about 0.5% to about 6.5% by molar ratio of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 30:1 to about 90:1 (w / w), about 70:1 to about 90:1 (w / w), about 75:1 to about 85:1 (w / w), or about 35:1 to about 85:1 (w / w). In some embodiments, the ratio of lipid to nucleic acid in the nanoparticle can be about 40:1 (w / w) to about 50:1 (w / w), or about 60:1 (w / w) to about 80:1 (w / w). In some embodiments of the preceding LNP, the lipid nanoparticle further comprises tannic acid at a tannic acid to nucleic acid ratio of about 7.5:1, or about 10:1, or about 12.5:1, or about 15:1.

[0401] In some embodiments, lipid nanoparticles are provided comprising about 50% by molar ratio of at least one compound of formula (II), about 38.5% by molar ratio of at least one structural lipid, about 10% by molar ratio of at least one phospholipid, about 1% by molar ratio of at least one PEGylated lipid, and about 0.5% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 40% to about 60% by molar ratio of at least one compound of Formula (II), about 28.5% to about 48.5% by molar ratio of at least one structured lipid, about 0.1% to about 20% by molar ratio of at least one phospholipid, about 0.1% to about 11% by molar ratio of at least one PEGylated lipid, and about 0.1% to about 10.5% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 45% to about 55% by molar ratio of at least one compound of Formula (II), about 33.5% to about 43.5% by molar ratio of at least one structured lipid, about 5% to about 15% by molar ratio of at least one phospholipid, about 0.5% to about 6% by molar ratio of at least one PEGylated lipid, and about 0.1% to about 5.5% by molar ratio of a targeting ligand comprising GalNac, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 70:1 to about 90:1 (w / w), or about 75:1 to about 85:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 80:1 (w / w). In some embodiments of the preceding LNPs, the lipid nanoparticles further comprise tannic acid in a ratio of tannic acid to nucleic acid of about 7.5:1, or about 10:1, or about 12.5:1, or about 15:1.

[0402] In some embodiments, lipid nanoparticles are provided, comprising at least one compound of formula (II) at a molar ratio of about 45%, at least one structural lipid at a molar ratio of about 45.75%, at least one phospholipid at a molar ratio of about 7.5%, at least one PEGylated lipid at a molar ratio of about 1.5%, and a targeting ligand comprising GalNac at a molar ratio of about 0.25%, wherein at least one nucleic acid comprises at least one DNA molecule and at least one RNA molecule.In some embodiments, the ratio of lipid to nucleic acid in nanoparticles can be about 50:1 (w / w).In some embodiments, lipid nanoparticles further comprise tannic acid, with the ratio of tannic acid to nucleic acid being about 7:1.

[0403] In some embodiments, lipid nanoparticles are provided, comprising at least one compound of formula (II) at a molar ratio of about 50%, at least one structural lipid at a molar ratio of about 41%, at least one phospholipid at a molar ratio of about 7.5%, at least one PEGylated lipid at a molar ratio of about 1%, and a targeting ligand comprising GalNac at a molar ratio of about 0.5%, wherein the at least one nucleic acid comprises at least one DNA molecule and at least one RNA molecule. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 50:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 60:1 (w / w). In some embodiments, the lipid nanoparticles further comprise tannic acid at a tannic acid to nucleic acid ratio of about 5:1, about 10:1, or about 15:1. In some embodiments, the lipid nanoparticles further comprise tannic acid at a tannic acid to total lipid ratio of about 0.2 or about 0.25.

[0404] In some embodiments, lipid nanoparticles are provided, comprising at least one compound of formula (II) at a molar ratio of about 45%, at least one structural lipid at a molar ratio of about 45.75%, at least one phospholipid at a molar ratio of about 7.5%, at least one PEGylated lipid at a molar ratio of about 1.5%, and a targeting ligand comprising GalNac at a molar ratio of about 0.25%, wherein the at least one nucleic acid comprises at least one DNA molecule and at least one RNA molecule.In some embodiments, the lipid nanoparticles can have a lipid to nucleic acid ratio of about 50:1 (w / w).In some embodiments, the lipid nanoparticles further comprise tannic acid at a tannic acid to nucleic acid ratio of about 5:1, about 10:1, or about 15:1.In some embodiments, the lipid nanoparticles further comprise tannic acid at a tannic acid to total lipid ratio of about 0.2 or about 0.25.

[0405] In some embodiments, lipid nanoparticles are provided, comprising at least one compound of formula (II) at a molar ratio of about 50%, at least one structural lipid at a molar ratio of about 38.5%, at least one phospholipid at a molar ratio of about 10%, and at least one PEGylated lipid at a molar ratio of about 1.5%, wherein at least one nucleic acid comprises at least one DNA molecule.In some embodiments, the ratio of lipid to nucleic acid in nanoparticles can be about 80:1 (w / w).In some embodiments of the preceding LNP, lipid nanoparticles further comprise proanthocyanidins at a proanthocyanidin to nucleic acid ratio of about 2.5:1, or about 5:1, or about 7.5:1, or about 10:1, or about 15:1, or about 20:1.

[0406] In some embodiments, lipid nanoparticles are provided, comprising at least one compound of formula (II) at a molar ratio of about 50%, at least one structural lipid at a molar ratio of about 38.5%, at least one phospholipid at a molar ratio of about 10%, and at least one PEGylated lipid at a molar ratio of about 1.5%, wherein the at least one nucleic acid comprises at least one DNA molecule.In some embodiments, the lipid to nucleic acid ratio in nanoparticles can be about 80:1 (w / w).In some embodiments of the preceding LNP, the lipid nanoparticles further comprise ellagic acid at an ellagic acid to nucleic acid ratio of about 2:5:1, or about 5:1, or about 10:1.

[0407] In some embodiments, lipid nanoparticles are provided, comprising at least one compound of formula (II) at a molar ratio of about 50%, at least one structural lipid at a molar ratio of about 38.5%, at least one phospholipid at a molar ratio of about 10%, and at least one PEGylated lipid at a molar ratio of about 1.5%, wherein the at least one nucleic acid comprises at least one DNA molecule.In some embodiments, the lipid to nucleic acid ratio in nanoparticles can be about 80:1 (w / w).In some embodiments of the preceding LNP, the lipid nanoparticles further comprise punicalagin at a punicalagin to nucleic acid ratio of about 2:5:1, or about 5:1, or about 10:1.

[0408] Pharmaceutical Compositions of the Present Disclosure In some embodiments, the present disclosure provides a pharmaceutical composition comprising at least one lipid nanoparticle of the present disclosure.In some embodiments, the present disclosure provides a pharmaceutical composition comprising at least one first nanoparticle of the present disclosure and at least one second nanoparticle of the present disclosure, wherein at least one first nanoparticle comprises at least one nucleic acid molecule encoding at least one transposase, and at least one second nanoparticle comprises at least one nucleic acid molecule encoding at least one transposon.In some embodiments, the at least one nucleic acid molecule encoding at least one transposase can be an RNA molecule (for example, an mRNA molecule), and the at least one nucleic acid molecule encoding at least one transposon can be a DNA molecule (for example, a DoggyBone DNA molecule or a DNA nanoplasmid).

[0409] In some embodiments, the present disclosure provides a composition comprising at least one cell contacted with at least one nanoparticle of the present disclosure. In some embodiments, the present disclosure provides a composition comprising at least one cell genetically modified using at least one nanoparticle of the present disclosure. In some embodiments, the present disclosure provides a composition comprising at least one cell genetically modified using any of the methods of the present disclosure.

[0410] In some embodiments, the present disclosure provides a pharmaceutical composition comprising at least one cell contacted with at least one nanoparticle of the present disclosure. In some embodiments, the present disclosure provides a pharmaceutical composition comprising at least one cell genetically modified using at least one nanoparticle of the present disclosure. In some embodiments, the present disclosure provides a pharmaceutical composition comprising at least one cell genetically modified using any of the methods of the present disclosure.

[0411] Methods of the present disclosure The present disclosure provides a method for delivering at least one nucleic acid to at least one cell, comprising contacting the at least one cell with at least one composition of the present disclosure.The present disclosure provides a method for delivering at least one nucleic acid to at least one cell, comprising contacting the at least one cell with at least one nanoparticle of the present disclosure.

[0412] In all of the methods, compositions, and kits of the present disclosure, at least one cell can be a liver cell, including, but not limited to, hepatocytes, hepatic stellate cells, Kupffer cells, or hepatic sinusoidal endothelial cells.

[0413] In some embodiments of any of the methods of the present disclosure, the cells can be in vivo, ex vivo, or in vitro. In some embodiments, any of the methods of the present disclosure can be applied in vivo, ex vivo, or in vitro.

[0414] The present disclosure provides a method for genetically modifying at least one cell, comprising contacting the at least one cell with at least one composition of the present disclosure.The present disclosure provides a method for genetically modifying at least one cell, comprising contacting the at least one cell with at least one nanoparticle of the present disclosure.

[0415] In some embodiments, genetically modifying a cell can include delivering at least one exogenous nucleic acid to a cell such that the cell expresses at least one protein that it would not otherwise normally express, or such that at least one cell expresses at least one protein at a level higher than the level at which the cell would otherwise normally express the at least one protein, or such that at least one cell expresses at least one protein at a level lower than the level at which the cell would otherwise normally express the at least one protein. In some embodiments, genetically modifying a cell can include delivering at least one exogenous nucleic acid to a cell such that the at least one exogenous nucleic acid is integrated into the genome of the at least one cell.

[0416] In some embodiments, the methods of the present disclosure can result in a plurality of cells, wherein at least about 1%, or at least about 2%, or at least about 3%, or at least about 4%, or at least about 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% of the cells of the plurality of cells express at least one protein encoded by at least one nucleic acid delivered to the plurality of cells via the nanoparticles of the present disclosure.

[0417] The present disclosure provides methods for treating at least one disease in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one composition of the present disclosure comprising at least one nucleic acid encoding a therapeutic protein.

[0418] The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering a therapeutically effective amount of at least one nanoparticle of the present disclosure comprising at least one nucleic acid encoding a therapeutic protein.

[0419] The present disclosure provides a method of treating at least one disease in a subject, comprising administering a therapeutically effective amount of cells, wherein the cells are contacted with at least one nanoparticle of the present disclosure, wherein the nanoparticle comprises at least one nucleic acid encoding a therapeutic protein.The present disclosure provides a method of treating at least one disease in a subject, comprising administering a therapeutically effective amount of cells, wherein the cells have been genetically modified using the compositions and / or methods of the present disclosure.

[0420] The present disclosure provides a method for treating a disease or disorder in a cell, tissue, organ, animal, or subject, comprising administering to or contacting with a cell, tissue, organ, animal, or subject a therapeutically effective amount of a composition disclosed herein. In one aspect, the subject is a mammal. Preferably, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0421] The present disclosure provides methods of treating at least one disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one of at least one composition disclosed herein comprising at least one nucleic acid encoding a therapeutic protein.

[0422] Without wishing to be bound by theory, it is hypothesized that the LNP compositions of the present disclosure target liver cells more effectively than other cells, thereby reducing off-target effects associated with other delivery compositions. In some embodiments, the LNP compositions provided herein that contain a targeting ligand result in less cytokine release than the same LNP composition that does not contain a targeting ligand. Cytokine release can be measured using any suitable method known in the art or described herein. For example, cytokine levels can be determined in the blood of subjects that have received an LNP composition that contains a targeting ligand using enzyme-linked immunosorbent assay (ELISA). Cytokine levels can then be compared to pre-treatment baseline levels.

[0423] The present disclosure provides a method for regulating or treating at least one malignant disease or disorder in a cell, tissue, organ, animal, or subject. In some embodiments, the at least one disease can be a malignant disease, including but not limited to cancer. In some embodiments, the at least one disease can be hemophilia A or hemophilia B. In some embodiments, the at least one disease can be a metabolic liver disorder (MLD). In some embodiments, the at least one disease can be a urea cycle disorder (UCD). MLD and / or UCD include, but are not limited to, N-acetylglutamate synthase (NAGS) deficiency, carbamoyl phosphate synthase I deficiency (CPSI deficiency), ornithine transcarbamylase (OTC) deficiency, argininosuccinate synthase deficiency (ASSD) (citrullinemia I), citrin deficiency (citrullinemia II), argininosuccinate lyase deficiency (argininosuccinic aciduria), arginase deficiency (hyperargininemia), ornithine translocase deficiency (HHH syndrome), methylmalonic acidemia (MMA), or a combination thereof.

[0424] The method of the present disclosure can be used to treat disease or disorder by using therapeutic transgene that encodes exogenous nucleic acid sequence or exogenous amino acid sequence.In this method, transgene is delivered to target cell, and mutated gene is replaced or mutated.The disease that can be treated by this method is generally caused by gene mutation, which causes protein not to be expressed or protein that does not function.Examples of therapeutic transgenes that can be delivered using the compositions disclosed herein include beta-thalassemia (HBB T87Q, BCL11A shRNA, IGF2BP1), sickle cell disease (HBB T87Q, BCL11A shRNA, IGF2BP1), hemophilia A (factor VIII), hemophilia B (factor IX), X-linked severe combined immunodeficiency (interleukin-2 receptor gamma (IL2RG)), hypolymphatic deficiency (tissue-nonspecific alkaline phosphatase (TNAP)), osteopetrosis (TCIRG1), glycogen storage disease type II (Pompe disease) (alpha-glucosidase (GAA)), alpha-galactosidase A deficiency (Fabry disease) (alpha-galactosidase A (GLA)), mucopolysaccharidosis type I (MPS I) (alpha-L-iduronidase (IDUA)), mucopolysaccharidosis type II (MPS II) (iduronate 2-sulfatase (IDS)), mucopolysaccharidosis type IIIA (MPS IIIA) (sulfoglycosamine sulfohydrolase (SGSH)), mucopolysaccharidosis type IIIB (MPS IIIB) (N-alpha-acetylglucosaminidase (NAGLU)), mucopolysaccharidosis type IV A (MPS IVA) (Morquio) (N-acetylgalactosamine-6-sulfate sulfatase (GALNS)), mucopolysaccharidosis type IV B (MPS IVB) β-galactosidase (GLB1) (β-galactosidase (GLB1)), cholesteryl ester storage disease (CESD) (lysosomal acid lipase (LIPA)), cystinosis (cystinosinin lysosomal cystine transporter (CTNS)), X-linked chronic granulomatous disease (X-CGD) (CYBB), Wiskott-Aldrich syndrome (WAS) (WAS), X-linked adrenoleukodystrophy (X-ALD) (ABCD1), metachromatic leukodystrophy (MLD) (ARSA), phenylketonuria (PAH), methylmalonic acidemia (MMUT), propionic acidemia (PCCA, PCCB), retinitis pigmentosa (RPE65), Usher syndrome (MYO7A), and Gaucher disease (GBA).

[0425] The methods of the present disclosure can optionally further include co-administration or combination therapy for treating such diseases or disorders, and administration of any composition or pharmaceutical composition disclosed herein further includes administration of at least one chemotherapeutic agent (e.g., alkylating agent, mitotic inhibitor, radiopharmaceutical) before and / or after the simultaneous administration.

[0426] nucleic acid molecule In some embodiments, the nucleic acid molecule can be a synthetic nucleic acid molecule. In some embodiments, the nucleic acid molecule can be a non-naturally occurring nucleic acid molecule. In some embodiments, the non-naturally occurring nucleic acid molecule can contain at least one non-naturally occurring nucleotide. The at least one non-naturally occurring nucleotide can be any non-naturally occurring nucleotide known in the art. In some embodiments, the nucleic acid molecule can be a modified nucleic acid molecule. In some embodiments, the modified nucleic acid molecule can contain at least one modified nucleotide. The at least one modified nucleotide can be any modified nucleic acid known in the art.

[0427] In some embodiments, mRNA molecules can be capped using any method and / or capping moiety known in the art. mRNA molecules can be capped with an m7G(5')ppp(5')G moiety. The m7G(5')ppp(5')G moiety is also referred to herein as "Cap0." mRNA molecules can be capped with a CleanCap® moiety. The CleanCap® moiety can include an m7G(5')ppp(5')(2'OMeA) (CleanCap® AG) moiety. The CleanCap® moiety can include an m7G(5')ppp(5')(2'OMeG) (CleanCap® GG) moiety. mRNA molecules can be capped with an anti-reverse cap analog (ARCA®) site. The ARCA® moiety can include an m7(3'-O-methyl)G(5')ppp(5')G moiety. The mRNA molecule can be capped with a CleanCap® 3'OMe moiety (CleanCap® + ARCA®).

[0428] In some embodiments, the mRNA molecule may comprise at least one modified nucleic acid.

[0429] At least one modified nucleic acid may contain 5-methoxyuridine (5moU). In some embodiments, at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, at least about 90%, or at least about 95%, or at least about 99% of the uridine bases in the mRNA molecule are 5-methoxyuridine bases. In some embodiments, all of the uridine bases in the mRNA molecule are 5-methoxyuridine bases. Without wishing to be bound by theory, 5-methoxyuridine may improve protein expression and reduce immunogenicity (see Li et al., Bioconjugate Chem. 2016, 27, 3, 849-853 and Vaidyanathan et al. Molecular Therapy-Nucleic Acids, 2018, 12, 530-542).

[0430] In some embodiments, the mRNA molecule may comprise at least one modified nucleic acid.

[0431] At least one modified nucleic acid contains N1-methylpseudouridine (me 1Ψ). In some embodiments, at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, at least about 90%, or at least about 95%, or at least about 99% of the uridine bases in the mRNA molecule are N1-methoxypseudouridine bases. Without wishing to be bound by theory, N1-methylpseudouridine may improve protein expression (see Li et al., Bioconjugate Chem. 2016, 27, 3, 849-853).

[0432] In some embodiments, the mRNA molecule may comprise at least one modified nucleic acid.

[0433] At least one modified nucleic acid may contain pseudouridine (Ψ). In some embodiments, at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, at least about 90%, or at least about 95%, or at least about 99% of the uridine bases in the mRNA molecule are pseudouridine bases. In some embodiments, all uridine bases in the mRNA molecule are pseudouridine bases. Without wishing to be bound by theory, pseudouridine may improve protein expression and reduce immunogenicity (see Li et al., Bioconjugate Chem. 2016, 27, 3, 849-853 and Vaidyanathan et al. Molecular Therapy-Nucleic Acids, 2018, 12, 530-542).

[0434] In some embodiments, the mRNA molecule may comprise at least one modified nucleic acid.

[0435] At least one modified nucleic acid may contain 5-methoxycytidine (5-MeC). In some embodiments, at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, at least about 90%, or at least about 95%, or at least about 99% of the cytidine bases in the mRNA molecule are 5-MeC bases. In some embodiments, all cytidine bases in the mRNA molecule are 5-MeC bases.

[0436] In some embodiments, the nucleic acid molecule may comprise a DNA molecule. Thus, in some embodiments, the lipid nanoparticle may comprise a DNA molecule. In some embodiments, the DNA molecule may be a circular DNA molecule, such as, but not limited to, a DNA plasmid or a DNA nanoplasmid. Thus, in some embodiments, the lipid nanoparticle may comprise a circular DNA molecule. In some embodiments, the lipid nanoparticle may comprise a Doggybone DNA molecule. In some embodiments, the lipid nanoparticle may comprise a DNA plasmid. In some embodiments, the lipid nanoparticle may comprise a DNA nanoplasmid. In some embodiments, the DNA molecule may be a linearized DNA molecule, such as, but not limited to, a linearized DNA plasmid or a linearized DNA nanoplasmid.

[0437] The DNA plasmid or DNA nanoplasmid may be at least about 0.25 kb in length, or at least about 0.5 kb, or at least about 0.75 kb, or at least about 1.0 kb, or at least about 1.25 kb, or at least about 1.5 kb, or at least about 1.75 kb, or at least about 2.0 kb, or at least about 2.25 kb, or at least about 2.5 kb, or at least about 2.75 kb, or at least about 3.0 kb, or at least about 3.25 kb, or at least about 3.5 kb, or at least about 3.5 kb. at least about 3.75 kb, or at least about 4.0 kb, or at least about 4.25 kb, or at least about 4.5 kb, or at least about 4.75 kb, or at least about 5.0 kb, or at least about 5.25 kb, or at least about 5.5 kb, or at least about 5.75 kb, or at least about 6.0 kb, or at least about 6.25 kb, or at least about 6.5 kb, or at least about 6.75 kb, or at least about 7.0 kb, or at least about 7.25 kb, or at least about 7.5 kb, or at least about 7.75 kb, or at least about 8.0 kb, or at least about 8.25 kb, or at least about 8.5 kb, or at least about 8.75 kb, or at least about 9.0 kb, or at least about 9.25 kb, or at least about 9.5 kb, or at least about 9.75 kb, or at least about 10.0 kb, or at least about 10.25 kb, or at least about 10.5 kb, or at least about 10.75 kb, or at least about 11.0 kb, or at least about 11.25 kb, or less It may be at least about 11.5 kb, or at least about 11.75 kb, or at least about 12 kb, or at least about 12.25 kb, or at least about 12.5 kb, or at least about 12.75 kb, or at least about 13.0 kb, or at least about 13.25 kb, or at least about 13.5 kb, or at least about 13.75 kb, or at least about 14.0 kb, or at least about 14.25 kb, or at least about 14.5 kb, or at least about 14.75 kb or at least about 15.0 kb.

[0438] In some embodiments, the nucleic acid molecule formulated in the lipid nanoparticles of the present disclosure can comprise at least one transgene sequence.In some embodiments, the transgene sequence can comprise a nucleotide sequence encoding at least one therapeutic protein.In some embodiments, the transgene sequence can comprise a nucleotide sequence encoding at least one transposase.In some embodiments, the transgene sequence can comprise a nucleotide sequence encoding at least one transposon.In some embodiments, the transposon can comprise a nucleotide sequence encoding at least one therapeutic protein.In some embodiments, the transposon can comprise a nucleotide sequence encoding at least one therapeutic protein and at least one protomer sequence, and the at least one therapeutic protein is operably linked to at least one promoter sequence.

[0439] In some aspects, the lipid nanoparticles of the present disclosure can be produced using a microfluidic mixing platform, hi some aspects, the microfluidic mixing platform can be a non-turbulent microfluidic mixing platform.

[0440] In some embodiments, a microfluidic mixing platform can produce lipid nanoparticles of the present invention by combining a miscible solvent phase containing the lipid components of the nanoparticle with an aqueous phase containing the cargo of the lipid nanoparticle (e.g., nucleic acid, DNA, mRNA, etc.) using a microfluidic device. In some embodiments, the miscible solvent phase and the aqueous phase are mixed in the microfluidic device under laminar flow conditions that do not allow the two phases to mix immediately. When the two phases move under laminar flow in a microfluidic channel, microscopic features within the channel allow for controlled, uniform mixing to produce the lipid nanoparticles of the present disclosure.

[0441] In some embodiments, microfluidic mixing platforms include, but are not limited to, NanoAssemblr® Spark (Precision NanoSystems), NanoAssemblr® Ignite™ (Precision NanoSystems), NanoAssemblr® Benchtop (Precision NanoSystems), NanoAssemblr® Blaze (Precision NanoSystems), or NanoAssemblr® GMP System (Precision NanoSystems).

[0442] In some embodiments, the lipid nanoparticles of the present disclosure can be produced using a microfluidic mixing platform that mixes at a rate of at least about 2.5 ml / min, or at least about 5 ml / min, or at least about 7.5 ml / min, or at least about 10 ml / min, or at least about 12.5 ml / min, or at least about 15 ml / min, or at least about 17.5 ml / min, or at least about 20 ml / min, or at least about 22.5 ml / min, or at least about 25 ml / min, or at least about 27.5 ml / min, or at least about 30 ml / min.

[0443] In some embodiments, the lipid nanoparticles of the present disclosure can be produced using a microfluidic mixing platform that mixes a miscible solvent phase with an aqueous phase in a ratio (solvent:water, v / v) of about 10:1, or about 9:1, or about 8:1, or about 7:1, or about 6:1, or about 5:1, or about 4:1, or about 3:1, or about 2:1, or about 1:1, or about 1:2, or about 1:3, or about 1:4, or about 1:5, or about 1:6, or about 1:7, or about 1:8, or about 1:9, or about 1:10.

[0444] piggyBac ITR sequences In some embodiments, the nucleic acid can comprise a piggyBac ITR sequence. In some embodiments, the nucleic acid can comprise a first piggyBac ITR sequence and a second piggyBac ITR sequence.

[0445] In some embodiments, the piggyBac ITR sequences can include any piggyBac ITR sequences known in the art.

[0446] In some embodiments of the methods of the present disclosure, the piggyBac ITR sequences, such as the first piggyBac ITR sequence and / or the second piggyBac ITR sequence in an AAV piggyBac transposon, may comprise, consist essentially of, or consist of Sleeping Beauty transposon ITRs, Helraiser transposon ITRs, Tol2 transposon ITRs, TcBuster transposon ITRs, or any combination thereof.

[0447] Transposition System In some embodiments, the nucleic acid may comprise a transposon or nanotransposon comprising a first nucleic acid sequence comprising: (a) a first inverted terminal repeat (ITR) or a sequence encoding the first ITR, (b) a second ITR or a sequence encoding the second ITR, and (c) an intra-ITR sequence or an intra-ITR encoding sequence, wherein the intra-ITR sequence comprises a transposon sequence or a sequence encoding the transposon.

[0448] In some embodiments, the nucleic acid may comprise a transposon or nanotransposon comprising a first nucleic acid sequence comprising: (a) a first inverted terminal repeat (ITR) or a sequence encoding the first ITR, (b) a second ITR or a sequence encoding the second ITR, and (c) an intra-ITR sequence or an intra-ITR coding sequence, wherein the intra-ITR sequence comprises a transposon sequence or a sequence encoding the transposon, and the second nucleic acid sequence comprises an inter-ITR sequence or an inter-ITR coding sequence, and the length of the inter-ITR sequence is 700 nucleotides or less.

[0449] The transposon or nanotransposon of the present disclosure can be a piggyBac™ (PB) transposon. In some embodiments where the transposon is a PB transposon, the transposase is a piggyBac™ (PB) transposase, a piggyBac-like (PBL) transposase, or a Super piggyBac™ (SPB) transposase. Preferably, the sequence encoding the SPB transposase is an mRNA sequence.

[0450] Non-limiting examples of PB transposons and PB, PBL and SPB transposases are described in detail in U.S. Patent Nos. 6,218,182; 6,962,810; 8,399,643 and PCT Publication No. WO2010 / 099296.

[0451] PB, PBL, and SPB transposases recognize transposon-specific inverted terminal repeats (ITRs) at the ends of transposons and insert their contents between the ITRs at the sequence 5'-TTAT-3' (TTAT target sequence) or the sequence 5'-TTAA-3' (TTAA target sequence) within the chromosomal site. The target sequences of the PB or PBL transposons are 5'-CTAA-3', 5'-TTAG-3', 5'-ATAA-3', 5'-TCAA-3', 5'AGTT-3', 5'-ATTA-3', 5'-GTTA-3', 5'-TTGA-3', 5'-TTTA-3', 5'-TTAC-3', 5'-ACTA-3', 5'-AGGG-3', 5'-CTAG-3', 5'-TGAA-3', 5'-AGGT-3', 5'-ATCA-3', 5'-CTCC-3', 5'-TAAA-3', 5'-TCTC-3', 5'TGAA-3', 5'-A AAT-3', 5'-AATC-3', 5'-ACAA-3', 5'-ACAT-3', 5'-ACTC-3', 5'-AGTG-3', 5'-ATAG-3', 5'-CAAA-3', 5'-CACA-3', 5'-CATA-3', 5'-CCAG-3', 5'-CCCA-3', 5'-CGTA-3', 5'-GTCC-3', 5'-TAAG-3', 5'-TCTA-3', 5'-TGAG-3', 5'-TGTT-3', 5'-TTCA-3', 5'-TTCT-3', and 5'-TTTT-3'. The PB or PBL transposon system does not impose any restrictions on the payload of the gene of interest that can be included between the ITRs.

[0452] Exemplary amino acid sequences of one or more PB, PBL, and SPB transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810; and U.S. Patent No. 8,399,643, each of which is incorporated by reference herein in its entirety for examples of transposases that may be used with the compositions and methods described herein. In some embodiments, the PB transposase comprises or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:1. In some embodiments, the PB transposase comprises the amino acid sequence of SEQ ID NO:1.

[0453] The PB or PBL transposase can comprise or consist of an amino acid sequence having two or more, three or more, or each of positions 30, 165, 282, and / or 538 of the sequence of SEQ ID NO: 1. The transposase can be an SPB transposase comprising or consisting of the amino acid sequence of SEQ ID NO: 1, wherein the amino acid substitution at position 30 can be a substitution of valine (V) for isoleucine (I), the amino acid substitution at position 165 can be a substitution of serine (S) for glycine (G), the amino acid substitution at position 282 can be a substitution of valine (V) for methionine (M), and the amino acid substitution at position 538 can be a substitution of lysine (K) for asparagine (N). In some embodiments, the SPB transposase comprises or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 2. In some embodiments, the SPB transposase comprises the amino acids set forth in SEQ ID NO: 2.

[0454] In certain embodiments, where the transposase comprises the above mutations at positions 30, 165, 282, and / or 538, the PB, PBL, and SPB transposases comprise the mutations at positions 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 401, 402, 403, 4 Examples of transposases that may further include one or more amino acid substitutions at positions 40, 421, 436, 456, 470, 486, 503, 552, 570, and 591 and that may be used in conjunction with the compositions and methods described herein are described in further detail in PCT Publication Nos. WO2019 / 173636 and WO2020 / 051374, each of which is incorporated by reference in its entirety.

[0455] In some embodiments, the PB transposase comprises or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 3. In some embodiments, the PB transposase comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0456] The PB or PBL transposase can comprise or consist of an amino acid sequence having two or more, three or more, or each of the amino acid substitutions at positions 29, 164, 281, and / or 537 of the sequence of SEQ ID NO: 3. The transposase can be an SPB transposase comprising or consisting of the amino acid sequence of SEQ ID NO: 3, wherein the amino acid substitution at position 29 can be a substitution of valine (V) for isoleucine (I), the amino acid substitution at position 164 can be a substitution of serine (S) for glycine (G), the amino acid substitution at position 281 can be a substitution of valine (V) for methionine (M), and the amino acid substitution at position 537 can be a substitution of lysine (K) for asparagine (N). In some embodiments, the SPB transposase comprises or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 4. In some embodiments, the SPB transposase comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0457] In certain embodiments, where the transposase comprises the above mutations at positions 29, 164, 281 and / or 537, the PB, PBL and SPB transposases comprise mutations at positions 2, 45, 81, 102, 118, 124, 176, 179, 184, 186, 199, 206, 208, 225, 234, 239, 240, 242, 257, 295, 297, 310, 314, 318, 326, 327, 330, 332, 334, 336, 338, 339, 340, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, Examples of transposases that may further include one or more amino acid substitutions at positions 39, 420, 435, 455, 469, 485, 502, 551, 569, and 590 and that may be used in conjunction with the compositions and methods described herein are described in further detail in PCT Publication Nos. WO2019 / 173636 and WO2020 / 051374, each of which is incorporated by reference in its entirety.

[0458] The PB, PBL, or SPB transposase can be isolated or derived from an insect, vertebrate, crustacean, or urochordate, as described in further detail in PCT Publication Nos. WO2019 / 173636 and PCT / US2019 / 049816. In a preferred embodiment, the PB, PBL, or SPB transposase is isolated or derived from the insect Trichoplusia ni (GenBank Accession No. AAA87375) or Bombyx mori (GenBank Accession No. BAD11135).

[0459] A hyperactive PB or PBL transposase is a transposase that is more active than the endogenous transposase from which it is derived. In a preferred embodiment, the hyperactive PB or PBL transposase is isolated or derived from Bombyx mori or Xenopus tropicalis. Examples of hyperactive PB or PBL transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810; U.S. Patent No. 8,399,643; and International Publication No. WO 2019 / 173636, each of which is incorporated by reference in its entirety for examples of transposases that can be used in conjunction with the compositions and methods described herein. A list of hyperactive amino acid substitutions is disclosed in U.S. Patent No. 10,041,077, which is incorporated by reference in its entirety for examples of amino acid substitutions that can be introduced into the transposases described herein. The transposon or nanotransposon of the present disclosure may be a Sleeping Beauty transposon. In some embodiments, when the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase (e.g., those disclosed in U.S. Pat. No. 9,228,180, which is incorporated by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein) or a hyperactive Sleeping Beauty (SB100X) transposase.

[0460] In some embodiments, PB or PBL is integration-deficient. Integration-deficient PB or PBL transposase is a transposase that can excise corresponding transposons, but integrates excised transposons less frequently than corresponding wild-type transposases. Examples of integration-deficient PB or PBL transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810; U.S. Patent No. 8,399,643 and International Publication No. WO 2019 / 173636, each of which is incorporated herein by reference in its entirety for examples of transposases that can be used in conjunction with the compositions and methods described herein. A list of integration-deficient amino acid substitutions is disclosed in U.S. Patent No. 10,041,077, which is incorporated herein by reference in its entirety for examples of amino acid substitutions that can be introduced into the transposases described herein.

[0461] In some embodiments, the PB or PBL transposase is fused to a nuclear localization signal. Examples of PB or PBL transposase fused to a nuclear localization signal are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810; U.S. Patent No. 8,399,643 and International Publication No. WO 2019 / 173636, each of which is incorporated herein by reference in its entirety for examples of transposases that can be used in conjunction with the compositions and methods described herein.

[0462] The transposon or nanotransposon of the present disclosure can be a Sleeping Beauty transposon.In some embodiments, when the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase (for example, the transposase disclosed in U.S. Patent No. 9,228,180, the entire contents of which are incorporated herein by reference, for examples of the transposase that can be used in conjunction with the compositions and methods described herein) or a hyperactive Sleeping Beauty (SB100X) transposase.

[0463] The transposon or nanotransposon of the present disclosure can be a Helraiser transposon. An exemplary Helraiser transposon is Helibat1. In some embodiments, when the transposon is a Helraiser transposon, the transposase is a Helitron transposase (e.g., as disclosed in International Publication No. 2019 / 173636, the entire contents of which are incorporated herein by reference, for examples of transposases that can be used in conjunction with the compositions and methods described herein).

[0464] The transposon or nanotransposon of the present disclosure can be a Tol2 transposon. In some embodiments, when the transposon is a Tol2 transposon, the transposase is a Tol2 transposase (for example, as disclosed in WO2019 / 173636).

[0465] The transposon or nanotransposon of the present disclosure can be a TcBuster transposon. In some embodiments, when the transposon is a TcBuster transposon, the transposase is a TcBuster transposase (e.g., those disclosed in International Publication No. 2019 / 173636, the entire contents of which are incorporated herein by reference, for examples of transposases that can be used in conjunction with the compositions and methods described herein). The TcBuster transposase can comprise or consist of a naturally occurring or non-naturally occurring amino acid sequence. The polynucleotide encoding the TcBuster transposase can comprise or consist of a naturally occurring amino acid sequence or a non-naturally occurring nucleic acid sequence.

[0466] In some embodiments, the mutant TcBuster transposase comprises one or more sequence mutations compared to the wild-type TcBuster transposase, which are described in further detail in PCT Publication Nos. WO2019 / 173636 and WO2020 / 051374, each of which is incorporated by reference in its entirety, for examples of transposases that may be used in conjunction with the compositions and methods described herein.

[0467] The cell delivery compositions (e.g., transposons) disclosed herein can include nucleic acid molecules encoding therapeutic proteins or therapeutic agents. Examples of therapeutic proteins include those disclosed in PCT Publication Nos. WO2019 / 173636 and WO2020 / 051374, each of which is incorporated herein by reference in its entirety for examples of transposases that can be used in conjunction with the compositions and methods described herein.

[0468] In some embodiments, the therapeutic protein may comprise a FVIII Nanoplasmid. An exemplary Nanoplasmid encoding a FVIII polypeptide is provided in SEQ ID NO: 9. Thus, the nucleic acids formulated in the nanoparticles of the present disclosure may comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 9.

[0469] In some embodiments, the therapeutic protein can comprise a propionyl-CoA carboxylase subunit alpha (PCCA) polypeptide. An exemplary transposon encoding a PCCA polypeptide is provided in SEQ ID NO: 10. Thus, the nucleic acids formulated in the nanoparticles of the present disclosure can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 10.

[0470] Gene editing The present disclosure provides a gene editing composition and / or a cell containing the gene editing composition. The gene editing composition may include nanoparticles containing nucleic acids, and the nucleic acid may include a sequence encoding a DNA binding domain and a sequence encoding a nuclease protein or its nuclease domain. The sequence encoding the nuclease protein or its nuclease domain may include a DNA sequence, an RNA sequence, or a combination thereof. The nuclease or its nuclease domain may include one or more of a CRISPR / Cas protein, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), and an endonuclease.

[0471] The nuclease or its nuclease domain may comprise a nuclease-inactivated Cas (dCas) protein and an endonuclease. The endonuclease may comprise a Clo051 nuclease or its nuclease domain. The gene editing composition may comprise a fusion protein. The fusion protein may comprise a nuclease-inactivated Cas9 (dCas9) protein and a Clo051 nuclease or a Clo051 nuclease domain. In some embodiments, the fusion protein may further comprise at least one nuclear localization signal (NLS). In some embodiments, the fusion protein may further comprise at least two NLSs. The gene editing composition may further comprise a guide sequence. The guide sequence may comprise an RNA sequence.

[0472] The transgene can include a nucleic acid sequence encoding a mini-Cas9 (Cas9) operably linked to an effector. The present disclosure provides fusion proteins comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a mini-Cas9 (Cas9). The mini-Cas9 constructs of the present disclosure can include an effector that comprises a Type IIS endonuclease.

[0473] The transgene can include a nucleic acid sequence encoding an inactivated small Cas9 (dSaCas9) operably linked to an effector. The transgene provides a nucleic acid sequence encoding a fusion protein comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule, where the effector comprises a small inactivated Cas9 (dSaCas9). The small inactivated Cas9 (dSaCas9) construct of the present disclosure can include an effector that comprises a type IIS endonuclease.

[0474] The transgene can include a nucleic acid sequence encoding an inactivated Cas9 (dCas9) operably linked to an effector. The transgene can include a nucleic acid sequence encoding a fusion protein comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule, where the effector comprises an inactivated Cas9 (dCas9). All inactivated Cas9 (dCas9) constructs of the present disclosure can include an effector that comprises a type IIS endonuclease.

[0475] The dCas9 may be isolated from or derived from Streptococcus pyogenes. The dCas9 may include dCas9 with substitutions at amino acid positions 10 and 840 that inactivate the catalytic site. In some embodiments, the substitutions are D10A and H840A.

[0476] The cell containing the gene editing composition can express the gene editing composition stably or transiently.Preferably, the gene editing composition is expressed transiently.The guide RNA can comprise a sequence complementary to the target sequence in the genomic DNA sequence.The target sequence in the genomic DNA sequence can be the target sequence in the safe harbor site of the genomic DNA sequence.

[0477] Gene editing compositions comprising Cas-CLOVER and methods of using these compositions for gene editing are described in detail in U.S. Patent Publication Nos. 2017 / 0107541, 2017 / 0114149, 2018 / 0187185, and U.S. Patent No. 10,415,024, each of which is incorporated by reference in its entirety for examples of gene editing systems that may be used in conjunction with the compositions and methods described herein. In some aspects, the Cas-CLOVER protein may comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO:11. In some embodiments, the Cas-CLOVER protein comprises the amino acid sequence set forth in SEQ ID NO:11.

[0478] Thus, the present disclosure provides any of the lipid nanoparticle compositions described herein, wherein the lipid nanoparticles comprise at least one genome editing composition, and the at least one genome editing composition comprises a) a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises (i) an inactivated Cas9 (dCas9) protein or its inactivated nuclease domain, (ii) a Clo051 protein or its nuclease domain, and b) at least one gRNA molecule. In some embodiments, the fusion protein may further comprise at least one NLS. In some embodiments, the at least one genome editing composition may comprise at least two gRNA molecules.

[0479] An exemplary nucleic acid sequence encoding the fusion protein is set forth in SEQ ID NO: 5. Accordingly, the nucleic acid molecules formulated in the lipid nanoparticles of the present disclosure can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 5.

[0480] Exemplary gRNA sequences are set forth in SEQ ID NOs: 6 and 7. Accordingly, gRNA molecules formulated in lipid nanoparticles of the present disclosure can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 6 or SEQ ID NO: 7.

[0481] Formulations, Dosages and Methods of Administration The present disclosure provides formulations, dosages and methods of administration of the compositions described herein.

[0482] The disclosed compositions and pharmaceutical compositions can further comprise at least one of any suitable auxiliary agent, such as, but not limited to, a diluent, a binder, a stabilizer, a buffer, a salt, a lipophilic solvent, a preservative, or an adjuvant. Pharmaceutically acceptable auxiliary agents are preferred. Non-limiting examples of such sterile solutions and methods for their preparation are well known in the art and are described, for example, but not limited to, in Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990 and in the "Physician's Desk Reference," 52nd ed., Medical Economics (Montvale, NJ) 1998. Pharmaceutically acceptable carriers can be routinely selected to suit the mode of administration, solubility, and / or stability of the composition, as is well known in the art or as described herein.

[0483] For example, the disclosed LNP compositions of the present invention can further comprise a diluent. In some embodiments, the diluent can be phosphate buffered saline (PBS).

[0484] Non-limiting examples of pharmaceutical excipients and additives suitable for use include proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars, such as alditols, aldonic acids, and esterified sugars; and polysaccharides or sugar polymers), which can be present alone or in combination and may comprise 1 to 99.99% by weight or volume, alone or in combination. Non-limiting examples of protein excipients include serum albumins, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acids / protein components that may also function as buffers include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine.

[0485] The composition can also contain a buffer or pH adjuster; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts such as citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, and phthalic acid salts, Tris, tromethamine hydrochloride, and phosphate buffers. Preferred buffers are organic acid salts such as citrate. In some embodiments, the buffer may be sucrose.

[0486] Many known and developed modes can be used to administer a therapeutically effective amount of the compositions or pharmaceutical compositions disclosed herein.Non-limiting examples of administration modes include bolus, buccal, injection, intra-articular, intrabronchial, intraperitoneal, intravesical, intrachondral, intracavitary, intracerebellar, intraventricular, intracavity, intracervical, intragastric, intrahepatic, intralesional, intramuscular, intramyocardial, intranasal, intraocular, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intravesical, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intratumoral, intravenous, intravesical, oral, parenteral, rectal, sublingual, subcutaneous, transdermal or intravaginal means.

[0487] The compositions of the present disclosure are intended for use in parenteral (subcutaneous, intramuscular, or intravenous) or any other administration, particularly in the form of a liquid solution or suspension; for vaginal or rectal administration, particularly in semi-solid forms such as, but not limited to, creams and suppositories; for buccal or sublingual administration, for example, but not limited to, in the form of tablets or capsules; or nasally, such as, but not limited to, in the form of powders, nasal sprays, or aerosols or certain pharmaceuticals; or with chemical enhancers such as dimethyl sulfoxide to modify skin structure or increase drug concentration in transdermal patches (Junginger, et al. In "Drug Permeation Enhancement;" Hsieh, D.S., Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994,), or by application of an electric field to create a transient transport pathway, such as electroporation, or to increase the mobility of charged drugs through the skin, such as iontophoresis, or by application of ultrasound, such as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (the above publications and patents are incorporated herein by reference in their entireties).

[0488] For parenteral administration, any composition disclosed herein may be formulated as a solution, suspension, emulsion, granules, powder, or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle, or provided separately. Parenteral formulations may contain common excipients such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalenes, etc. Aqueous or oily suspensions for injection can be prepared according to known methods using appropriate emulsifiers or wetting agents and suspending agents. Injectable preparations can be non-toxic, non-oral diluents such as aqueous solutions, sterile injectable solutions, or suspensions in solvents. Usable vehicles or solvents include water, Ringer's solution, isotonic saline, etc., and sterile fixed oils can be used as common solvents or suspension media. For these purposes, any type of fixed oil and fatty acid can be used, including natural, synthetic, or semi-synthetic fatty oils or fatty acids; natural, synthetic, or semi-synthetic mono-, di-, or triglycerides. Parenteral administration is known in the art and includes, but is not limited to, conventional injection means, the gas-pressurized needleless injection device described in U.S. Pat. No. 5,851,198, and the laser perforation device described in U.S. Pat. No. 5,839,446, each of which is incorporated herein by reference in its entirety for examples of injection devices that may be used in conjunction with the compositions and methods described herein.

[0489] For pulmonary administration, the compositions or pharmaceutical compositions described herein are preferably delivered in a particle size effective to reach the lower respiratory tract of the lungs or paranasal sinuses. The compositions or pharmaceutical compositions can be delivered by any of a variety of inhalation or nasal devices known in the art for administering therapeutic agents by inhalation. These devices, which can deposit aerosolized formulations in a patient's paranasal sinuses or alveoli, include metered-dose inhalers, nebulizers (e.g., jet nebulizers, ultrasonic nebulizers, etc.), dry powder generators, sprayers, and the like. All such devices can employ formulations suitable for administering the compositions or pharmaceutical compositions described herein as aerosols. Such aerosols can be composed of solutions (both aqueous and non-aqueous) or solid particles. In metered-dose inhalers (MDIs), a propellant, a composition or pharmaceutical composition described herein, and excipients or other additives are contained in a canister as a mixture with a liquefied compressed gas. Actuation of a metering valve releases the mixture as an aerosol. A more detailed description of pulmonary administration and formulations and related devices is disclosed in PCT Publication No. WO2019 / 049816, which is incorporated herein by reference in its entirety, for examples of transposases that may be used in conjunction with the compositions and methods described herein.

[0490] For absorption through mucosal surfaces, the composition comprises an emulsion comprising a plurality of submicron particles, a mucoadhesive polymer, a bioactive peptide, and an aqueous continuous phase, which promotes absorption through mucosal surfaces by achieving mucoadhesion of the emulsion particles (see, for example, U.S. Pat. No. 5,514,670, incorporated herein by reference in its entirety). Mucosal surfaces suitable for application of the emulsions of the present disclosure include the corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, gastric, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, such as suppositories, may contain excipients such as polyalkylene glycols, petrolatum, cocoa butter, etc. Formulations for nasal administration are solid and may contain excipients such as lactose, and may be aqueous or oily solutions of nasal drops. For buccal administration, excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, and the like (for examples, see U.S. Pat. No. 5,849,695, which is incorporated herein by reference in its entirety). A more detailed description of mucosal administration and formulations is disclosed in PCT Publication No. WO2019 / 049816, each of which is incorporated herein by reference in its entirety for examples of formulations that may be used in conjunction with the compositions and methods described herein.

[0491] For transdermal administration, the compositions or pharmaceutical compositions disclosed herein are encapsulated in a delivery device such as liposomes or polymer nanoparticles, microparticles, microcapsules, or microspheres (collectively referred to as microparticles unless otherwise specified).Several suitable devices are known, including microparticles made from synthetic polymers, such as polyhydroxy acids, such as polylactic acid, polyglycolic acid, and their copolymers, polyorthoesters, polyanhydrides, and polyphosphazenes, and natural polymers, such as collagen, polyamino acids, albumin, and other proteins, alginic acid, and other polysaccharides (see, for example, U.S. Patent No. 5,814,599, each of which is incorporated herein by reference in its entirety).A more detailed description of transdermal administration, formulations, and suitable devices is disclosed in PCT Publication No. WO2019 / 049816, which is incorporated herein by reference in its entirety for examples of formulations and devices that can be used in conjunction with the compositions and methods described herein.

[0492] It may be desirable to administer the disclosed compounds to a subject over an extended period of time, for example, from one week to one year from a single administration. A variety of sustained release, depot, and implant formulations are available.

[0493] Suitable dosages are well known in the art.See, for example, Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, California (2000); Nursing 2001 Handbook of Drugs, 21st Edition, Springhouse Corp., Springhouse, Pa., 2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc., Upper Saddle River, NJ. Preferred doses can optionally include about 0.1-99 and / or 100-500 mg / kg / administration, or any range, value, or fraction thereof, or can achieve a serum concentration of about 0.1-5000 μg / ml per single or multiple administrations, or per any range, value, or fraction thereof. Preferred dosage ranges for the compositions or pharmaceutical compositions disclosed herein are from about 1 mg / kg to about 3, about 6, or about 12 mg / kg of subject body weight.

[0494] Alternatively, the dosage may vary depending on known factors such as the pharmacodynamic properties of the particular agent and its mode and route of administration, the age, health, and weight of the recipient, the nature and extent of the symptoms, type of concurrent treatment, frequency of treatment, and the desired effect.

[0495] As a non-limiting example, human or animal treatment may be provided as a single or periodic dose of about 0.1-100 mg / kg per day, or any range, value, or fraction thereof, of a composition or pharmaceutical composition disclosed herein, at least once on days 1-40, or alternatively or additionally, at least once on weeks 1-52, or alternatively or additionally, at least once on years 1-20, or any combination thereof, using a single dose, an infusion, or multiple doses.

[0496] In embodiments where the composition administered to a subject in need thereof is a modified cell disclosed herein, the cells are about 1 x 10 3 ~1×10 15 1 x 10 cells 3 ~1×10 15 cells, approximately 1 x 10 4 ~1×10 12 cells; approximately 1 x 10 5 ~1×10 10 cells; approximately 1 x 10 6 ~1×10 9 cells; approximately 1 x 10 6 ~1×10 8 cells; approximately 1 x 10 6 ~1×10 7 cells; or approximately 1 x 10 6 ~25×10 6 In one embodiment, the cells may be about 5 x 10 6 ~25×10 6 It is administered in individual cells.

[0497] A more detailed description of the pharmaceutically acceptable excipients, formulations, dosages, and methods of administration of the disclosed compositions and pharmaceutical compositions is disclosed in PCT Publication No. WO2019 / 04981, which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein.

[0498] The present disclosure provides for the use of the disclosed compositions and pharmaceutical compositions for the treatment of a disease or disorder in a cell, tissue, organ, animal, or subject, as known in the art or as described herein, e.g., by administering or contacting a cell, tissue, organ, animal, or subject with a therapeutically effective amount of the composition or pharmaceutical composition. In some embodiments, the subject is a mammal. Preferably, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0499] The present disclosure provides a method for regulating or treating at least one malignant disease or disorder in a cell, tissue, organ, animal or subject. Non-limiting examples of malignant diseases or disorders include cancer and liver diseases or disorders.

[0500] Any of the methods can include administering an effective amount of any of the compositions or pharmaceutical compositions disclosed herein to a cell, tissue, organ, animal, or subject in need of such modulation, treatment, or therapy. Such methods can optionally further include co-administration or combination therapy for treating such a disease or disorder, and the administration of any of the compositions or pharmaceutical compositions disclosed herein can further include administering at least one chemotherapeutic agent (e.g., alkylating agent, mitotic inhibitor, radiopharmaceutical) before and / or after the simultaneous administration.

[0501] In some embodiments, after administration, the subject does not develop graft versus host (GvH) and / or host versus graft (HvG). In certain embodiments, administration is systemic. Systemic administration can be by any means known in the art and described in detail herein. Preferably, systemic administration is intravenous injection or infusion. In certain embodiments, administration is local. Local administration can be by any means known in the art and described in detail herein. Preferably, local administration is by intratumoral, intraspinal, intraventricular, intraocular, or intraosseous injection or infusion.

[0502] In some embodiments, the therapeutically effective dose is a single dose. In some embodiments, the single dose is one of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 doses or any number therebetween that are produced simultaneously. In some embodiments, when the composition is autologous or allogeneic cells, the dose is sufficient to allow the cells to engraft and / or persist for a sufficient time to treat the disease or disorder.

[0503] In some embodiments of the treatment methods described herein, treatment may be modified or terminated. Specifically, in embodiments in which the composition used for treatment comprises an inducible pro-apoptotic polypeptide, apoptosis can be selectively induced in cells by contacting the cells with an inducer. Treatment may be modified or terminated, for example, in response to signs of recovery, signs of reduced disease severity / progression, signs of disease remission / cessation, and / or the occurrence of adverse events. In some embodiments, the method includes administering an inhibitor of the inducer to inhibit the modification of the cell therapy, thereby restoring the function and / or effectiveness of the cell therapy (e.g., if signs or symptoms of the disease recur or increase in severity and / or if adverse events resolve).

[0504] Nucleic acid construction The isolated nucleic acids of the present disclosure can be produced using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and / or (d) combinations thereof, as are known in the art.

[0505] The nucleic acid may conveniently contain sequences in addition to the polynucleotide of the present disclosure. For example, a multicloning site containing one or more endonuclease restriction sites may be inserted into the nucleic acid to facilitate isolation of the polynucleotide. Also, a translatable sequence may be inserted to facilitate isolation of the translated polynucleotide of the present disclosure. For example, a hexa-histidine marker sequence provides a convenient means for purifying the protein of the present disclosure. The nucleic acid of the present disclosure, excluding the coding sequence, may optionally be a vector, adapter, or linker for cloning and / or expressing the polynucleotide of the present disclosure.

[0506] Additional sequences can be added to such cloning and / or expression sequences to optimize function in cloning and / or expression, to aid in isolation of the polynucleotide, or to improve introduction of the polynucleotide into cells. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra; or Sambrook, supra.)

[0507] Recombinant methods for constructing nucleic acids The isolated nucleic acid compositions of the present disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methodologies known to those skilled in the art. In some embodiments, oligonucleotide probes that selectively hybridize to the polynucleotides of the present disclosure under stringent conditions are used to identify the desired sequence in a cDNA or genomic DNA library. The isolation of RNA, the construction of cDNA and genomic libraries are well known to those skilled in the art. (See, for example, Ausubel, supra; or Sambrook, supra).

[0508] Nucleic Acid Screening and Isolation Methods Probes based on the polynucleotide sequences of the present disclosure can be used to screen cDNA or genomic libraries. Probes can be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes from the same or different organisms. Those skilled in the art will understand that varying degrees of hybridization stringency can be employed in the assay, and that either the hybridization or wash medium can be stringent. The more stringent the hybridization conditions, the higher the degree of complementarity between the probe and target must be for duplex formation to occur. The degree of stringency can be controlled by one or more of temperature, ionic strength, pH, and the presence of a partially denaturing solvent, such as formamide. For example, hybridization stringency can be conveniently varied by changing the polarity of the reaction solution, e.g., by manipulating the formamide concentration within the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding varies depending on the stringency of the hybridization medium and / or wash medium. The degree of complementarity is optimally 100%, or 70-100%, or any range or value therebetween, although it should be understood that minor sequence differences between the probe and primer can be compensated for by reducing the stringency of the hybridization and / or wash solutions.

[0509] Methods for amplifying RNA or DNA are well known in the art and can be used in accordance with the present disclosure without undue experimentation, based on the teachings and guidance provided herein.

[0510] Known methods for amplifying DNA or RNA include polymerase chain reaction (PCR) and related amplification processes (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188 (Mullis et al.); 4,795,699 and 4,921,794 (Tabor et al.); 5,142,033 (Innis); 5,122,464 (Wilson et al.); 5,091,310 (Innis); 5,066,584 (Gyllensten et al.); 4,88 Nos. 9,818 (Gelfand et al.); 4,994,370 (Silver et al.); 4,766,067 (Biswas); 4,656,134 (Ringold), and RNA-mediated amplification using antisense RNA against a target sequence as a template for double-stranded DNA synthesis (U.S. Pat. No. 5,130,238 (Malek et al.), trade name NASBA), the entire contents of which are incorporated herein by reference. (See, for example, Ausubel, supra; or Sambrook, supra.)

[0511] For example, polymerase chain reaction (PCR) technology can be used to directly amplify the sequences of the polynucleotides and related genes of the present disclosure from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods are also useful for, for example, cloning nucleic acid sequences encoding proteins to be expressed, generating nucleic acids to be used as probes to detect the presence of desired mRNA in a sample, nucleic acid sequencing, and other purposes. Examples of techniques sufficient to guide one of skill in the art through in vitro amplification methods can be found in Berger, supra; Sambrook, supra; Ausubel, supra; Mullis, et al., U.S. Pat. No. 4,683,202 (1987); and Innis, et al., PCR Protocols: A Guide to Methods and Applications, Eds., Academic Press Inc., San Diego, Calif. (1990). Commercially available kits for genomic PCR amplification are known in the art. See, for example, the Advantage-GC Genomic PCR Kit (Clontech). Additionally, to improve the yield of long PCR products, for example, T4 gene 32 protein (Boehringer Mannheim) can be used.

[0512] Synthetic methods for constructing nucleic acids The isolated nucleic acids of the present disclosure can also be prepared by direct chemical synthesis using known methods (see, for example, Ausubel, et al., supra). Chemical synthesis generally produces a single-stranded oligonucleotide, which can be converted into double-stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. Those skilled in the art will recognize that chemical synthesis of DNA can be limited to sequences of about 100 bases or more, but that longer sequences can be obtained by ligating shorter sequences.

[0513] Recombinant Expression Cassette The present disclosure further provides a recombinant expression cassette comprising the nucleic acid of the present disclosure. The nucleic acid sequence of the present disclosure can be used to construct a recombinant expression cassette that can be introduced into at least one desired host cell. The recombinant expression cassette typically consists of the polynucleotide of the present disclosure operably linked to a transcription initiation regulatory sequence that directs the transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct the expression of the nucleic acid of the present disclosure.

[0514] In some embodiments, isolated nucleic acids that function as promoters, enhancers, or other elements can be introduced into appropriate locations (upstream, downstream, or in an intron) of non-heterologous forms of the polynucleotides of the disclosure to up- or down-regulate expression of the polynucleotides of the disclosure. For example, endogenous promoters can be altered in vivo or in vitro by mutation, deletion, and / or substitution.

[0515] Expression vectors and host cells The present disclosure also relates to vectors comprising the isolated nucleic acid molecules of the present disclosure, and host cells genetically engineered with the recombinant vectors, as known in the art (see, e.g., Sambrook, et al., supra; Ausubel, et al., supra, each of which is incorporated herein by reference).

[0516] The polynucleotide can optionally be ligated to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and transduced into host cells.

[0517] The DNA insert must be operably linked to a suitable promoter. The expression construct further contains a transcription initiation site, a termination site, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct preferably includes translation initiation at a start and stop codon (e.g., UAA, UGA, or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG being preferred for mammalian or eukaryotic expression.

[0518] Expression vectors preferably, but optionally, include at least one selectable marker, such as, but not limited to, ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / Geneticin (neo gene), DHFR (encoding dihydrofolate reductase and conferring resistance to methotrexate), mycophenolic acid, or glutamine synthetase (GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739), blasticidin (bsd gene), resistance genes for eukaryotic cell culture, and ampicillin, zeocin (Sh bla gene) for culturing in E. coli and other bacteria or prokaryotes. The host cells may contain genes encoding the following: bla gene, puromycin (pac gene), hygromycin B (hygB gene), G418 / Geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline resistance genes (the above patents are incorporated herein by reference in their entireties). Appropriate culture media and conditions for the above host cells are known in the art. Suitable vectors will be readily apparent to those skilled in the art. Introduction of vector constructs into host cells can be accomplished by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other known methods. Such methods are described in the art, such as Sambrook, supra, chapters 1-4 and 16-18; Ausubel, supra, chapters 1, 9, 13, 15, and 16.

[0519] The expression vector preferably includes at least one selectable cell surface marker for isolating cells modified by the disclosed compositions and methods, but this is optional. The selectable cell surface markers of the present disclosure consist of a surface protein, glycoprotein, or group of proteins that distinguish a cell or a subset of cells from another defined subset of cells. Preferably, the selectable cell surface marker distinguishes cells modified by the disclosed compositions or methods from cells not modified by the disclosed compositions or methods. Examples of such cell surface markers include, but are not limited to, "cluster designator" or "classification determinant" proteins (often abbreviated as "CD"), such as truncated or full-length forms of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or combinations thereof. Cell surface markers also include the suicide gene marker RQR8 (Philip B et al. Blood. 2014 Aug 21;124(8):1277-87).

[0520] Preferably, but optionally, the expression vector includes at least one selectable drug resistance marker for isolating cells modified by the compositions and methods of the present disclosure. The selectable drug resistance markers of the present disclosure may include wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[0521] Those of skill in the art are familiar with the numerous expression systems available for expression of nucleic acid molecules encoding the proteins of the present disclosure.

[0522] definition As used throughout this disclosure, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes a plurality of such methods, reference to "a dose" includes a reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0523] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend to some extent on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" means within one or more standard deviations. Alternatively, "about" can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When particular values ​​are described in this application and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range of the particular value.

[0524] In the chemical formula shown herein: TIFF2026503550000143.tif10170Indicates where a functional group is attached to another part of the molecule. Definitions of specific functional groups and chemical terms are explained in more detail below.

[0525] Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds as being within its scope, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, racemic mixtures thereof, and other mixtures thereof. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are intended to be included in the present invention.

[0526] Isomeric mixtures containing various isomer ratios can be utilized in accordance with the present invention. For example, when only two isomers are combined, mixtures containing isomer ratios of 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 are all contemplated by the present invention. Those skilled in the art will readily appreciate that similar ratios are contemplated for more complex isomer mixtures.

[0527] For example, if a specific enantiomer of a compound of the invention is desired, it can be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary, the resulting diastereomeric mixture separated, and the auxiliary cleaved to provide the pure desired enantiomer. Alternatively, if the molecule contains a basic functional group such as an amino group or an acidic functional group such as a carboxyl group, diastereomeric salts can be formed with an appropriate optically active acid or base, and the diastereomers thus formed can then be separated by fractional crystallization or chromatographic means well known in the art, followed by recovery of the pure enantiomer.

[0528] Those skilled in the art will appreciate that synthetic methods such as those described herein utilize various protecting groups. As used herein, the term "protecting group" refers to the temporary blocking of a specific functional moiety, such as O, S, or N, so that a reaction can be selectively carried out at another reactive site in a multifunctional compound. In certain embodiments, the protecting group reacts selectively and efficiently to give a protected substrate that is stable for the anticipated reaction; the protecting group should be selectively removable and efficient with readily available, preferably non-toxic, reagents that do not attack other functional groups; the protecting group forms an easily separable derivative (more preferably, without creating a new stereogenic center); and the protecting group has minimal additional functionality to avoid further reactive sites. As detailed herein, protecting groups for oxygen, sulfur, nitrogen, and carbon can be utilized.

[0529] The term "aliphatic," as used herein, includes both saturated and unsaturated, straight-chain (i.e., unbranched), branched, acyclic, cyclic, or polycyclic aliphatic hydrocarbons, which are optionally substituted with one or more functional groups. As will be understood by those skilled in the art, "aliphatic," as used herein, is intended to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term "alkyl" includes straight-chain, branched, and cyclic alkyl groups. A similar convention applies to other general terms, such as "alkenyl," "alkynyl," and the like. Furthermore, as used herein, the terms "alkyl," "alkenyl," "alkynyl," and the like, encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, "lower alkyl" is used to refer to alkyl groups (cyclic, acyclic, substituted, unsubstituted, branched, or unbranched) having 1 to 6 carbon atoms.

[0530] In certain embodiments, the alkyl, alkenyl, and alkynyl groups used in the present invention contain 1-18 aliphatic carbon atoms. In certain embodiments, the alkyl, alkenyl, and alkynyl groups used in the present invention contain 1-15 aliphatic carbon atoms. In certain other embodiments, the alkyl, alkenyl, and alkynyl groups used in the present invention contain 1-10 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups used in the present invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups used in the present invention contain 1-6 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups used in the present invention contain 1-4 carbon atoms. Exemplary aliphatic groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, cyclopropyl-CH-cyclopropyl, vinyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, --CH-cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, cyclopentyl, --CH-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, and --CH-cyclohexyl moieties, which may also bear one or more substituents. Alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

[0531] As used herein, the term "alkyl" refers to a saturated, straight-chain (e.g., unbranched) or branched-chain aliphatic group having 1 to 18 carbon atoms. Thus, "alkyl" includes C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, ​​C47, C48, C49, C50, C51, C52, C5 10 , C 11 and C 12Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, and the like.

[0532] The term "alkylene" refers to a divalent alkyl group. The monovalent alkyl groups described above can become alkylene by removing a second hydrogen atom from the alkyl. As defined herein, alkylene also refers to C1-C 18 It may also be alkylene. Furthermore, alkylene may also be C1 to C 12 It may be alkylene. Typical alkylene groups include, but are not limited to, -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH2CH(CH3)-, -CH2C(CH3)2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and the like.

[0533] The term "alkenyl" refers to an unsaturated, straight-chain or optionally branched-chain aliphatic group having 2 to 18 carbon atoms and one or more carbon-carbon double bonds. Thus, "alkenyl" includes C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, ​​C47, C48, C49, C50, C51, C52, C53 10 , C 11 and C 12 Alkenyl groups include, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.

[0534] The term "alkynyl" refers to an unsaturated, straight-chain or optionally branched-chain aliphatic group having 2 to 18 carbon atoms and one or more carbon-carbon triple bonds. Thus, "alkynyl" includes C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, ​​C47, C48, C49, C50, C51, C52, C53 10 , C 11 and C 12 Representative alkynyl groups include ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

[0535] As used herein, the term "aryl" refers to a C6-C6 alkyl group consisting of 1 to 3 aromatic rings. 14 An aromatic moiety, optionally substituted. Thus, "aryl" refers to C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , and C 14 Exemplary aryl groups include C6-C 10 Aryl groups. Particular aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, fluorenyl, and the like.

[0536] As used herein, the term "cycloalkyl" includes saturated and partially unsaturated cyclic hydrocarbon groups containing 3 to 12 carbon atoms. Thus, "cycloalkyl" includes C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26 10 , C 11 and C 12 Representative cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0537] As used herein, the term "hydroxyalkyl" refers to an -alkyl-OH or an alkyl chain substituted with at least one -OH.

[0538] As used herein, the term "halo" or "halogen" refers to fluoro, chloro, bromo, and iodo.

[0539] It will be understood that the compounds of any one of the formulas disclosed herein, and any pharmaceutically acceptable salts thereof, include stereoisomers, mixtures of stereoisomers, and polymorphs of all isomeric forms of said compounds.

[0540] The term "independently selected" is used herein to indicate that the R groups can be the same or different.

[0541] The terms "substituted" and "substituent," as used herein, whether preceded by the term "optionally" or not, refer to the ability to change one functional group to another, provided that the valences of all atoms are maintained, as would be understood by one of ordinary skill in the art. When more than one position in a given structure may be substituted with more than one substituent selected from a specified group, the substituents may be the same or different at every position. Substituents may also be further substituted (e.g., a substituent on an aryl group may have another substituent, such as another aryl group further substituted with fluorine at one or more positions).

[0542] The present disclosure provides isolated or substantially purified polynucleotide or protein compositions. An "isolated" or "purified" polynucleotide or protein, or a biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its natural environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material and culture medium when produced by recombinant techniques, and is substantially free of chemical precursors and other chemicals when chemically synthesized. Optimally, an "isolated" polynucleotide is free of sequences that naturally flank the polynucleotide in the genomic DNA of the organism from which the polynucleotide is derived (i.e., sequences located at the 5' and 3' ends of the polynucleotide, optimally, protein-encoding sequences). For example, in various embodiments, an isolated polynucleotide may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When a protein of the disclosure or a biologically active portion thereof is recombinantly produced, optimally, the culture medium contains less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

[0543] The present disclosure provides fragments and variants of the disclosed DNA sequences, as well as proteins encoded by these DNA sequences. As used throughout this disclosure, the term "fragment" refers to a portion of a DNA sequence or a portion of an amino acid sequence, and thus the protein encoded thereby. A fragment of a DNA sequence consisting of a coding sequence may encode a protein fragment that retains the biological activity of the native protein, as described herein, and thus retains DNA recognition or binding activity to a target DNA sequence. Alternatively, a fragment of a DNA sequence useful as a hybridization probe generally does not encode a protein that retains biological activity or promoter activity. Thus, a fragment of a DNA sequence can range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide of the present disclosure.

[0544] The nucleic acids or proteins of the present disclosure can be constructed by a modular approach, involving preassembly of monomeric and / or repeating units in a target vector, which can then be assembled into a final destination vector. Polypeptides of the present disclosure can be constructed by a modular approach by preassembling repeating units in a target vector, which can be composed of repeating monomers of the present disclosure, which can then be assembled into a final destination vector. The present disclosure provides polypeptides produced by this method, and nucleic acid sequences encoding these polypeptides. The present disclosure provides host organisms and cells containing nucleic acid sequences encoding the polypeptides produced by this modular approach.

[0545] The term "antibody" is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies) and antibody compositions with multiple epitopic specificities. It is also within the scope of the present specification to use natural or synthetic analogs, variants, alleles, homologs, and orthologs of the present antibodies defined herein (collectively referred to herein as "analogs"). Thus, according to one aspect of the present specification, the term "antibodies herein" in the broadest sense also includes such analogs. Generally, such analogs may have one or more amino acid residues substituted, deleted, and / or added compared to the antibodies defined herein.

[0546] The term "comprising" is intended to mean that the compounds, compositions, and methods include the recited elements, but do not exclude others. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements that are essential to the combination for the purpose described. Thus, a composition consisting essentially of the elements defined herein does not exclude trace amounts of contaminants or inert carriers. "Consisting of" is intended to mean excluding more than trace amounts of other components and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0547] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression includes splicing of the mRNA in a eukaryotic cell.

[0548] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. A gene product can be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, microRNA, structural RNA, or other types of RNA) or a protein produced by translation of mRNA. Gene products also include RNAs modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0549] "Modulation" or "regulation" of gene expression refers to a change in the activity of a gene. Modulation of expression includes, but is not limited to, gene activation and gene repression.

[0550] The term "operatively linked" or its equivalents (e.g., "linked operatively") means that two or more molecules are positioned relative to each other so that they can interact in a manner that affects the function attributed to one or both molecules or a combination thereof.

[0551] Non-covalently linked components, as well as methods for making and using non-covalently linked components, are disclosed. The various components can take a variety of different forms, as described herein. For example, non-covalently linked (i.e., operably linked) proteins can be used to allow for transient interactions, avoiding one or more problems in the art. The ability of non-covalently linked components, such as proteins, to associate and dissociate allows for functional association only, or primarily, under circumstances where such association is required for the desired activity. Linkage need only be for a period of time sufficient to achieve the desired effect.

[0552] A method for targeting a protein to a specific locus in the genome of an organism is disclosed. The method may include providing a DNA localization component and providing an effector molecule, wherein the DNA localization component and the effector molecule can be operably linked via a non-covalent linkage.

[0553] A "target site" or "target sequence" is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind when conditions sufficient for binding are present.

[0554] The term "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked. A description of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid can encompass the complementary strand of a described single strand. The nucleic acids of the present disclosure also encompass substantially identical nucleic acids and their complements that retain the same structure or encode the same protein.

[0555] A probe of the present disclosure may consist of a single-stranded nucleic acid capable of hybridizing to a target sequence under stringent hybridization conditions. Thus, a nucleic acid of the present disclosure may refer to a probe that hybridizes under stringent hybridization conditions.

[0556] The nucleic acids of the present disclosure may be single-stranded or double-stranded. The nucleic acids of the present disclosure may be predominantly single-stranded or may contain double-stranded sequences. The nucleic acids of the present disclosure may be predominantly double-stranded or may contain single-stranded sequences. The nucleic acids of the present disclosure may include genomic DNA, cDNA, RNA, or hybrids thereof. The nucleic acids of the present disclosure may contain a combination of deoxyribonucleotides and ribonucleotides. The nucleic acids of the present disclosure may contain a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. The nucleic acids of the present disclosure may be synthesized to contain unnatural amino acid modifications. The nucleic acids of the present disclosure may be obtained by chemical synthesis or recombinant methods.

[0557] The nucleic acids of the present disclosure may have either their entire base sequence or a portion thereof that does not occur in nature. The nucleic acids of the present disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not occur in nature, and the entire nucleic acid sequence may not occur in nature. The nucleic acids of the present disclosure may contain one or more overlapping sequences, inverted sequences, or repeated sequences, and as a result, the sequence does not occur in nature, and the entire nucleic acid sequence may not occur in nature. The nucleic acids of the present disclosure may contain modified nucleotides, artificial nucleotides, or synthetic nucleotides that do not occur in nature, and the entire nucleic acid sequence may not occur in nature.

[0558] Given the redundancy in the genetic code, more than one nucleotide sequence may encode a particular protein, and all such nucleotide sequences are contemplated herein.

[0559] As used throughout this disclosure, the term "operably linked" refers to the expression of a gene under the control of a spatially linked promoter. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. Changes in the distance between the promoter and the gene can be accommodated without impairing the function of the promoter.

[0560] As used throughout this disclosure, the term "promoter" refers to a synthetic or naturally occurring molecule capable of conferring, activating, or enhancing expression of a nucleic acid in a cell. A promoter can contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter can also contain distal enhancer or repressor elements, located as far away as thousands of base pairs from the transcription start site. Promoters are derived from viruses, bacteria, fungi, plants, insects, animals, etc. Promoters can constitutively or differentially regulate the expression of genetic components with respect to the cell, tissue or organ in which expression occurs, or the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, EF-1 alpha promoter, CAG promoter, SV40 early promoter or SV40 late promoter, and CMV IE promoter.

[0561] As used throughout this disclosure, the term "substantially complementary" refers to a first sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

[0562] As used throughout this disclosure, the term "substantially identical" refers to a first and second sequence or nucleic acids that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, where the first sequence is substantially complementary to the complement of the second sequence.

[0563] As used throughout this disclosure, the term "variant," when used to describe a nucleic acid, refers to (i) a portion or fragment of a referenced nucleotide sequence; (ii) a complement of the referenced nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to the referenced nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, its complement, or a sequence substantially identical thereto.

[0564] As used throughout this disclosure, the term "vector" refers to a nucleic acid sequence containing an origin of replication. Vectors include viral vectors, bacteriophages, bacterial artificial chromosomes, and yeast artificial chromosomes. Vectors can be DNA or RNA vectors. Vectors can be self-replicating extrachromosomal vectors, preferably DNA plasmids. Vectors are composed of amino acids and DNA sequences, RNA sequences, or a combination of both DNA and RNA sequences.

[0565] As used throughout this disclosure, the term "variant," when used to describe a peptide or polypeptide, refers to a peptide or polypeptide that differs in amino acid sequence by amino acid insertions, deletions, or conservative substitutions, but retains at least one biological activity. A variant can also refer to a protein having an amino acid sequence substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity.

[0566] Conservative amino acid substitutions, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are recognized in the art as typically resulting in minor changes. These minor changes can be identified, in part, by considering the hydropathic index of an amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid takes into account its hydrophobicity and charge. Protein function can be maintained even when substituted with an amino acid having a similar hydropathic index. In some embodiments, amino acids with a hydropathic index of ±2 are substituted. Amino acid hydrophilicity can also be used to identify substitutions that maintain the biological function of a protein. Considering the hydrophilicity of amino acids in the context of a peptide allows for calculation of the peptide's greatest local average hydrophilicity, a useful index that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101 is incorporated herein by reference in its entirety.

[0567] Substitution of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, e.g., immunogenicity. Substitutions can be made with amino acids whose hydrophilicity values ​​are within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are affected by the specific side chain of that amino acid. Consistent with this observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids, particularly their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0568] As used herein, "conservative" amino acid substitutions can be defined as shown in Tables A, B, or C below. In some embodiments, fusion polypeptides and / or nucleic acids encoding such fusion polypeptides contain conservative substitutions introduced by modifying a polynucleotide encoding a polypeptide of the present disclosure. Amino acids can be classified according to their physical properties and contribution to the secondary and tertiary structure of proteins. A conservative substitution is the replacement of one amino acid with another amino acid with similar properties. Exemplary conservative substitutions are shown in Table 1.

[0569] [Table 1]

[0570] Alternatively, conservative amino acids can be grouped as described in Table 2 by Lehninger (Biochemistry, Second Edition; Worth Publishers, Inc. NY, NY (1975), pp. 71-77).

[0571] [Table 2]

[0572] Alternatively, exemplary conservative substitutions are shown in Table 3. [Table 3]

[0573] It should be understood that the polypeptides of the present disclosure are intended to include polypeptides having one or more insertions, deletions, substitutions, or any combination thereof, of amino acid residues, as well as polypeptides having modifications other than insertions, deletions, or substitutions of amino acid residues. A polypeptide or nucleic acid of the present disclosure may contain one or more conservative substitutions.

[0574] As used throughout this disclosure, the term "two or more" of the foregoing amino acid substitutions refers to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more of the described amino acid substitutions. The term "two or more" can refer to 2, 3, 4, or 5 of the described amino acid substitutions.

[0575] The polypeptides and proteins of the present disclosure may have either their entire nucleotide sequence or a portion thereof that is not naturally occurring. The polypeptides and proteins of the present disclosure may contain one or more mutations, substitutions, deletions, or insertions that are not naturally occurring, and the entire amino acid sequence may not be naturally occurring. The polypeptides and proteins of the present disclosure may contain one or more duplicated, inverted, or repeated sequences, such that the sequence is not naturally occurring and the entire amino acid sequence may not be naturally occurring. The polypeptides and proteins of the present disclosure may contain modified, artificial, or synthetic amino acids that are not naturally occurring, and the entire amino acid sequence may not be naturally occurring.

[0576] As used throughout this disclosure, "sequence identity" can be determined using a standalone executable BLAST engine program (bl2seq) for blasting two sequences, which can be obtained from the National Center for Biotechnology Information (NCBI) ftp site using default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; incorporated herein by reference in its entirety). The term "identical" or "identity," when used in the context of two or more nucleic acid or polypeptide sequences, refers to a specific percentage of identical residues over a specific region of each sequence. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions where identical residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are of different lengths, or if the alignment produces sequences with one or more offset ends, and the specified comparison region contains only a single sequence, the residues of the single sequence are included in the denominator but not in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identification can be done manually or using computer alignment algorithms such as BLAST and BLAST 2.0.

[0577] As used throughout this disclosure, the term "endogenous" refers to a nucleic acid or protein sequence that is naturally associated with the target gene or host cell into which it is introduced.

[0578] As used throughout this disclosure, the term "exogenous" refers to a nucleic acid or protein sequence that is not naturally associated with the target gene or host cell into which it is introduced, and includes non-naturally occurring multiple copies of a naturally occurring nucleic acid, e.g., a DNA sequence, or a naturally occurring nucleic acid sequence located in a non-naturally occurring genomic location.

[0579] The present disclosure provides a method for introducing a polynucleotide construct containing a DNA sequence into a host cell. By "introducing" it is meant presenting the polynucleotide construct to the cell in such a way that the polynucleotide construct can access the interior of the host cell. The method of the present disclosure does not depend on a particular method for introducing the polynucleotide construct into the host cell, but only on the polynucleotide construct accessing the interior of one cell of the host. Methods for introducing polynucleotide constructs into bacteria, plants, fungi, and animals are known in the art, including, but not limited to, stable transformation, transient transformation, and virus-mediated methods. [Example]

[0580] For the following examples, compound numbers are assigned to compounds of formula (I) and (II) of the present disclosure according to the following: TIFF2026503550000147.tif255170TIFF2026503550000148.tif255170TIFF2026503550000149.tif245170 TIFF2026503550000150.tif224170TIFF2026503550000151.tif216170TIFF2026503550000152.tif214170 TIFF2026503550000153.tif236170TIFF2026503550000154.tif221170TIFF2026503550000155.tif251170 TIFF2026503550000156.tif252170TIFF2026503550000157.tif241170TIFF2026503550000158.tif149170

[0581] Example 1 - Preparation of Compound No. 1 Compound No. 1 was prepared according to general scheme (A). The crude material was purified by flash chromatography on silica gel using DCM / EtOAC. 1H NMR (500MHz, CDCl3): δ3.87(d,8H,J=9.8Hz),2.77(t,8H,J=9.8Hz),2.43(t,12H,J=4.8Hz),2.28(m,4H),2.18(s,3H),1.78- 1.74(m,14H),1.58-1.49(m,20H),1.31-1.22(m,16H),1.16-1.11(m,14H),0.97-0.88(m,8H),0.86(d,24H,J=9.8Hz).MS:m / z 1155.2(M+H).

[0582] Example 2 - Preparation of Compound No. 2 Compound No. 2 was prepared according to general scheme (A). The crude material was purified by flash chromatography on silica gel using DCM / EtOAC. 1 H NMR (500MHz, CDCl3): δ3.87(d,8H,J=6Hz),2.77(t,8H,J=6Hz),2.43(t,12H,J=4.8Hz),2.19(m,4H), 2.18(s,3H),1.77-1.61(m,30H),1.25-1.19(m,20H),1.18-1.12(m,32H),0.98-0.82(m,24H).MS:m / z 1371.2(M+H).

[0583] Example 3 - Preparation of Compound No. 3 Compound No. 3 was prepared according to general scheme (C). The crude was purified by silica gel flash column chromatography using 4% MeOH / CH2Cl2. Light brown oil, 0.91 g; yield: 85%. 1 H NMR(499MHz,CDCl3)δ4.25(s,16H),2.76(t,J=7.2Hz,8H),2.46(t,J=7.3Hz,11H),2.35-2.12(m,10H),1.98-1.92( m,8H),1.83-1.77(m,8H),1.66-1.53(m,6H),1.40(qd,J=13.3,3.4Hz,8H),1.32-1.14(m,36H),0.96-0.84(m,20H).

[0584] Example 4 - Preparation of Compound No. 4 Compound No. 4 was prepared according to general scheme (C). The crude was purified by silica gel flash column chromatography using 4% MeOH / CH2Cl2. Light brown oil, 0.80 g; yield: 67%. 1 H NMR(499MHz,CDCl3)δ4.25(s,16H),2.79(t,J=7.2Hz,8H),2.59-2.38(m,15H),2.30-2.15(m,7H),1.99 -1.91(m,8H),1.84-1.77(m,8H),1.40(qd,J=13.1,3.5Hz,8H),1.34-1.12(m,37H),0.97-0.82(m,20H).

[0585] Example 5 - Preparation of Compound No. 5 Compound No. 5 was prepared according to general scheme (C). The crude material was purified by silica gel flash column chromatography using 4% MeOH / CHCl. ​​Light brown oil, 0.136 mg; yield: 60%; H NMR (499 MHz, CDCl) δ 4.26 (s, 16H), 2.77 (t, J = 7.2 Hz, 8H), 2.50-2.40 (m, 12H), 2.36-2.25 (m, 12H), 2.18 (s, 3H), 1.71-1.55 (m, 33H), 1.35-1.28 (m, 8H), 1.23-1.12 (m, 23H), 0.89-0.80 (m, 8H).

[0586] Example 6 - Preparation of Compound No. 6 Compound No. 6 was prepared according to general scheme (C). The crude material was purified by silica gel flash column chromatography using 4% MeOH / CHCl. ​​Light brown oil, 0.101 mg; yield: 45%; H NMR (499 MHz, CDCl) δ 4.26 (s, 16H), 2.79 (t, J = 7.2 Hz, 8H), 2.58-2.40 (m, 15H), 2.32 (t, J = 7.5 Hz, 8H), 2.22 (s, 3H), 1.71-1.56 (m, 29H), 1.34-1.10 (m, 32H), 0.89-0.80 (m, 8H).

[0587] Example 7 - Preparation of Compound No. 7 Compound No. 7 was prepared according to general scheme (D). The crude was purified by column chromatography (MeOH / DCM). 1 H NMR(499MHz,CDCl3)δ3.98(d,J=7.2Hz,5H),3.89(dd,J=6.6,2.2Hz,11H),2.77(t,J=7.4Hz,8H),2.43(t,J=7.3Hz,12H),2.28(t,J=7.5Hz,12H),2.17 (s,3H),1.85-1.78(m,2H),1.67-1.49(m,34H),1.46-1.36(m,6H),1.23-1. 15(m,10H),1.06-0.94(m,12H),0.88(d,J=6.6Hz,24H).MS actual value 1386.8[M+H] + , calculated value for [C79H139N3O16=1386.02].

[0588] Example 8 - Preparation of Compound No. 8 Compound No. 8 was prepared according to general scheme (D). The crude was purified by column chromatography (MeOH / DCM). 1 H NMR(500MHz,CDCl3)δ3.98(d,J=7.2Hz,5H),3.89(dd,J=6.6,1.8Hz,11H),2.77(t,J= 7.3Hz,8H),2.43(t,J=7.3Hz,12H),2.29(q,J=7.1Hz,12H),2.17(s,3H),1.85-1.75( m,14H),1.61(h,J=7.6Hz,18H),1.58-1.49(m,4H),1.45-1.37(m,4H),1.31(ddt,J=1 1.2,8.0,5.3Hz,16H),1.06-0.93(m,12H),0.89(t,J=6.9Hz,12H).MS actual value 1330.5[M+H] + , calculated value for [C75H131N3O16=1329.95].

[0589] Example 9 - Preparation of Compound No. 9 Compound No. 9 was prepared according to general scheme (D). The crude was purified by column chromatography (MeOH / DCM). 1H NMR(500MHz,CDCl3)δ3.98(d,J=7.2Hz,5H),3.89(dd,J=6.5,1.8Hz,11H),2.76(t,J =7.3Hz,8H),2.43(t,J=7.2Hz,12H),2.30(q,J=9.8Hz,12H),1.94(s,3H),1.83-1.7 6(m,12H),1.64-1.56(m,18H),1.59-1.50(m,5H,1.45-1.37(m,5H),1.34-1 .22(m,40H),1.06-0.93(m,12H),0.88(t,J=6.9Hz,12H).MS actual value 1498.6[M+H] + , calculated value for [C87H155N3O16=1498.14].

[0590] Example 10 - Preparation of Compound No. 10 Compound No. 10 was prepared according to general scheme (D). The crude was purified by column chromatography (MeOH / DCM). 1 H NMR(500MHz,CDCl3)δ4.05(td,J=6.8,2.1Hz,16H),2.76(t,J=7.3Hz,8H),2. 43(td,J=7.3,3.0Hz,12H),2.28(dd,J=8.9,5.8Hz,4H),2.17(d,J=7.0Hz,12 H),1.80-1.73(m,3H),1.73-1.65(m,16H),1.64-1.55(m,24H),1.32(s,32H) ,1.37-1.20(m,8H),1.20-1.08(m,4H),1.02-0.90(m,8H).MS actual value 1442.5[M+H] + , calculated value for [C83H147N3O16=1442.08].

[0591] Example 11 - Preparation of Compound No. 11 Compound No. 11 was prepared according to general scheme (D). The crude was purified by column chromatography (MeOH / DCM). 1H NMR(499MHz,CDCl3)δ4.05(td,J=6.8,1.6Hz,16H),2.77(t,J=7.3Hz,8H),2.43(td,J=7.0,2.2Hz,12H),2.29(q,J=7.0Hz,12H),2.1 8(s,3H),1.74-1.63(m,16H),1.61-1.54(m,32H),1.52(dt,J=8.7,7.0Hz,8H),1.32(m,24H),0.94-0.83(m,8H).MS actual value 1498.6[M+H] + , calculated value for [C87H155N3O16=1498.1].

[0592] Example 12 - Preparation of Compound No. 12 Compound No. 12 was prepared according to general scheme (D). The crude was purified by column chromatography (MeOH / DCM). 1 H NMR (500MHz, CDCl3): δ5.27-5.32(m,8H),3.88(d,16H,J=9.8Hz),2.77(t,8H,J=12Hz),2.43(t,8H,J=9.8Hz),2.23(t,8H,J=9.8Hz),2.18(s,3H) ,2.06-2.02(dd,8H,J=3Hz,9.8Hz),1.80-1.82(m,16H),1.62-1.60(m,34 H),1.36-1.26(m,56H),1.01-0.97(m,14H),0.89(d,12H,J=3Hz).MS:m / z 1988.1(M+H).

[0593] Example 13 - Preparation of Compound No. 13 Compound No. 13 was prepared according to general scheme (B).

[0594] Example 14 - Preparation of Compound No. 14 Compound No. 14 was prepared according to general scheme (B).

[0595] Example 15 - Preparation of Compound No. 15 Compound No. 15 was prepared according to general scheme (E).

[0596] Following the general protocol for amine alkylation described in General Scheme E.1, amine H2 (17 mg) was combined with C6C25C (300 mg) and DIPEA (200 μL) in THF / CH3CN (1:1, 1.0 mL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent. Brown oil, 119 mg (58%); LC-MS: Rt 7.867 min, m / z calculated [M+H]: 738.55, found 738.4.

[0597] Example 16 - Preparation of Compound No. 16 Compound No. 16 was prepared according to general scheme (E).

[0598] Following the general protocol for amine alkylation described in General Scheme E.1, amine H3 (17 mg) was combined with C6C25C (290 mg) and DIPEA (200 μL) in THF / CH3CN (1:1, 1.0 mL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent. Brown oil, 134 mg (79%); LC-MS: Rt 7.883 min, m / z calculated [M+H]: 752.6, found 752.4.

[0599] Example 17 - Preparation of Compound No. 17 Compound No. 17 was prepared according to general scheme (E).

[0600] Following the general protocol for amine alkylation described in General Scheme E.1, amine H4 (22 mg) was combined with C6C25C (310 mg) and DIPEA (200 μL) in THF / CH3CN (1:1, 1.0 mL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent. Brown oil, 101 mg (53%); LC-MS: Rt 7.900 min, m / z calculated [M+H]: 766.58, found 766.4.

[0601] Example 18 - Preparation of Compound No. 18 Compound No. 18 was prepared according to general scheme (E).

[0602] Following the general protocol for amine alkylation described in General Scheme E.1, amine H2 (16.7 mg) was combined with 5CC3 (192 mg) and DIPEA (110 μL) in THF / CH3CN (1:1, 1.0 mL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent. Brown oil, 88 mg (60%); 1 H NMR(499MHz,CDCl3)δ4.10(t,J=6.3Hz,4H),3.55(t,J=5.3Hz,2H),2.64-2.48(m,6H),2.21(tt,J=12.2,3.6Hz,2H),1.98-1.91(m,4H),1. 83-1.74(m,8H),1.40(qd,J=13.1,3.4Hz,4H),1.33-1.13(m,19H),0.95-0.85(m,10H);LC-MS:Rt7.617 min, m / z calculated value [M+H]:538.45,actual value 538.2.

[0603] Example 19 - Preparation of Compound No. 19 Compound No. 19 was prepared according to general scheme (E).

[0604] Following the general protocol for amine alkylation described in General Scheme E.1, amine H3 (25.7 mg) was combined with 5CC3 (322 mg) and DIPEA (175 μL) in THF / CH3CN (1:1, 1.0 mL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent. Brown oil, 108 mg (57%); LC-MS: Rt 7.583 min, m / z calculated [M+H]: 552.46, found 552.2.

[0605] Example 20 - Preparation of Compound No. 20 Compound No. 20 was prepared according to general scheme (E).

[0606] Following the general protocol for amine alkylation described in General Scheme E.1, amine H4 (30.7 mg) was combined with 5CC3 (334 mg) and DIPEA (180 μL) in THF / CHCN (1:1, 1.0 mL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent. Brown oil, 134 mg (71%); LC-MS: Rt 7.583 min, m / z calculated [M+H]: 566.48, found 566.2.

[0607] Example 21 - Preparation of Compound No. 21 Compound No. 21 was prepared according to general scheme (E).

[0608] Following the general protocol for amine alkylation described in General Scheme E.1, amine H2 (23 mg) was combined with 5CC7 (372 mg) and DIPEA (200 μL) in THF / CH3CN (1:1, 1.0 mL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent. Brown oil, 161 mg (72%); LC-MS: Rt 7.983 min, m / z calculated [M+H]: 594.51, found 594.4.

[0609] Example 22 - Preparation of Compound No. 22 Compound No. 22 was prepared according to general scheme (E).

[0610] Following the general protocol for amine alkylation described in General Scheme E.1, amine H3 (29 mg) was combined with 5CC7 (413 mg) and DIPEA (200 μL) in THF / CHCN (1:1, 1.0 mL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent. Brown oil, 154 mg (66%); LC-MS: Rt 8.000 min, m / z calculated [M+H]: 608.52, found 608.2.

[0611] Example 23 - Preparation of Compound No. 23 Compound No. 23 was prepared according to general scheme (E).

[0612] Following the general protocol for amine alkylation described in General Scheme E.1, amine H4 (28 mg) was combined with 5CC7 (363 mg) and DIPEA (200 μL) in THF / CHCN (1:1, 1.0 mL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent. Brown oil, 122 mg (62%); LC-MS: Rt 8.000 min, m / z calculated [M+H]: 622.54, found 622.2.

[0613] Example 24 - Preparation of Compound No. 24 Compound No. 24 was prepared according to general scheme (E).

[0614] Following the general protocol for amine alkylation described in General Scheme E.1, amine H2 (25 mg) was combined with 5CC7 (370 mg) and DIPEA (200 μL) in THF / CH3CN (1:1, 1.0 mL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent. Brown oil, 161 mg (61%); LC-MS: Rt 8.283 min, m / z calculated [M+H]: 650.57, found 650.4.

[0615] Example 25 - Preparation of Compound No. 25 Compound No. 25 was prepared according to general scheme (E).

[0616] Following the general protocol for amine alkylation described in General Scheme E.1, amine H3 (28 mg) was combined with 5CC7 (399 mg) and DIPEA (200 μL) in THF / CHCN (1:1, 1.0 mL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent. Brown oil, 180 mg (73%); LC-MS: Rt 8.267 min, m / z calculated [M+H]: 664.59, found 664.4.

[0617] Example 26 - Preparation of Compound No. 26 Compound No. 26 was prepared according to general scheme (E).

[0618] Following the general protocol for amine alkylation described in General Scheme E.1, amine H4 (27 mg) was combined with 5CC7 (332 mg) and DIPEA (200 μL) in THF / CHCN (1:1, 1.0 mL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent. Brown oil, 142 mg (69%); LC-MS: Rt 8.267 min, m / z calculated [M+H]: 678.6, found 679.0.

[0619] Example 27 - Preparation of Compound No. 27 Compound No. 27 was prepared according to general scheme (E).

[0620] Following the general protocol for amine alkylation described in General Scheme E.1, amine H2 (19 mg) was reacted with C6CyO in THF / CH3CN (1:1, 1.0 mL). 6,10 (470 mg) and DIPEA (200 μL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent. Brown oil, 210 mg (70%); LC-MS: Rt 9.383 min, m / z calculated [M+H]: 962.80, found 963.0.

[0621] Example 28 - Preparation of Compound No. 28 Compound No. 28 was prepared according to general scheme (E).

[0622] Following the general protocol for amine alkylation described in General Scheme E.1, amine H3 (15 mg) was reacted with C6CyO in THF / CH3CN (1:1, 1.0 mL). 6,10 (304 mg) and DIPEA (200 μL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent. Brown oil, 125 mg (64%); LC-MS: Rt 9.383 min, m / z calculated [M+H]: 976.82, found 977.0.

[0623] Example 29 - Preparation of Compound No. 29 Compound No. 29 was prepared according to general scheme (E).

[0624] Following the general protocol for amine alkylation described in General Scheme E.1, amine H4 (16 mg) was reacted with C6CyO in THF / CH3CN (1:1, 1.0 mL). 6,10 (275 mg) and DIPEA (200 μL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent. Brown oil, 92 mg (52%); LC-MS: Rt 9.583 min, m / z calculated [M+H]: 990.83, found 991.0.

[0625] Example 30 - Preparation of Compound No. 30 Compound No. 30 was prepared according to general scheme (E).

[0626] Following the general protocol for amine alkylation described in General Scheme E.1, amine H2 (11 mg) was reacted with C5Cy in THF / CH3CN (1:1, 0.8 mL). 8,9 (289 mg) and DIPEA (150 μL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent. Brown oil, 92 mg (50%); LC-MS: Rt 9.583 min, m / z calculated [M+H]: 1018.86, found 1019.0.

[0627] Example 31 - Preparation of Compound No. 31 Compound No. 31 was prepared according to general scheme (E).

[0628] Following the general protocol for amine alkylation described in General Scheme E.1, amine H3 (10 mg) was reacted with C5Cy in THF / CH3CN (1:1, 0.8 mL). 8,9 (254 mg) and DIPEA (150 μL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent. Brown oil, 71 mg (52%); LC-MS: Rt 9.533 min, m / z calculated [M+H]: 1032.88, found 1033.0.

[0629] Example 32 - Preparation of Compound No. 32 Compound No. 32 was prepared according to general scheme (E).

[0630] Following the general protocol for amine alkylation described in General Scheme E.1, amine H4 (16 mg) was reacted with C5Cy in THF / CH3CN (1:1, 0.8 mL). 8,9 (283 mg) and DIPEA (150 μL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent. Brown oil, 124 mg (65%); LC-MS: Rt 9.567 min, m / z calculated [M+H]: 1046.89, found 1047.0.

[0631] Example 33 - Preparation of Compound No. 33 Compound No. 33 was prepared according to general scheme (E).

[0632] Following the general protocol for amine alkylation described in General Scheme E.1, H2NHC75C (70 mg) was reacted with C6CyO in THF / CH3CN (1:1, 0.8 mL). 6,10 (160 mg) and DIPEA (150 μL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent. Brown oil, 96 mg (62%); LC-MS: Rt 9.000 min, m / z calculated [M+H]: 806.69, found 807.0.

[0633] Example 34 - Preparation of Compound No. 34 Compound No. 34 was prepared according to general scheme (E).

[0634] Following the general protocol for amine alkylation described in General Scheme E.1, H2NHC75C (71 mg) was converted to C5CyO 8,9 (162 mg) and DIPEA (150 μL) were combined in THF / CH3CN (1:1, 0.8 mL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent. Brown oil, 102 mg (65%); LC-MS: Rt 9.133 min, m / z calculated [M+H]: 835.73, found 835.8.

[0635] Example 35 - Preparation of Compound No. 35 Compound No. 35 was prepared according to general scheme (E).

[0636] Following the general protocol for amine alkylation described in General Scheme E.1, amine 404 (19 mg) was combined with 5CC3 (264 mg) and DIPEA (120 μL) in THF / CH3CN (1:1, 1.0 mL). After the reaction, the crude product was purified with 4% MeOH / DCM eluent. Colorless oil, 23 mg (16%); LC-MS: Rt 7.583 min, m / z calculated [M+H]: 1098.95, found 1099.

[0637] Example 36 - Preparation of Compound No. 36 Compound No. 36 was prepared according to general scheme (E).

[0638] Following the general protocol for amine alkylation described in General Scheme E.1, amine H2 (12 mg) was combined with BC6B5C (292 mg) and DIPEA (200 μL) in THF / CH3CN (1:1, 0.8 mL). After the reaction, the crude product was purified with 6% MeOH / DCM eluent. Brown oil, 142 mg (61%); 1 H NMR(499MHz,CDCl3)δ4.12(t,J=5.5Hz,12H),3.55(s,2H),2.61(s,2H),2.49(s,3H) ),2.39(p,J=6.0Hz,2H),2.32(t,J=7.5Hz,4H),2.22(tt,J=12.2,3.6Hz,4H),1.98 -1.91(m,8H),1.80(dt,J=15.1,3.2Hz,8H),1.64(q,J=7.6Hz,4H),1.53-1.13(m,5 3H),0.88(q,J=6.8Hz,20H);LC-MS:Rt9.625min,m / z calculated value [M+H]:1186.91,actual value 1186.80.

[0639] Example 37 - Preparation of Compound No. 37 Compound No. 37 was prepared according to general scheme (E).

[0640] Following the general protocol for amine alkylation described in General Scheme E.1, amine H3 (22 mg) was combined with BC6B5C (442 mg) and DIPEA (200 μL) in THF / CHCN (1:1, 0.8 mL). After the reaction, the crude product was purified with 6% MeOH / DCM eluent. Brown oil, 127 mg (36%); 1 H NMR(499MHz,CDCl3)δ4.12(t,J=4.8Hz,12H),3.81-3.75(m,2H),2.65(s,2H),2.3 9(td,J=12.8,6.8Hz,5H),2.32(t,J=7.5Hz,4H),2.22(tt,J=12.2,3.6Hz,4H),1. 98-1.91(m,8H),1.84-1.76(m,8H),1.72-1.60(m,7H),1.50(s,4H),1.44-1.13(m ,49H),0.95-0.84(m,20H);LC-MS:Rt9.504 min,m / z calculated value [M+H]:1200.92,actual value 1200.75.

[0641] Example 38 - Preparation of Compound No. 38 Compound No. 38 was prepared according to general scheme (E).

[0642] Following the general protocol for amine alkylation described in General Scheme E.1, amine H4 (10 mg) was combined with BC6B5C (179 mg) and DIPEA (100 μL) in THF / CHCN (1:1, 0.8 mL). After the reaction, the crude product was purified with 6% MeOH / DCM eluent. Brown oil, 27 mg (20%); LC-MS: Rt 9.481 min, m / z calculated [M+H]: 1214.94, found 1214.75.

[0643] Example 39 - Preparation of Compound No. 39 Compound No. 39 was prepared according to general scheme (E).

[0644] Following the general protocol for amine alkylation described in General Scheme E.1, the amine N-(4-aminobutyl)acetamide A4 (10 mg) was combined with B6B5C (117 mg) and DIPEA (60 μL) in THF / CHCN (1:1, 0.8 mL). After the reaction, the crude product was purified with an 8% MeOH / DCM eluent. Brown oil, 35 mg (36%); LC-MS: Rt 9.469 min, m / z calculated [M+H]: 1255.96, found 1255.75.

[0645] Example 40 - Preparation of Compound No. 40 Compound No. 40 was prepared according to general scheme (B). 1 H NMR(500MHz,CDCl3)δ4.12-3.91(m,13H),3.89(d,J=6.5Hz,6H),3.43-3.33(m,2H),3.13(d,J=16.1Hz,6H),2.29(t,J=7.6H) z,8H),1.96-1.71(m,22H),1.68-1.47(m,19H),1.46-1.24(m,22H),1.06-0.94(m,11H),0.89(t,J=6.8Hz,12H).MS(ESI):C 70 H 119 NO 17 Calculated value for [M+H] + 1246.8, measured value 1247.0. Example 41 - Preparation of Compound No. 41

[0646] Compound No. 41 was prepared according to general scheme (B). 1 H NMR(500MHz,CDCl3)δ4.13-3.92(m,11H),3.89(d,J=6.5Hz,6H),3.43-3.33( m,3H),3.14(s,3H),2.94(s,2H),2.76(s,3H),2.29(t,J=7.6Hz,8H),1.97-1. 73(m,23H),1.67-1.57(m,14H),1.53(tq,J=8.3,4.4Hz,6H),1.41(q,J=7.3Hz ,7H),1.37-1.25(m,17H),1.06-0.94(m,12H),0.93-0.85(m,12H).MS(ESI):C 69 H117 NO 16 Calculated value for [M+H] + 1216.8, measured value 1216.6.

[0647] Example 42 - Preparation of Compound No. 42 Compound No. 42 was prepared according to general scheme (B). 1 H NMR(500MHz,CDCl3)δ4.05-3.83(m,9H),3.38(t,J=7.1Hz,2H),3.22-2.99(m,5H),1.91(dd,J=10.4,5.3Hz,4H),1.89-1.66(m,19H), 1.54(ddq,J=26.6,13.3,6.7Hz,9H),1.39-1.22(m,10H),1.24-1.08(m,19H),1.01-0.88(m,12H),0.86(d,J=6.6Hz,26H).MS(ESI):C 66 H 119 Calculated value for NO9 [M+H] + 1070.9, measured value 1071.1.

[0648] Example 43 - Preparation of Compound No. 43 Compound No. 43 was prep...

Claims

1. Compounds of formula (I): Formula (I) or a salt thereof, wherein A is, Each B independently: where * indicates the bond to A and ** indicates the bond to C, Each C independently: n is an integer ranging from 2 to 6; a is an integer ranging from 1 to 5; b is an integer ranging from 1 to 5; each y is independently an integer ranging from 1 to 10; Each R １ may independently include one or more C ３ ~C １２ unbranched C optionally substituted with cycloalkyl １ ~C １８ is alkyl; Each R １ ' are independently unbranched C １ ~C １８ alkylene; R ３ C optionally substituted with one or more hydroxyl １ ~C １０ Alkyl or -NH-(C=O)-(C １ ~C ６ or a salt thereof.

2. A is, The compound of claim 1.

3. Each C is 3. The compound of claim 1 or 2.

4. Each C is 3. The compound of claim 1 or 2.

5. A is, Each C is The compound according to any one of claims 1 to 4.

6. A is, Each C is The compound according to any one of claims 1 to 4.

7. Each R １ But C ４ The compound of any one of claims 1 to 6, which is alkyl.

8. Each R １ but, The compound of claim 7.

9. Each R １ 'But C １ The compound of any one of claims 1 to 8, which is alkylene.

10. Each R １ 'But C ２ The compound of any one of claims 1 to 8, which is alkylene.

11. Each R １ 'But C ４ The compound of any one of claims 1 to 8, which is alkylene.

12. The compound according to any one of claims 1 to 11, wherein a is 1.

13. The compound according to any one of claims 1 to 11, wherein b is 1.

14. The compound according to any one of claims 1 to 13, wherein a is 1 and b is 1.

15. The compound according to any one of claims 1 to 11, wherein a is 2.

16. The compound according to any one of claims 1 to 11, wherein b is 2.

17. The compound according to any one of claims 1 to 16, wherein a is 2 and b is 2.

18. The compound of any one of claims 1 to 17, wherein n is 4.

19. The compound of any one of claims 1 to 18, wherein y is 1.

20. The compound of any one of claims 1 to 18, wherein y is 7.

21. A compound selected from:

22. Compound of formula (II): Formula (II) or a salt thereof, During the ceremony, A is, Each B independently: where * indicates the bond to A and ** indicates the bond to C, Each C independently: or C １ ~C １８ is alkyl; n is an integer ranging from 2 to 6; a is an integer ranging from 1 to 5; b is an integer ranging from 1 to 5; Each R １ But independently, C １ ~C １８ Alkyl or C ２ ~C １８ alkenyl, C １ ~C １８ Alkyl or C ２ ~C １８ Alkenyl is one or more C ３ ~C １２ optionally substituted with cycloalkyl; Each R １ ' are independently unbranched C １ ~C １８ alkylene; R ３ is (i) C optionally substituted with one or more hydroxyl １ ~C １０ Alkyl, —NH—(C═O)—(C １ ~C ６ alkyl) or phenyl, or (ii) cyclohexyl or -(C １ ~C ６ alkylene)-hydroxyl; Each Y is independently Here, *** is R １ indicates a bond to each p is independently an integer ranging from 0 to 3; each q is independently 0 or 1; A compound or a salt thereof, wherein each z is independently 0 or 1.

23. A is, 23. The compound of claim 22.

24. A is, 23. The compound of claim 22.

25. A is, 23. The compound of claim 22.

26. Each B, wherein * represents a bond to A and ** represents a bond to C.

27. Each B, wherein * represents a bond to A and ** represents a bond to C.

28. Each C is The compound according to any one of claims 22 to 27.

29. Each C is The compound according to any one of claims 22 to 27.

30. Each C is The compound according to any one of claims 22 to 27.

31. Each C is The compound according to any one of claims 22 to 27.

32. Each C is or C １ ~C １８ The compound of any one of claims 22 to 27, which is alkyl.

33. Each Y is Here, *** is R １ 33. The compound of any one of claims 22 to 32, exhibiting binding to:

34. Each Y is Here, *** is R １ 33. The compound of any one of claims 22 to 32, exhibiting binding to:

35. Each Y is The compound according to any one of claims 22 to 32.

36. Each R １ But C １ ~C １８ The compound of any one of claims 22 to 35, which is alkyl.

37. Each C is Here, *** is R １ and each R １ is C １ ~C １８ The compound of any one of claims 22 to 36, which is alkyl.

38. Each C is Each R １ is C １ ~C １８ The compound of any one of claims 22 to 36, which is alkyl.

39. Each R １ but, The compound according to any one of claims 22 to 38.

40. Each R １ but, The compound according to any one of claims 22 to 38.

41. Each R １ but, The compound according to any one of claims 22 to 38.

42. Each R １ but, The compound according to any one of claims 22 to 38.

43. Each R １ but, The compound according to any one of claims 22 to 38.

44. Each R １ But one or more C ３ ~C １２ Cycloalkyl-substituted C １ ~C １８ The compound of any one of claims 22 to 38, which is alkyl.

45. Each R １ but, The compound according to any one of claims 22 to 44.

46. Each R １ But C ２ ~C １８ The compound of any one of claims 22 to 35, which is alkenyl.

47. Each C is Here, *** is R １ and each R １ is C ２ ~C １８ The compound of any one of claims 22 to 46, which is alkenyl.

48. Each R １ but, 48. The compound according to any one of claims 22 to 47.

49. Each C is Each R １ is C １ ~C １８ The compound of any one of claims 22 to 36, which is alkyl.

50. Each R １ but, 50. The compound according to any one of claims 22 to 49.

51. Each R １ but, 50. The compound according to any one of claims 22 to 49.

52. Each C is Here, *** is R １ and each R １ is C １ ~C １８ The compound of any one of claims 22 to 36, which is alkyl.

53. Each R １ but, The compound according to any one of claims 22 to 52.

54. 54. The compound of any one of claims 22 to 53, wherein a is 2.

55. 54. The compound of any one of claims 22 to 53, wherein b is 2.

56. 56. The compound of any one of claims 22 to 55, wherein a is 2 and b is 2.

57. 57. The compound of any one of claims 22 to 56, wherein z is 1.

58. 57. The compound of any one of claims 22 to 56, wherein z is 0.

59. The compound of any one of claims 22 to 58, wherein p is 0.

60. 59. The compound of any one of claims 22 to 58, wherein p is 1.

61. The compound of any one of claims 22 to 58, wherein p is 3.

62. R ３ is CH ３ The compound according to any one of claims 22 to 61,

63. R ３ is substituted with one or more hydroxyl groups １ ~C １０ The compound of any one of claims 22 to 61, which is alkyl.

64. R ３ is one or more hydroxyl or -(C １ ~C ６ The compound of any one of claims 22 to 61, which is cyclohexyl substituted with alkylene)-hydroxyl.

65. R ３ but, 62. The compound according to any one of claims 22 to 61.

66. 66. The compound of any one of claims 22 to 65, wherein n is 4.

67. A compound selected from:

68. A composition comprising at least one lipid nanoparticle containing at least one compound of formula (I) according to any one of claims 1 to 21.

69. the at least one lipid nanoparticle comprises about 40.75% by molar of the at least one compound of formula (I); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 51.75% by molar ratio; About 5% by molar ratio of DOPC, and further comprising about 2.5% by molar ratio of DMG-PEG2000; 69. The composition of claim 68, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 120:1 (w / w).

70. the at least one lipid nanoparticle comprises about 40% to about 46% by molar ratio of the at least one compound of formula (I); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is about 45.9% to about 51.8% cholesterol by molar ratio; DOPC in a molar ratio of about 4.9% to about 7%, and further comprising about 2% to about 3% by molar ratio of DMG-PEG2000; 69. The composition of claim 68, wherein the ratio of lipid to nucleic acid in said at least one nanoparticle is from about 80:1 (w / w) to about 120:1 (w / w).

71. the at least one lipid nanoparticle comprises about 54% to about 60% by molar ratio of the at least one compound of formula (I); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is about 30% to about 36% cholesterol by molar ratio; DOPC in a molar ratio of about 2.8% to about 7%, and further comprising about 3% by molar ratio of DMG-PEG2000; 69. The composition of claim 68, wherein the ratio of lipid to nucleic acid in said at least one nanoparticle is from about 60:1 (w / w) to about 100:1 (w / w).

72. the at least one lipid nanoparticle comprises about 40.75% by molar of the at least one compound of formula (I); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 51.75% by molar ratio; About 5% by molar ratio of DOPC, and further comprising about 2.5% by molar ratio of DMG-PEG2000; 69. The composition of claim 68, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 120:1 (w / w).

73. the at least one lipid nanoparticle comprises about 40.8% to about 45.9% by molar ratio of the at least one compound of formula (I); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is about 45.9% to about 53.8% cholesterol by molar ratio; DOPC in a molar ratio of about 0% to about 6.2%, and further comprising about 2% to about 2.5% by molar ratio of DMG-PEG2000; 69. The composition of claim 68, wherein the ratio of lipid to nucleic acid in said at least one nanoparticle is from about 40:1 (w / w) to about 50:1 (w / w).

74. the at least one lipid nanoparticle comprises about 54.2% to about 60% by molar ratio of the at least one compound of formula (I); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is about 38% to about 39.5% cholesterol by molar ratio; DOPC in a molar ratio of about 0% to about 3.9%, and further comprising about 2% to about 2.4% by molar ratio of DMG-PEG2000; 69. The composition of claim 68, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 50:1 (w / w).

75. A composition comprising at least one lipid nanoparticle comprising at least one compound of formula (II) according to any one of claims 22 to 67.

76. the at least one lipid nanoparticle comprises about 40.75% by molar of the at least one compound of formula (II); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 51.75% by molar ratio; About 5% by molar ratio of DOPC, and further comprising about 2.5% by molar ratio of DMG-PEG2000; 76. The composition of claim 75, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w / w).

77. the at least one lipid nanoparticle comprises about 43.17% by molar of the at least one compound of formula (II); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 43.17% by molar ratio; About 11.96% DOPC by molar ratio, and further comprising about 1.7% by molar ratio of DMG-PEG2000; 76. The composition of claim 75, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w / w).

78. the at least one lipid nanoparticle comprises about 50% by molar ratio of the at least one compound of formula (II); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 38.5% by molar ratio; About 10% by molar ratio of DOPC, DSPC, or DPPC, and further comprising about 1.5% by molar ratio of DMG-PEG2000; 76. The composition of claim 75, wherein the ratio of lipid to nucleic acid in the at least one type of nanoparticle is about 40:1 (w / w), about 50:1 (w / w), about 60:1 (w / w), or about 80:1 (w / w).

79. the at least one lipid nanoparticle comprises about 50% by molar ratio of the at least one compound of formula (II); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 38% by molar ratio About 10% by molar ratio of DOPC, DSPC, or DPPC, and further comprising about 2% by molar ratio of DMG-PEG2000; 76. The composition of claim 75, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 40:1 (w / w) or about 50:1 (w / w).

80. the at least one lipid nanoparticle comprises about 54% by molar of the at least one compound of formula (II); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 35% by molar ratio About 10% by molar ratio of DOPC, and further comprising about 1% by molar ratio of DMG-PEG2000; 76. The composition of claim 75, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w / w).

81. the at least one lipid nanoparticle comprises about 40.8% to about 54% by molar ratio of the at least one compound of formula (II); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is about 35% to about 51.8% cholesterol by molar ratio; DOPC in a molar ratio of about 5% to about 12%, and further comprising about 1% to about 2.5% by molar ratio of DMG-PEG2000; 76. The composition of claim 75, wherein the ratio of lipid to nucleic acid in said at least one nanoparticle is from about 80:1 to about 100:1 (w / w).

82. the at least one lipid nanoparticle comprises about 50% by molar ratio of the at least one compound of formula (II); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 38.5% by molar ratio; Approximately 10% by molar ratio of DSPC, About 1.25% molar ratio of DMG-PEG2000; and further comprising a targeting ligand comprising about 0.25% GalNac by molar ratio; 76. The composition of claim 75, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 80:1 (w / w).

83. the at least one lipid nanoparticle comprises about 50% by molar ratio of the at least one compound of formula (II); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 38.5% by molar ratio; Approximately 10% by molar ratio of DSPC, About 1.2% by molar ratio of DMG-PEG2000; and further comprising a targeting ligand comprising about 0.3% GalNac by molar ratio; 76. The composition of claim 75, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 50:1 (w / w).

84. the at least one lipid nanoparticle comprises about 50% by molar ratio of the at least one compound of formula (II); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 38.5% by molar ratio; Approximately 10% by molar ratio of DSPC, About 1% molar ratio of DMG-PEG2000; and further comprising a targeting ligand comprising about 0.5% GalNac by molar ratio; 76. The composition of claim 75, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 50:1 (w / w) or about 80:1 (w / w).

85. the at least one lipid nanoparticle comprises about 45% by molar of the at least one compound of formula (II); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 42.5% by molar ratio; about 10% by molar ratio of DSPC, and further comprising about 2.5% by molar ratio of DMG-PEG2000; 76. The composition of claim 75, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 60:1 (w / w).

86. the at least one lipid nanoparticle comprises about 45% by molar of the at least one compound of formula (II); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 42.5% by molar ratio; Approximately 10% by molar ratio of DSPC, About 2.25% molar ratio of DMG-PEG2000; and further comprising a targeting ligand comprising about 0.25% GalNac by molar ratio; 76. The composition of claim 75, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 60:1 (w / w).

87. the at least one lipid nanoparticle comprises about 45% by molar of the at least one compound of formula (II); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 42.5% by molar ratio; Approximately 10% by molar ratio of DSPC, About 2% by molar ratio of DMG-PEG2000; and further comprising a targeting ligand comprising about 0.5% GalNac by molar ratio; 76. The composition of claim 75, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 60:1 (w / w).

88. the at least one lipid nanoparticle comprises about 50% by molar ratio of the at least one compound of formula (II); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 38% by molar ratio Approximately 10% by molar ratio of DSPC, About 1.7% molar ratio of DMG-PEG2000; and further comprising a targeting ligand comprising about 0.3% GalNac by molar ratio; 76. The composition of claim 75, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 50:1 (w / w).

89. the at least one lipid nanoparticle comprises about 50% by molar ratio of the at least one compound of formula (II); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 38% by molar ratio Approximately 10% by molar ratio of DSPC, About 1.5% by molar ratio of DMG-PEG2000; and further comprising a targeting ligand comprising about 0.5% GalNac by molar ratio; 76. The composition of claim 75, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 50:1 (w / w).

90. the at least one lipid nanoparticle comprises about 50% by molar ratio of the at least one compound of formula (II); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 38.5% by molar ratio; About 10% by molar ratio of DOPC, and About 1.5% by molar ratio of DMG-PEG2000; and Further containing tannic acid; the ratio of lipid to nucleic acid in said at least one nanoparticle is about 80:1 (w / w); 76. The composition of claim 75, wherein the ratio of tannic acid to nucleic acid in the at least one nanoparticle is about 10:

1.

91. the at least one lipid nanoparticle comprises about 50% by molar ratio of the at least one compound of formula (II); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 38.5% by molar ratio; About 10% DOPC by molar ratio, Approximately 1% molar ratio of DMG-PEG2000; a targeting ligand comprising about 0.5% GalNac by molar ratio; and Further containing tannic acid; the ratio of lipid to nucleic acid in said at least one nanoparticle is about 80:1 (w / w); 76. The composition of claim 75, wherein the ratio of tannic acid to nucleic acid in the at least one nanoparticle is about 10:

1.

92. the at least one lipid nanoparticle comprises about 45% by molar of the at least one compound of formula (II); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 45.75% by molar ratio; About 7.5% by molar ratio of DOPC, and Approximately 1.5% molar ratio of DMG-PEG2000; a targeting ligand comprising about 0.25% GalNac by molar ratio; and Further containing tannic acid; the ratio of lipid to nucleic acid in said at least one nanoparticle is about 50:1 (w / w); 76. The composition of claim 75, wherein the ratio of tannic acid to nucleic acid in the at least one type of nanoparticle is about 5:1, about 7:1, about 10:1, or about 15:

1.

93. the at least one lipid nanoparticle comprises about 50% by molar ratio of the at least one compound of formula (II); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 41% by molar ratio About 7.5% by molar ratio of DOPC, and Approximately 1% molar ratio of DMG-PEG2000; a targeting ligand comprising about 0.5% GalNac by molar ratio; and Further containing tannic acid; the lipid to nucleic acid ratio in said at least one nanoparticle is about 50:1 (w / w) or about 60:1 (w / w); 76. The composition of claim 75, wherein the ratio of tannic acid to nucleic acid in the at least one nanoparticle is about 5:1, about 10:1, or about 15:

1.

94. the at least one lipid nanoparticle comprises about 50% by molar ratio of the at least one compound of formula (II); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 38.5% by molar ratio; About 10% by molar ratio of DOPC, and About 1.5% by molar ratio of DMG-PEG2000; and Further containing proanthocyanidins; the ratio of lipid to nucleic acid in said at least one nanoparticle is about 80:1 (w / w); 76. The composition of claim 75, wherein the ratio of proanthocyanidin to nucleic acid in the at least one nanoparticle is about 2.5:1 or about 5:

1.

95. the at least one lipid nanoparticle comprises about 50% by molar ratio of the at least one compound of formula (II); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 38.5% by molar ratio; About 10% by molar ratio of DOPC, and About 1.5% by molar ratio of DMG-PEG2000; and Further containing ellagic acid; the ratio of lipid to nucleic acid in said at least one nanoparticle is about 80:1 (w / w); 76. The composition of claim 75, wherein the ratio of ellagic acid to nucleic acid in the at least one type of nanoparticle is about 2.5:1, about 5:1, or about 10:

1.

96. the at least one lipid nanoparticle comprises about 50% by molar ratio of the at least one compound of formula (II); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle is Cholesterol, approximately 38.5% by molar ratio; About 10% by molar ratio of DOPC, and About 1.5% by molar ratio of DMG-PEG2000; and Further comprising punicalagin; the ratio of lipid to nucleic acid in said at least one nanoparticle is about 80:1 (w / w); 76. The composition of claim 75, wherein the ratio of punicalagin to nucleic acid in the at least one nanoparticle is about 2.5:

1.

97. The composition of any one of claims 68 to 96, wherein the RNA molecule is an mRNA molecule, preferably said mRNA molecule further comprising a 5'-CAP.

98. 98. The composition of any one of claims 68 to 97, wherein the at least one RNA molecule comprises a nucleic acid sequence encoding at least one transposase, preferably wherein the transposase is a piggyBac™ (PB) transposase, a piggyBac-like (PBL) transposase, a Super piggyBac™ (SPB) transposase polypeptide, a Sleeping Beauty transposase, a Hyperactive Sleeping Beauty (SB100X) transposase, a Helitron transposase, a Tol2 transposase, a TcBuster transposase, or a mutant TcBuster transposase.

99. The composition of any one of claims 68 to 98, wherein the DNA molecule is a circular DNA molecule, a DoggyBone DNA molecule, a DNA plasmid, a DNA nanoplasmid or a linearized DNA molecule.

100. 100. The composition of any one of claims 68 to 99, wherein said at least one DNA molecule comprises a nucleic acid sequence encoding at least one transposon.

101. The composition of any one of claims 68 to 100, wherein the at least one nucleic acid molecule comprises a nucleic acid sequence encoding at least one therapeutic protein.

102. 102. The composition of any one of claims 68 to 101, wherein the at least one nucleic acid molecule comprises a nucleic acid sequence encoding at least one transposon, and the transposon comprises a nucleic acid sequence encoding at least one therapeutic protein.

103. A pharmaceutical composition comprising the composition of any one of claims 68 to 102 and at least one pharmaceutically acceptable excipient or diluent.

104. 104. A method for delivering at least one nucleic acid to at least one cell, the method comprising contacting said at least one cell with at least one composition of any one of claims 68 to 103.

105. 105. A method of genetically modifying at least one cell, the method comprising contacting said at least one cell with at least one composition according to any one of claims 68 to 104.

106. 106. The method of claim 104 or 105, wherein the at least one cell is a liver cell.

107. 107. The method of claim 106, wherein the liver cells are hepatocytes, hepatic stellate cells, Kupffer cells, or hepatic sinusoidal endothelial cells.

108. 108. At least one cell modified according to the method of any one of claims 105 to 107.

109. 109. A method of treating at least one disease or disorder in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of at least one composition of any one of claims 101 to 103 or at least one cell of claim 108.

110. 110. The method of claim 109, wherein the at least one disease or disorder is a liver disease or disorder.

111. 111. The composition of any one of Claims 68-110, wherein the at least one RNA molecule comprises a nucleic acid sequence encoding a fusion protein, the fusion protein comprising (i) an inactivated Cas9 (dCas9) protein or an inactivated nuclease domain thereof, and (ii) a Clo051 protein or a nuclease domain thereof.

112. 112. The composition of claim 111, further comprising at least one guide RNA molecule.

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