Lipidoid compounds and related compositions and uses

Abstract

This disclosure provides compositions comprising lipidoid compounds, methods for preparing such compositions, and uses of these compositions in gene delivery applications.

Description

Related applications

[0001] This application claims priority and benefits of U.S. Provisional Application No. 63 / 580,080, filed on 1 September 2023, and U.S. Provisional Application No. 63 / 610,676, filed on 15 December 2023. The contents of each of the aforementioned patent applications are incorporated herein by reference in their entirety.

[0002] Sequence List The sequence listing XML related to this application is provided electronically in XML format and is incorporated herein by reference. The XML file containing the sequence listing XML is named "POTH-088_001WO_SeqList.xml". The XML file is 25,048 bytes in size, created on August 29, 2024, and filed electronically through the USPTO Patent Center. [Technical Field]

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

[0004] 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 harmful side effects in patients. The present disclosure provides improved compositions, methods and kits for delivering nucleic acids to various cell types including hepatocytes in vivo, ex vivo and in vitro. More specifically, the present disclosure provides improved lipid nanoparticle compositions and methods of using the same. These lipid nanoparticle compositions and methods enable delivery of nucleic acids to cells with high efficiency and low toxicity. Accordingly, the compositions and methods of the present disclosure are widely applicable to many diverse fields including gene therapy. Summary of the Invention

[0005] In some aspects, novel compounds are provided. In one aspect, the novel compound is of formula (I)

[0006] which is the compound shown in TIFF2026530178000002.tif30170 or a salt thereof, wherein: Z is the structure shown in TIFF2026530178000003.tif73170 or a covalent bond; X a , X b and X c are each independently NH or O; Y a , Y b , Y c , Y d , Y e and Y f are each independently C or O; r, s and t are each independently an integer ranging from 1 to 9; and R 2a , R 2b , R 2c , R2d , R 2e and R 2f Each of these is independent of C1~C 18 Alkyl, C2~C 18 Alkenyl, -(cyclohexyl)-(C1~C 18 Alkyl),-(cyclohexyl)-(C2~C 18 Alkenyl) or -(cyclohexyl)-(CH2-O-CO)-(C1~C 18 Alkyl) and the above C1~C 18 Alkyl or C2-C 18 The alkenyl is optionally substituted with one or more cyclohexyl groups.

[0007] In another embodiment, the novel compound is a compound of formula (II): TIFF2026530178000004.tif43170 or a salt thereof, During the ceremony: G is TIFF2026530178000005.tif17170 or The filename is TIFF2026530178000006.tif20170; Y a , Y b , Y c and Y d Each of them is independently either C or O; Each of r and s is an integer in the range of 1 to 9, independently of the others; R1 is H or C1-C3 alkyl; and R 2a , R 2b , R 2c and R 2d Each of these is independent of C1~C 18 Alkyl or C2-C 18 Alkenyl, -(cyclohexyl)-(C1~C 18 Alkyl),-(cyclohexyl)-(C2~C 18 Alkenyl), or -(cyclohexyl)-(CH2-O-CO)-(C1~C 18 Alkyl) and the above C1~C 18 Alkyl or C2-C18 The alkenyl is optionally substituted with one or more cyclohexyl groups.

[0008] In some embodiments, novel lipid nanoparticles ("LNPs") containing novel compounds are provided. In one embodiment, the novel compound is a compound of formula (I). In another embodiment, the novel compound is a compound of formula (II). In yet another embodiment, the novel compound is a compound of formula (II').

[0009] In some embodiments, pharmaceutical compositions are provided, comprising the composition of the present disclosure and at least one pharmaceutically acceptable excipient or diluent.

[0010] In some embodiments, a method is 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.

[0011] In some embodiments, a method is provided for genetically modifying at least one cell, comprising contacting the at least one cell with at least one composition of the present disclosure.

[0012] In some embodiments, a method is provided for treating at least one disease or disorder in a subject requiring treatment of at least one disease or disorder, comprising administering at least one therapeutically effective amount of at least one composition of the present disclosure to the subject.

[0013] In some embodiments, a method is 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.

[0014] In some embodiments, cells modified by the method of the present disclosure are provided.

[0015] Any aspect and / or embodiment described herein may be combined with any other aspect and / or embodiment described herein.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which this disclosure belongs. Wherever context explicitly indicates otherwise, singular terms include plural forms; for example, “a,” “an,” and “the” are singular or plural, and “or” is understood to be inclusive. For example, “an element” means one or more elements. Throughout this specification, the word “comprising,” or variations such as “comprises” or “comprising,” is understood to mean including the element, integer, or process, or group of elements, integers, or processes described, but not to mean the exclusion of any other element, integer, or process, or group of elements, integers, or processes. "Approximately" can be understood as being 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 evident from the context, all figures provided herein are qualified with this term "approximately."

[0017] Methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, but preferred methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. References cited herein do not claim to be prior art of the claimed invention. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, materials, methods, and examples are illustrative and not intended to be limiting. Other features and advantages of this disclosure will become apparent from the detailed description and claims below. [Modes for carrying out the invention]

[0018] This disclosure provides novel lipidoid compounds, novel lipid nanoparticle compositions (LNPs) comprising novel lipidoid compounds, methods for preparing LNPs, and methods for using them. In non-limiting examples, the compositions and methods of this limited disclosure can be used for gene delivery. In non-limiting examples, the compositions and methods of this disclosure may be broadly 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 non-limiting examples, the compositions and methods of this disclosure may be broadly used to deliver nucleic acids and induce the expression of secreted therapeutic proteins.

[0019] Compounds of the Disclosure In one embodiment, this disclosure relates to formula (I) We provide the compound or salt of TIFF2026530178000007.tif30170, During the ceremony: Z is TIFF2026530178000008.tif73170 or covalent; X a , X b and X c Each of them is independently either NH or O; Y a , Y b , Y c , Y d , Y e and Y f Each of them is independently either C or O; Each of r, s, and t is independently an integer in the range of 1 to 9; and R 2a , R 2b , R 2c , R 2d , R 2e and R 2f Each of these is independent of C1~C 18 Alkyl, C2~C 18 Alkenyl, -(cyclohexyl)-(C1~C 18Alkyl),-(cyclohexyl)-(C2~C 18 Alkenyl) or -(cyclohexyl)-(CH2-O-CO)-(C1~C 18 Alkyl) and the above C1~C 18 Alkyl or C2-C 18 The alkenyl is optionally substituted with one or more cyclohexyl groups.

[0020] In some embodiments, the novel compound is of formula (I) We provide the compound or salt of TIFF2026530178000009.tif29170, During the ceremony: Z is TIFF2026530178000010.tif18170 or covalent; X a and X b Each of them is independently either NH or O; Y a , Y b , Y c and Y d Each of them is independently either C or O; Each of r and s is independently an integer in the range of 1 to 9; and R 2a , R 2b , R 2c and R 2d Each of these is independent of C1~C 18 Alkyl, C2~C 18 Alkenyl, -(cyclohexyl)-(C1~C 18 Alkyl), or -(cyclohexyl)-(C2~C 18 Alkenyl) and the above C1~C 18 Alkyl or C2-C 18 The alkenyl is optionally substituted with one or more cyclohexyl groups.

[0021] In some embodiments, Z is The filename is TIFF2026530178000011.tif14170.

[0022] In some embodiments, Z is TIFF2026530178000012.tif19170.

[0023] In some embodiments, Z is TIFF2026530178000013.tif31170.

[0024] In some embodiments, Z is TIFF2026530178000014.tif38170.

[0025] In some embodiments, Z is TIFF2026530178000015.tif23170.

[0026] In some embodiments, Z is a covalent bond.

[0027] In some embodiments, X a , X b and X c are each NH.

[0028] In some embodiments, X a , X b and X c are each O.

[0029] In some embodiments, Y a , Y b , Y c , Y d , Y e and Y f are each C.

[0030] In some embodiments, Y a , Y b , Y c , Y d , Y e and Y f are each O.

[0031] In some embodiments, each of r, s and t is 3.

[0032] In some embodiments, R 2a , R 2b , R 2c , R 2d , R 2e and R 2f are each C1-C 18 alkyl.

[0033] In some embodiments, X a and X b are each NH.

[0034] In some embodiments, X a and X b are each O.

[0035] In some embodiments, Y a , Y b , Y c and Y d are each C.

[0036] In some embodiments, Y a , Y b , Y c and Y d are each O.

[0037] In some embodiments, r and s are each 3.

[0038] In some embodiments, R 2a , R 2b , R 2c and R 2d are each C1-C 18 alkyl.

[0039] In some embodiments, R 2a , R 2b , R 2c and R 2d are each C1-C 18 alkyl substituted with one or more cyclohexyl. In some embodiments, R 2a , R2b , R 2c and R 2d Each of them is, The filename is TIFF2026530178000016.tif16170.

[0040] In some embodiments, R 2a , R 2b , R 2c and R 2d Each of them is -(cyclohexyl)-(C1~C 18 It is alkyl. In some embodiments, R 2a , R 2b , R 2c and R 2d Each of them is, The filename is TIFF2026530178000017.tif13170.

[0041] In some embodiments, R 2a , R 2b , R 2c , R 2d、 R 2e , and R 2f Each of them is -(cyclohexyl)-(CH2-O-CO)-(C1~C 18 It is alkyl.

[0042] In some embodiments, Z is TIFF2026530178000018.tif14170X a and X b Each of them is NH. In some embodiments, Y a , Y b , Y c and Y d Each of them is C. In some embodiments, Y a , Y b , Y c and Y d Each of them is O. In some embodiments, each of r and s is 3. In some embodiments, R 2a , R 2b , R 2c and R 2d Each of these is C1~C18 It is alkyl. In some embodiments, R 2a , R 2b , R 2c and R 2d Each of these is a C1-C substituted with one or more cyclohexyl groups. 18 It is alkyl. In some embodiments, R 2a , R 2b , R 2c and R 2d Each of them is, The file is TIFF2026530178000019.tif16170. In some embodiments, R 2a , R 2b , R 2c and R 2d Each of them is, The filename is TIFF2026530178000020.tif13170.

[0043] In some embodiments, Z is TIFF2026530178000021.tif14170, X a and X b Each of them is O. In some embodiments, Y a , Y b , Y c and Y d Each of these is C. In some embodiments, Y a , Y b , Y c and Y d Each of them is O. In some embodiments, each of r and s is 3. In some embodiments, R 2a , R 2b , R 2c and R 2d Each of these is C1~C 18 It is alkyl. In some embodiments, R 2a , R 2b , R 2c and R 2d Each of these is a C1-C substituted with one or more cyclohexyl groups. 18 It is alkyl. In some embodiments, R 2a , R 2b, R 2c and R 2d Each of them is, This is TIFF2026530178000022.tif16170. In some embodiments, R 2a , R 2b , R 2c and R 2d Each of them is, The filename is TIFF2026530178000023.tif13170.

[0044] In some embodiments, Z is a covalent bond, and X a and X b Each of them is O. In some embodiments, Y a , Y b , Y c and Y d Each of these is C. In some embodiments, Y a , Y b , Y c and Y d Each of them is O. In some embodiments, each of r and s is 3. In some embodiments, R 2a , R 2b , R 2c and R 2d Each of these is C1~C 18 It is alkyl. In some embodiments, R 2a , R 2b , R 2c and R 2d Each of these is a C1-C substituted with one or more cyclohexyl groups. 18 It is alkyl. In some embodiments, R 2a , R 2b , R 2c and R 2d Each of them is, This is TIFF2026530178000024.tif16170. In some embodiments, R 2a , R 2b , R 2c and R 2d Each of them is, The filename is TIFF2026530178000025.tif13170.

[0045] In some embodiments, Z is TIFF2026530178000026.tif19170, X a and X b Each of them is NH. In some embodiments, Y a , Y b , Y c and Y d Each of them is C. In some embodiments, Y a , Y b , Y c and Y d Each of them is O. In some embodiments, each of r and s is 3. In some embodiments, R 2a , R 2b , R 2c and R 2d Each of these is C1~C 18 It is alkyl. In some embodiments, R 2a , R 2b , R 2c and R 2d Each of these is a C1-C substituted with one or more cyclohexyl groups. 18 It is alkyl. In some embodiments, R 2a , R 2b , R 2c and R 2d Each of them is, This is TIFF2026530178000027.tif16170. In some embodiments, R 2a , R 2b , R 2c and R 2d Each of them is, The filename is TIFF2026530178000028.tif13170.

[0046] In some embodiments, Z is TIFF2026530178000029.tif31170, X a and X b Each of them is NH. In some embodiments, Y a , Y b, Y c , Y d , Y e and Y f Each of these is C. In some embodiments, each of r, s and t is 3. In some embodiments, R 2a , R 2b , R 2c , R 2d , R 2e and R 2f Each of these is C1~C 18 It is alkyl.

[0047] In some embodiments, Z is TIFF2026530178000030.tif38170, X a , X b and X c Each of them is NH. In some embodiments, Y a , Y b , Y c , Y d , Y e and Y f Each of these is C. In some embodiments, each of r, s and t is 3. In some embodiments, R 2a , R 2b , R 2c , R 2d , R 2e and R 2f Each of these is C1~C 18 It is alkyl.

[0048] In some embodiments, Z is TIFF2026530178000031.tif23170, X a and X b Each of them is O. In some embodiments, Y a , Y b , Y c and Y d Each of them is C. In some embodiments, each of r and s is 3. In some embodiments, R 2a , R 2b , R 2c and R2d Each of these is C1~C 18 It is alkyl.

[0049] In some embodiments, the compound of formula (I) is The compound is selected from TIFF2026530178000032.tif249170 and TIFF2026530178000033.tif91170.

[0050] In one embodiment, this disclosure relates to formula (II) We provide the compound or salt of TIFF2026530178000034.tif43170, During the ceremony: G is TIFF2026530178000035.tif17170 or The filename is TIFF2026530178000036.tif20170; Y a , Y b , Y c and Y d Each of them is independently either C or O; Each of r and s is an integer in the range of 1 to 9, independently of the others; R1 is H or C1-C3 alkyl; and R 2a , R 2b , R 2c and R 2d Each of these is independent of C1~C 18 Alkyl, C2~C 18 Alkenyl, -(cyclohexyl)-(C1~C 18 Alkyl),-(cyclohexyl)-(C2~C 18 Alkenyl), or -(cyclohexyl)-(CH2-O-CO)-(C1~C 18 Alkyl) and the above C1~C 18 Alkyl or C2-C 18 The alkenyl is optionally substituted with one or more cyclohexyl groups.

[0051] In one embodiment, this disclosure relates to formula (II') We provide the compound or salt of TIFF2026530178000037.tif39170, During the ceremony: Y a , Y b , Y c and Y d Each of them is independently either C or O; Each of r and s is an integer in the range of 1 to 9, independently of the others; R1 is H or C1-C3 alkyl; and R 2a , R 2b , R 2c and R 2d Each of these is independent of C1~C 18 Alkyl or C2-C 18 It is an alkenyl, and the above C1~C 18 Alkyl or C2-C 18 The alkenyl is optionally substituted with one or more cyclohexyl groups.

[0052] In some embodiments, G is The filename is TIFF2026530178000038.tif17170.

[0053] In some embodiments, G is The filename is TIFF2026530178000039.tif17170.

[0054] In some embodiments, G is The filename is TIFF2026530178000040.tif20170.

[0055] In some embodiments, Y a , Y b , Y c and Y d Each of them is C.

[0056] In some embodiments, Y a , Y b , Y c and Y d Each of them is O.

[0057] In some embodiments, r and s are each 3.

[0058] In some embodiments, R1 is H.

[0059] In some embodiments, R 2a , R 2b , R 2c and R 2d Each of these is C1~C 18 It is alkyl.

[0060] In some embodiments, R 2a , R 2b , R 2c and R 2d Each of these is a C1-C substituted with one or more cyclohexyl groups. 18 It is alkyl. In some embodiments, R 2a , R 2b , R 2c and R 2d Each of them is, The filename is TIFF2026530178000041.tif16170.

[0061] In some embodiments, R 2a , R 2b , R 2c and R 2d Each of them is -(cyclohexyl)-(CH2-O-CO)-(C1~C 18 It is alkyl.

[0062] In some embodiments, the compound of formula (II) or formula (II') is The compound is selected from TIFF2026530178000042.tif245170 and TIFF2026530178000043.tif79170.

[0063] It will be understood that any one compound of any of the formulas disclosed herein and any pharmaceutically acceptable salt thereof include stereoisomers of the compound, mixtures of stereoisomers, and polymorphs of all isomeric forms.

[0064] On the other hand, it will be understood that the compounds disclosed herein may be presented without a specified configuration (e.g., without a specified stereochemistry). Such presentations are intended to encompass all available isomers, tautomers, regioisomers, and stereoisomers of the compound. In some embodiments herein, a presentation of a compound without a specific configuration is intended to refer to each of the available isomers, tautomers, regioisomers, and stereoisomers of the compound, or any mixture thereof.

[0065] It should be understood that any compound of any formula described herein includes the compound itself, as well as their salts and, optionally, their solvates. Salts may be formed, for example, between an anion on a substituted compound disclosed herein and a positively charged group (e.g., amino). Suitable anions include chlorides, bromides, iodides, sulfates, bisulfates, sulfamates, nitrates, phosphates, citrates, methanesulfons, trifluoroacetates, glutamates, glucurons, glutarates, malates, maleates, succinates, fumarates, tartrates, tosylates, salicylates, lactates, naphthalenesulfons, and acetates (e.g., trifluoroacetates).

[0066] In any of the formulas described herein, where "-" is used to indicate a connection between two variables (e.g., A and B), it will be understood that the connection can be one or more covalent bonds.

[0067] General method for preparing compounds of formula (I), formula (II), or formula (II') of the present disclosure Compounds of formula (I), formula (II), 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.

[0068] General synthesis procedure for lipidoids (A) To a solution of N-Boc pentanoic acid 1 (10 g, 46 mmol) and N-Boc 1,4-diamine 2 (8.66 g, 1.0 equivalent) in anhydrous dichloromethane (DCM, 200 ml), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 10.6 g, 1.2 equivalents), N,N-diisopropylethylamine (DIEA, 12.7 ml, 1.5 equivalents), and 4-dimethylaminopyridine (DMAP, 280 mg, catalyst) were sequentially added at room temperature. The resulting reaction mixture was stirred overnight at room temperature. The reaction mixture was then quenched with saturated NaHCO3 (150 ml) and extracted three times with DCM to obtain the crude product, which was recrystallized with 10 MeOH in DCM to obtain a white solid (13 g, 75% yield).

[0069] Boc-protected diamine 3 (2 g) was dissolved in 2 ml of DCM, followed by the addition of 2 ml of TFA at room temperature. The resulting reaction mixture was stirred at room temperature for 1-2 hours until TLC showed no residual starting material. The lower layer was then separated, and any remaining trace amounts of TFA were stripped off to obtain the crude diamine TFA salt (3 g).

[0070] Crude diamine (500 mg, containing TFA) was mixed with R-substituted epoxide 5 (4.0 equivalents) in the presence of 2.0 equivalents of Cs2CO3. The suspension was prepared at 78°C. ° The mixture was heated in C for 48 hours. The residue was passed through a silica gel column to obtain the desired product 6 as a clear oil (yield approximately 35%).

[0071] General procedure for the synthesis of lipidoid (B) TIFF2026530178000045.tif248170 intermediate 8

[0072] A mixture of 1,2-epoxydodecane (5g, 27.2 mmol) and N-Boc1,4-diamine (2.55g, 0.5 equivalents) is used. °The mixture was heated overnight in 1C. After both LC-MS and TLC indicated that the reaction was complete, the reaction mixture was redissolved in 50% trifluoroacetic acid (TFA) in 20 ml of DCM, and the resulting reaction mixture was stirred at room temperature for 2-3 hours until the starting materials disappeared. After removing the solvent, the crude product was extracted with DCM and saturated NaHCO3, and this was applied to the next step without further purification.

[0073] Intermediate 10

[0074] To a mixture of disulfide acid 9 (500 mg, 2.1 mmol) and N-OH succinimide (289 mg, 2.4 equivalents) in anhydrous DCM (20 ml), EDCI (1.05 g, 2.6 equivalents), DIEA (812 mg, 3.0 equivalents), and catalyst DMAP (25 mg) were sequentially added at room temperature. The resulting reaction mixture was stirred at room temperature for 24 hours, quenched with saturated NaHCO3 (20 ml), the solvent was stripped, and the crude product was extracted with DCM and brine. The residue was applied to the next step without further purification.

[0075] Intermediate 15 or 16

[0076] Mix cyclohexyl alcohol 13 or 14 with solid NaOH (2.0 equivalents), and steep the resulting suspension for 30 minutes at 45°C. ° The mixture was heated to C, followed by the addition of tetrabutylammonium bromide (0.05 equivalents) and epichlorohydrin (2.0 equivalents). The reaction suspension was then heated to 50°C. ° The mixture was vigorously stirred in 1C for 24 hours. The reaction mixture was cooled, and the residue was extracted with cold ÃO and H₂O. After drying over Na₂SO₄, the crude product was purified by flash column to obtain the desired product 15 or 16 in 76% yield.

[0077] Intermediate 18

[0078] Diol 17 was mixed with succinic anhydride (2.1 equivalents) in DCM, and subsequently treated with DMAP (2.1 equivalents) at room temperature. The resulting reaction mixture was stirred for 18 hours, and EDCI (2.3 equivalents), DMAP (2.3 equivalents), and N-OH succinate (2.1 equivalents) were added sequentially. The reaction mixture was stirred overnight at room temperature. The reaction was quenched with saturated NaHCO3 and extracted three times with DCM. The combined organic layers were dried and concentrated to obtain the crude product, which was purified by silica gel flash chromatography to obtain the desired bis-NHS ester 18 in 45% yield.

[0079] compound 1 TIFF2026530178000046.tif34170

[0080] To a solution of crude NHS ester 10 (100 mg, 0.23 mmol) in anhydrous THF (5 ml), intermediate 8 (313 mg, 3.0 equivalents) and DIEA (127 μL, 3.0 equivalents) were added. The resulting reaction mixture was stirred at room temperature for 24 hours. After removing the solvent, the residue was passed through a silica gel column to obtain the desired product, compound 1 (105 mg, 41% yield), as a clear oil.

[0081] 1 H NMR(500MHz,CDCl3)δ7.26(br,1H), 7.13(br,1H), 4.01-3.84(m,4H), 3.32-3.25(m,6H), 3.13-3.05(m,6H), 2.94-2.93(m,4H), 2.74- 2.69(m,4H), 2.37-2.34(m,4H), 2.05-1.99(m,4H), 1.82-1.80(m,4H), 1.62-1.37(m,20H), 1.34-1.20(m,62H), 0.88(t,J=10Hz,12H). MS(ESI): Calculated value C 64 H 130 N4O6S2[M+H] + Regarding 1114.9, the measured value was 1116.3.

[0082] compound 2 TIFF2026530178000047.tif27170

[0083] A mixture of 1,2-epoxydodecane (500 mg, 2.7 mmol) and 4-OH-butylamine (120 mg, 0.5 equivalents) is used in 80 ° Intermediate 12 was obtained by heating overnight in 14C. After both LC-MS and TLC indicated that the reaction was complete, the reaction mixture was redissolved in anhydrous DCM (100 ml), followed by the sequential addition of disulfide acid 9 (385 mg, 0.6 equivalents), EDCI (674 mg, 1.3 equivalents), and DIEA (0.75 ml, 1.5 equivalents). The resulting reaction mixture was stirred at room temperature for 24 hours until the starting materials were gone. The reaction mixture was quenched with saturated NaHCO3, the solvent was removed, and the crude product was extracted with DCM, which was then subjected to silica gel purification.

[0084] 1 H NMR(500MHz,CDCl3)δ4.11-4.09(m,4H), 3.62-3.60(m,4H), 2.62(s,4H), 2.59-2.53(m,6H), 2.42-2.37(m,6H), 1.6 3-1.53(m,6H), 1.51-1.45(m,6H), 1.44-1.40(m,4H), 1.39-1.36(m,8H), 1.33-1.25(m,68H), 0.88(t,J=10Hz,12H). MS(ESI): Calculated value C 64 H 128 N2O8S2[M+H] + Regarding 1116.9, the measured value was 1118.2.

[0085] compound 3 TIFF2026530178000048.tif40170

[0086] A mixture of triol 12 (300 mg, 0.656 mmol) and succinic anhydride (72 mg, 1.1 equivalents) in anhydrous DCM (5 ml) was treated with DIEA (151 μL, 1.2 equivalents) overnight at room temperature. To the resulting reaction mixture, EDCI (163 mg, 1.3 equivalents), then 0.9 equivalents of triol 12 (270 mg), DIEPA (150 μL, 1.2 equivalents), and a catalytic amount of DMAP were sequentially added. After stirring the reaction mixture for 24 hours, the desired product, compound 3, was obtained as a colorless oil (108 mg, 17%) by silica gel chromatography.

[0087] 1 H NMR (500MHz, CDCl3) δ4.09-4.07(m,4H), 3.62-3.60(m,4H), 2.73-2.70(t,J=10Hz,4H), 2.59-2.53(m,4H) ), 2.45-2.38(m,10H), 2.04-2.0(m,4H), 1.65-1.40(m,14H), 1.33-1.25(m,64H), 0.88(t,J=10Hz,12H). MS(ESI):C 60 H 120 N2O8[M+H] + The calculated value is 996.9, and the measured value is 998.1.

[0088] General procedure for the synthesis of lipidoid (C) TIFF2026530178000049.tif126170TIFF2026530178000050.tif131170 Intermediate 20

[0089] Following the same procedure as for intermediate 18, intermediate 20 was obtained as a grayish-white solid in 41% yield and was then treated with diamine intermediate 8 in DMF in the presence of TEA at room temperature. After workup and extraction with DCM / saturated NaHCO3, the crude product was obtained. By silica gel chromatography, the desired oily compound 17 was obtained in 28% yield.

[0090] Intermediate 21

[0091] To a solution of intermediate 8 in dehydrated DCM (0.5 M), acrylic chloride (6 equivalents) was added dropwise in the presence of DIEA (10 equivalents) in an ice bath. The resulting reaction mixture was stirred overnight at room temperature, quenched with saturated NaHCO3, and subsequently extracted with DCM. After concentration, the crude product was treated with 3N NaHCO3 in a solvent mixture of MeOH / H2O until most of the starting material had disappeared. The resulting reaction mixture was neutralized with HCl and extracted with DCM, and after purification by silica gel chromatography, the desired intermediate 21 was obtained in 67% yield.

[0092] compound 16

[0093] A solvent-free mixture of intermediate 21 (2.2 equivalents) and intermediate 8 was incubated in a sealed tube for 48 hours at 90°C. ° The mixture was heated to C. Compound 16 was purified using silica gel in a yield of 35%.

[0094] compound 19

[0095] Following the same procedure as for intermediate 20, intermediate 23 was directly applied to the subsequent esterification without further separation by using DMF as the solvent for the preparation of intermediate 23 and EDCI as the coupling reagent in the presence of DMAP. Compound 19 was obtained in 15% yield over two steps.

[0096] General procedure for the synthesis of lipidoids (D) TIFF2026530178000051.tif125170 intermediate 25

[0097] The solvent-free reaction of the starting material 24 solution with 2.0 equivalents of powdered NaOH was carried out for 50 minutes. ° The mixture was heated at 1°C for 1 hour, and then epichlorohydrin (2.0 equivalents) and the catalyst tetrabutylammonium bromide (TBAB) (2.0 equivalents) were added. The resulting reaction mixture was stirred overnight at the same temperature. The reaction mixture was diluted with DCM and subsequently filtered through a celite pad to obtain crude intermediate 25, which was purified by silica gel chromatography in 56% yield.

[0098] Intermediate 27

[0099] Intermediate 25 was treated with camphorsulfonic acid (CSA, 0.1 eq) in a mixture of solvents DCM and MeOH (1 / 1) for 24 hours, followed by neutralization and concentration to obtain a crude product, which was purified by chromatography to give the desired alcohol 26. Coupling of 26 with hexanoic acid was performed according to a standard protocol using EDCI and DMAP in DCM. 1 H NMR (500 MHz, CDCl3) δ 4.00-3.98 (d, J=10 Hz, 1H), 3.89-3.88 (d, J=5 Hz, 1H), 3.72-3.69 (m, 1H), 3.43-3.27 (m, 3H), 3.15-3.12 (m, 1H), 2.79 (t, J=5 Hz, 1H), 2.61-2.59 (dd, J=5 Hz, 10 Hz, 1H), 2.29 (t, 5 Hz, 2H), 1.85-1.78 (m, 3H), 1.65-1.50 (m, 6H), 1.43-1.27 (m, 5H), 1.01-0.95 (m, 2H), 0.89 (t, J=5 Hz, 3H). MS (ESI): C 17 H 30 O4[M+Na] + calcd. 321.2, found 321.1.

[0100] Intermediates 30 and 31

[0101] The starting amine and Intermediate 27 (2.2 eq) were combined, and the solvent-free reaction was carried out at 85 ° °C to 90 ° °C for 24 to 48 hours to give the desired diol 28 or 29 in 30% to 40% yield after chromatographic purification. The diol was treated with TFA (5.0 eq) in DCM for 2 to 3 hours at room temperature to give amine 30 and acid 31 after purification by silica gel chromatography.

[0102] Compound 20

[0103] Acid 31 was mixed with EDCI and alcohol in DCM and stirred at room temperature for 30 minutes in the presence of molecular sieves. Then, amine 30 and DIEA were added via syringe. The resulting reaction mixture was stirred for 8 hours and work-up according to a standard protocol. After purification by chromatography, compound 20 was obtained in 45% yield.

[0104] General procedure for the synthesis of lipidoids (E) TIFF2026530178000052.tif110170 intermediate 34

[0105] A mixture of azido-butanol (1.1g) and octylaldehyde (0.3 equivalents, 500mg) is used. ° The mixture was heated in 1C for 5 hours and purified with silica gel to obtain the desired product di-N3 C10 (510 mg, 43%), which was then treated with Pd-C in methanol for 48 hours to obtain diamine 34 in the presence of an H2 balloon. 1 H NMR(500MHz,CDCl3)δ4.45(t,J=5Hz,1H), 3.60-3.58(m,2H), 3.44-3.41(m,2H), 2.74( m,4H), 1.98-1.95(m,8H), 1.63-1.52(m,6H), 1.29-1.26(m,14H), 0.89(t,J=5Hz,3H).

[0106] compound 21

[0107] Diamine 34 epoxide and 88 ° The reaction was carried out in 1C for 24 hours, and compound 21 was obtained in 35% yield after silica gel chromatography.

[0108] Intermediate 37

[0109] To a solution of 4-OH 1-Boc-butylamine (189 mg, 1 mmol) in acetonitrile (5 mL), pyridine (240 μL, 3 mmol) and 4-nitrophenyl chloroformate (280 mg, 1.4 mmol) were added at room temperature. After stirring for 4 hours, 4-NH21-Boc-butylamine (380 mg, 2 mmol) was added, and the resulting reaction mixture was stirred for a further 2 hours. The reaction mixture was extracted in DCM (50 mL), washed with water, dried over anhydrous sodium sulfate, and concentrated under vacuum. The crude residue was purified using silica gel chromatography (10% RINKAN in hexane) to obtain intermediate 37 (141 mg, yield 35%) as a clear oil. 1 H NMR (500MHz, CDCl3) δ4.07-4.04(m,2H), 3.19-2.89(m,6H), 1.63-1.51(m,8H), 1.44(s,18H). MS(ESI):C 19 H 37 N3O6[M+H] + The calculated value is 404.3, and the measured value is 404.1.

[0110] compound 22

[0111] Carbamate diamine 37 was redissolved in DCM, followed by treatment with 10 equivalents of 4N HCl in dioxane. After stirring overnight at room temperature to remove the solvent, diamine 38HCl salt was obtained as a white solid. Following a standard epoxide ring-opening protocol, the diamine was heated with 1,2-epoxydodecane, and compound 22 was obtained in 31% yield after column purification.

[0112] Lipid nanoparticles disclosed herein This disclosure provides lipid nanoparticles (LNPs) comprising one or more compounds of formula (I), formula (II'), and / or formula (II). In addition to one or more compounds of formula (I), formula (II'), and / or formula (II), the LNPs of this disclosure may include one or more additional LNP components as described below.

[0113] In some embodiments, the LNP of the Disclosure may contain, in molar proportion, 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% of at least one compound of the Disclosure. In some embodiments, at least one compound is at least one compound of formula (I), formula (II'), or formula (II) as described herein. In some embodiments, at least one compound of the present disclosure is a mixture of two or more compounds of formula (I), formula (II'), or formula (II).

[0114] In some embodiments, the LNP of the Disclosure may contain, in molar proportion, at least one compound of the Disclosure in amounts of about 2.5%, or about 5%, or about 7.5%, or about 10%, or about 12.5%, or about 15%, or about 17.5%, or about 20%, or about 22.5%, or about 25%, or about 27.5%, or about 30%, or about 32.5%, or about 35%, or about 37.5%, or about 40%, or about 42.5%, or about 45%, or about 47.5%, or about 50%, or about 52.5%, or about 55%, or about 57.5%, or about 60%, or about 62.5%, or about 65%, or about 67.5%, or about 70%. In some embodiments, at least one compound is at least one compound of formula (I), formula (II'), or formula (II) as described herein. In some embodiments, at least one compound of the present disclosure is a mixture of two or more compounds of formula (I), formula (II'), or formula (II).

[0115] structural lipids

[0116] 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% of at least one structural lipid.

[0117] In some embodiments, the structural lipid may be a steroid. In some embodiments, the structural lipid may be a sterol. In some embodiments, the structural lipid may include cholesterol. In some embodiments, the structural lipid may include ergosterol. In some embodiments, the structural lipid may be a phytosterol.

[0118] In some embodiments, at least one structural lipid is a mixture of two structural lipids.

[0119] Phospholipids

[0120] 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% of at least one phospholipid.

[0121] As used herein, the term “phospholipid” is used in its broadest sense to refer to any amphiphilic molecule comprising a polar (hydrophilic) head group containing a phosphate and two hydrophobic fatty acid chains. In some embodiments, the phospholipid may contain dioleoylphosphatidylethanolamine (DOPE). In some embodiments, the phospholipid may contain 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments, the phospholipid may contain 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some embodiments, the phospholipids are DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPA-NA (1,2-dielcyl-sn-glycero-3-phosphate (sodium salt)), DEPC (1,2-dielcyl-sn-glycero-3-phosphocholine), DEPE (1,2-dielcyl-sn-glycero-3-phosphoethanolamine), DEPG-NA (1,2-dielcyl Coil-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-phosphocholine), 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-N A(1,2-dimiristoyl-sn-glycero-3-phosphate (sodium salt)), DMPC(1,2-dimiristoyl-sn-glycero-3-phosphocholine), DMPE(1,2-dimiristoyl-sn-glycero-3-phosphoethanolamine), DMPG-NA(1,2-dimiristoyl-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-glycerol) 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)), DPPC(1,2-dipalmitoyl-sn-glycero-3-phospho Choline), 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-distearoyaloyl 1,2-distearoyl-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), bovine 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-phosphoethanolamine), 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.

[0122] PEGylated lipid

[0123] In some aspects, the LNP can further comprise at least about 0.25 mol%, or at least about 0.5 mol%, or at least about 0.75 mol%, or at least about 1.0 mol%, or at least about 2.5 mol%, or at least about 5 mol%, or at least about 7.5 mol%, or at least about 10 mol% of the PEGylated lipid, based on molar ratio.

[0124] As used herein, the term “PEGylated lipid” is used to refer to any lipid that has been modified (e.g., covalently linked) to at least one polyethylene glycol molecule. In some embodiments, the PEGylated lipid may include 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (hereinafter referred to as “DMG-PEG2000” or “DMG-PEG”).

[0125] In some embodiments, at least one PEGylated lipid is a mixture of two PEGylated lipids.

[0126] Exemplary LNP composition

[0127] The following are exemplary LNP compositions of the present disclosure, comprising at least one compound of formula (I), formula (II'), and / or formula (II), at least one structural lipid, at least one PEGylated lipid, and at least one phospholipid.

[0128] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain, in molar ratio, at least one compound of formula (I) in about 35% to about 50%, at least one structural lipid in about 37.5% to about 56%, at least one phospholipid in about 5% to about 12%, and at least one PEGylated lipid in about 1% to about 2.5%.

[0129] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain, by molar ratio, at least one compound of formula (I) in about 35%, at least one structural lipid in about 56%, at least one phospholipid in about 7.5%, and at least one PEGylated lipid in about 1.5%.

[0130] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain, by molar ratio, at least one compound of formula (I) in about 50%, at least one structural lipid in about 38.5%, at least one phospholipid in about 10%, and at least one PEGylated lipid in about 1.5%.

[0131] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain, by molar ratio, at least one compound of formula (I) in about 50%, at least one structural lipid in about 37.5%, at least one phospholipid in about 10%, and at least one PEGylated lipid in about 2.5%.

[0132] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain, by molar ratio, at least 40% of at least one compound of formula (I), by molar ratio at least 52.5% of at least one structural lipid, by molar ratio at least 5% of at least one phospholipid, and by molar ratio at least 2.5% of at least one PEGylated lipid.

[0133] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain, by molar ratio, at least 45% of at least one compound of formula (I), by molar ratio at least 45.5% of at least one structural lipid, by molar ratio at least 7.5% of at least one phospholipid, and by molar ratio at least 2% of at least one PEGylated lipid.

[0134] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain, by molar ratio, at least 43.17% of at least one compound of formula (I), by molar ratio at least 43.17% of at least one structural lipid, by molar ratio at least 11.96% of at least one phospholipid, and by molar ratio at least 1.7% of at least one PEGylated lipid.

[0135] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain, by molar ratio, at least one compound of formula (I) in about 45%, at least one structural lipid in about 43%, at least one phospholipid in about 10%, and at least one PEGylated lipid in about 2%.

[0136] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain, by molar ratio, at least 45% of at least one compound of formula (I), by molar ratio at least 46% of at least one structural lipid, by molar ratio at least 7.5% of at least one phospholipid, and by molar ratio at least 1.5% of at least one PEGylated lipid.

[0137] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain, by molar ratio, at least one compound of formula (I) in about 50%, at least one structural lipid in about 39%, at least one phospholipid in about 10%, and at least one PEGylated lipid in about 1%.

[0138] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain, by molar ratio, at least one compound of formula (I) in about 50%, at least one structural lipid in about 41.5%, at least one phospholipid in about 7.5%, and at least one PEGylated lipid in about 1%.

[0139] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain, by molar ratio, at least 40% of at least one compound of formula (I), by molar ratio at least 48.5% of at least one structural lipid, by molar ratio at least 10% of at least one phospholipid, and by molar ratio at least 1.5% of at least one PEGylated lipid.

[0140] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain at least one compound of formula (II) in molar ratios of about 35% to about 50%, at least one structural lipid in molar ratios of about 37% to about 59%, at least one phospholipid in molar ratios of about 5% to about 10%, and at least one PEGylated lipid in molar ratios of about 1% to about 3%.

[0141] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain, by molar ratio, at least one compound of formula (II) in about 35%, at least one structural lipid in about 59%, at least one phospholipid in about 5%, and at least one PEGylated lipid in about 1%.

[0142] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain, by molar ratio, at least one compound of formula (II) at about 50%, at least one structural lipid at about 41.5%, at least one phospholipid at about 7.5%, and at least one PEGylated lipid at about 1%.

[0143] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain, by molar ratio, at least one compound of formula (II) in about 50%, at least one structural lipid in about 39%, at least one phospholipid in about 10%, and at least one PEGylated lipid in about 1%.

[0144] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain, by molar ratio, at least one compound of formula (II) in about 42.5%, at least one structural lipid in about 51.5%, at least one phospholipid in about 5%, and at least one PEGylated lipid in about 1%.

[0145] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain at least one compound of formula (II') in molar ratios of about 35% to about 50%, at least one structural lipid in molar ratios of about 37% to about 59%, at least one phospholipid in molar ratios of about 5% to about 10%, and at least one PEGylated lipid in molar ratios of about 1% to about 3%.

[0146] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain, by molar ratio, at least one compound of formula (II'), by molar ratio at least 59% of at least one structural lipid, by molar ratio at least 5% of at least one phospholipid, and by molar ratio at least 1% of at least one PEGylated lipid.

[0147] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain, by molar ratio, at least one compound of formula (II') in about 50%, at least one structural lipid in about 41.5%, at least one phospholipid in about 7.5%, and at least one PEGylated lipid in about 1%.

[0148] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain, by molar ratio, at least one compound of formula (II'), by molar ratio at least 39% of at least one structural lipid, by molar ratio at least 10% of at least one phospholipid, and by molar ratio at least 1% of at least one PEGylated lipid.

[0149] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain, by molar ratio, at least one compound of formula (II'), by molar ratio at least 42.5%, by molar ratio at least one structural lipid, by molar ratio at least 5%, by molar ratio at least one phospholipid, and by molar ratio at least 1% of at least one PEGylated lipid.

[0150] In some embodiments of the LNP described above, the compound of formula (I), formula (II'), or formula (II) contained in the LNP composition is one of compound numbers 1 to 14.

[0151] In some embodiments, the compound of formula (I), formula (II'), or formula (II) is one of the compound numbers 1 to 14.

[0152] In some embodiments of the LNP described above, the compound of formula (I), formula (II'), or formula (II) contained in the LNP composition is one of compound numbers 1 to 22.

[0153] In some embodiments, the compound of formula (I), formula (II'), or formula (II) is one of compound numbers 1 to 22.

[0154] In some embodiments, the structural lipid can be cholesterol.

[0155] In some embodiments, phospholipids are DOPE.

[0156] In some embodiments, the phospholipid is a DSPC.

[0157] In some embodiments, phospholipids are DOPCs.

[0158] In some embodiments, the phospholipid is DPPC.

[0159] In some embodiments, the phospholipid may be a mixture of DSPC and DOPC. In some embodiments, the mixture of DSPC and DOPC may contain DSPC and DOPC in a 1:1 ratio (for example, an LNP containing 10% phospholipid may contain 5% DOPC and 5% DSPC).

[0160] In some earlier embodiments of LNPs, the PEGylated lipid may be DMG-PEG2000.

[0161] In some embodiments, the structural lipid may be cholesterol, the phospholipid may be DOPE, and the PEGylated lipid may be DMG-PEG2000.

[0162] In some embodiments, the structural lipid may be cholesterol, the phospholipid may be DOPC, and the PEGylated lipid may be DMG-PEG2000.

[0163] In some embodiments, the structural lipid may be cholesterol, the phospholipid may be DSPC, and the PEGylated lipid may be DMG-PEG2000.

[0164] In some embodiments, the structural lipid may be cholesterol, the phospholipid may be DPPC, and the PEGylated lipid may be DMG-PEG2000.

[0165] In some embodiments, the structural lipid may be cholesterol, the phospholipid may be a mixture of DSPC and DOPC, and the PEGylated lipid may be DMG-PEG2000. In some embodiments, the mixture of DSPC and DOPC may contain DSPC and DOPC in a 1:1 ratio (for example, an LNP containing 10% phospholipid may contain 5% DOPC and 5% DSPC).

[0166] Table 1A shows further exemplary LNP compositions of this disclosure. Table 1A TIFF2026530178000053.tif254170TIFF2026530178000054.tif255170TIFF2026530178000055.tif255170TIFF2026530178000056.tif99170

[0167] In some embodiments of the LNP compositions described in Tables 1A to 1C, the compound of formula (I), formula (II), or formula (II') is one of compound numbers 1 to 22.

[0168] In some embodiments of the LNP compositions described in Tables 1A to 1C, the structural lipid may be cholesterol.

[0169] In some embodiments of the LNP compositions described in Tables 1A-1C, the phospholipid is DOPE.

[0170] In some embodiments, including the LNP compositions described in Tables 1A to 1C, the phospholipid is DSPC.

[0171] In some embodiments, including the LNP compositions described in Tables 1A to 1C, the phospholipid is DOPC.

[0172] In some embodiments, including the LNP compositions described in Tables 1A to 1C, the phospholipid is DPPC.

[0173] In some embodiments of the preceding LNPs, including the LNP compositions described in Table 1, the PEGylated lipid may be DMG-PEG2000.

[0174] In some embodiments of the LNP compositions described in Tables 1A to 1C, the structural lipid may be cholesterol, the phospholipid may be DOPE, and the PEGylated lipid may be DMG-PEG2000.

[0175] In some embodiments of the LNP compositions described in Tables 1A to 1C, the structural lipid may be cholesterol, the phospholipid may be DOPC, and the PEGylated lipid may be DMG-PEG2000.

[0176] In some embodiments of the LNP compositions described in Tables 1A to 1C, the structural lipid may be cholesterol, the phospholipid may be DSPC, and the PEGylated lipid may be DMG-PEG2000.

[0177] In some embodiments of the LNP compositions described in Tables 1A to 1C, the structural lipid may be cholesterol, the phospholipid may be DPPC, and the PEGylated lipid may be DMG-PEG2000.

[0178] Targeted ligand

[0179] In some embodiments, the LNPs, including those listed in Table 1A, may further include at least one targeted ligand.

[0180] In some embodiments, LNPs are present in a molar ratio 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%. It may further contain %, 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 targeted ligands.

[0181] A targeted ligand can be any ligand that, compared to a species without such a ligand, results in enhanced affinity for a selected target, such as a molecule, cell, or cell type, such as a cellular or organ compartment, tissue, organ, or region of the body.

[0182] Without adhering to any particular theory, compositions containing targeted lipids are well-tolerated, provide an appropriate therapeutic index, and consequently, treatment of patients with an effective amount of the composition is associated with an improvement in the patient's toxicity and / or risk profile compared to treatment of patients with an effective amount of the composition without the targeted ligand.

[0183] In some embodiments, the targeted ligand derives enhanced affinity to the liver or hepatocytes, such as liver cells. A non-limiting example of a targeted ligand that derives enhanced affinity to the liver or hepatocytes is GalNac (n-acetyl-galactosamine). Accordingly, in some embodiments, the present disclosure provides compositions comprising a targeted ligand containing GalNac.

[0184] In some embodiments, the targeting ligand containing GalNac may be a PEGylated GalNac molecule. In some embodiments, the PEGylated GalNac molecule may be Tri-GalNac-PEG2000-DSPE (hereinafter referred to as "GalNac-PEG"), whose structure is shown below. TIFF2026530178000057.tif40170.

[0185] Accordingly, in some embodiments, the Disclosure provides LNPS comprising GalNac-PEG. In some embodiments, the targeted ligand may also include a group of targeting agents, e.g., tissue targeting agents. A non-limiting example of the group of targeting agents may be a polyvalent GalNac molecule. Accordingly, in some embodiments, the Disclosure provides LNP compositions comprising a targeted ligand comprising a polyvalent GalNac. A non-limiting example of a polyvalent GalNac molecule is GalNac-PEG.

[0186] Table 1B shows exemplary LNP compositions of the present disclosure comprising at least one compound of formula (I), formula (II), or formula (II'), at least one structural lipid, at least one PEGylated lipid, and at least one phospholipid, as well as a targeted ligand containing at least one GalNac. Table 1B TIFF2026530178000058.tif212170

[0187] In some embodiments, including LNPS shown in Table 1B, the targeting ligand containing GalNac is GalNac-PEG.

[0188] nucleic acid molecule

[0189] In some embodiments, the lipid nanoparticles of this disclosure, including those listed in Tables 1A and 1B, may further contain at least one nucleic acid. In some embodiments, the lipid nanoparticles may contain multiple nucleic acid molecules. In some embodiments, at least one nucleic acid or multiple nucleic acid molecules may be formulated in the lipid nanoparticles.

[0190] Therefore, the lipid nanoparticles may include at least one nucleic acid, at least one compound of the disclosure, at least one structural lipid, at least one phospholipid, and at least one PEGylated lipid. In some embodiments, the lipid nanoparticles may further include at least one targeted ligand.

[0191] In some embodiments, at least one nucleic acid molecule 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 nanoplasmid.

[0192] In some embodiments of the preceding LNP, at least one nucleic acid molecule 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 may be 5-methylcytidine.

[0193] In some embodiments, at least one RNA molecule is a guide RNA (gRNA) molecule.

[0194] In some embodiments, at least one nucleic acid may include both an mRNA molecule and a guide RNA (gRNA) molecule. That is, the LNPs of this disclosure may include both an mRNA molecule and a gRNA molecule. In some embodiments where the LNP includes both an mRNA molecule and a gRNA molecule, the mRNA molecule includes at least one nucleic acid sequence encoding a fusion protein, the fusion protein including (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.

[0195] In some embodiments where the LNP contains both mRNA and gRNA molecules, the mRNA:gRNA ratio may be approximately 1:2, or approximately 1:3, or approximately 1:4, or approximately 1:5, or approximately 1:6, or approximately 1:7, or approximately 1:8, or approximately 1:9, or approximately 1:10, or approximately 1:1, or approximately 2:1, or approximately 3:1, or approximately 4:1, or approximately 5:1, or approximately 6:1, or approximately 7:1, or approximately 8:1, or approximately 9:1, or approximately 10:1.

[0196] In some embodiments, at least one nucleic acid molecule may include at least one RNA molecule and at least one DNA molecule. That is, the LNPs of this disclosure may include both RNA and DNA molecules.

[0197] In some embodiments, the LNPs of this disclosure may include both RNA molecules and DNA molecules, wherein the RNA molecule includes at least one nucleic acid sequence encoding a transposase, and the DNA molecule includes at least one nucleic acid sequence including a transposon. In some embodiments, the transposase may be any of the transposases described herein. In some embodiments, the transposon may be a transposon comprising at least one nucleic acid sequence encoding an FVIII polypeptide.

[0198] In some embodiments of the present disclosure, where the LNP comprises both RNA (e.g., mRNA) and DNA, the RNA to DNA ratio (RNA:DNA) in the LNP may 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.

[0199] In some embodiments, lipid nanoparticles may contain lipids and nucleic acids in a specific ratio (weight / weight).

[0200] In some embodiments, lipid nanoparticles containing at least one nucleic acid are arranged in a ratio of approximately 5:1 to approximately 15:1, or approximately 10:1 to approximately 20:1, or approximately 15:1 to approximately 25:1, or approximately 20:1 to approximately 30:1, or approximately 25:1 to approximately 35:1, or approximately 30:1 to approximately 40:1, or approximately 35:1 to approximately 45:1, or approximately 40:1 to approximately 50:1, or approximately 45:1 to approximately 55:1, or approximately 50:1 to approximately 60:1, or approximately 55:1 to approximately 65:1, or approximately 60:1 to approximately 70:1, or approximately 65:1 to approximately 75:1, or approximately 70:1 to approximately 80:1, or approximately 75:1 to approximately 85 Lipids and nucleic acids can be included in a ratio of :1, or approximately 80:1 to 90:1, or approximately 85:1 to 95:1, or approximately 90:1 to 100:1, or approximately 95:1 to 105:1, or approximately 100:1 to 110:1, or approximately 105:1 to 115:1, or approximately 110:1 to 120:1, or approximately 115:1 to 125:1, or approximately 120:1 to 130:1, or approximately 125:1 to 135:1, or approximately 130:1 to 140:1, or approximately 135:1 to 145:1, or approximately 140:1 to 150:1 (lipids:nucleic acids (weight / weight)).

[0201] In some embodiments, lipid nanoparticles containing at least one nucleic acid are in a ratio of approximately 5:1, or approximately 10:1, or approximately 15:1, or approximately 20:1, or approximately 25:1, or approximately 30:1, or approximately 35:1, or approximately 40:1, or approximately 45:1, or approximately 50:1, or approximately 55:1, or approximately 60:1, or approximately 65:1, or approximately 70:1, or approximately 75:1, or approximately 80: Lipids and nucleic acids can be included in a ratio of 1, or approximately 85:1, or approximately 90:1, or approximately 95:1, or approximately 100:1, or approximately 105:1, or approximately 110:1, or approximately 115:1, or approximately 120:1, or approximately 125:1, or approximately 130:1, or approximately 135:1, or approximately 140:1, or approximately 145:1, or approximately 150:1 (lipids:nucleic acids (weight / weight)).

[0202] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain lipids and nucleic acids in a ratio of about 10:1, or about 25:1, or about 40:1 (lipids:nucleic acids (weight / weight)).

[0203] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain lipids and nucleic acids 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 (weight / weight)).

[0204] Further features of the nucleic acid molecules of this disclosure are provided herein.

[0205] Polyphenol additives

[0206] In some embodiments, the lipid nanoparticles of this disclosure, including those listed in Tables 1A and 1B, may further contain at least one polyphenol (also referred to herein as a “polyphenol additive”).

[0207] As used herein, the term “polyphenol” is used to refer to any compound having at least two phenol subunits, where phenol is an aromatic ring having at least one hydroxyl substituent as defined herein. Polyphenols include compounds having at least two phenol subunits, e.g., flavonoids, catechins, anthocyanins, stilbenes, and ellagic acid. Polyphenols also include compounds having at least three phenol subunits, e.g., proanthocyanidins, tannins, and punicalagines.

[0208] Accordingly, the lipid nanoparticles may include at least one compound of the present disclosure, at least one structural lipid, at least one phospholipid, at least one PEGylated lipid, and at least one polyphenol. In some embodiments, the lipid nanoparticles may further include at least one nucleic acid, at least one targeted ligand, or any combination thereof.

[0209] In some embodiments, at least one polyphenol is malvidin 3-o-(6"-p-coumaroyl-glucoside), delphinidin 3-o-(6"-acetyl-galactoside), cyanidin 3-o-(6"-acetyl-galactoside), cyanidin 3-o-galactoside, cyanidin 3-o-glucoside, cyanidin 3-o-rutinoside, cyanidin 3-o-sophoroside, pelargonidin 3-o-glucoside, cyanidin 3-o-(6"-malonyl-glucoside), peonidin, peonidin 3-o-glucoside, peonidin 3-o-rutinoside, pelargonidin 3-o-rutinoside, pelargonidin, cyanidin, malvidin 3,5-o-diglucoside, cyanidin 3-o-glucosyl-rutinoside, pelargonidin 3-o-sophoroside, pelargonidin 3-o-glucosyl-rutinoside, cyanidin 3-o-(6"-succinyl-glucoside), pelargonidin 3-o-(6"-succinyl-glucoside), delphinidin 3-o-galactoside, delphinidin 3-o-glucoside, delphinidin 3-o-arabinoside, petunidine 3-o-galactoside, cyanidin 3-o-arabinoside, petunidine 3-o-glucoside, peonidin 3-o-galacto Side, petunidine 3-o-arabinoside, malvidin 3-o-glucoside, malvidin 3-o-arabinoside, delphinidin 3-o-(6"-acetyl-glucoside), petunidine 3-o-(6"-acetyl-galactoside), peonidin 3-o-(6"-acetyl-galactoside), cyanidin 3-o-(6"-acetyl-glucoside), malvidin 3-o-(6"-acetyl-galactoside), petunidine 3-o-(6"-acetyl-glucoside), malvidin 3-o-(6"-acetyl-glucoside), peonidin 3-o-( 6"-acetyl-glucoside), pelargonidin 3-o-arabinoside, delphinidin 3-o-rutinoside, cyanidin 3-o-sambubioside, pelargonidin 3-o-(6"-malonyl-glucoside), peonidin 3-o-(6"-p-coumaroyl-glucoside), cyanidin 3-o-xyloside, malvidin 3-o-galactoside, peonidin 3-o-arabinoside, petunidine 3-o-rutinoside, delphinidin 3-o-xyloside, petunidine 3-o-(6"-p-coumaroyl-glucoside), pelargonidin 3-o- Galactosid, pelargonidin 3-o-sambubioside, delphinidin 3-o-sambubioside, cyanidin 3-o-xylosyl-rutinoside, biticin a, delphinidin 3-o-(6"-p-coumaroyl-glucoside), pigment a, cyanidin 3-o-(6"-p-coumaroyl-glucoside), cyanidin 3-o-sambubioside 5-o-glucoside, cyanidin 3-o-(6"-caffeoyl-glucoside), cyanidin 3,5-o-diglucoside, pinotin a, delphinidin 3,5-o-diglucoside, pelargonidin 3,5-o-diglucoside, malvidin 3-o-(6"-caffeoyl-glucoside), cyanidin 3-o-(6"-dioxalyl-glucoside), delphinidin 3-o-glucosyl-glucoside, cyanidin 3-o-(6"-malonyl-3"-glucosyl-glucoside), delphinidin 3-o-feruloyl-glucoside, petunidine 3,5-o-diglucoside, petunidine 3-o-rhamnoside, cyanidin 3-o-diglucoside-5-o-glucoside, peonidin 3-o-diglucoside-5-o-glucoside, Onidin 3-o-(2-o-(6-o-(e)-caffeoyl-d-glucosyl)-d-glucoside)-5-od-glucoside, peonidin 3-o-sophoroside, peonidin 3-o-sambubioside, peonidin 3-o-sambubioside-5-o-glucoside, peonidin 3-o-xyloside, 4'-o-methylcyanidin 3-od-glucoside, cyanidin 3-o-(3",6"-o-dimalonyl-glucoside), 4-o-methyldelphinidin 3-od-glucoside, isopeonidin 3-o-arabinoside, i Sopeonidin 3-o-galactoside, isopeonidin 3-o-glucoside, isopeonidin 3-o-rutinoside, isopeonidin 3-o-sambubioside, isopeonidin 3-o-xyloside, cyanidin 3-o-(2-o-(6-o-(e)-caffeoyl-d-glucoside)-d-glucoside)-5-od-glucoside, 4'-o-methyldelphinidin 3-o-rutinoside, butein, xanthohumol, phloretin, phloretin 2'-o-xylosyl-glucoside, 3-hydroxyphloretin 2' -o-Xylosyl-glucoside, 3-hydroxyphloretin 2'-o-glucoside, phloretin 2'-o-glucuronide, dihydroquercetin 3-o-rhamnoside, dihydroquercetin, dihydromyricetin 3-o-rhamnoside, (+)-catechin, (-)-epicatechin, (+)-gallocatechin, (-)-epigallocatechin, (-)-epicatechin 3-o-gallate, (-)-epigallocatechin 3-o-gallate, theaflavin, theaflavin 3-o-gallate, theaflavin 3'-o-gallate, theaflavin 3,3'-o-digallate, (+)-gallocatechin 3-o-gallate, (+)-catechin 3-o-gallate, procyanidin dimer bl, procyanidin dimer b2, procyanidin dimer b3, procyanidin dimer b4, procyanidin dimer b5, procyanidin dimer b7, prodelfinidin dimer b3, procyanidin trimer cl, procyanidin trimer eec, procyanidin trimer t2, procyanidin trimer c2, prodelfinidin Din trimer gc-gc-c, prodelphinidin trimer gc-cc, prodelphinidin trimer c-gc-c, (-)-epicatechin-(2a-7)(4a-8)-epicatechin 3-o-galactoside, cinnamtannin a2, (+)-catechin 3-o-glucose, 3'-o-methylepicatechin, 4'-o-methyl-(-)-epicatechin 3'-o-glucuronide, epicatechin 3'-o-glucuronide, 3'-o-methylcatechin, 4',4"-o-dimethylepigallocatechin 3-o-gallate, 4'-o-methylepigallocatechin, 4"-o-methylepigallocatechin 3-o-gallate, 4'-o-methylepicatechin, epigallocatechin 3-o-gallate-7-o-glucoside-4"-o-glucuronide, (-)-epigallocatechin 3-o-glucuronide, 3'-o-methyl-(-)-epicatechin 7-o-glucuronide, epicatechin 7-o-glucuronide, (-)-epigallocatechin 3'-o-glucuronide, (-)-epi Gallocatechin 7-o-glucuronide, 4'-o-methyl-(-)-epigallocatechin 3'-o-glucuronide, 4'-o-methyl-(-)-epigallocatechin 7'-o-glucuronide, naringenin, eriodictyol, hesperetin, eriocitrin, hesperidin, naringin, naringin, neoeriocitrin, neohesperidin, ponsillin, dzimin, naringin 4'-o-glucoside, naringin 4'-o-glucoside, naringin 6'-malonate, isosakuranetin, naringe Naringenin 7-o-glucoside, pinosembrin, 8-prenylnaringenin, 6-prenylnaringenin, 6-geranylnaringenin, isoxanthohumol, eriodictiol 7-o-glucoside, sakuranetin, hesperetin 3'-o-glucuronide, hesperetin 7-o-glucuronide, hesperetin 3'-sulfate, homoeriodictiol, naringenin 4'-o-glucuronide, naringenin 5-o-glucuronide, naringenin 7-o-glucuronide, hesperetin 3',7- o-diglucuronide, hesperetin 5,7-o-diglucuronide, apigenin, luteolin, diosmin, isoleuforin, neodiosmin, leuforin, sinensetin, nobiletin, tangeretin, luteolin 7-o-diglucuronide, chrysin, luteolin 7-o-rutinoside, tetramethylscutellain, luteolin 7-o-glucoside, apigenin 7-o-glucoside, apigenin 6,8-di-c-glucoside, apigenin 6,8-c-arabinoside-c-glucoside, apigenin 6,8-c-galactoside-c-arabinoside, luteolin 7-o-glucuronide, apigenin 7-o-glucuronide, luteolin 7-o-malonyl-glucoside, luteolin 6-c-glucoside, luteolin 7-o-(2-apiosyl-glucoside), luteolin 7-o-(2-apiosyl-6-malonyl)-glucoside, apigenin 7-o-apiosyl-glucoside, 7,3',4'-trihydroxyflavone, 7,4'-dihydroxyflavone, geraldone, baicalein, apigenin 6-c-glucoside, hispizuline, silcimaritin, 5,6-dihydroxy-7,8,3',4'-Tetramethoxyflavone, Pebrerin, Gardenin b, Nepetin, Jaseocidine, Silicineol, Eupatrin, 6-Hydroxyltheolin, 6-Hydroxyltheolin 7-o-Rhamnoside, Scutellain, Apigenin 7-o-(6"-Malonylapiosyl-Glucoside), Chrysoeriol 7-o-Apiosyl-Glucoside, Chrysoeriol 7-o-(6"-Malonylapiosyl-Glucoside), Chrysoeriol 7-o-Glucoside, Chrysoeriol 7-o-(6"-Malonyl-Glucoside), Apigenin 7-o-Diglyceride Clonide, Leuforin 4'-o-glucoside, Kaempferol, Quercetin, Quercetin 3-o-galactoside, Quercetin 3-o-glucoside, Quercetin 3-o-xyloside, Quercetin 3-o-rhamnoside, Quercetin 3-o-rutinoside, Quercetin 3-o-sophoroside, Quercetin 3-o-arabinoside, Quercetin 3-o-xylosyl-glucuronide, Isorhamnetin 3-o-glucoside 7-o-rhamnoside, Isorhamnetin 3-o-rutinoside, Kaempferol 3-o-glucuronide, Isorhamnetin 7-o-rhamnoside, Quercetin 3,4'-o-diglucoside, myricetin 3-o-rutinoside, myricetin, morin, kaemperia, myricetin 3-o-galactoside, myricetin 3-o-glucoside, quercetin 3-o-glucosyl-xyloside, quercetin 3-o-acetyl-rhamnoside, kaemperolf 3-o-galactoside, galangin, isorhamnetin, kaemperolf 3-o-glucoside, kaemperolf 3-o-rutinoside, kaemperolf 3-o-glucosyl-rhamnosyl-galactoside, kaemperolf 3-o-glucosyl-rhamnosyl-glucoside, quercetin 3-o-g Glucosyl-rhamnosyl-galactoside, quercetin 3-o-glucosyl-rhamnosyl-glucoside, rhamnetin, isorhamnetin 3-o-glucoside, myricetin 3-o-rhamnoside, quercetin 3-o-rhamnosyl-galactoside, quercetin 3-o-glucuronide, isorhamnetin 3-o-glucuronide, myricetin 3-o-arabinoside, quercetin 7,4'-o-diglucoside, quercetin 4'-o-glucoside, isorhamnetin 4'-o-glucoside, 3,7-dimethylquercetin, kaempferol 3-o-sophoroside, kaempferol 3,7-o-diglucoside, Kaempferol 3-o-sophoroside 7-o-glucoside, Quercetin 3-o-(6"-malonyl-glucoside), Kaempferol 3-o-(6"-malonyl-glucoside), Kaempferol 3-o-rhamnoside, Quercetin 3-o-(6"-malonyl-glucoside) 7-o-glucoside, Patsuretin 3-o-glucosyl-(1->6)-[apiosyl(1->2)]-glucoside, Spinacetin 3-o-glucosyl-(1-> 6)-[apiosyl(1->2)]-glucoside, patsuretin 3-o-(2"-feruroylglucosyl)(1->6)-[apiosyl(1->2)]-glucoside, spinacetin 3-o-(2"-p-coumaroylglucosyl)(1->6)-[apiosyl(1->2)]-glucoside, spinacetin 3-o-(2"-feruroylglucosyl)(1->6)-[apiosyl(1->2)]-glucoside, spinacetin 3-o-glucosyl-(, 1->6)-Glucoside, Jaseidine 4'-o-glucuronide, 5,3',4'-Trihydroxy-3-methoxy-6:7-methylenedioxyflavone 4'-o-glucuronide, 5,4'-Dihydroxy-3,3'-Dimethoxy-6:7-methylenedioxyflavone 4'-o-glucuronide, Kaemperoll 3-o-xylosyl-glucoside, Kaemperoll 3-o-acetyl-glucoside, Quercetin 3-o-xylosyl-rutinoside, Kaemperoll 3-o-xylosyl-rutinoside, Kaemperoll 7-o-glucoside, Kaemperoll 3-o-galact 7-o-rhamnoside, Kaempferol 3-o-(6"-acetyl-galactoside)7-o-rhamnoside, Quercetin 3-o-galactoside 7-o-rhamnoside, Quercetin 3-o-(6"-acetyl-galactoside)7-o-rhamnoside, Kaempferol 3-o-(2"-rhamnosyl-galactoside)7-o-rhamnoside, Kaempferol 3-o-(2"-rhamnosyl-6"-acetyl-galactoside)7-o-rhamnoside, 6,8-dihydroxykaempferol, isorhamnetin 3-o-galactoside, Quercetin 3-o-rhamnosyl-rhamnosyl-gluco Side, Kaempferol 3-o-rhamnosyl-rhamnosyl-glucoside, methylgalangin, Kaempferol 3,7,4'-o-triglucoside, 3-methoxynobiletin, 3-methoxysinensetin, quercetin 3'-o-glucuronide, quercetin 3'-sulfate, quercetin 4'-o-glucuronide, isorhamnetin 4'-o-glucuronide, daidzein, formononetin, genistein, biochanin a, glycitein, glycin, 6"-o-acetyldaidzin, 6"-o-malonylgenistin, daidzin, genistin, 6"-o-acetylgenistin Tin, 6"-o-acetylglycinin, 6"-o-malonyl daidzin, 6"-o-malonyl glycinin, 2',7-dihydroxy-4',5'-dimethoxyisoflavon, 2-dehydro-o-desmethylangolencine, 2'-hydroxyformononetin, 3',4',7-trihydroxyisoflavan, 3',4',7-trihydroxyisoflavanone, 3'-hydroxydaidzein, 3'-hydroxy-o-desmethylangolencine, 4',6,7-trihydroxyisoflavanone, 4',7-dihydroxy-3'-methoxyisoflavan, 4',7-Dihydroxy-6-methoxyisoflavan, 4'-o-methylequol, 5,6,7,3',4'-pentahydroxyisoflavon, 5,6,7,4'-tetrahydroxyisoflavon, 5,7,8,3',4'-pentahydroxyisoflavon, 5,7,8,4'-tetrahydroxyisoflavon, 5'-hydroxy-o-desmethylangolencine, 5'-methoxy-o-desmethylangolencine, 6,7,3',4'-tetrahydroxyisoflavon, 6,7,4'-trihydroxyiso Flavonoids, 6'-hydroxyangolencine, 6'-hydroxy-o-desmethylangolencine, 7,8,3',4'-tetrahydroxyisoflavonoid, 7,8,4'-trihydroxyisoflavonoid, angolencine, calicosine, daidzein 4'-o-glucuronide, daidzein 7-o-glucuronide, dihydrobiochanin a, dihydrodaidzein, dihydrodaidzein 7-o-glucuronide, dihydroformononetin, dihydrogenistein, dihydroglycitein, equol, formononetin 7 -o-glucuronide, genistein 4',7-o-diglucuronide, genistein 4'-o-glucuronide, genistein 5-o-glucuronide, genistein 7-o-glucuronide, glycitein 4'-o-glucuronide, glycitein 7-o-glucuronide, copalin, o-desmethylangolencine, orobol, prunetin, pseudobaptigenin, puerarin, daidzin 4'-o-glucuronide, irisolidone 7-o-glucuronide, tectorigenin 7-sulfate, tectorigenin 4'-sulfate Rufate, Irisolidone, Tectorigenin, Tectoridine, 5,7-Dihydroxy-8,4'-Dimethoxyisoflavones, Isotectorigenin, Equol 7-o-glucuronide, Equol 4'-o-glucuronide, 3',4',5,7-Tetrahydroxyisoflavanone, 3'-o-Methylequol, 6-o-Methylequol, 3'-Hydroxygenistein, 6-Hydroxydihydrodaidzein, 3'-Hydroxyequol, Cis-4-Hydroxyequol, 4'-Methoxy-2',37-Trihydroxyisoflavanone, Irilon, Bestiton, Sachibanone, Butin, 3'-Hydroxymelanetin, Melanetin, Stebenin, Violanon, Isoliquitigenin, Dalbergin, 3'-o-Methylviolanon, 8-Hydroxydihydrodaidzein, Secoisolariciresinol, Matairesinol, Lariciresinol, Pinoresinol, Syringaresinol, Isolalariciresinol, Arctigenin, Trache Rogenin, Mediolesinol, 1-Acetoxypinolesinol, Sesamin, Sesamolin, Sesamolinol, Sesaminol, Sesamol, 7-Hydroxymatairesinol, Isohydroxymatairesinol, Secoisolaralicyresinol-Sesquilignan, Cyclolariciresinol, 7-Oxomatailesinol, Todrolactol a, Conidendrin, 7-Hydroxysecoisolaralicyresinol, Nortrachelogenin, Lari Silesinol-sesquilignan, anhydro-secoisolaricilesinol, dimethylmateilesinol, episesamine, episesaminol, enterodiol, enterolactone, sesaminol 2-o-triglucoside, cisandrin, gomisin d, cisandrol b, tigroylgomisin h, cisanghenol, cisanthelin a, gomisin m2, deoxycisandrin, cisandrin b, cisandrin c, 2-hydroxyenterodiol, 4-hydroxyenterodiol, 6-hydroxyenterodiol, 2-hydroxyenterolactone, 4-hydroxyenterolactone, 6-hydroxyenterolactone, 2'-hydroxyenterolactone, 4'-hydroxyenterolactone, 6'-hydroxyenterolactone, 5-hydroxyenterolactone, 7-hydroxyenterolactone, 4-ethylbenzoic acid, glycine, 1,3,5-Trimethoxybenzene, vanilloyl glycine, 4-vinylguaiacol, 4-ethylguaiacol, 4-vinylsyringol, 5-heneycosenylresorcinol, 5-heneycosylresorcinol, 5-heptadecylresorcinol, 5-nonadecenylresorcinol, 5-nonadecenylresorcinol, 5-pentacosenylresorcinol, 5-pentacosylresorcinol, 5-pentadecylresorcinol, 5- Tricosenylresorcinol, 5-tricosylresorcinol, 3-methylcatechol, 4-methylcatechol, 4-ethylcatechol, 4-vinylphenol, 4-ethylphenol, curcumin, demethoxycurcumin, bisdemethoxycurcumin, bergapten, psoralen, xanthototoxin, isopimpinelin, syringal aldehyde, protocatecaldehyde, vanillin, 4-hydroxybenzaldehyde, gallic acid aldehyde, p-anisaldehyde, vanillin 4-sulfate, 3-methoxyacetophenone, 2,3-dihydroxy-1-guaiasylpropanone, paeonol, 2,4-dihydroxyacetophenone 5-sulfate, 2-hydroxy-4-methoxyacetophenone 5-sulfate, resacetophenone, noraciliol, ferulaldehyde, synapaldehyde, coumarin, melane, scopoletin, esculetin , Esculin, Umbelliferone, 4-Hydroxycoumarin, Uroritin A 3,8-O-Diglucuronide, Uroritin A, Uroritin B, Uroritin B 3-O-Glucuronide, Uroritin C, 2-Methoxy-5-Prop-1-Enylphenol, Anethole, Eugenol, Acetyl Eugenol, [6]-Gingerol, Estragol, Guaiacol, Juglon, 1,4-Naphthoquinone, Carnosic Acid, Rosmanol, Carnosol, Epirosmanol, Rosmazial, Thymol, Carvacrol, Tyrosol, Hydroxytyrosol, 3,4-DHPEA-AC, P-HPEA-AC, Oleuropein, Demethyloleuropein, 3,4-DHPEA-EA, Ligustroside, 3,4-dhpea-eda, hydroxytyrosol 4-o-glucoside, oleoside dimethyl ester, oleoside 11-methyl ester, p-hpea-eda, p-hpea-ea, oleuropein-aglycone, ligstroside-aglycone, tyrosol 4-sulfate, coumestrol, catechol, pyrogallol, florin, phenol, arbutin, 3,4-dihydroxyphenyl glycol, lithospermic acid, salvianolic acid b, salvianolic acid c, salvianolic acid d, salvianolic acid g, isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate, ellagic acid glucoside, Protocatechuic acid, gallic acid, vanillic acid, gentisic acid, ellagic acid, 4-hydroxybenzoic acid, syringic acid, 5-o-galloylquinic acid, ellagic acid arabinoside, ellagic acid acetyl-xyloside, ellagic acid acetyl-arabinoside, benzoic acid, 2-hydroxybenzoic acid, 3-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 4-o-glucoside of 4-hydroxybenzoic acid, protocatechuic acid 4-o-glucoside, gallic acid 4-o-glucoside, 3,5-dihydroxybenzoic acid, 2,6-dihydroxybenzoic acid, 3-o-gallate of gallic acid, ethyl gallate, valoneic acid (acid) dilactone, galloyl glucose, lambertianin c, sanguine h-6, punicalagin, gallagic acid, 3-o-methylgallic acid, 4-o-methylgallic acid, 3,4-o-dimethylgallic acid, punicalin, 4-hydroxyhippuric acid, 3-hydroxyhippuric acid, 2-hydroxyhippuric acid, hippuric acid, paeoniflorin, vanillic acid 4-sulfate, p-coumaric acid, 5-p-coumaroylquinic acid, 4-p-coumaroylquinic acid, caffeic acid, feruloyl glucose, ferulic acid, caffeoyltartaric acid, rosmarinic acid, o-coumaric acid, m-coumaric acid, sinapic acid, p-coumaroyl glucose, p-coumaroylquinic acid, 3-caffeoylquinic acid, beruvascoside, 4-caffeoylquinic acid, p-coumaroyltartaric acid, 2,5-di-s-glutathioneylcaphthalate, feruloyl tartaric acid, ethyl caffeate, cinnamoyl glucose, 5-caffeoylquinic acid, 3-p-coumaroylquinic acid, 2-s-glutathioneylcaphthalate, 5-feruloylquinic acid, 4-feruloylquinic acid, 3-feruloylquinic acid, 5-sinapoylquinic acid, 4-sinapoylquinic acid, 3-sinapoylquinic acid, 3,5-dicaffeoylquinic acid, isoferulic acid, caffeoyl glucose, 4-o-glucoside p-coumarate, 4-o-glucoside caffeate, 4-o-glucoside ferulic acid, p-coumaroyl tartaric acid glucoside, ethyl p-coumarate, hydroxycaffeate, Chicoric acid, 5-5'-dehydrodiferulic acid, 5-8'-dehydrodiferulic acid, 1,2-disinapoylgenthiobiose, 1-sinapoyl-2-feruloylgenthiobiose, 1,2-diferuloylgenthiobiose, 1,2,2'-tricinapoylgenthiobiose, 1,2'-disinapoyl-2-feruloylgenthiobiose, 1-sinapoyl-2,2'-diferuloylgenthiobiose, 1,2,2'-triferuloylgenthiobiose, 8-o-4'-dehydrodiferulic acid, 5-8'-benzofurandehydrodiferulic acid, 3,4-dicaffeoylquinic acid, 3,4-diferuloylquinic acid, 3,5-diferuloylquinic acid, 1,5-Dicaffeoylquinic acid, 4,5-Dicaffeoylquinic acid, Abenanthramide 2p, Abenanthramide 2c, Abenanthramide 2f, p-Coumaroylmalic acid, p-Coumaroylglycolic acid, Cinnamic acid, Caffeoylaspartic acid, p-Coumaroyltyrosine, Synapin, Abenanthramide K, 24-Methylcholesterol ferlate, 24-Methylcholesterol ferlate, 24-Methyllatosterol ferlate, Stigmamanol ferlate, Sitosterol ferlate, Scotenol (s Chottenol ferlate, 24-methylenecholestanol ferlate, 3-o-methylrosmarinic acid, feruloyl glycine, isoferulic acid 3-o-glucuronide, isoferulic acid 3-sulfate, ferulic acid 4-sulfate, ferulic acid 4-o-glucuronide, caffeic acid 4-sulfate, caffeic acid 3-sulfate, feruloyl cl-glucuronide, isoferuloyl cl-glucuronide, caffeic acid 3-o-glucuronide, caffeic acid 4-o-glucuronide, caffeyl cl-gluc Ronide, 1,5-diferuloylquinic acid, 1-caffeoyl-5-feruloylquinic acid, 1-feruloyl-5-caffeoylquinic acid, 3,4-dihydroxyphenylacetic acid, 4-hydroxyphenylacetic acid, homovanillic acid, homoveratolic acid, methoxyphenylacetic acid, 3-hydroxyphenylacetic acid, 2-hydroxyphenylacetic acid, phenacetyl (phenacetyl)glycine, phenylacetic acid, 4-hydroxymandelic acid, 2-hydroxy-2-phenylacetic acid, homovanillic acid 4-sulfate, dihydro-p-couma Acid, dihydrocaffeic acid, 3-hydroxy-3-(3-hydroxyphenyl)propionic acid, 3-(3,4-dihydroxyphenyl)-2-methoxypropionic acid, 3-hydroxyphenylpropionic acid, 4-sulfate dihydroferulic acid, 3-o-glucuronide dihydrocaffeic acid, 3-sulfate dihydrocaffeic acid, dihydroferulic acid, 4-o-glucuronide dihydroferulic acid, dihydrosinapic acid, dihydroferuloylglycine, danchence, 3-methoxy-4-hydroxyphenyllactic acid, 3,4-Dihydroxyphenyl lactate methyl ester, hydroxydanchence, 3-phenylpropionic acid, 3-hydroxy-4-methoxyphenyl lactate, 4-hydroxyphenyl-2-propionic acid, 5-(3'-methoxy-4'-hydroxyphenyl)-γ-valerolactone, 4-hydroxy-(3',4'-dihydroxyphenyl)valeric acid, 5-(3',4'-dihydroxyphenyl)-valeric acid, 5-(3',4'-dihydroxyphenyl)-γ-valerolactone, 5- (3',4',5'-trihydroxyphenyl)-γ-valerolactone, 5-(3',5'-dihydroxyphenyl)-γ-valerolactone, 3-hydroxyphenylvaleric acid, 5-(3',5'-dihydroxyphenyl)-γ-valerolactone 3-o-glucuronide, trans-resveratrol, piceatannol, ε-viniferin, pterostilbene, δ-viniferin, palidol, piceatannol 3-o-glucoside, pinosylvin, resveratrol 5-o-glucuronide Glucoside, resveratrol, resveratrol 3-o-glucoside, 3,4,5,4'-tetramethoxystilbene, 3'-hydroxy-3,4,5,4'-tetramethoxystilbene, 4'-hydroxy-3,4,5-trimethoxystilbene, 4-hydroxy-3,5,4'-trimethoxystilbene, cis-resveratrol 3-o-glucuronide, cis-resveratrol 3-sulfate, cis-resveratrol 4'-o-glucuronide, cis-resveratrol Trans-resveratrol 4'-sulfate, trans-resveratrol 3-sulfate, trans-resveratrol 3,5-disulfate, trans-resveratrol 3,4'-disulfate, trans-resveratrol 3-o-glucuronide, trans-resveratrol 3-sulfate, trans-resveratrol 4'-o-glucuronide, trans-resveratrol 4'-sulfate, dihydroresveratrol, tannic acid, or any combination thereof.

[0210] In some embodiments, at least one polyphenol is selected from tannic acid, proanthocyanidin, punicalagin, ellagic acid, theaflavin 3,3'-digalate, pentagalloyl glucose, catechin gallate, 1,3,6-tri-O-galloyl-beta-D-glucose, terimagrandin II, 1,2,3,6-tetragalloyl glucose, or geraniin.

[0211] In some embodiments, at least one polyphenol is tannic acid.

[0212] In some embodiments, lipid nanoparticles may contain polyphenols and nucleic acids in a specific ratio (weight / weight).

[0213] In some embodiments, lipid nanoparticles containing polyphenols 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 Polyphenols and nucleic acids may be included in a ratio of approximately 8.5:1, or approximately 9:1, or approximately 9.5:1, or approximately 10:1, or approximately 10.5:1, or approximately 11:1, or approximately 11.5:1, or approximately 12:1, or approximately 12.5:1, or approximately 13:1, or approximately 13.5:1, or approximately 14:1, or approximately 14.5:1, or approximately 15:1, or approximately 15.5:1, or approximately 16:1, or approximately 16.5:1, or approximately 17:1, or approximately 17.5:1, or approximately 18:1, or approximately 18.5:1, or approximately 19:1, or approximately 19.5:1, or approximately 20:1 (polyphenols:nucleic acids).

[0214] In some embodiments, lipid nanoparticles comprising tannic acid and at least one nucleic acid may contain tannic acid and nucleic acid in 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 (weight / weight)). In some embodiments, the at least one nucleic acid may include DNA.

[0215] In some embodiments, lipid nanoparticles may contain polyphenols and lipids in a specific ratio (weight / weight).

[0216] In some embodiments, lipid nanoparticles, including those shown in Tables 1A and 1B, may contain polyphenols and lipids in polyphenol:lipid ratios of approximately 0.08:1, 0.1:1, 0.15:1, 0.17:1, 0.2:1, 0.22:1, 0.25:1, or 0.3:1 by weight / weight.

[0217] Exemplary LNPs in this disclosure

[0218] The following is an example LNP of the present disclosure.

[0219] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, in molar ratio, at least 35% to about 50% of at least one compound of formula (I), in molar ratio at least 37.5% to about 56% of at least one structural lipid, in molar ratio at least 5% to about 12% of at least one phospholipid, and in molar ratio at least 1% to about 2.5% 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, in molar ratio at least 25% to about 60% of at least one compound of formula (I), in molar ratio at least 27.5% to about 66% of at least one structural lipid, in molar ratio at least 0.1% to about 22% of at least one phospholipid, and in molar ratio at least 0.1% to about 12.5% ​​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 disclosure provides lipid nanoparticles comprising, in molar ratio, about 30% to about 55% of at least one compound of formula (I), in molar ratio, about 32.5% to about 61% of at least one structural lipid, in molar ratio, about 1% to about 17% of at least one phospholipid, and in molar ratio, about 1% to about 7.5% of at least one PEGylated lipid, wherein 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 may be about 40:1 to about 80:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be approximately 60:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be approximately 70:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be approximately 80:1 (w / w).

[0220] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 35% of at least one compound of formula (I), by molar ratio at least 56% of at least one structural lipid, by molar ratio at least 7.5% of at least one phospholipid, and by molar ratio at least 1.5% 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, by molar ratio at least 25% to about 45% of at least one compound of formula (I), by molar ratio at least 46% to about 66% of at least one structural lipid, by molar ratio at least 0.1% to about 17.5% of at least one phospholipid, and by molar ratio at least 0.1% to about 11.5% 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 disclosure provides lipid nanoparticles comprising, in molar ratio about 30% to about 40% at least one compound of formula (I), in molar ratio about 51% to about 61% at least one structural lipid, in molar ratio about 1% to about 12.5% ​​at least one phospholipid, and in molar ratio about 1% to about 6.5% at least one PEGylated lipid, wherein 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 may be about 40:1 (w / w) to about 60:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w).

[0221] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 50% of at least one compound of formula (I), by molar ratio at least 38.5% of at least one structural lipid, by molar ratio at least 10% of at least one phospholipid, and by molar ratio at least 1.5% 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, by molar ratio at least 40% to about 60% of at least one compound of formula (I), by molar ratio at least 28.5% to about 48.5% of at least one structural lipid, by molar ratio at least 0.1% to about 20% of at least one phospholipid, and by molar ratio at least 0.1% to about 11.5% 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 disclosure provides lipid nanoparticles comprising, in molar ratio about 45% to about 55%, at least one compound of formula (I), in molar ratio about 33.5% to about 43.5%, at least one structural lipid, in molar ratio about 1% to about 15%, at least one phospholipid, and in molar ratio about 1% to about 6.5%, at least one PEGylated lipid, wherein 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 may be about 50:1 (w / w) to about 90:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 60:1 (w / w) or about 80:1 (w / w).

[0222] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 50% of at least one compound of formula (I), by molar ratio at least 37.5% of at least one structural lipid, by molar ratio at least 10% of at least one phospholipid, and by molar ratio at least 2.5% 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, by molar ratio at least 40% to about 60% of at least one compound of formula (I), by molar ratio at least 27.5% to about 47.5% of at least one structural lipid, by molar ratio at least 0.1% to about 20% of at least one phospholipid, and by molar ratio at least 0.1% to about 12.5% ​​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 disclosure provides lipid nanoparticles comprising, in molar ratio about 45% to about 55%, at least one compound of formula (I), in molar ratio about 32.5% to about 42.5%, at least one structural lipid, in molar ratio about 1% to about 15%, at least one phospholipid, and in molar ratio about 1% to about 7.5%, at least one PEGylated lipid, wherein 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 may be about 30:1 (w / w) to about 50:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 (w / w).

[0223] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 40% of at least one compound of formula (I), by molar ratio at least 52.5% of at least one structural lipid, by molar ratio at least 5% of at least one phospholipid, and by molar ratio at least 2.5% 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, by molar ratio at least 30% to about 50% of at least one compound of formula (I), by molar ratio at least 42.5% to about 62.5% of at least one structural lipid, by molar ratio at least 0.1% to about 15% of at least one phospholipid, and by molar ratio at least 0.1% to about 12.5% ​​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 disclosure provides lipid nanoparticles comprising, in molar ratio about 35% to about 45% at least one compound of formula (I), in molar ratio about 47.5% to about 57.5% at least one structural lipid, in molar ratio about 1% to about 10% at least one phospholipid, and in molar ratio about 1% to about 7.5% at least one PEGylated lipid, wherein at least one nucleic acid comprises at least one RNA molecule (e.g., 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 may be about 30:1 (w / w) to about 50:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 (w / w).

[0224] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 45% of at least one compound of formula (I), by molar ratio at least 45.5% of at least one structural lipid, by molar ratio at least 7.5% of at least one phospholipid, and by molar ratio at least 2% 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, by molar ratio at least 35% to about 55% of at least one compound of formula (I), by molar ratio at least 35.5% to about 55.5% of at least one structural lipid, by molar ratio at least 0.1% to about 17.5% of at least one phospholipid, and by molar ratio at least 0.1% to about 12% 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 disclosure provides lipid nanoparticles comprising, in molar ratio about 40% to about 50% at least one compound of formula (I), in molar ratio about 40.5% to about 50.5% at least one structural lipid, in molar ratio about 1% to about 12.5% ​​at least one phospholipid, and in molar ratio about 1% to about 7% at least one PEGylated lipid, wherein 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 may be about 40:1 (w / w) to about 60:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w).

[0225] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 43.17% of at least one compound of formula (I), by molar ratio at least 43.17% of at least one structural lipid, by molar ratio at least 11.96% of at least one phospholipid, and by molar ratio at least 1.7% 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, by molar ratio at least 33.17% to about 53.17% of at least one compound of formula (I), by molar ratio at least 33.17% to about 53.17% of at least one structural lipid, by molar ratio at least 1.96% to about 21.96% of at least one phospholipid, and by molar ratio at least 0.1% to about 11.7% 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 disclosure provides lipid nanoparticles comprising, in molar ratio about 38.17% to about 48.17%, at least one compound of formula (I), in molar ratio about 38.17% to about 48.17%, at least one structural lipid, in molar ratio about 6.96% to about 16.96%, at least one phospholipid, and in molar ratio about 1% to about 6.7%, at least one PEGylated lipid, wherein at least one nucleic acid comprises at least one RNA molecule (e.g., 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 may be about 70:1 (w / w) to about 90:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 80:1 (w / w).

[0226] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 45% of at least one compound of formula (I), by molar ratio at least 43% of at least one structural lipid, by molar ratio at least 10% of at least one phospholipid, and by molar ratio at least 2% 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, by molar ratio at least 35% to about 55% of at least one compound of formula (I), by molar ratio at least 33% to about 53% of at least one structural lipid, by molar ratio at least 0.1% to about 20% of at least one phospholipid, and by molar ratio at least 0.1% to about 12% 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 disclosure provides lipid nanoparticles comprising, in molar ratio, about 40% to about 50% of at least one compound of formula (I), in molar ratio, about 38% to about 48% of at least one structural lipid, in molar ratio, about 1% to about 15% of at least one phospholipid, and in molar ratio, about 1% to about 7% of at least one PEGylated lipid, wherein 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 may be about 70:1 (w / w) to about 90:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 80:1 (w / w).

[0227] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 45% of at least one compound of formula (I), by molar ratio, at least 46% of at least one structural lipid, by molar ratio, at least 7.5% of at least one phospholipid, and by molar ratio, at least 1.5% 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, by molar ratio, at least 35% to about 55% of at least one compound of formula (I), by molar ratio, at least 36% to about 56% of at least one structural lipid, by molar ratio, at least 0.1% to about 17.5% of at least one phospholipid, and by molar ratio, at least 0.1% to about 11.5% 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 disclosure provides lipid nanoparticles comprising, in molar ratio about 40% to about 50% at least one compound of formula (I), in molar ratio about 41% to about 51% at least one structural lipid, in molar ratio about 1% to about 12.5% ​​at least one phospholipid, and in molar ratio about 1% to about 6.5% at least one PEGylated lipid, wherein 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 may be about 50:1 (w / w) to about 70:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 60:1 (w / w).

[0228] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 50% of at least one compound of formula (I), by molar ratio at least 39% of at least one structural lipid, by molar ratio at least 10% of at least one phospholipid, and by molar ratio at least 1% 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, by molar ratio at least 40% to about 60% of at least one compound of formula (I), by molar ratio at least 29% to about 49% of at least one structural lipid, by molar ratio at least 0.1% to about 20% of at least one phospholipid, and by molar ratio at least 0.1% to about 11% 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 disclosure provides lipid nanoparticles comprising, in molar ratio, about 45% to about 55% of at least one compound of formula (I), in molar ratio, about 34% to about 44% of at least one structural lipid, in molar ratio, about 1% to about 15% of at least one phospholipid, and in molar ratio, about 1% to about 6% of at least one PEGylated lipid, wherein 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 may be about 50:1 (w / w) to about 70:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 60:1 (w / w).

[0229] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 50% of at least one compound of formula (I), by molar ratio at least 41.5% of at least one structural lipid, by molar ratio at least 7.5% of at least one phospholipid, and by molar ratio at least 1% 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, by molar ratio at least 40% to about 60% of at least one compound of formula (I), by molar ratio at least 31.5% to about 51.5% of at least one structural lipid, by molar ratio at least 0.1% to about 17.5% of at least one phospholipid, and by molar ratio at least 0.1% to about 11% 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 disclosure provides lipid nanoparticles comprising, in molar ratio about 45% to about 55%, at least one compound of formula (I), in molar ratio about 36.5% to about 46.5%, at least one structural lipid, in molar ratio about 1% to about 12.5%, at least one phospholipid, and in molar ratio about 1% to about 6%, at least one PEGylated lipid, wherein 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 may be about 40:1 (w / w) to about 60:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w).

[0230] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 40% of at least one compound of formula (I), by molar ratio at least 48.5% of at least one structural lipid, by molar ratio at least 10% of at least one phospholipid, and by molar ratio at least 1.5% 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, by molar ratio at least 30% to about 50% of at least one compound of formula (I), by molar ratio at least 38.5% to about 58.5% of at least one structural lipid, by molar ratio at least 0.1% to about 20% of at least one phospholipid, and by molar ratio at least 0.1% to about 11.5% 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 disclosure provides lipid nanoparticles comprising, in molar ratio about 35% to about 45% at least one compound of formula (I), in molar ratio about 43.5% to about 53.5% at least one structural lipid, in molar ratio about 1% to about 10% at least one phospholipid, and in molar ratio about 1% to about 6.5% at least one PEGylated lipid, wherein at least one nucleic acid comprises at least one RNA molecule (e.g., 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 may be about 70:1 (w / w) to about 90:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 80:1 (w / w).

[0231] In some embodiments, lipid nanoparticles are provided, comprising at least one compound of formula (II) in molar ratios of about 35% to about 50%, at least one structural lipid in molar ratios of about 37% to about 59%, at least one phospholipid in molar ratios of about 5% to about 10%, and at least one PEGylated lipid in molar ratios of about 1% to about 3%, wherein at least one nucleic acid comprises at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the disclosure provides lipid nanoparticles comprising at least one compound of formula (II) in molar ratios of about 25% to about 60%, at least one structural lipid in molar ratios of about 27% to about 69%, at least one phospholipid in molar ratios of about 0.1% to about 20%, and at least one PEGylated lipid in molar ratios of about 0.1% to about 13%, wherein at least one nucleic acid comprises at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio about 30% to about 55% at least one compound of formula (II), in molar ratio about 32% to about 64% at least one structural lipid, in molar ratio about 1% to about 15% at least one phospholipid, and in molar ratio about 0.5% to about 8% at least one PEGylated lipid, wherein at least one nucleic acid comprises at least one RNA molecule (e.g., 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 may be about 40:1 to about 80:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be approximately 60:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be approximately 70:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be approximately 80:1 (w / w).

[0232] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 35% of at least one compound of formula (II), by molar ratio at least 59% of at least one structural lipid, by molar ratio at least 5% of at least one phospholipid, and by molar ratio at least 1% 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, by molar ratio at least 25% to about 45% of at least one compound of formula (II), by molar ratio at least 49% to about 69% of at least one structural lipid, by molar ratio at least 0.1% to about 15% of at least one phospholipid, and by molar ratio at least 0.1% to about 11% 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 disclosure provides lipid nanoparticles comprising, in molar ratio, about 30% to about 40% of at least one compound of formula (II), in molar ratio, about 54% to about 64% of at least one structural lipid, in molar ratio, about 1% to about 10% of at least one phospholipid, and in molar ratio, about 1% to about 6% of at least one PEGylated lipid, wherein 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 may be about 50:1 (w / w) to about 70:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 60:1 (w / w).

[0233] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 50% of at least one compound of formula (II), by molar ratio at least 41.5% of at least one structural lipid, by molar ratio at least 7.5% of at least one phospholipid, and by molar ratio at least 1% 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, by molar ratio at least 40% to about 60% of at least one compound of formula (II), by molar ratio at least 31.5% to about 51.5% of at least one structural lipid, by molar ratio at least 0.1% to about 17.5% of at least one phospholipid, and by molar ratio at least 0.1% to about 11% 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 disclosure provides lipid nanoparticles comprising, in molar ratio about 45% to about 55%, at least one compound of formula (II), in molar ratio about 36.5% to about 46.5%, at least one structural lipid, in molar ratio about 1% to about 12.5%, at least one phospholipid, and in molar ratio about 1% to about 6%, at least one PEGylated lipid, wherein 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 may be about 50:1 (w / w) to about 70:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 60:1 (w / w).

[0234] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 50% of at least one compound of formula (II), by molar ratio at least 39% of at least one structural lipid, by molar ratio at least 10% of at least one phospholipid, and by molar ratio at least 1% 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, by molar ratio at least 40% to about 60% of at least one compound of formula (II), by molar ratio at least 29% to about 49% of at least one structural lipid, by molar ratio at least 0.1% to about 20% of at least one phospholipid, and by molar ratio at least 0.1% to about 11% 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 disclosure provides lipid nanoparticles comprising, in molar ratio about 45% to about 55%, at least one compound of formula (II), in molar ratio about 34% to about 44%, at least one structural lipid, in molar ratio about 1% to about 10%, at least one phospholipid, and in molar ratio about 1% to about 6%, at least one PEGylated lipid, wherein 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 may be about 40:1 (w / w) to about 60:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w).

[0235] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 42.5% of at least one compound of formula (II), by molar ratio at least 51.5% of at least one structural lipid, by molar ratio at least 5% of at least one phospholipid, and by molar ratio at least 1% 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, by molar ratio at least 32.5% to about 52.5% of at least one compound of formula (II), by molar ratio at least 41.5% to about 61.5% of at least one structural lipid, by molar ratio at least 0.1% to about 15% of at least one phospholipid, and by molar ratio at least 0.1% to about 11% 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 disclosure provides lipid nanoparticles comprising, in molar ratio about 37.5% to about 47.5%, at least one compound of formula (II), in molar ratio about 46.5% to about 56.5%, at least one structural lipid, in molar ratio about 1% to about 10%, at least one phospholipid, and in molar ratio about 1% to about 6%, at least one PEGylated lipid, wherein 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 may be about 30:1 (w / w) to about 50:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 (w / w).

[0236] In some embodiments, lipid nanoparticles are provided, comprising at least one compound of formula (II') in a molar ratio of about 35% to about 50%, at least one structural lipid in a molar ratio of about 37% to about 59%, at least one phospholipid in a molar ratio of about 5% to about 10%, and at least one PEGylated lipid in a molar ratio of about 1% to about 3%, wherein at least one nucleic acid comprises at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the disclosure provides lipid nanoparticles comprising at least one compound of formula (II') in a molar ratio of about 25% to about 60%, at least one structural lipid in a molar ratio of about 27% to about 69%, at least one phospholipid in a molar ratio of about 0.1% to about 20%, and at least one PEGylated lipid in a molar ratio of about 0.1% to about 13%, wherein at least one nucleic acid comprises at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio about 30% to about 55% at least one compound of formula (II'), in molar ratio about 32% to about 64% at least one structural lipid, in molar ratio about 1% to about 15% at least one phospholipid, and in molar ratio about 0.5% to about 8% at least one PEGylated lipid, wherein at least one nucleic acid comprises at least one RNA molecule (e.g., 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 may be about 40:1 to about 80:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be approximately 60:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be approximately 70:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be approximately 80:1 (w / w).

[0237] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 35% of at least one compound of formula (II'), by molar ratio at least 59% of at least one structural lipid, by molar ratio at least 5% of at least one phospholipid, and by molar ratio at least 1% 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, by molar ratio at least 25% to about 45% of at least one compound of formula (II'), by molar ratio at least 49% to about 69% of at least one structural lipid, by molar ratio at least 0.1% to about 15% of at least one phospholipid, and by molar ratio at least 0.1% to about 11% 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 disclosure provides lipid nanoparticles comprising, in molar ratio about 30% to about 40% at least one compound of formula (II'), in molar ratio about 54% to about 64% at least one structural lipid, in molar ratio about 1% to about 10% at least one phospholipid, and in molar ratio about 1% to about 6% at least one PEGylated lipid, wherein at least one nucleic acid comprises at least one RNA molecule (e.g., 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 may be about 50:1 (w / w) to about 70:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 60:1 (w / w).

[0238] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 50% of at least one compound of formula (II'), by molar ratio at least 41.5% of at least one structural lipid, by molar ratio at least 7.5% of at least one phospholipid, and by molar ratio at least 1% 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, by molar ratio at least 40% to about 60% of at least one compound of formula (II'), by molar ratio at least 31.5% to about 51.5% of at least one structural lipid, by molar ratio at least 0.1% to about 17.5% of at least one phospholipid, and by molar ratio at least 0.1% to about 11% 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 disclosure provides lipid nanoparticles comprising, in molar ratio about 45% to about 55%, at least one compound of formula (II'), in molar ratio about 36.5% to about 46.5%, at least one structural lipid, in molar ratio about 1% to about 12.5%, at least one phospholipid, and in molar ratio about 1% to about 6%, at least one PEGylated lipid, wherein 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 may be about 50:1 (w / w) to about 70:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 60:1 (w / w).

[0239] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 50% of at least one compound of formula (II'), by molar ratio at least 39% of at least one structural lipid, by molar ratio at least 10% of at least one phospholipid, and by molar ratio at least 1% 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, by molar ratio at least 40% to about 60% of at least one compound of formula (II'), by molar ratio at least 29% to about 49% of at least one structural lipid, by molar ratio at least 0.1% to about 20% of at least one phospholipid, and by molar ratio at least 0.1% to about 11% 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 disclosure provides lipid nanoparticles comprising, in molar ratio about 45% to about 55%, at least one compound of formula (II'), in molar ratio about 34% to about 44%, at least one structural lipid, in molar ratio about 1% to about 10%, at least one phospholipid, and in molar ratio about 1% to about 6%, at least one PEGylated lipid, wherein 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 may be about 40:1 (w / w) to about 60:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w).

[0240] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 42.5% of at least one compound of formula (II'), by molar ratio at least 51.5% of at least one structural lipid, by molar ratio at least 5% of at least one phospholipid, and by molar ratio at least 1% 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, by molar ratio at least 32.5% to about 52.5% of at least one compound of formula (II'), by molar ratio at least 41.5% to about 61.5% of at least one structural lipid, by molar ratio at least 0.1% to about 15% of at least one phospholipid, and by molar ratio at least 0.1% to about 11% 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 disclosure provides lipid nanoparticles comprising, in molar ratio, about 37.5% to about 47.5% of at least one compound of formula (II'), in molar ratio, about 46.5% to about 56.5% of at least one structural lipid, in molar ratio, about 1% to about 10% of at least one phospholipid, and in molar ratio, about 1% to about 6% of at least one PEGylated lipid, wherein 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 may be about 30:1 (w / w) to about 50:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 (w / w).

[0241] In some embodiments, the nucleic acid molecule is a DNA molecule. Accordingly, the disclosure provides lipid nanoparticles comprising, in molar ratio, about 35% to about 50% of at least one compound of formula (I), in molar ratio, about 37.5% to about 56% of at least one structural lipid, in molar ratio, about 5% to about 12% of at least one phospholipid, and in molar ratio, about 1% to about 2.5% of at least one PEGylated lipid, wherein at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio, about 25% to about 60% of at least one compound of formula (I), in molar ratio, about 27.5% to about 66% of at least one structural lipid, in molar ratio, about 0.1% to about 22% of at least one phospholipid, and in molar ratio, about 0.1% to about 12.5% ​​of at least one PEGylated lipid, wherein at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio, about 30% to about 55% of at least one compound of formula (I), in molar ratio, about 32.5% to about 61% of at least one structural lipid, in molar ratio, about 1% to about 17% of at least one phospholipid, and in molar ratio, about 1% to about 7.5% of at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 to about 80:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be approximately 50:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be approximately 60:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be approximately 70:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be approximately 80:1 (w / w).

[0242] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 35% of at least one compound of formula (I), by molar ratio at least 56% of at least one structural lipid, by molar ratio at least 7.5% of at least one phospholipid, and by molar ratio at least 1.5% 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, by molar ratio at least 25% to about 45% of at least one compound of formula (I), by molar ratio at least 46% to about 66% of at least one structural lipid, by molar ratio at least 0.1% to about 17.5% of at least one phospholipid, and by molar ratio at least 0.1% to about 11.5% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio, about 30% to about 40% of at least one compound of formula (I), in molar ratio, about 51% to about 61% of at least one structural lipid, in molar ratio, about 1% to about 12.5% ​​of at least one phospholipid, and in molar ratio, about 1% to about 6.5% of at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 (w / w) to about 60:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w).

[0243] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 50% of at least one compound of formula (I), by molar ratio at least 38.5% of at least one structural lipid, by molar ratio at least 10% of at least one phospholipid, and by molar ratio at least 1.5% 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, by molar ratio at least 40% to about 60% of at least one compound of formula (I), by molar ratio at least 28.5% to about 48.5% of at least one structural lipid, by molar ratio at least 0.1% to about 20% of at least one phospholipid, and by molar ratio at least 0.1% to about 11.5% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the Disclosure provides lipid nanoparticles comprising, in molar ratio about 45% to about 55%, at least one compound of formula (I), in molar ratio about 33.5% to about 43.5%, at least one structural lipid, in molar ratio about 1% to about 15%, at least one phospholipid, and in molar ratio about 1% to about 6.5%, at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w) to about 90:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 60:1 (w / w) or about 80:1 (w / w).

[0244] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 50% of at least one compound of formula (I), by molar ratio at least 37.5% of at least one structural lipid, by molar ratio at least 10% of at least one phospholipid, and by molar ratio at least 2.5% 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, by molar ratio at least 40% to about 60% of at least one compound of formula (I), by molar ratio at least 27.5% to about 47.5% of at least one structural lipid, by molar ratio at least 0.1% to about 20% of at least one phospholipid, and by molar ratio at least 0.1% to about 12.5% ​​of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio about 45% to about 55%, at least one compound of formula (I), in molar ratio about 32.5% to about 42.5%, at least one structural lipid, in molar ratio about 1% to about 15%, at least one phospholipid, and in molar ratio about 1% to about 7.5%, at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 30:1 (w / w) to about 50:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 (w / w).

[0245] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 40% of at least one compound of formula (I), by molar ratio at least 52.5% of at least one structural lipid, by molar ratio at least 5% of at least one phospholipid, and by molar ratio at least 2.5% 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, by molar ratio at least 30% to about 50% of at least one compound of formula (I), by molar ratio at least 42.5% to about 62.5% of at least one structural lipid, by molar ratio at least 0.1% to about 15% of at least one phospholipid, and by molar ratio at least 0.1% to about 12.5% ​​of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio, about 35% to about 45% of at least one compound of formula (I), in molar ratio, about 47.5% to about 57.5% of at least one structural lipid, in molar ratio, about 1% to about 10% of at least one phospholipid, and in molar ratio, about 1% to about 7.5% of at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 30:1 (w / w) to about 50:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 (w / w).

[0246] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 45% of at least one compound of formula (I), by molar ratio at least 45.5% of at least one structural lipid, by molar ratio at least 7.5% of at least one phospholipid, and by molar ratio at least 2% 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, by molar ratio at least 35% to about 55% of at least one compound of formula (I), by molar ratio at least 35.5% to about 55.5% of at least one structural lipid, by molar ratio at least 0.1% to about 17.5% of at least one phospholipid, and by molar ratio at least 0.1% to about 12% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio, about 40% to about 50% of at least one compound of formula (I), in molar ratio, about 40.5% to about 50.5% of at least one structural lipid, in molar ratio, about 1% to about 12.5% ​​of at least one phospholipid, and in molar ratio, about 1% to about 7% of at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 (w / w) to about 60:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w).

[0247] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 43.17% of at least one compound of formula (I), by molar ratio, at least 43.17% of at least one structural lipid, by molar ratio, at least 11.96% of at least one phospholipid, and by molar ratio, at least 1.7% 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, by molar ratio, at least 33.17% to about 53.17% of at least one compound of formula (I), by molar ratio, at least 33.17% to about 53.17% of at least one structural lipid, by molar ratio, at least 1.96% to about 21.96% of at least one phospholipid, and by molar ratio, at least 0.1% to about 11.7% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio, about 38.17% to about 48.17% of at least one compound of formula (I), in molar ratio, about 38.17% to about 48.17% of at least one structural lipid, in molar ratio, about 6.96% to about 16.96% of at least one phospholipid, and in molar ratio, about 1% to about 6.7% of at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 70:1 (w / w) to about 90:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 80:1 (w / w).

[0248] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 45% of at least one compound of formula (I), by molar ratio, at least 43% of at least one structural lipid, by molar ratio, at least 10% of at least one phospholipid, and by molar ratio, at least 2% 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, by molar ratio, at least 35% to about 55% of at least one compound of formula (I), by molar ratio, at least 33% to about 53% of at least one structural lipid, by molar ratio, at least 0.1% to about 20% of at least one phospholipid, and by molar ratio, at least 0.1% to about 12% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio, about 40% to about 50% of at least one compound of formula (I), in molar ratio, about 38% to about 48% of at least one structural lipid, in molar ratio, about 1% to about 15% of at least one phospholipid, and in molar ratio, about 1% to about 7% of at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 70:1 (w / w) to about 90:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 80:1 (w / w).

[0249] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 45% of at least one compound of formula (I), by molar ratio, at least 46% of at least one structural lipid, by molar ratio, at least 7.5% of at least one phospholipid, and by molar ratio, at least 1.5% 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, by molar ratio, at least 35% to about 55% of at least one compound of formula (I), by molar ratio, at least 36% to about 56% of at least one structural lipid, by molar ratio, at least 0.1% to about 17.5% of at least one phospholipid, and by molar ratio, at least 0.1% to about 11.5% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio, about 40% to about 50% of at least one compound of formula (I), in molar ratio, about 41% to about 51% of at least one structural lipid, in molar ratio, about 1% to about 12.5% ​​of at least one phospholipid, and in molar ratio, about 1% to about 6.5% of at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w) to about 70:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 60:1 (w / w).

[0250] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 50% of at least one compound of formula (I), by molar ratio, at least 39% of at least one structural lipid, by molar ratio, at least 10% of at least one phospholipid, and by molar ratio, at least 1% 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, by molar ratio, at least 40% to about 60% of at least one compound of formula (I), by molar ratio, at least 29% to about 49% of at least one structural lipid, by molar ratio, at least 0.1% to about 20% of at least one phospholipid, and by molar ratio, at least 0.1% to about 11% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio, about 45% to about 55% of at least one compound of formula (I), in molar ratio, about 34% to about 44% of at least one structural lipid, in molar ratio, about 1% to about 15% of at least one phospholipid, and in molar ratio, about 1% to about 6% of at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w) to about 70:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 60:1 (w / w).

[0251] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 50% of at least one compound of formula (I), by molar ratio at least 41.5% of at least one structural lipid, by molar ratio at least 7.5% of at least one phospholipid, and by molar ratio at least 1% 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, by molar ratio at least 40% to about 60% of at least one compound of formula (I), by molar ratio at least 31.5% to about 51.5% of at least one structural lipid, by molar ratio at least 0.1% to about 17.5% of at least one phospholipid, and by molar ratio at least 0.1% to about 11% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio, about 45% to about 55% of at least one compound of formula (I), in molar ratio, about 36.5% to about 46.5% of at least one structural lipid, in molar ratio, about 1% to about 12.5% ​​of at least one phospholipid, and in molar ratio, about 1% to about 6% of at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 (w / w) to about 60:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w).

[0252] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 40% of at least one compound of formula (I), by molar ratio at least 48.5% of at least one structural lipid, by molar ratio at least 10% of at least one phospholipid, and by molar ratio at least 1.5% 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, by molar ratio at least 30% to about 50% of at least one compound of formula (I), by molar ratio at least 38.5% to about 58.5% of at least one structural lipid, by molar ratio at least 0.1% to about 20% of at least one phospholipid, and by molar ratio at least 0.1% to about 11.5% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio, about 35% to about 45% of at least one compound of formula (I), in molar ratio, about 43.5% to about 53.5% of at least one structural lipid, in molar ratio, about 1% to about 10% of at least one phospholipid, and in molar ratio, about 1% to about 6.5% of at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 70:1 (w / w) to about 90:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 80:1 (w / w).

[0253] In some embodiments, lipid nanoparticles are provided, comprising at least one compound of formula (II) in a molar ratio of about 35% to about 50%, at least one structural lipid in a molar ratio of about 37% to about 59%, at least one phospholipid in a molar ratio of about 5% to about 10%, and at least one PEGylated lipid in a molar ratio of about 1% to about 3%, wherein at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising at least one compound of formula (II) in a molar ratio of about 25% to about 60%, at least one structural lipid in a molar ratio of about 27% to about 69%, at least one phospholipid in a molar ratio of about 0.1% to about 20%, and at least one PEGylated lipid in a molar ratio of about 0.1% to about 13%, wherein at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio, about 30% to about 55% of at least one compound of formula (II), in molar ratio, about 32% to about 64% of at least one structural lipid, in molar ratio, about 1% to about 15% of at least one phospholipid, and in molar ratio, about 0.5% to about 8% of at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 to about 80:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be approximately 50:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be approximately 60:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be approximately 70:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be approximately 80:1 (w / w).

[0254] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 35% of at least one compound of formula (II), by molar ratio at least 59% of at least one structural lipid, by molar ratio at least 5% of at least one phospholipid, and by molar ratio at least 1% 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, by molar ratio at least 25% to about 45% of at least one compound of formula (II), by molar ratio at least 49% to about 69% of at least one structural lipid, by molar ratio at least 0.1% to about 15% of at least one phospholipid, and by molar ratio at least 0.1% to about 11% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio, about 30% to about 40% of at least one compound of formula (II), in molar ratio, about 54% to about 64% of at least one structural lipid, in molar ratio, about 1% to about 10% of at least one phospholipid, and in molar ratio, about 1% to about 6% of at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w) to about 70:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 60:1 (w / w).

[0255] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 50% of at least one compound of formula (II), by molar ratio at least 41.5% of at least one structural lipid, by molar ratio at least 7.5% of at least one phospholipid, and by molar ratio at least 1% 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, by molar ratio at least 40% to about 60% of at least one compound of formula (II), by molar ratio at least 31.5% to about 51.5% of at least one structural lipid, by molar ratio at least 0.1% to about 17.5% of at least one phospholipid, and by molar ratio at least 0.1% to about 11% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio, about 45% to about 55% of at least one compound of formula (II), in molar ratio, about 36.5% to about 46.5% of at least one structural lipid, in molar ratio, about 1% to about 12.5% ​​of at least one phospholipid, and in molar ratio, about 1% to about 6% of at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w) to about 70:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 60:1 (w / w).

[0256] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 50% of at least one compound of formula (II), by molar ratio at least 39% of at least one structural lipid, by molar ratio at least 10% of at least one phospholipid, and by molar ratio at least 1% 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, by molar ratio at least 40% to about 60% of at least one compound of formula (II), by molar ratio at least 29% to about 49% of at least one structural lipid, by molar ratio at least 0.1% to about 20% of at least one phospholipid, and by molar ratio at least 0.1% to about 11% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio about 45% to about 55%, at least one compound of formula (II), in molar ratio about 34% to about 44%, at least one structural lipid, in molar ratio about 1% to about 10%, at least one phospholipid, and in molar ratio about 1% to about 6%, at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 (w / w) to about 60:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w).

[0257] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 42.5% of at least one compound of formula (II), by molar ratio at least 51.5% of at least one structural lipid, by molar ratio at least 5% of at least one phospholipid, and by molar ratio at least 1% 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, by molar ratio at least 32.5% to about 52.5% of at least one compound of formula (II), by molar ratio at least 41.5% to about 61.5% of at least one structural lipid, by molar ratio at least 0.1% to about 15% of at least one phospholipid, and by molar ratio at least 0.1% to about 11% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio, about 37.5% to about 47.5% of at least one compound of formula (II), in molar ratio, about 46.5% to about 56.5% of at least one structural lipid, in molar ratio, about 1% to about 10% of at least one phospholipid, and in molar ratio, about 1% to about 6% of at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 30:1 (w / w) to about 50:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 (w / w).

[0258] In some embodiments, lipid nanoparticles are provided, comprising at least one compound of formula (II') in a molar ratio of about 35% to about 50%, at least one structural lipid in a molar ratio of about 37% to about 59%, at least one phospholipid in a molar ratio of about 5% to about 10%, and at least one PEGylated lipid in a molar ratio of about 1% to about 3%, wherein at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising at least one compound of formula (II') in a molar ratio of about 25% to about 60%, at least one structural lipid in a molar ratio of about 27% to about 69%, at least one phospholipid in a molar ratio of about 0.1% to about 20%, and at least one PEGylated lipid in a molar ratio of about 0.1% to about 13%, wherein at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio, about 30% to about 55% of at least one compound of formula (II'), in molar ratio, about 32% to about 64% of at least one structural lipid, in molar ratio, about 1% to about 15% of at least one phospholipid, and in molar ratio, about 0.5% to about 8% of at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 to about 80:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be approximately 50:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be approximately 60:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be approximately 70:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be approximately 80:1 (w / w).

[0259] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 35% of at least one compound of formula (II'), by molar ratio at least 59% of at least one structural lipid, by molar ratio at least 5% of at least one phospholipid, and by molar ratio at least 1% 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, by molar ratio at least 25% to about 45% of at least one compound of formula (II'), by molar ratio at least 49% to about 69% of at least one structural lipid, by molar ratio at least 0.1% to about 15% of at least one phospholipid, and by molar ratio at least 0.1% to about 11% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio about 30% to about 40%, at least one compound of formula (II'), in molar ratio about 54% to about 64%, at least one structural lipid, in molar ratio about 1% to about 10%, at least one phospholipid, and in molar ratio about 1% to about 6%, at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w) to about 70:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 60:1 (w / w).

[0260] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 50% of at least one compound of formula (II'), by molar ratio at least 41.5% of at least one structural lipid, by molar ratio at least 7.5% of at least one phospholipid, and by molar ratio at least 1% 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, by molar ratio at least 40% to about 60% of at least one compound of formula (II'), by molar ratio at least 31.5% to about 51.5% of at least one structural lipid, by molar ratio at least 0.1% to about 17.5% of at least one phospholipid, and by molar ratio at least 0.1% to about 11% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio about 45% to about 55%, at least one compound of formula (II'), in molar ratio about 36.5% to about 46.5%, at least one structural lipid, in molar ratio about 1% to about 12.5%, at least one phospholipid, and in molar ratio about 1% to about 6%, at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w) to about 70:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 60:1 (w / w).

[0261] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 50% of at least one compound of formula (II'), by molar ratio at least 39% of at least one structural lipid, by molar ratio at least 10% of at least one phospholipid, and by molar ratio at least 1% 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, by molar ratio at least 40% to about 60% of at least one compound of formula (II'), by molar ratio at least 29% to about 49% of at least one structural lipid, by molar ratio at least 0.1% to about 20% of at least one phospholipid, and by molar ratio at least 0.1% to about 11% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio about 45% to about 55%, at least one compound of formula (II'), in molar ratio about 34% to about 44%, at least one structural lipid, in molar ratio about 1% to about 10%, at least one phospholipid, and in molar ratio about 1% to about 6%, at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 (w / w) to about 60:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 50:1 (w / w).

[0262] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise, by molar ratio, at least 42.5% of at least one compound of formula (II'), by molar ratio at least 51.5% of at least one structural lipid, by molar ratio at least 5% of at least one phospholipid, and by molar ratio at least 1% 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, by molar ratio at least 32.5% to about 52.5% of at least one compound of formula (II'), by molar ratio at least 41.5% to about 61.5% of at least one structural lipid, by molar ratio at least 0.1% to about 15% of at least one phospholipid, and by molar ratio at least 0.1% to about 11% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the disclosure provides lipid nanoparticles comprising, in molar ratio, about 37.5% to about 47.5% of at least one compound of formula (II'), in molar ratio, about 46.5% to about 56.5% of at least one structural lipid, in molar ratio, about 1% to about 10% of at least one phospholipid, and in molar ratio, about 1% to about 6% of at least one PEGylated lipid, wherein at least one nucleic acid comprises 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 nanoplasmid. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 30:1 (w / w) to about 50:1 (w / w). In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 40:1 (w / w).

[0263] In some embodiments, lipid nanoparticles are provided comprising, in molar ratio, at least 42.5% of at least one compound of formula (I), in molar ratio, at least 45.75% of at least one structural lipid, in molar ratio, at least 10% of at least one phospholipid, in molar ratio, at least 1.5% of at least one PEGylated lipid, and in molar ratio, at least 0.25% of at least one targeted ligand containing GalNac, 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 the nanoparticle may be about 50:1 (w / w).

[0264] In some embodiments, lipid nanoparticles are provided comprising, in molar ratio, at least 35% of at least one compound of formula (I), in molar ratio, at least one structural lipid, in molar ratio, at least 10% of at least one phospholipid, in molar ratio, at least 2% of at least one PEGylated lipid, and in molar ratio, at least 0.25% of at least one targeted ligand containing GalNac, 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 the nanoparticle may be about 60:1 (w / w).

[0265] In some embodiments, lipid nanoparticles are provided, comprising at least one compound of formula (II) in molar ratio of about 40%, at least one structural lipid in molar ratio of about 50.2%, at least one phospholipid in molar ratio of about 7.5%, at least one PEGylated lipid in molar ratio of about 2%, and at least one targeted ligand containing GalNac in molar ratio of about 0.3%, wherein at least one nucleic acid contains at least one DNA molecule. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 60:1 (w / w). In some embodiments of the preceding LNPs, the lipid nanoparticles further contain tannic acid in a tannic acid-to-lipid ratio of about 0.15, or about 2, or about 0.22.

[0266] In some embodiments, lipid nanoparticles are provided, comprising at least one compound of formula (II') in molar ratio of about 40%, at least one structural lipid in molar ratio of about 50.2%, at least one phospholipid in molar ratio of about 7.5%, at least one PEGylated lipid in molar ratio of about 2%, and at least one targeted ligand containing GalNac in molar ratio of about 0.3%, wherein at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the lipid-to-nucleic acid ratio in the nanoparticles may be about 60:1 (w / w). In some embodiments of the preceding LNPs, the lipid nanoparticles further contain tannic acid in a tannic acid-to-lipid ratio of about 0.15, or about 2, or about 0.22.

[0267] Composition of the present disclosure This disclosure provides compositions comprising at least one lipid nanoparticle of the Disclosure. The compositions of the Disclosure may further comprise any number of additional components.

[0268] Pharmaceutical compositions of the present disclosure In some embodiments, the Disclosure provides a pharmaceutical composition comprising at least one lipid nanoparticle of the Disclosure. In some embodiments, the Disclosure provides a pharmaceutical composition comprising at least one first nanoparticle of the Disclosure and at least one second nanoparticle of the 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 may be an RNA molecule (e.g., an mRNA molecule), and the at least one nucleic acid molecule encoding at least one transposon may be a DNA molecule (e.g., a DoggyBone DNA molecule or a DNA nanoplasmid).

[0269] In some embodiments, the Disclosure provides a composition comprising at least one cell in contact with at least one nanoparticle of the Disclosure. In some embodiments, the Disclosure provides a composition comprising at least one cell genetically modified using at least one nanoparticle of the Disclosure. In some embodiments, the Disclosure provides a composition comprising at least one cell genetically modified using any method of the Disclosure.

[0270] In some embodiments, the Disclosure provides a pharmaceutical composition comprising at least one cell in contact with at least one nanoparticle of the Disclosure. In some embodiments, the Disclosure provides a pharmaceutical composition comprising at least one cell genetically modified using at least one nanoparticle of the Disclosure. In some embodiments, the Disclosure provides a pharmaceutical composition comprising at least one cell genetically modified using any method of the Disclosure.

[0271] Method of Disclosure This disclosure provides a method for delivering at least one nucleic acid to at least one cell, comprising contacting at least one cell with at least one composition of this disclosure. This disclosure provides a method for delivering at least one nucleic acid to at least one cell, comprising contacting at least one cell with at least one nanoparticle of this disclosure.

[0272] In all methods, compositions, and kits of this disclosure, at least one cell may be a hepatocyte. Hepatocytes include, but are not limited to, hepatocytes, hepatic stellate cells, Kupffer cells, or hepatic sinusoidal endothelial cells.

[0273] In some embodiments of any method of the present disclosure, cells may be present in vivo, ex vivo, or in vitro. In some embodiments, any method of the present disclosure can be applied in vivo, ex vivo, or in vitro.

[0274] This disclosure provides a method for genetically modifying at least one cell, comprising contacting at least one cell with at least one composition of this disclosure. This disclosure provides a method for genetically modifying at least one cell, comprising contacting at least one cell with at least one nanoparticle of this disclosure.

[0275] In some embodiments, genetic modification of a cell may include delivering at least one exogenous nucleic acid to the cell so that the cell expresses at least one protein that would not otherwise normally be expressed, or so 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 at least one protein, or so that the cell expresses at least one protein at a level lower than the level at which the cell would otherwise normally express. In some embodiments, genetic modification of a cell may include delivering at least one exogenous nucleic acid to the cell so that at least one exogenous nucleic acid is incorporated into the genome of at least one cell.

[0276] In some embodiments, a plurality of cells can be obtained by the method of the present disclosure, and 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 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.

[0277] This disclosure provides a method for treating at least one disease in a subject, comprising administering to the subject at least one therapeutically effective amount of at least one composition of this disclosure, comprising at least one nucleic acid encoding a therapeutic protein. In some embodiments, the subject is human. The terms “subject” and “patient” are used interchangeably herein. This disclosure provides a method for treating at least one disease in a subject, comprising administering to the subject at least one therapeutically effective amount of at least one nanoparticle of this disclosure, comprising at least one nucleic acid encoding a therapeutic protein.

[0278] While we do not wish to be bound by theory, we hypothesize that the LNP compositions of this disclosure target hepatocytes more effectively than other cells, thereby reducing off-target effects associated with other delivery compositions.

[0279] In some embodiments, LNP compositions provided herein containing a targeted ligand result in less cytokine release than the same LNP compositions without the targeted 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 a subject that has received the LNP composition containing the targeted ligand using an enzyme-linked immunosorbent assay (ELISA). The cytokine levels can then be compared to baseline levels before treatment.

[0280] This disclosure provides a method for treating at least one disease in a subject, comprising administering a therapeutically effective dose of cells, wherein the cells are in contact with at least one nanoparticle of this disclosure comprising at least one nucleic acid encoding a therapeutic protein. This disclosure provides a method for treating at least one disease in a subject, comprising administering a therapeutically effective dose of cells, wherein the cells are genetically modified using the compositions and / or methods of this disclosure. In some embodiments, the at least one disease may be a malignant disease, including but not limited to cancer. In some embodiments, the at least one disease may be a metabolic hepatic disorder (MLD). In some embodiments, the at least one disease may 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 combinations thereof.

[0281] The methods described herein may be used to treat a disease or disorder by the use of a therapeutic transgene encoding an exogenous nucleic acid sequence or an exogenous amino acid sequence. In such a method, the transgene is delivered to a target cell to replace or repair a mutated gene. Diseases that can be treated in such a way are generally caused by mutations in genes that result in the non-expression or non-functional expression of a protein.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)), hypophosphatasia (tissue-nonspecific alkaline phosphatase (TNAP)), petrosis osteogenesis (TCIRG1), type II glycogen storage disorder (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 IVA (MPS IVA) (Morquio) (N-acetylgalactosamine-6-sulfate sulfatase (GALNS)), Mucopolysaccharidosis type IVB (MPS IVB) Examples include beta-galactosidase (GLB1), cholesteryl ester storage disorder (CESD) (lysosomal acid lipase (LIPA)), cystinosis (cystinosine lysosomal cystine transporter (CTNS)), X-linked chronic granulomatous disease (X-CGD) (CYBB), Wiscott-Aldrich syndrome (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).

[0282] The methods of the present disclosure may optionally further include co-administration or combination therapy for treating such diseases or disorders, and the administration of any composition or pharmaceutical composition disclosed herein may further include administration before, concurrently with, and / or after administration of at least one chemotherapeutic agent (e.g., alkylating agent, fission inhibitor, radiopharmaceutical).

[0283] nucleic acid molecule In some embodiments, the nucleic acid molecule may be an RNA molecule. Therefore, in some embodiments, the lipid nanoparticles may contain at least one RNA molecule. At least one RNA molecule may be encapsulated within the lipid nanoparticles. In some embodiments, the RNA molecule may be an mRNA molecule. In some embodiments, the lipid nanoparticles may contain at least one mRNA molecule. The mRNA molecule may be encapsulated within the lipid nanoparticles.

[0284] In some embodiments, the nucleic acid molecule may be a synthetic nucleic acid molecule. In some embodiments, the nucleic acid molecule may be a nucleic acid molecule that does not exist in nature. In some embodiments, a nucleic acid molecule that does not exist in nature may contain at least one nucleic acid molecule that does not exist in nature. The at least one nucleic acid molecule that does not exist in nature may be any nucleic acid molecule that is known in the art. In some embodiments, the nucleic acid molecule may be a modified nucleic acid molecule. In some embodiments, a modified nucleic acid molecule may contain at least one modified nucleic acid molecule that does not exist in nature. The at least one modified nucleic acid molecule may be any modified nucleic acid molecule that is known in the art.

[0285] 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, also referred to herein as "Cap0". mRNA molecules can be capped with a CleanCap® moiety, which may include an m7G(5')ppp(5')(2'OMeA)(CleanCap®AG) moiety. CleanCap® moieties may also include an m7G(5')ppp(5')(2'OMeG)(CleanCap®GG) moiety. mRNA molecules can be capped with an anti-reverse cap analog (ARCA®) moiety, which may include an m7(3'-O-methyl)G(5')ppp(5')G moiety. mRNA molecules can be capped with the CleanCap® 3'OMe moiety (CleanCap® + ARCA®).

[0286] In some embodiments, the mRNA molecule may contain at least one modified nucleic acid.

[0287] 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%, or 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. While we do not wish 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).

[0288] In some embodiments, the mRNA molecule may contain at least one modified nucleic acid.

[0289] At least one modified nucleic acid is N1-methylpseudolidine (me 1Ψ) may include. 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%, or at least about 90%, or at least about 95%, or at least about 99% of the uridine bases in the mRNA molecule. In some embodiments, all uridine bases in the mRNA molecule are N1-methylpseudridine bases. While we do not wish to be constrained by theory, N1-methylpseudridine may improve protein expression (see Li et al., Bioconjugate Chem. 2016, 27, 3, 849-853).

[0290] In some embodiments, the mRNA molecule may contain at least one modified nucleic acid.

[0291] 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%, or at least about 90%, or at least about 95%, or at least about 99% of the uridine bases in the mRNA molecule. In some embodiments, all uridine bases in the mRNA molecule are pseudouridine bases. While we do not wish 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).

[0292] In some embodiments, the mRNA molecule may contain at least one modified nucleic acid.

[0293] At least one modified nucleic acid contains 5-methylcytidine (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%, or 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.

[0294] In some embodiments, nucleic acid molecules can include DNA molecules. Therefore, in some embodiments, lipid nanoparticles can include DNA molecules. In some embodiments, the DNA molecule may be, but is not limited to, a circular DNA molecule such as a DNA plasmid or a DNA nanoplasmid. Therefore, in some embodiments, lipid nanoparticles can include circular DNA molecules. In some embodiments, lipid nanoparticles can include Doggybone DNA molecules. In some embodiments, lipid nanoparticles can include DNA plasmids. In some embodiments, lipid nanoparticles can include DNA nanoplasmids. In some embodiments, the DNA molecule may be, but is not limited to, a linearized DNA molecule such as a linearized DNA plasmid or a linearized DNA nanoplasmid.

[0295] DNA plasmids or DNA nanoplasmids are short in length of at least approximately 0.25kb, or at least approximately 0.5kb, or at least approximately 0.75kb, or at least approximately 1.0kb, or at least approximately 1.25kb, or at least approximately 1.5kb, or at least approximately 1.75kb, or at least approximately 2.0kb, or at least approximately 2.25kb, or at least approximately 2.5kb, or at least approximately 2.75kb, or at least approximately 3.0kb, or at least approximately 3.25kb, or at least approximately 3.5kb, or less At least approximately 3.75kb, or at least approximately 4.0kb, or at least approximately 4.25kb, or at least approximately 4.5kb, or at least approximately 4.75kb, or at least approximately 5.0kb, or at least approximately 5.25kb, or at least approximately 5.5kb, or at least approximately 5.75kb, or at least approximately 6.0kb, or at least approximately 6.25kb, or at least approximately 6.5kb, or at least approximately 6.75kb, or at least approximately 7.0kb, or at least approximately 7.25kb, or at least approximately 7.5kb, Or at least about 7.75kb, or at least about 8.0kb, or at least about 8.25kb, or at least about 8.5kb, or at least about 8.75kb, or at least about 9.0kb, or at least about 9.25kb, or at least about 9.5kb, or at least about 9.75kb, or at least about 10.0kb, or at least about 10.25kb, or at least about 10.5kb, or at least about 10.75kb, or at least about 11.0kb, or at least about 11.25kb, or less It could be at least about 11.5kb, or at least about 11.75kb, or at least about 12kb, or at least about 12.25kb, or at least about 12.5kb, or at least about 12.75kb, or at least about 13.0kb, or at least about 13.25kb, or at least about 13.5kb, or at least about 13.75kb, or at least about 14.0kb, or at least about 14.25kb, or at least about 14.5kb, or at least about 14.75kb, or at least about 15.0kb.

[0296] In some embodiments, nucleic acid molecules formulated in lipid nanoparticles of the present disclosure may include at least one transgene sequence. In some embodiments, the transgene sequence may include a nucleotide sequence encoding at least one therapeutic protein. In some embodiments, the transgene sequence may include a nucleotide sequence encoding at least one transposase. In some embodiments, the transgene sequence may include a nucleotide sequence encoding at least one transposon. In some embodiments, the transposon may include a nucleotide sequence encoding at least one therapeutic protein. In some embodiments, the transposon may include a nucleotide sequence encoding at least one therapeutic protein and at least one promoter sequence, wherein the at least one therapeutic protein is operably ligated to at least one promoter sequence.

[0297] In some embodiments, the lipid nanoparticles of this disclosure may be produced using a microfluidic mixing platform. In some embodiments, the microfluidic mixing platform may be a non-turbulent microfluidic mixing platform.

[0298] In some embodiments, a microfluidic mixing platform can generate lipid nanoparticles of the Disclosure by combining a miscible solvent phase containing the lipid components of the nanoparticles with an aqueous phase containing the cargo of the lipid nanoparticles (e.g., nucleic acids, DNA, mRNA, etc.) using a microfluidic device. In some embodiments, the miscible solvent phase and the aqueous phase are mixed within the microfluidic device under laminar flow conditions where the two phases cannot be immediately mixed. As the two phases move laminarly through a microfluidic channel, the microscopic features within the channel enable controlled and uniform mixing for generating the lipid nanoparticles of the Disclosure.

[0299] In some embodiments, the microfluidic mixing platform may include, but is 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).

[0300] In some embodiments, the lipid nanoparticles of the Disclosure may be produced using a microfluidic mixing platform, which 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.

[0301] In some embodiments, the lipid nanoparticles of the Disclosure may be produced using a microfluidic mixing platform, which mixes a miscible solvent phase with an aqueous phase in a ratio 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 (solvent:water, v / v).

[0302] piggyBac ITR array In some embodiments, the nucleic acid may include a piggyBac ITR sequence. In some embodiments, the nucleic acid may include a first piggyBac ITR sequence and a second piggyBac ITR sequence.

[0303] In some embodiments, the piggyBac ITR sequence may include any piggyBac ITR sequence known in the art.

[0304] In some aspects of the methods of the present disclosure, the piggyBac ITR sequences, such as a first piggyBac ITR sequence and / or a second piggyBac ITR sequence in an AAV piggyBac transposon, may include, essentially consist of, or be composed of, a Sleeping Beauty transposon ITR, a Helraiser transposon ITR, a Tol2 transposon ITR, a TcBuster transposon ITR, or any combination thereof.

[0305] Transposition System In some embodiments, the nucleic acid may include a transposon or nanotransposon comprising a first nucleic acid sequence comprising: (a) a first inverted end sequence (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 a sequence encoding an intra-ITR, wherein the intra-ITR sequence comprises a transposon sequence or a sequence encoding a transposon.

[0306] In some embodiments, the nucleic acid may comprise a transposon or nanotransposon comprising a first nucleic acid sequence including: (a) a first inverted terminal sequence (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 a sequence encoding an intra-ITR, where the intra-ITR sequence comprises a transposon sequence or a sequence encoding a transposon, and the second nucleic acid sequence comprises an inter-ITR sequence or a sequence encoding an inter-ITR, the length of which the inter-ITR sequence is 700 nucleotides or less.

[0307] The transposons or nanotransposons of this disclosure may be piggyBac(PB) transposons. 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.

[0308] Non-limiting examples of PB transposons and PB, PBL, and SPB transposases are described herein by reference in U.S. Patent No. 6,218,182; U.S. Patent No. 6,962,810; U.S. Patent No. 8,399,643 and International Publication No. 2010 / 099296, each of which is incorporated herein by reference in its entirety.

[0309] PB, PBL, and SPB transposases recognize the transposon-specific inverted end sequence (ITR) at the end of a transposon and insert their contents between the ITRs of the chromosomal sequence 5'-TTAT-3' (TTAT target sequence) or the chromosomal sequence 5'-TTAA-3' (TTAA target sequence). The target sequences of 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'-T TAC-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'-AAAT- These transposons may include, or consist of, 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'. There are no restrictions on the payload for the target gene that can be included between ITRs in PB or PBL transposon systems.

[0310] 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, and each of the examples of transposases that may be used in the compositions and methods described herein is incorporated herein by reference in whole. In a preferred embodiment, a PB transposase contains or comprises an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the amino acid sequence of Sequence ID No. 1.

[0311] PB or PBL transposases may contain or consist of an amino acid sequence having two or more, three or more, or each of these amino acid substitutions at positions 30, 165, 282, and / or 538 of the sequence of SEQ ID NO: 1. A transposase may be an SPB transposase containing or consisting of the amino acid sequence of the sequence of SEQ ID NO: 1, where the amino acid substitution at position 30 may be a substitution of isoleucine (I) with valine (V), the amino acid substitution at position 165 may be a substitution of glycine (G) with serine (S), the amino acid substitution at position 282 may be a substitution of methionine (M) with valine (V), and the amino acid substitution at position 538 may be a substitution of asparagine (N) with lysine (K). In a preferred embodiment, the SPB transposase contains or comprises an amino acid sequence that is 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: 2.

[0312] In certain embodiments of the transposase containing the above mutations at positions 30, 165, 282 and / or 538, the PB, PBL and SPB transposases are transposases of SEQ ID NO: 1 or SEQ ID NO: 2, 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, 340, The transposases may further include one or more amino acid substitutions at positions 421, 436, 456, 470, 486, 503, 552, 570, and 591. Examples of transposases that can be used in the compositions and methods described herein are described in more detail in PCT Publication Nos. 2019 / 173636 and 2020 / 051374, each of which is incorporated herein by reference in whole.

[0313] In a preferred embodiment, the PB transposase contains or comprises an amino acid sequence that is 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: 3.

[0314] PB or PBL transposases may contain or consist of an amino acid sequence having two or more, three or more, or each of, amino acid substitutions at positions 29, 164, 281, and / or 537 of the sequence of Sequence ID No. 3. The transposase may be an SPB transposase containing or consisting of the amino acid sequence of Sequence ID No. 3, where the amino acid substitution at position 29 may be a substitution of isoleucine (I) with valine (V), the amino acid substitution at position 164 may be a substitution of glycine (G) with serine (S), the amino acid substitution at position 281 may be a substitution of methionine (M) with valine (V), and the amino acid substitution at position 537 may be a substitution of asparagine (N) with lysine (K). In a preferred embodiment, the SPB transposase contains or comprises an amino acid sequence that is 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: 4.

[0315] In certain embodiments of the transposase containing the above mutations at positions 29, 164, 281 and / or 537, the PB, PBL and SPB transposases are found 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, and 339 of the sequence of SEQ ID NO: 3 or SEQ ID NO: 4. The transposases may further include one or more amino acid substitutions at positions 420, 435, 455, 469, 485, 502, 551, 569, and 590, and each of the transposases that may be used in the compositions and methods described herein is described in more detail in PCT Publication Nos. 2019 / 173636 and 2020 / 051374, the whole of which is incorporated herein by reference.

[0316] PB, PBL, or SPB transposases may be isolated or derived from insects, vertebrates, crustaceans, or tunicates, as described in detail in International Publication Nos. 2019 / 173636 and International Publication Nos. 2020 / 051374, each of which is incorporated herein by reference in its entirety, for examples of transposases that may be used in the compositions and methods described herein. In preferred embodiments, PB, PBL, or SPB transposases are isolated from or derived from the insects Trichoplusia ni (GenBank accession number AAA87375) or Bombyx mori (GenBank accession number BAD11135).

[0317] Highly active PB or PBL transposases are transposases that are more active than the naturally occurring variants from which they are derived. In preferred embodiments, highly active PB or PBL transposases are isolated or derived from Bombyx mori or Xenopus tropicalis. Examples of highly active 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. 2019 / 173636, each of which is incorporated herein by reference in whole for examples of transposases that may be used in conjunction with the compositions and methods described herein. A list of highly active amino acid substitutions is disclosed in U.S. Patent No. 10,041,077, which is incorporated herein by reference in whole for examples of amino acid substitutions that may be introduced into the transposases disclosed herein. The transposons or nanotransposons of this disclosure may be Sleeping Beauty transposons. In some embodiments, when the transposon is a Sleeping Beauty transposon, the transposase is either a Sleeping Beauty transposase (for example, as disclosed in U.S. Patent No. 9,228,180, which is incorporated herein by reference in its entirety, for examples of transposases that may be used in the compositions and methods described herein) or a highly active Sleeping Beauty (SB100X) transposase.

[0318] In some embodiments, PB or PBL transposases are integration defects. Integration-defective PB or PBL transposases are transposases that can excise the corresponding transposon but incorporate the excised transposon at a lower frequency than the corresponding wild-type transposase. Examples of integration-defective 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. 2019 / 173636, each of which, with reference to examples of transposases that may be used in the compositions and methods described herein, is incorporated herein by reference in whole. A list of integration-defective amino acid substitutions is disclosed in U.S. Patent No. 10,041,077, which, with reference to examples of amino acid substitutions that may be introduced into the transposases described herein, is incorporated herein by whole.

[0319] In some embodiments, the PB or PBL transposase is fused to a nuclear localization signal. Examples of PB or PBL transposases 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. 2019 / 173636, and examples of transposases that may be used in conjunction with the compositions and methods described herein are incorporated herein by reference in whole.

[0320] The transposons or nanotransposons of this disclosure may be Sleeping Beauty transposons. In some embodiments, when the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase (for example, those disclosed in U.S. Patent No. 9,228,180, which is incorporated herein by reference in whole, for examples of transposases that may be used in the compositions and methods described herein) or a highly active Sleeping Beauty (SB100X) transposase.

[0321] The transposons or nanotransposons of this disclosure may be Helraiser transposons. An example of a Helraiser transposon is Helibat1. In some embodiments, when the transposon is a Helraiser transposon, the transposase is a Helitron transposase (for example, disclosed in International Publication No. 2019 / 173636, which is incorporated herein by reference in its entirety, for examples of transposons that may be used in the compositions and methods described herein).

[0322] The transposons or nanotransposons of this disclosure may be Tol2 transposons. In some embodiments, when the transposon is a Tol2 transposon, the transposase is a Tol2 transposase (for example, as disclosed in International Publication No. 2019 / 173636, which is incorporated herein by reference in its entirety, for examples of transposons and nanotransposons that may be used in conjunction with the compositions and methods described herein).

[0323] The transposons or nanotransposons of this disclosure may be TcBuster transposons. In some embodiments, when the transposon is a TcBuster transposon, the transposase is a TcBuster transposase or a highly active TcBuster transposase (for example, as disclosed in International Publication 2019 / 173636). The TcBuster transposase may or may consist of a naturally occurring amino acid sequence or a non-naturally occurring amino acid sequence. The polynucleotide encoding the TcBuster transposase may or may consist of a naturally occurring nucleic acid sequence or a non-naturally occurring nucleic acid sequence.

[0324] In some embodiments, the mutant TcBuster transposase contains one or more sequence mutations compared to the wild-type TcBuster transposase, each of which is further described in PCT Publications WO2019 / 173636 and WO2020 / 051374, which are incorporated herein by reference in their entirety, for examples of transposases that may be used in the compositions and methods described herein.

[0325] The cell delivery compositions (e.g., transposons) disclosed herein may include nucleic acid molecules encoding therapeutic proteins or therapeutic agents. Examples of therapeutic proteins are those disclosed in PCT Publications WO2019 / 173636 and WO2020 / 051374, and each of the examples of therapeutic proteins that may be used in the compositions and methods described herein is incorporated herein by reference in whole.

[0326] Gene editing systems This disclosure provides a gene editing composition and / or cells containing the gene editing composition. The gene editing composition may include nanoparticles containing nucleic acids, the nucleic acids including 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 the following: CRISPR / Cas proteins, transcriptional activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and endonucleases.

[0327] The nuclease or its nuclease domain may include a nuclease-inactivating Cas(dCas) protein and an endonuclease. The endonuclease may include a Clo051 nuclease or its nuclease domain. The gene editing composition may include a fusion protein. The fusion protein may include a nuclease-inactivating Cas9(dCas9) protein and a Clo051 nuclease or a Clo051 nuclease domain. In some embodiments, the fusion protein may further include at least one nuclear localization signal (NLS). In some embodiments, the fusion protein may further include at least two NLSs. The gene editing composition may further include a guide sequence. The guide sequence may include an RNA sequence.

[0328] The transgene may include a nucleic acid sequence encoding a small Cas9 (Cas9) operably linked to an effector. The disclosure provides a fusion protein comprising, essentially comprising, or comprising a DNA localization component and an effector molecule, wherein the effector comprises a small Cas9 (Cas9). The small Cas9 constructs of the disclosure may include an effector comprising an IIS-type endonuclease.

[0329] The transgene may include a nucleic acid sequence encoding an inactivated, small Cas9 (dSaCas9) operably linked to an effector. The transgene may include a nucleic acid sequence encoding a fusion protein comprising, essentially comprising, or comprising a DNA localization component and an effector molecule, wherein the effector comprises a small, inactivated Cas9 (dSaCas9). The small, inactivated Cas9 (dSaCas9) constructs of this disclosure may include an effector comprising an IIS-type endonuclease.

[0330] The transgene may include a nucleic acid sequence encoding inactivated Cas9 (dCas9) operably linked to an effector. The transgene may include a nucleic acid sequence encoding a fusion protein comprising, essentially comprising, or comprising a DNA localization component and an effector molecule, wherein the effector may include a nucleic acid sequence containing inactivated Cas9 (dCas9). The inactivated Cas9 (dCas9) constructs of the present disclosure may include an effector containing an IIS-type endonuclease.

[0331] dCas9 may be isolated from or derived from Streptococcus pyogenes. dCas9 may include dCas9 having amino acid substitutions at positions 10 and 840 that inactivate the catalytic site. In some embodiments, these substitutions are D10A and H840A.

[0332] Cells containing a gene editing composition can stably or transiently express the gene editing composition. Preferably, the gene editing composition is expressed transiently. The guide RNA may contain a sequence complementary to the target sequence in the genomic DNA sequence. The target sequence in the genomic DNA sequence may be a target sequence within a safe harbor region of the genomic DNA sequence.

[0333] Gene editing compositions containing Cas-CLOVER, and methods for using these compositions for gene editing, are described in detail in U.S. Patent Publications 2017 / 0107541, 2017 / 0114149, 2018 / 0187185, and U.S. Patent No. 10,415,024, each of which is incorporated herein by reference in whole, with respect to examples of gene editing compositions that may be used in the compositions and methods described herein. In some embodiments, the Cas-CLOVER protein contains, essentially, or may contain an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same amino acid sequence as SEQ ID NO: 5 or 6.

[0334] Accordingly, this disclosure provides any of the lipid nanoparticle compositions described herein, the lipid nanoparticle comprising at least one genome editing composition, the at least one genome editing composition comprising 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.

[0335] Formulation, dosage, and mode of administration This disclosure provides formulations, dosages, and methods of administration of the compositions described herein.

[0336] The disclosed compositions and pharmaceutical compositions may further include, but are not limited to, at least one of any suitable adjuvants, such as diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, and adjuvants. Pharmaceutically acceptable adjuvants are preferred. Non-limiting examples of such sterile solutions and methods for their preparation are well known in the art, including, but are limited to, Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990 and "Physician's Desk Reference," 52nd ed., Medical Economics (Montvale, NJ) 1998. Pharmaceutically acceptable carriers suitable for the dosage, solubility, and / or stability of the compositions can be routinely selected, are well known in the art, or are described herein.

[0337] For example, the LNP compositions disclosed in this disclosure may further include a diluent. In some compositions, the diluent may be phosphate-buffered saline (PBS).

[0338] Non-limiting examples of pharmaceutical excipients and additives suitable for use include proteins, peptides, amino acids, lipids, and carbohydrates (e.g., monosaccharides, sugars including di-, tri-, tetra-, and oligosaccharides, derivatized sugars such as alditol, aldonic acid, and esterified sugars, and polysaccharides or sugar polymers), which can exist alone or in combination, and which together constitute 1 to 99.99% by weight or volume. Non-limiting examples of protein excipients include serum albumins such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, and casein. Representative amino acid / protein components that can also function as buffers include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, and aspartame. One preferred amino acid is glycine.

[0339] The composition may also contain a buffer or pH adjuster, typically a salt prepared from an organic acid or base. Typical buffers include organic acid salts such as citric acid, ascorbic acid, gluconic acid, carbonate, tartaric acid, succinic acid, acetic acid, and phthalic acid salts, as well as Tris, tromethamine hydrochloride, and phosphate buffer. Preferred buffers are organic acid salts such as citrate. In some embodiments, the buffer may also contain sucrose.

[0340] Many known and developed methods can be used to administer a therapeutically effective amount of the composition or pharmaceutical composition disclosed herein. Non-limiting examples of methods of administration include bolus, buccal, infusion, intra-articular, intra-bronchial, intraperitoneal, intra-articular capsule, intra-cartilage, intracavitary, intra-abdominal, intraventricular, intracolonic, intracerebral, intra-stomal, intrahepatic, intrafocal, intramuscular, intramyocardial, intranasal, intraocular, intraosseous, intraosteal, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intranephrine, intraretinal, intraspinal, synovial, intrathoracic, intrauterine, intratumoral, intravenous, intrabladder, oral, parenteral, rectal, sublingual, subcutaneous, percutaneous, or vaginal methods.

[0341] The compositions of this disclosure are particularly for use in parenteral (subcutaneous, intramuscular, or intravenous) or any other administration in the form of liquid solutions or suspensions; particularly for use in vaginal or rectal administration in semi-solid forms such as creams and suppositories, but not limited to these; for buccal or sublingual administration in the form of tablets or capsules, but not limited to these; or nasally in the form of powders, nasal sprays or aerosols or certain drugs, but not limited to these; 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, DS, Eds., pp. 59-90 (Marcel Dekker, Inc. New York) They may be prepared for transdermal use, such as gels, ointments, lotions, suspensions, or patch delivery systems, by means of creating a transient transport pathway such as electroporation, or by applying an electric field to increase the mobility of charged drugs through the skin such as iontophoresis, or by applying ultrasound such as sonophoresis (U.S. Patents No. 4,309,989 and No. 4,767,402) (the above publications and patents are fully incorporated herein by reference), but not limited to the following:

[0342] For parenteral administration, any composition disclosed herein may be formulated in conjunction with a pharmaceutically acceptable parenteral vehicle as a solution, suspension, emulsion, particles, powder, or lyophilized powder, or may be provided separately from the parenteral vehicle. Formulations for parenteral administration may contain, as common excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, plant-derived oils, hydrogenated naphthalene, etc. Aqueous or oily suspensions for injection may be prepared according to known methods using appropriate emulsifiers or humectants and suspending agents. Injectable preparations may be non-toxic, orally unadministerable diluents such as aqueous solutions, sterile injection solutions, or suspensions in solvents. Suitable vehicles or solvents include water, Ringer's solution, isotonic saline, etc., and sterile non-volatile oils may be used as common solvents or suspension solvents. For these purposes, all kinds of non-volatile oils and fatty acids may be used, including natural, synthetic, or semi-synthetic fatty oils or fatty acids; natural, synthetic, or semi-synthetic mono-, di-, or tri-glycerides. Parenteral administration is known in the art and, without limitation, includes conventional injection methods, gas-pressurized needleless injection devices such as those described in U.S. Patent No. 5,851,198, and laser puncture devices such as those described in U.S. Patent No. 5,839,446.

[0343] For pulmonary administration, preferably, the compositions or pharmaceutical compositions described herein are delivered in a particle size effective to reach the lungs or the lower airways of the sinuses. The compositions or pharmaceutical compositions may be delivered by any of the various inhalation or nasal devices known in the art for the administration of therapeutic agents by inhalation. These devices, which can deposit aerosolized formulations into the sinuses or alveoli of a patient, include metered-dose inhalers, nebulizers (e.g., jet nebulizers, ultrasonic nebulizers, etc.), dry powder generators, nebulizers, etc. All such devices can use formulations suitable for administration to aerosolize the compositions or pharmaceutical compositions described herein. Such aerosols may consist of solutions (both aqueous and non-aqueous) or solid particles. In a metered-dose inhaler (MDI), a propellant, the compositions or pharmaceutical compositions described herein, and excipients or other additives are contained in a canister as a mixture including liquefied compressed gas. The mixture is released as an aerosol by the operation of a metering valve. A more detailed description of pulmonary administration, formulations and related devices is disclosed in International Publication No. 2019 / 049816.

[0344] For absorption via the mucosal surface, the composition comprises an emulsion containing a plurality of submicron particles, a mucosal-adhering polymer, a bioactive peptide, and an aqueous continuous phase, which promotes absorption via the mucosal surface by achieving mucosal adhesion of the emulsion particles (U.S. Patent No. 5,514,670). Suitable mucosal surfaces for application of the emulsion of this disclosure include the cornea, conjunctiva, cheek, sublingual, nose, vagina, lung, stomach, intestine, and rectal administration routes. Formulations for vaginal or rectal administration, such as suppositories, may contain excipients such as polyalkylene glycol, petrolatum, and cocoa butter. Formulations for nasal administration are solid and may contain excipients such as lactose, and may be an aqueous or oily solution of the nasal spray. For buccal administration, excipients include sugars, calcium stearate, magnesium stearate, and pregelatinized starch (U.S. Patent No. 5,849,695). A more detailed description of mucosal administration and formulations is disclosed in International Publication No. 2019 / 049816.

[0345] For transdermal administration, the compositions or pharmaceutical compositions disclosed herein are encapsulated in delivery devices such as liposomes or polymer nanoparticles, microparticles, microcapsules, or microspheres (collectively referred to as microparticles unless otherwise specified). Several suitable devices are known, such as microparticles made from polyhydroxy acids such as polylactic acid, polyglycolic acid and its copolymers, polyorthoesters, polyanhydrides and polyphosphazenes, as well as synthetic polymers such as natural polymers such as collagen, polyamino acids, albumin and other proteins, alginates and other polysaccharides, and combinations thereof (U.S. Patent No. 5,814,599). A more detailed description of transdermal administration, formulations and suitable devices is disclosed in International Publication No. 2019 / 049816.

[0346] It may be desirable to deliver the disclosed compound to the subject in a single dose over a long period, for example, from one week to one year. Various sustained-release formulations, depot formulations, and implant formulations can be used.

[0347] Suitable dosages are well known in the art. For example, see 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, Calif. (2000), Nursing 2001 Handbook of Drugs, 21st edition, Springhouse Corp., Springhouse, Pa., 2001, and Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc., Upper Saddle River, NJ. Preferred doses may optionally include about 0.1 to 99 and / or 100 to 500 mg / kg / administer, or any range, value or fraction thereof, or a serum concentration of about 0.1 to 5000 μg / ml can be achieved per single or multiple administrations, or per any range, value or fraction thereof. Preferred dose 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 the subject's body weight.

[0348] Alternatively, the dosage may be adjusted depending on known factors such as the pharmacodynamic properties of the particular drug, its mode and route of administration, the recipient's age, health status, and weight, the nature and severity of symptoms, the type of concurrent treatment, the frequency of treatment, and the desired effect.

[0349] As a non-limiting example, human or animal treatments may be provided using single doses, infusion doses or repeated doses, as a single or periodic dose of about 0.1 to 100 mg / kg per day or any range, value, or fraction thereof of the compositions or pharmaceutical compositions disclosed herein, at least once between days 1 to 40, or alternatively or additionally, at least once between weeks 1 to 52, or alternatively or additionally, at least once over 1 to 20 years, or any combination thereof.

[0350] In embodiments in which the composition administered to a subject requiring administration of the composition is a modified cell disclosed herein, the cell is approximately 1 × 10 3 ~1 × 10 15 Individual cells; 1 × 10 3 ~1 × 10 15 Each cell is approximately 1 x 10⁻⁶ 4 ~1 × 10 12 Individual cells; approximately 1 × 10⁻⁶ 5 ~1 × 10 10 Individual cells; approximately 1 × 10⁻⁶ 6 ~1 × 10 9 Individual cells; approximately 1 × 10⁻⁶ 6 ~1 × 10 8 Individual cells; approximately 1 × 10⁻⁶ 6 ~1 × 10 7 A single cell; or about 1 × 10⁻⁶ 6 ~25×10 6 It can be a single cell. In one embodiment, the cell is approximately 5 × 10 6 ~25×10 6 It is administered as individual cells.

[0351] A more detailed description of the disclosed compositions and pharmaceutically acceptable excipients, formulations, dosages, and methods of administration of the pharmaceutical compositions is disclosed in International Publication No. 2019 / 04981.

[0352] This disclosure provides the use of the disclosed composition or pharmaceutical composition for treating a disease or disorder in a cell, tissue, organ, animal, or subject, as known in the art or as described herein, for example, by administering or contacting a therapeutically effective amount of the composition or pharmaceutical composition to a cell, tissue, organ, animal, or subject. In one embodiment, the subject is a mammal. Preferably, the subject is a human. The terms “subject” and “patient” are used interchangeably herein.

[0353] This disclosure provides methods for modulating or treating at least one malignant disease or disorder in cells, tissues, organs, animals, or subjects. Non-limiting examples of malignant diseases or disorders include cancer and liver disease or disorder.

[0354] Any method may include administering an effective amount of any composition or pharmaceutical composition disclosed herein to cells, tissues, organs, animals or subjects requiring such adjustment, treatment or therapy. Such methods may optionally further include co-administration or combination therapy for treating such disease or disorder, and the administration of any composition or pharmaceutical composition disclosed herein may further include administering at least one chemotherapeutic agent (e.g., alkylating agent, mitotic inhibitor, radiopharmaceutical) before, concurrently with, and / or after the administration.

[0355] In some embodiments, the subject does not develop graft-versus-host (GvH) and / or host-versus-graft (HvG) after administration. In some embodiments, the administration is systemic. Systemic administration may be any means known in the art and described in detail herein. Preferably, systemic administration is intravenous injection or intravenous infusion. In some embodiments, the administration is local. Local administration may be any means known in the art and described in detail herein. Preferably, local administration is by intratumoral injection or infusion, intraspinal injection or infusion, intraventricular injection or infusion, intraocular injection or infusion, or intraosseous injection or infusion.

[0356] In some embodiments, the therapeutically effective dose is a single dose. In some embodiments, the single dose is one dose from 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 in between, which are manufactured simultaneously. In some embodiments, if the composition is autologous cells or allogeneic cells, the dose is an amount sufficient for the cells to engraft and / or persist for a sufficient time to treat the disease or disorder.

[0357] In some embodiments of the treatment methods described herein, the treatment may be modified or terminated. Specifically, in embodiments in which the composition used for the treatment comprises an inducible pro-apoptotic polypeptide, apoptosis may be selectively induced in cells by contacting them with the inducer. The treatment may be modified or terminated, for example, in response to signs of recovery, signs of reduced disease severity / progression, signs of remission / cessation of disease, 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 (for example, when signs or symptoms of the disease recur or increase in severity, and / or when adverse events resolve).

[0358] Nucleic acid construction The isolated nucleic acids of this disclosure can be prepared using (a) recombinant methods, (b) synthesis techniques, (c) purification techniques, and / or (d) a combination thereof, as is well known in the art.

[0359] Nucleic acids may conveniently contain sequences in addition to the polynucleotides of the Disclosure. For example, a multicloning site containing one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in the isolation of the polynucleotides. Translationable sequences may also be inserted to aid in the isolation of the translated polynucleotides of the Disclosure. For example, a hexa-histidine marker sequence provides a convenient means of purifying the proteins of the Disclosure. Any nucleic acid of the Disclosure, excluding the coding sequence, may optionally be a vector, adapter, or linker for cloning and / or expression of the polynucleotides of the Disclosure.

[0360] To optimize function in cloning and / or expression, to aid in the isolation of polynucleotides, or to improve the introduction of polynucleotides into cells, additional sequences can be added to such cloning and / or expression sequences. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art (see, for example, Ausubel or Sambrook above).

[0361] Recombination methods for constructing nucleic acids The isolated nucleic acid compositions of this disclosure, e.g., 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 this disclosure under stringent conditions are used to identify desired sequences in cDNA or genomic DNA libraries. The isolation of RNA, as well as the construction of cDNA and genomic libraries, are well known to those skilled in the art (see, for example, Ausubel or Sambrook above).

[0362] Nucleic acid screening and isolation methods The polynucleotide sequence-based probes of this disclosure can be used to screen cDNA libraries or genomic libraries. The probes are 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 various degrees of hybridization stringency can be employed in the assay, and that either the hybridization medium or the washing medium can be stringent. As the hybridization conditions become more stringent, a higher degree of complementarity between the probe and the target is required for double-strand formation to occur. The degree of stringency can be controlled by one or more of the following: temperature, ionic strength, pH, and the presence of a partially denaturing solvent such as formamide. For example, the stringency of hybridization can be conveniently altered by changing the polarity of the reaction mixture, for example, by manipulating the concentration of formamide 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 washing medium. The degree of complementarity is optimally 100%, or 70-100%, or a range or value within that range. However, it should be understood that slight sequence differences in the probe and primer can be compensated for by reducing the stringency of the hybridization medium and / or washing solution.

[0363] Methods for amplifying RNA or DNA are well known in the art and can be used in accordance with this disclosure without excessive experimentation, based on the teachings and guidelines presented herein.

[0364] Known methods for amplifying DNA or RNA include polymerase chain reaction (PCR) and related amplification processes (e.g., U.S. Patent 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), and 5,066,584 (G)). Examples include, but are not limited to, the works of Ausubel et al. (Yylensten et al.), Gelfand et al. (Gelfand et al.), Silver et al. (Silver et al.), Biswas (Biswas), and Ringold (Ringold), as well as RNA-mediated amplification using antisense RNA against a target sequence as a template for double-stranded DNA synthesis (US Patent No. 5,130,238 (Malek et al.), trade name NASBA), and the entire contents of those references are incorporated herein by reference. (See, for example, Ausubel or Sambrook).

[0365] For example, polymerase chain reaction (PCR) technology can be used to directly amplify the sequences of the polynucleotides and related genes of this disclosure from a genomic DNA or cDNA library. PCR and other in vitro amplification methods are also useful for purposes such as cloning nucleic acid sequences encoding proteins to be expressed, creating 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 those skilled in the art through in vitro amplification methods can be found in Berger, Sambrook (n.), Ausubel (n.), Mullis (n.), et al., U.S. Patent 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). Commercial kits for genomic PCR amplification are known in the art. See, for example, the Advantage-GC Genomic PCR Kit (Clontech). Furthermore, to improve the yield of longer PCR products, for example, the T4 gene 32 protein (Boehringer Mannheim) can be used.

[0366] Synthesis methods for constructing nucleic acids The isolated nucleic acids of this disclosure can also be prepared by direct chemical synthesis using known methods (see, for example, Ausubel et al.). Chemical synthesis generally yields single-stranded oligonucleotides, which can be converted to double-stranded DNA by hybridization with a complementary sequence or by polymerization using DNA polymerase with the single strand as a template. Those skilled in the art will understand that while the chemical synthesis of DNA is limited to sequences of approximately 100 bases or more, longer sequences can be obtained by ligating shorter sequences.

[0367] Recombinant expression cassette The disclosure further provides recombinant expression cassettes comprising the nucleic acids of the disclosure. The nucleic acid sequences of the disclosure can be used to construct recombinant expression cassettes that can be introduced into at least one desired host cell. The recombinant expression cassette typically consists of the polynucleotides of the disclosure operably ligated to transcription initiation regulatory sequences that direct the transcription of the polynucleotides in the intended host cell. Both heterogeneous and non-heterogeneous (i.e., endogenous) promoters can be employed to direct the expression of the nucleic acids of the disclosure.

[0368] In some embodiments, isolated nucleic acids functioning as promoters, enhancers, or other elements may be introduced at appropriate locations (upstream, downstream, or in an intron) of a non-heterogeneous form of the polynucleotide of the Disclosure to control the expression of the polynucleotide of the Disclosure up or down. For example, an endogenous promoter can be altered in vivo or in vitro by mutation, deletion, and / or substitution.

[0369] Expression vectors and host cells This disclosure also relates to vectors containing isolated nucleic acid molecules of this disclosure, and to host cells genetically engineered with recombinant vectors, as is well known in the art. See, for example, Sambrook, et al. and Ausubel, et al., respectively, which are fully incorporated herein by reference.

[0370] Polynucleotides can be optionally conjugated to vectors containing selectable markers for replication in a host. Generally, plasmid vectors are introduced in precipitates such as calcium phosphate precipitates or in complexes with charged lipids. If the vector is a virus, it can be packaged in vitro using a suitable packaging cell line and transduced into host cells.

[0371] The DNA insert must be operablely ligated to an appropriate promoter. The expression construct further includes transcription start and end points, and a ribosome binding site for translation in the transcribed region. The coding portion of the mature transcript expressed by the construct preferably includes a translation start codon at the start of the mRNA to be translated, and preferably has a stop codon (e.g., UAA, UGA, or UAG) appropriately positioned at the end, with UAA and UAG preferred for expression in mammalian or eukaryotic cells.

[0372] The expression vector preferably, but optionally, includes at least one selectable marker. Such markers include, but are not limited to, ampicillin, zeosin (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 synthase (GS, U.S. Patent Nos. 5,122,464, 5,770,359, and 5,827,739), blasticidine (bsd gene), resistance genes for eukaryotic cell culture, and ampicillin, zeosin (Sh Examples include the 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 fully incorporated herein by reference). Suitable 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. The introduction of vector constructs into host cells can be carried out 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, for example, in Sambrook, Chapters 1-4 and 16-18, and Ausubel, Chapters 1, 9, 13, 15, and 16.

[0373] The expression vector preferably, but optionally, includes at least one selectable cell surface marker for isolating cells modified by the compositions and methods of the present disclosure. The selectable cell surface markers of the present disclosure consist of surface proteins, glycoproteins, or groups of proteins that distinguish cells or subsets of cells from another defined subset of cells. Preferably, the selectable cell surface markers distinguish cells modified by the compositions or methods of the present disclosure from cells that have not been modified by the compositions or methods of the present disclosure. Examples of such cell surface markers include, but are not limited to, “cluster designation” or “classification determinant” proteins (often abbreviated as “CD”) such as cleaved or full-length forms of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or combinations thereof. Cell surface markers include the suicide gene marker RQR8 (Philip B et al. Blood. 2014 Aug 21;124(8):1277-87).

[0374] The expression vector preferably, but optionally, 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.

[0375] Those skilled in the art will be familiar with the numerous expression systems available for expressing nucleic acid molecules encoding the proteins of this disclosure.

[0376] definition When used throughout this disclosure, the singular forms "a," "an," and "the" include multiple references unless the context explicitly indicates otherwise. Thus, for example, a reference to "a method" includes multiple such methods, and a reference to "a dose" includes one or more doses and their equivalents known to those skilled in the art.

[0377] The terms “about” or “approximately” mean that a particular value is within an acceptable margin of error, as determined by those skilled in the art, and this depends to some extent on the method by which the value is measured or determined, e.g., on the limitations of the measurement system. For example, “about” means within one or more standard deviations. Alternatively, “about” could mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Or, particularly with respect to biological systems or processes, the term could mean within one order of magnitude of the value, preferably up to five times, and more preferably up to two times. Where a particular value is described in this application and claims, unless otherwise specified, the term “about” should be assumed to mean within an acceptable margin of error of that particular value.

[0378] In the chemical formulas shown in this specification, mark TIFF2026530178000059.tif10170 shows the position where a functional group attaches to another part of the molecule. Definitions of specific functional groups and chemical terms are explained in detail below.

[0379] Certain compounds in this disclosure may exist in the form of specific geometric or stereoisomers. This disclosure intends to include all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, racemic mixtures thereof, and other mixtures thereof, within the scope of this disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, and mixtures thereof, are intended to be included in this disclosure.

[0380] Isomer mixtures containing various isomer ratios can be utilized in accordance with this disclosure. 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 intended by this disclosure. Those skilled in the art will readily understand that similar ratios are intended for more complex isomer mixtures.

[0381] For example, if a specific enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary agent, the resulting diastereomer mixture can be separated, and the auxiliary groups can be 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, a diastereomer salt can be formed using a suitable optically active acid or base, and then the diastereomer thus formed can be separated by fractional crystallization or chromatographic means well known in the art, after which the pure enantiomer can be recovered.

[0382] Those skilled in the art will understand that the synthetic methods described herein utilize various protecting groups. As used herein, the term “protecting group” means temporarily blocking a specific functional group moiety, such as O, S, or N, to allow a reaction to proceed selectively at another reaction site in a polyfunctional compound. In certain embodiments, the protecting group reacts selectively and in good yield to provide a protected substrate stable for the expected reaction; the protecting group should be selectively and in good yield by readily available, preferably non-toxic, reagents that do not attack other functional groups; the protecting group forms a readily separable derivative (more preferably without generating a new steric center); and the protecting group has minimal additional functional groups to avoid further reaction sites. Protecting groups of oxygen, sulfur, nitrogen, and carbon can be utilized, as detailed herein.

[0383] The term “aliphatic,” as used herein, includes both saturated and unsaturated, linear (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 portions. Thus, as used herein, the term “alkyl” includes linear, branched, and cyclic alkyl groups. A similar convention applies to other common terms such as “alkenyl” and “alkynyl.” Furthermore, as used herein, the terms “alkyl,” “alkenyl,” and “alkynyl” 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.

[0384] In certain embodiments, the alkyl group, alkenyl group, and alkynyl group contain 1 to 18 aliphatic carbon atoms. In certain embodiments, the alkyl group, alkenyl group, and alkynyl group contain 1 to 15 aliphatic carbon atoms. In certain other embodiments, the alkyl group, alkenyl group, and alkynyl group contain 1 to 10 aliphatic carbon atoms. In yet another embodiment, the alkyl group, alkenyl group, and alkynyl group contain 1 to 8 aliphatic carbon atoms. In yet another embodiment, the alkyl group, alkenyl group, and alkynyl group contain 1 to 6 aliphatic carbon atoms. In yet another embodiment, the alkyl group, alkenyl group, and alkynyl group contain 1 to 4 carbon atoms. Examples of aliphatic groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, --CH2-cyclopropyl, vinyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, --CH2-cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, cyclopentyl, --CH2-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, --CH2-cyclohexyl moieties, and these may also have one or more substituents. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, and 1-methyl-2-buten-1-yl. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), and 1-propynyl.

[0385] As used herein, the term "alkyl" refers to a saturated linear or branched aliphatic group having 1 to 18 carbon atoms, and therefore "alkyl" refers to C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 and C 12It includes the group. Examples 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, and dodecyl.

[0386] The term "alkylene" refers to a divalent alkyl group. The monovalent alkyl groups described above can become alkylenes by removing a second hydrogen atom from the alkyl group. As defined herein, alkylenes are also C1-C 18 Alkylenes are also C1~C 12 Alkylenes are also acceptable. Typical alkylene groups include, but are not limited to, -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH2CH(CH3)-, -CH2C(CH3)2-, -CH2CH2CH2-, and -CH2CH2CH2CH2-.

[0387] The term "alkenyl" refers to an unsaturated linear or optionally branched aliphatic group having 2 to 18 carbon atoms and one or more carbon-carbon double bonds. Thus, "alkenyl" refers to C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 and C 12 It includes the group. Examples of alkenyl groups include ethenyl, propenyl, butenyl, and 1-methyl-2-buten-1-yl.

[0388] The term "alkynyl" refers to an unsaturated linear or optionally branched aliphatic group having 2 to 18 carbon atoms and one or more carbon-carbon triple bonds. Thus, "alkynyl" refers to C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 and C 12 It includes the group. Representative alkynyl groups include ethynyl, 2-propynyl (propargyl), and 1-propynyl.

[0389] As used herein, the term "aryl" refers to a C6-C6 compound consisting of 1-3 aromatic rings. 14 The aromatic part is arbitrarily substituted. Thus, "aryl" is C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , and C 14 Contains cyclic hydrocarbon groups. Exemplary aryl groups are C6-C6 10 This refers to an aryl group. Examples of aryl groups include phenyl, naphthyl, anthracenyl, and fluorenyl, but the group is not limited to these.

[0390] As used herein, the term “cycloalkyl” includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbon atoms. Thus, “cycloalkyl” includes C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 and C 12 It contains a cyclic hydrocarbon group. Typical cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0391] As used herein, the term “hydroxyalkyl” refers to an alkyl chain substituted with an alkyl-OH group or at least one alkyl-OH group.

[0392] As used herein, the terms "halo" or "halogen" refer to fluoro, chloro, bromo, and iodine.

[0393] It will be understood that any one compound of any of the formulas disclosed herein and any pharmaceutically acceptable salt thereof include stereoisomers of the compound, mixtures of stereoisomers, and polymorphs of all isomeric forms.

[0394] In this specification, the term "independently selected" is used to indicate that the R groups may be the same or different.

[0395] As used herein, the terms “substituted” and “substituent,” whether preceded by the term “arbitrarily,” refer to the ability to change one functional group to another, provided that the valence of all atoms is maintained, as understood by those skilled in the art. Where two or more positions in any structure may be substituted with two or more substituents selected from a particular group, the substituents may be the same or different at any position. Furthermore, substituents may be further substituted (for example, substituents on an aryl group may have other substituents, such as another aryl group further substituted with fluorine at one or more positions).

[0396] This disclosure provides isolated or substantially purified polynucleotide or protein compositions. “Isolated” or “purified” polynucleotides or proteins, or their biologically active portions, substantially or essentially contain no components that would normally accompany or interact with polynucleotides or proteins found in their naturally occurring environments. Therefore, isolated or purified polynucleotides or proteins, if produced by recombinant technology, substantially contain no other cellular material or culture medium, and if chemically synthesized, substantially contain no chemical precursors or other chemicals. Optimally, an “isolated” polynucleotide does not contain sequences naturally adjacent to it in the genomic DNA of the organism from which it originates (i.e., sequences located at the 5' and 3' ends of the polynucleotide (optimally, protein-coding sequences)). For example, in various embodiments, an isolated polynucleotide may contain approximately 5 kb, approximately 4 kb, approximately 3 kb, approximately 2 kb, approximately 1 kb, approximately 0.5 kb, or less than approximately 0.1 kb of nucleotide sequences naturally adjacent to it in the genomic DNA of the cell from which it originates. Substantially cellular protein-free protein preparations include those containing approximately 30%, approximately 20%, approximately 10%, approximately 5%, or less than approximately 1% (dry weight) of contaminating protein. When the proteins of this disclosure or their biologically active portions are recombinantly produced, the culture medium optimally contains approximately 30%, approximately 20%, approximately 10%, approximately 5%, or less than approximately 1% (dry weight) of chemical precursors or non-protein-of-interest chemicals.

[0397] This disclosure provides disclosed DNA sequence fragments and variants, as well as proteins encoded by these DNA sequences. Where used throughout this disclosure, the term “fragment” refers to a portion of a DNA sequence, or a portion of an amino acid sequence, and by extension, the protein encoded thereby. A DNA sequence fragment consisting of a coding sequence may encode a protein fragment that retains the biological activity of the native protein and therefore retains DNA recognition or binding activity to a target DNA sequence, as described herein. Alternatively, DNA sequence fragments useful as hybridization probes generally do not encode a protein that retains biological activity or promoter activity. Therefore, DNA sequence fragments may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides to the full-length polynucleotides of this disclosure.

[0398] The nucleic acids or proteins of this disclosure can be constructed by a modular approach, which involves pre-assembling monomer units and / or repeating units in a target vector and then assembling them into a final target vector. The polypeptides of this disclosure can be constructed by a modular approach, which involves pre-assembling repeating units in a target vector that can be composed of repeating monomers of this disclosure and then assembled into a final target vector. This disclosure provides polypeptides produced by this method and nucleic acid sequences encoding these polypeptides. This disclosure provides host organisms and cells containing nucleic acid sequences encoding polypeptides produced by this modular approach.

[0399] The term “antibody” is used in its broadest sense and specifically includes single monoclonal antibodies (including agonist and antagonist antibodies) and antibody compositions having multiepitope specificity. The use of natural or synthetic analogs, variants, alleles, homologs, and orthologues (collectively referred to herein as “analogs”) of antibodies as defined herein is also within the scope of this specification. Therefore, according to certain aspects of this specification, the term “antibody as defined herein” in its broadest sense also includes such analogs. Generally, such analogs may have one or more amino acid residues substituted, deleted, and / or added compared to antibodies as defined herein.

[0400] The term “comprising” is intended to mean that a composition and method includes the elements described but does not exclude others. “Consisting essentially of,” when used to define a composition and method, means excluding other elements that are essentially important to the combination when used for the intended purpose. Thus, a composition consisting essentially of the components defined herein does not exclude trace amounts of contaminants or inert carriers. “Consisting of” means excluding elements and substantial method steps that are more than trace amounts of other components. The embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0401] 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 the splicing of mRNA in eukaryotic cells.

[0402] "Gene expression" is the process of converting the information contained in a gene into a gene product. A gene product can be a direct transcript 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 the translation of mRNA. Gene products also include RNA modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins modified by processes such as methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation, and glycosylation.

[0403] The "regulation" or "control" of gene expression refers to a change in gene activity. Regulation of expression includes, but is not limited to, gene activation and gene repression.

[0404] The term "operatively linked" or its synonym (e.g., "linked operatively") means that two or more molecules are positioned relative to each other in such a way that they can interact with one or both molecules or a combination thereof to influence the function attributable to each of them.

[0405] Components linked by non-covalent bonds, and methods for producing and using non-covalently linked components are disclosed. Various components can take on a variety of different forms, as described herein. For example, non-covalently linked (i.e., operably linked) proteins can be used to enable transient interactions that circumvent one or more problems in the art. The ability of non-covalently linked components, such as proteins, to associate and dissociate allows for functional associations only under circumstances where such association is required for the desired activity, or primarily functional associations. The linkage only needs to last long enough to achieve the desired effect.

[0406] A method for inducing a protein to a specific gene locus in the genome of an organism is disclosed. This method may include a step of providing a DNA localization component and a step of providing an effector molecule, the DNA localization component and the effector molecule being operablely linked via non-covalent linkage.

[0407] A "target site" or "target sequence" is a nucleic acid sequence that defines the portion of the nucleic acid to which a binding molecule will bind, provided that sufficient conditions for binding are present.

[0408] The terms “nucleic acid,” “oligonucleotide,” or “polynucleotide” refer to at least two nucleotides linked by a covalent bond. A single-stranded description also defines the sequence of the complementary strand. Thus, a nucleic acid may encompass the complementary strand of a described single-stranded strand. The nucleic acids of this disclosure also encompass substantially identical nucleic acids and their complements that retain the same structure or encode the same protein.

[0409] The probes of this disclosure may include single-stranded nucleic acids that can hybridize to a target sequence under stringent hybridization conditions. Therefore, the nucleic acids of this disclosure may refer to probes that hybridize under stringent hybridization conditions.

[0410] The nucleic acids of this disclosure may be single-stranded or double-stranded. The nucleic acids of this disclosure may contain double-stranded sequences even if the majority of the molecule is single-stranded. The nucleic acids of this disclosure may contain single-stranded sequences even if the majority of the molecule is double-stranded. The nucleic acids of this disclosure may include genomic DNA, cDNA, RNA, or hybrids thereof. The nucleic acids of this disclosure may include combinations of deoxyribonucleotides and ribonucleotides. The nucleic acids of this disclosure may include combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. The nucleic acids of this disclosure may be synthesized to include non-natural amino acid modifications. The nucleic acids of this disclosure may be obtained by chemical synthesis or recombinant methods.

[0411] The nucleic acids disclosed herein may have their entire base sequence or any part thereof that does not exist in nature. The nucleic acids disclosed herein may contain one or more mutations, substitutions, deletions, or insertions that do not exist in nature, and the entire nucleic acid sequence may not exist in nature. The nucleic acids disclosed herein may contain one or more duplicate, inverted, or repeat sequences that do not exist in nature, and as a result, the entire nucleic acid sequence may not exist in nature. The nucleic acids disclosed herein may contain modified nucleotides, artificial nucleotides, or synthetic nucleotides that do not exist in nature, and the entire nucleic acid sequence may not exist in nature.

[0412] When the genetic code contains redundancy, multiple nucleotide sequences can encode a particular protein. All such nucleotide sequences are assumed herein.

[0413] 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 originates. Changes in the distance between the promoter and the gene can be adapted without impairing the function of the promoter.

[0414] As used throughout this disclosure, the term “promoter” refers to a synthetic or naturally occurring molecule that can confer, activate, or enhance the expression of nucleic acids within a cell. Promoters may include one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. Promoters may also include distal enhancer or repressor elements located thousands of base pairs away from the transcription start site. Promoters may originate from viruses, bacteria, fungi, plants, insects, animals, and the like. Promoters can constitutively or differentially control the expression of gene components with respect to cells, tissues or organs in which expression occurs, or developmental stages 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.

[0415] As used throughout this disclosure, the term “substantially complementary” means 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 the hybridization of two sequences under stringent hybridization conditions.

[0416] As used throughout this disclosure, the term “substantially identical” means that a first sequence and a second sequence are substantially complementary to the complement of a second sequence, with respect to nucleic acids, that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical across regions 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, respectively.

[0417] As used throughout this disclosure, the term “Variant” means, when used to describe a nucleic acid, (i) a portion or fragment of a referenced nucleotide sequence; (ii) a complement to a referenced nucleotide sequence or a portion thereof; (iii) a nucleic acid substantially identical to a referenced nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions with a referenced nucleic acid, its complement, or a sequence substantially identical thereto.

[0418] As used throughout this disclosure, the term “vector” refers to a nucleic acid sequence containing an origin of replication. Examples of vectors include viral vectors, bacteriophages, bacterial artificial chromosomes, and yeast artificial chromosomes. A vector may be either a DNA vector or an RNA vector. A vector may be a self-replicating extrachromosomal vector, preferably a DNA plasmid. A vector may contain amino acids and a DNA sequence, an RNA sequence, or a combination of both DNA and RNA sequences.

[0419] As used throughout this disclosure, the term “variant” means, when used to describe a peptide or polypeptide, a peptide or polypeptide having a different amino acid sequence due to an amino acid insertion, deletion, or conserved substitution, but retaining at least one biological activity. A variant may also mean a protein having a substantially identical amino acid sequence to a reference protein having an amino acid sequence that retains at least one biological activity.

[0420] Conservative substitutions of amino acids, i.e., substitution of an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are typically recognized in the art as involving only minor changes. These small changes can be partially identified by considering the hydrophobicity index of amino acids, as understood in the art. Kyte et al., J.Mol.Biol.157:105-132 (1982). The hydrophobicity index of an amino acid takes into account its hydrophobicity and charge. Substitution with amino acids having similar hydrophobicity indices can maintain the function of the protein. In some embodiments, amino acids with hydrophobicity indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that maintain the biological function of the protein. By considering the hydrophilicity of amino acids in the context of peptides, it is possible to calculate the maximum local mean hydrophilicity of the peptide, which is a useful indicator that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101 is fully incorporated herein by reference.

[0421] By substituting amino acids with similar hydrophilicity values, peptides with preserved biological activity, such as immunogenicity, can be obtained. Substitutions can be performed with amino acids whose hydrophilicity values ​​are within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by its specific side chain. Consistent with this observation, it is understood that amino acid substitutions suitable for biological function depend on the relative similarity of the amino acids, particularly their side chains, as revealed by their hydrophobicity, hydrophilicity, charge, size, and other properties.

[0422] As used herein, “conservative” amino acid substitutions may be defined as shown in Tables A, B, or C below. In some embodiments, fusion polypeptides and / or nucleic acids encoding such fusion polypeptides include conservative substitutions introduced by modifying the polynucleotides encoding the polypeptides of this disclosure. Amino acids can be classified by their physical properties and their contribution to the secondary and tertiary structures of proteins. A conservative substitution is the replacement of one amino acid with another amino acid having similar properties. Exemplary conservative substitutions are shown in Table 1C. Table 1C - Conservative Substitution I TIFF2026530178000060.tif45170

[0423] Alternatively, conserved amino acids can be classified as shown in Table 2, as described by Lehninger (Biochemistry, Second Edition, Worth Publishers, Inc. NY, NY (1975), pp. 71-77). Table 2 - Conservative Substitutions II TIFF2026530178000061.tif72170

[0424] As alternatives, exemplary conservative substitutions are shown in Table 3. Table 3 - Conservative Substitutions III TIFF2026530178000062.tif148170

[0425] It should be understood that the polypeptides of this 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. The polypeptides or nucleic acids of this disclosure may contain one or more conservative substitutions.

[0426] As used throughout this disclosure, the term “two or more” of the aforementioned amino acid substitutions means two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, thirteenth, twenty, or more of the listed amino acid substitutions. The term “two or more” may mean two, three, four, or five of the listed amino acid substitutions.

[0427] The polypeptides and proteins of this disclosure may have sequences, either entirely or in part, that do not exist in nature. The polypeptides and proteins of this disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not exist in nature, resulting in an entire amino acid sequence that does not exist in nature. The polypeptides and proteins of this disclosure may contain one or more duplicate, inverted, or repeat sequences, resulting in a sequence that does not exist in nature, resulting in an entire amino acid sequence that does not exist in nature. The polypeptides and proteins of this disclosure may contain modified amino acids, artificial amino acids, or synthetic amino acids that do not exist in nature, resulting in an entire amino acid sequence that does not exist in nature.

[0428] Where used throughout this disclosure, “sequence identity” may be determined using a standalone executable BLAST engine program for blasting two sequences (bl2seq) with default parameters, which can be obtained from the National Center for Biotechnology Information (NCBI) ftp site (the entire text is incorporated herein by reference; Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250). Where used in the context of two or more nucleic acid or polypeptide sequences, the term “identical” or “sequence” refers to a specific percentage of the same residues across a particular region of each sequence. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a given region, determining the number of positions where identical residues appear in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the given region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences have different lengths, or if alignment generates sequences with one or more ends shifted, 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. Identity can be performed manually or using computer sequencing algorithms such as BLAST or BLAST 2.0.

[0429] 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 the host cell into which it is introduced.

[0430] As used throughout this disclosure, the term “exogenous” means nucleic acids or protein sequences that naturally accompany a target gene or the host cell into which it is introduced, and includes naturally occurring nucleic acids that do not naturally exist, such as DNA sequences or naturally occurring nucleic acid sequences located at naturally occurring genomic locations.

[0431] This disclosure provides a method for introducing a polynucleotide construct containing a DNA sequence into a host cell. “Introducing” means presenting the polynucleotide construct to the cell in a manner that allows access to the interior of the host cell. The method of this disclosure does not depend on a specific method for introducing the polynucleotide construct into a host cell, but only on the polynucleotide construct accessing the interior of a single host cell. Methods for introducing polynucleotide constructs into bacteria, plants, fungi, and animals are known in the art, but are not limited to stable transformation methods, transient transformation methods, and virus-mediated methods.

[0432] Examples For the following examples, compound numbers are assigned to the compounds of formula (I) and (II) in this disclosure according to the following: TIFF2026530178000063.tif224170TIFF2026530178000064.tif241170TIFF2026530178000065.tif221170

[0433] Example 1 - Preparation of Compound No. 1 Compound number 1 was prepared according to general scheme (A). The crude product was purified by silica gel flash column chromatography. 1 H NMR(500MHz,CDCl3)δ7.26(br,1H), 7.13(br,1H), 4.01-3.84(m,4H), 3.32-3.25(m,6H), 3.13-3.05(m,6H), 2.94-2.93(m,4H), 2.74- 2.69(m,4H), 2.37-2.34(m,4H), 2.05-1.99(m,4H), 1.82-1.80(m,4H), 1.62-1.37(m,20H), 1.34-1.20(m,62H), 0.88(t,J=10Hz,12H). MS(ESI): Calculated value C 64 H 130 N4O6S2[M+H] + Regarding 1114.9, the measured value was 1116.3.

[0434] Example 2 - Preparation of Compound No. 2 Compound number 2 was prepared according to general scheme (A). The crude product was purified by silica gel flash column chromatography. 1 H NMR(500MHz,CDCl3)δ4.11-4.09(m,4H), 3.62-3.60(m,4H), 2.62(s,4H), 2.59-2.53(m,6H), 2.42-2.37(m,6H), 1.6 3-1.53(m,6H), 1.51-1.45(m,6H), 1.44-1.40(m,4H), 1.39-1.36(m,8H), 1.33-1.25(m,68H), 0.88(t,J=10Hz,12H). MS(ESI):C 64 H 128 N2O8S2[M+H] + The calculated value is 1116.9, and the measured value is 1118.2.

[0435] Example 3 - Preparation of Compound No. 3 Compound number 3 was prepared according to general scheme (A). The crude product was purified by silica gel flash column chromatography. 1 H NMR (500MHz, CDCl3) δ4.09-4.07(m,4H), 3.62-3.60(m,4H), 2.73-2.70(t,J=10Hz,4H), 2.59-2.53(m,4H) ), 2.45-2.38(m,10H), 2.04-2.0(m,4H), 1.65-1.40(m,14H), 1.33-1.25(m,64H), 0.88(t,J=10Hz,12H). MS(ESI):C 60 H 120 N2O8[M+H] + The calculated value is 996.9, and the measured value is 998.1.

[0436] Example 4 - Preparation of Compound No. 4 Compound number 4 was prepared according to general scheme (A). 1H NMR(500MHz,CDCl3)δ6.5(br,2H), 4.05-4.02(m,4H), 3.72-3.39(m,16H), 3.28-3.25(m,4H), 2.96-2.85(m,16H), 2 .72-2.65(m,8H), 2.33-2.31(m,4H), 2.05-2.01(m,4H), 1.69-1.50(m,30H), 1.35-1.10(m,34H)-0.82-0.91(m,8H). MS(ESI):C 68 H 130 N4O 10 For S2[M+H]+, the calculated value was 1227.9, and the measured value was 1228.6.

[0437] Example 5 - Preparation of Compound No. 5 Compound number 5 was prepared according to general scheme (A). 1 H NMR(500MHz,CDCl3)δ6.5(br,2H), 3.98(m,4H), 3.40-3.38(m,8H)3.27-3.24(m,12H), 2.84-2.66(m,20 H), 2.38-2.30(m,20H), 2.38-2.30(m,4H), 2.03-1.48(m,16H), 1.25-1.15(m,45H), 0.89-0.85(m,39H). MS(ESI):C 88 H 170 N4O 10 S2[M+H] + The calculated value is 1508.2, and the measured value is 1508.9.

[0438] Example 6 - Preparation of Compound No. 6 Compound number 6 was prepared according to the general scheme (A). MS(ESI):C 72 H 146 N4O6S2[M+H] + The calculated value is 1228.1, and the measured value is 1227.8.

[0439] Example 7 - Preparation of Compound No. 7 Compound number 7 was prepared according to general scheme (A). 1H NMR (500MHz, CDCl3) δ7.01-7.1(br,2H), 4.28(s,4H), 4.03-3.97(m,4H)3.72-3.64(m,4H), 3.27(br,6H), 3.13-2.97(m,8H) ), 2.71-2.65(m,4H), 2.50-2.49(m,4H), 1.59-1.57(m,6H), 1.447-1.36(m,16H), 1.31-1.25(m,64H), 0.89-0.85(m,12H). MS(ESI):C 66 H 130 N4O 10 [M+H] + The calculated value is 1140.1, and the measured value is 1140.6.

[0440] Example 8 - Preparation of Compound No. 8 Compound number 8 was prepared according to general scheme (B). 1 H NMR (500MHz, DMSO): δ7.7(br,1H), 4.27-4.21(br,4H), 4.43(m,4H), 3.17(m,2H), 2.49-2.02(m,8H), 1.37-1.24(m,84H), 0.85(m,12H). MS(ESI):C 57 H 117 N3O5[M+H] + The calculated value was 924.9, and the measured value was 926.2.

[0441] Example 9 - Preparation of Compound No. 9 Compound number 9 was prepared according to the general scheme (B). MS(ESI):C 41 H 85 N3O5[M+H] + The calculated value is 700.6, and the measured value is 701.3.

[0442] Example 10 - Preparation of Compound No. 10 Compound number 10 was prepared according to the general scheme (B). MS(ESI):C 65 H 133 N3O5[M+H] + The calculated value is 1037, and the measured value is 1037.6.

[0443] Example 11 - Preparation of Compound No. 11 Compound number 11 was prepared according to the general scheme (B). MS(ESI):C 73 H 149 N3O5[M+H] + The calculated value is 1149.1, and the measured value is 1149.7.

[0444] Example 12 - Preparation of Compound No. 12 Compound number 12 was prepared according to the general scheme (B). MS(ESI):C 69 H 141 N3O9[M+H] + The calculated value is 1157.1, and the measured value is 1157.5.

[0445] Example 13 - Preparation of Compound No. 13 Compound number 13 was prepared according to the general scheme (B). MS(ESI):C 53 H 109 N3O9[M+H] + The calculated value was 932.8, and the measured value was 933.2.

[0446] Example 14 - Preparation of Compound No. 14 Compound number 14 was prepared according to the general scheme (B). MS(ESI):C 61 H 117 N3O9[M+H] + The calculated value was 1036.9, and the measured value was 1037.2.

[0447] Example 15 - Preparation of Compound No. 15 Compound number 15 was prepared according to the general scheme (B). MS(ESI):C 74 H 146 N4O 10 [M+H] + The calculated value is 1252.1, and the measured value is 1252.6.

[0448] Example 16 - Preparation of Compound No. 16 Compound number 16 was prepared according to the general scheme (C). MS(ESI):C 90 H 184 N6O8[M+H]+ The calculated value is 1478.4, and the measured value is 1479.1.

[0449] Example 17 - Preparation of Compound No. 17 Compound number 17 was prepared according to the general scheme (C). MS(ESI):C 100 H 196 N6O 15 [M+H] + The calculated value is 1722.5, and the measured value is 1723.3.

[0450] Example 18 - Preparation of Compound No. 18 Compound number 18 was prepared according to the general scheme (C). MS(ESI):C 112 H 220 N6O 15 [M+H] + The calculated value is 1890.7, and the measured value is 1890.4.

[0451] Example 19 - Preparation of Compound No. 19 Compound number 19 was prepared according to the general scheme (C). 1 H NMR(500MHz,CDCl3)δ4.67(m,2H), 4.26-3.81(m,12H), 3.27-3.02(m,12H), 2.55(s,8H), 2.22 -2.15(m,4H), 2.05-1.97(m,4H), 1.79-1.55(m,8H), 1.71-1.17(m,72H), 0.89-0.85(m,12H). MS(ESI):C 72 H 136 N4O 14 [M+H] + The calculated value is 1282.2, and the measured value is 1282.1.

[0452] Example 20 - Preparation of Compound No. 20 Compound number 20 was prepared according to the general scheme (D). MS(ESI):C 77 H 141 N3O 17 [M+H] + The calculated value is 1381, and the measured value is 1381.5.

[0453] Example 21 - Preparation of Compound No. 21 Compound number 21 was prepared according to the general scheme (E). 1 H NMR (500MHz, CDCl3) δ4.44(t,J=10Hz,1H), 3.64-3.42(m,8H), 2.91-2.74(m,12H), 1.59-1.26(m,100H), 0.89(t,J=5Hz,15H). MS(ESI):C 66 H 136 N2O6[M+H] + The calculated value is 1054, and the measured value is 1054.2.

[0454] Example 22 - Preparation of Compound No. 22 Compound number 22 was prepared according to the general scheme (E). 1 H NMR (500MHz, CDCl3) δ5.12(br,1H), 4.08-4.07(m,2H), 3.65-3.68(m,4H), 2.72-2.45(m,12H), 1.75-1.26(m,80H), 0.86(t,J=5Hz,12H). MS(ESI):C 57 H 117 N3O6[M+H] + The calculated value is 940.9, and the measured value is 941.1.

[0455] Example 23 - Preparation and in vivo screening of LNPs of the Disclosure containing mRNA or DNA The following are non-limiting examples providing exemplary methods for formulating multiple multi-component LNP compositions comprising exemplary compounds of formula (I), formula (II'), or formula (II) and mRNA or DNA.

[0456] To formulate LNP, an LNP composition was prepared by combining compound number 8, phospholipid DOPC, structural lipid cholesterol (Chol), and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (DMG-PEG2000; Avanti Polar Lipids, Alabaster, Alabama, USA).

[0457] Each 25 mg / ml stock solution was prepared by solubilizing the lipids in 200-proof HPLC-grade ethanol, and the stock solutions were stored at -80°C until formulation. During formulation, the lipid stock solution was equilibrated to room temperature for a short time, and then 50-55°C. ° It was placed on a hot plate maintained at a temperature range of 1°C. Then, a high-temperature lipid stock solution was added to obtain the desired final molar percentage.

[0458] A 1 mg / ml solution of the desired nucleic acid to be incorporated into the LNP was added to 150 mM sodium acetate buffer (pH 5.2) to prepare a stock solution, which was kept on ice. The ethanol phase was vigorously mixed with the nucleic acid in the sodium acetate phase using a Precision Nanoassemblr instrument.

[0459] Next, the obtained LNP composition was transferred to a Repligen Float-A-Lyzer dialysis device (Spectrum Chemical Mfg. Corp, California, USA) having a molecular weight cutoff (MWCO) of 8-10 kDa, and 4 phosphate-buffered saline (PBS) (dialysis fluid:dialysis buffer volume at least 1:200 v / v), pH 7.4. ° The LNPs were treated by dialysis overnight in 1C (or alternatively, at least 4 hours at room temperature) to remove 25% ethanol and achieve complete buffer exchange. In some experiments, the LNPs were concentrated by ultracentrifugation at approximately 4100 × g in an Amicon® Ultra-4 centrifugal filter unit MWCO-30kDa (Millipore Sigma, USA). The LNPs were then sterilized until further use was permitted. ° Saved in C.

[0460] In one experiment, the LNP composition of this disclosure, containing compound number 8 and the RNA encoding firefly luciferase (TriLink BioTechnologies), was prepared as described above. The LNP composition is shown in Table 4. Table 4 TIFF2026530178000066.tif102170

[0461] Adult female BALB / C mice (n=3 / group) were intravenously administered 0.5-0.7 mg / kg of 5'-CleanCap--fLuciferase mRNA (TriLink Biotech) containing the LNP composition shown in Table 4. One group of mice was treated with a vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control.

[0462] In another experiment, the LNP composition of this disclosure, containing compound number 8 and DNA encoding firefly luciferase (Nature Technology Corporation), was prepared as described above. The LNP composition is shown in Table 5. Table 5 TIFF2026530178000067.tif89170

[0463] Using the IVIS Lumina in vivo imaging system (Perkin Elmer) according to the manufacturer's instructions, the location and extent of luciferase expression in anesthetized mice were determined by bioluminescence imaging (BLI) at 4 hours for mRNA and at 48 hours for DNA in treated and control mice. Briefly, mice were anesthetized with isoflurane in oxygen and placed supine on a heated stage. Subsequently, D-luciferin (Perkin-Elmer number 122799) was administered intravenously to the mice, and BLI was performed. The mRNA results are shown in Table 6, and the DNA results are shown in Table 7. Table 6 TIFF2026530178000068.tif114170Table 7 TIFF2026530178000069.tif101170

[0464] Visual analysis of BLI images revealed that BLI signals were primarily located in the liver. Therefore, as shown in Tables 6 and 7, the LNP compositions of this disclosure successfully delivered mRNA or DNA to liver cells in vivo, and the encoded transgenes were expressed in the liver cells.

[0465] Example 2: Preparation and in vivo screening of LNPs of the Disclosure, including 4-mRNA. The following are non-limiting examples of exemplary methods for formulating multiple multicomponent LNP compositions comprising exemplary compounds of formula (I), formula (II'), or formula (II) and mRNA.

[0466] An LNP composition of this disclosure, containing one of compound numbers 1 to 14 and RNA encoding firefly luciferase (TriLink BioTechnologies), was prepared as described in Example 23. The LNP composition is shown in Table 8. Table 8 TIFF2026530178000070.tif67170

[0467] Adult female BALB / C mice (n=3 / group) were intravenously administered 0.5 mg / kg of 5'-CleanCap--fLuciferase mRNA (TriLink Biotech) containing the LNP composition shown in Table 8. One group of mice was treated with a vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control.

[0468] Using the IVIS Lumina in vivo imaging system (Perkin-Elmer) according to the manufacturer's instructions, the location and extent of luciferase expression in anesthetized mice were determined at 4 hours by bioluminescence imaging (BLI) of treated and control mice. Briefly, mice were anesthetized with isoflurane in oxygen and placed supine on a heated stage. Subsequently, D-luciferin (Perkin-Elmer number 122799) was administered intravenously to the mice, and BLI was performed. The results are shown in Table 9. Table 9 TIFF2026530178000071.tif74170

[0469] As demonstrated in this embodiment, the LNP composition of this disclosure successfully delivered mRNA in vivo primarily to liver cells, as shown by visual BLI analysis, and the encoded transgene was subsequently expressed by the cells.

[0470] Example 2: Preparation and in vivo screening of LNPs of the Disclosure, including 5-mRNA. The LNP composition of this disclosure, containing compound number 1 and RNA encoding firefly luciferase (TriLink BioTechnologies), was prepared as described in Example 23. The LNP composition is shown in Table 10. Table 10 TIFF2026530178000072.tif48170

[0471] Mice were treated with an LNP composition and subjected to BLI as described in Example 23. One group of mice was treated with a vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control. The results are shown in Table 11. Table 11 TIFF2026530178000073.tif55170

[0472] As demonstrated in this embodiment, the LNP composition of this disclosure successfully delivered mRNA in vivo primarily to liver cells, as shown by visual BLI analysis, and the encoded transgene was subsequently expressed by the cells.

[0473] Example 26: Preparation and in vivo screening of LNPs of the present disclosure, including mRNA. An LNP composition of this disclosure, containing one of compound numbers 1 to 14 and RNA encoding firefly luciferase (TriLink BioTechnologies), was prepared as described in Example 23. The LNP composition is shown in Table 12. Table 12 TIFF2026530178000074.tif46170

[0474] Mice were treated with an LNP composition and subjected to BLI as described in Example 23. One group of mice was treated with a vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control. The results are shown in Table 13. Table 13 TIFF2026530178000075.tif49170

[0475] As demonstrated in this embodiment, the LNP composition of this disclosure successfully delivered mRNA in vivo primarily to liver cells, as shown by visual BLI analysis, and the encoded transgene was subsequently expressed by the cells.

[0476] Example 27: Preparation and in vivo screening of LNPs of the Disclosure containing mRNA An LNP composition of this disclosure, containing one of compound numbers 1 to 14 and RNA encoding firefly luciferase (TriLink BioTechnologies), was prepared as described in Example 23. The LNP compositions are shown in Table 14. Table 14 TIFF2026530178000076.tif74170

[0477] Mice were treated with an LNP composition and subjected to BLI as described in Example 23. One group of mice was treated with a vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control. The results are shown in Table 15. Table 15 TIFF2026530178000077.tif77170

[0478] As demonstrated in this embodiment, the LNP composition of this disclosure successfully delivered mRNA in vivo primarily to liver cells, as shown by visual BLI analysis, and the encoded transgene was subsequently expressed by the cells.

[0479] Example 28 - The LNP composition of this disclosure delivers RNA to the liver with high specificity in vivo. This embodiment demonstrates the ability of the LNP composition of the present disclosure to deliver Cas-CLOVER mRNA targeted by a pair of gRNAs against the psk9 gene to the liver, resulting in subsequent in vivo gene editing of the psk9 gene. As will be understood by those skilled in the art, the psk9 protein is secreted by hepatocytes and binds to the LDL receptor, inducing its internalization and lysosomal degradation, resulting in an increase in circulating levels of LDL cholesterol.

[0480] In two separate experiments, each group of adult female BALB / C mice (n=2 / group) was intravenously co-administered with mRNA encoding 5'-CleanCap-5MeC-Cas-CLOVER (SEQ ID NO: 7) and a pair of gRNAs (SEQ ID NOs: 8 and 9) targeted to the first exon of the mouse pcsk9 gene.

[0481] In the first experiment, mRNA and gRNA molecules were incorporated into the LNP composition of this disclosure containing compound number 8, as shown in Table 16. All uridine residues in the mRNA were N1-methylpseudridine. Table 16 TIFF2026530178000078.tif41170

[0482] Each group of mice was administered a dose (0.5 mg / kg) of Cas-CLOVER mRNA and a pair of pcsk9 gRNA co-encapsulated in the LNP composition of this disclosure. One group of mice was treated with a vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control. Cationic lipid ssPalmO-Ph-P4C2: Another group of mice was treated with a benchmark LNP composition containing JPEG2026530178000079.jpg30170, as described in Akita et al., (2020) Biol. Phar. Bull. 43:1617-1625.

[0483] In the second experiment, mRNA and gRNA molecules were incorporated into the LNP composition of this disclosure containing compound number 1, as shown in Table 17. All uridine residues in the mRNA were N1-methylpseudridine. Table 17 TIFF2026530178000080.tif62170

[0484] Each group of mice was administered a dose (0.5 mg / kg) of Cas-CLOVER mRNA and a pair of pcsk9 gRNA co-encapsulated in the LNP composition of this disclosure. One group of mice was treated with a vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control.

[0485] Seven days after administration, blood was collected from the subjects. Briefly, after euthanasia, 500 μL of blood was collected by cardiac puncture using a 2 ml syringe and a 25 G needle, transferred to a microcentrifuge tube, incubated at room temperature for 1 hour, and then centrifuged at 1500 g for 15 minutes to separate the cell fraction from the serum. The serum fraction (200 μL) was transferred to a new tube and stored at -80°C until further analysis.

[0486] Serum levels of pcsk9 protein were measured in mice 7 days after administration. The results for the first experiment are shown in Table 18 and for the second experiment in Table 19. Briefly, Pcsk9 in each serum sample was determined using a mouse Pcsk9 ELISA kit (Biolegend) according to the manufacturer's instructions. All serum samples were assayed in triplicate, and the results were expressed as a percentage of Pcsk9 levels compared to Pcsk9 levels in PBS-treated mice. Table 18 TIFF2026530178000081.tif51170Table 19 TIFF2026530178000082.tif74170

[0487] The results of these experiments demonstrate that Cas-CLOVER mRNA delivered by the LNP compositions of this disclosure is effective for editing the pcsk9 gene in liver in vivo. Furthermore, Cas-CLOVER mRNA delivered by certain LNP compositions of this disclosure was far more effective in editing the pcsk9 gene in liver in vivo compared to a benchmark LNP composition.

[0488] Example 29 - In vivo delivery of co-encapsulated mRNA and DNA to the liver by LNPs The following are non-limiting examples that provide exemplary methods for formulating multiple multi-component LNP compositions, including exemplary compounds of formula (I) and co-encapsulated mRNA and DNA.

[0489] These experiments demonstrate that luciferase expression was achieved through successful delivery of the two-component DNA / RNA system via the LNP composition to liver cells, resulting in transposition of the luciferase transgene facilitated by SPB.

[0490] Adult BALB / C mice (n=3) were administered a single co-encapsulated LNP containing both the firefly luciferase transposon and SPB. The composition of the co-encapsulated LNP is shown in Table 20 and was prepared as described in Example 23, incorporating nanoplasmid DNA (SEQ ID NO: 10) containing mRNA encoding the active SPB and the transposon. All cytidine residues in the mRNA were 5-methylcytidine (5-MeC). Table 20 TIFF2026530178000083.tif47170

[0491] Mice were treated with LNPs (Large Nucleotide Polypeptides) co-encapsulated in a 1:2 mRNA:DNA ratio at a dose of either 0.5 mg / kg or 1.0 mg / kg. One group of mice was treated with a vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control.

[0492] The location and degree of luciferase expression in treated mice and control mice were determined as described in Example 15, and the results are shown in Table 21. Table 21 TIFF2026530178000084.tif49170

[0493] This example demonstrates, as shown by visual BLI analysis, that the LNP composition of this disclosure successfully delivered a two-component DNA / RNA system to liver cells, and that the desired transgene was stably integrated into the genome without episomalization.

[0494] Example 30 - Improved tolerability of LNP-mediated delivery of co-encapsulated mRNA and DNA to the liver in vivo. The following are non-limiting examples demonstrating that the lipid nanoparticle compositions of the present disclosure, containing GalNac, can be used to deliver co-encapsulated mRNA and DNA to liver cells in vivo with improved tolerability compared to benchmark LNP compositions containing GalNac or LNP compositions of the present disclosure that do not contain GalNac.

[0495] As described in Example 29, the luciferase expression in this example indicates that the two-component DNA / RNA system of the LNP composition was successfully delivered to liver cells, resulting in transposition of the luciferase transgene promoted by SPB.

[0496] Adult BALB / C mice (n=3) were administered a single co-encapsulated LNP containing both the firefly luciferase transposon and SPB. The composition of the co-encapsulated LNP is shown in Table 22 and was prepared as described in Example 23, incorporating nanoplasmid DNA (SEQ ID NO: 10) containing mRNA encoding the active SPB and the transposon. All cytidine residues in the mRNA were 5-methylcytidine (5-MeC). Table 22 TIFF2026530178000085.tif115170

[0497] Mice were treated with co-encapsulated LNPs containing mRNA and DNA in a 1:2 mRNA:DNA ratio at a dose of 1.0 mg / kg. One group of mice was treated with a vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control. Another group of mice was treated with the following structure, prepared as described in PCT application number PCT / US2023 / 061005: The cells were treated with a benchmark LNP composition containing the cationic lipid HMA-404 having TIFF2026530178000086.tif46170.

[0498] The location and degree of luciferase expression in treated and control mice were determined as described in Example 23, and the results are shown in Table 23. The LNP composition of this disclosure successfully delivered a two-component DNA / RNA system to liver cells, as indicated by the luciferase expression levels compared to the vehicle, as shown in Table 23. Table 23 TIFF2026530178000087.tif125170

[0499] The levels of five inflammatory cytokines present in the serum were evaluated for each tested concentration 4 hours after LNP administration. Briefly, serum samples were prepared as described in the liver enzyme analysis section, and serum concentrations of each cytokine were measured using a commercially available ELISA kit (e.g., R&D Systems Quantikine ELISA kit). Table 24 shows the levels of the inflammatory cytokines interleukin-6 (IL-6), interferon-gamma (INF-γ), tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1 beta (MIP-1β) in mice treated with the LNP of this disclosure compared to vehicles treated at 4 hours. Table 24: Cytokine concentration (pg / ml) TIFF2026530178000088.tif120170

[0500] The results of this embodiment demonstrate that the addition of GalNac to the LNP compositions of the present disclosure for administering co-encapsulated mRNA and DNA to hepatocytes improved in vivo tolerability compared to the LNP compositions of the present disclosure without GalNac. The LNP compositions of the present disclosure with GalNac (e.g., LNP IDs A.3, A.9, A.10, A.11, A.12) showed comparable or higher luciferase expression and comparable or even reduced cytokine induction. Furthermore, the addition of GalNac to certain LNP compositions in this disclosure resulted in significantly lower cytokine levels compared to the same LNP compositions without GalNac (for example, compare LNP ID.A.3 and A.9 with LNP ID.A.13; LNP ID.A.6 with LNP ID.A.2; LNP ID.A.11 and A.12 with LNP ID.A.1; LNP ID.A.8 with LNP ID.A.7; and LNP ID.A.10 with LNP ID.A.1).

[0501] Example 31 - In vivo delivery of co-encapsulated mRNA and DNA to the liver by LNPs This experiment demonstrates that FVIII expression was achieved through successful delivery of the two-component DNA / RNA system via the LNP composition to liver cells, resulting in transposition of the FVIII transgene facilitated by SPB.

[0502] Wild-type C57BL / 6 adult mice (N=4) were administered a single co-encapsulated LNP containing both the FVIII transposon and SPB. Table 25 shows the composition of the co-encapsulated LNPs incorporating mRNA encoding the active SPB and TTR-FVIII nanoplasmid DNA (SEQ ID NO: 9). All cytidine residues in the mRNA were 5-methylcytidine (5-MeC). Table 25 TIFF2026530178000089.tif32170

[0503] Mice in each group received co-encapsulated LNPs (Large Nucleotide Plasma) containing mRNA and DNA in a 1:2 mRNA:DNA ratio at a dose of 0.75 mg / kg or 1 mg / kg. One group of mice was treated with a vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control.

[0504] Prior to administration, mice were placed under isoflurane anesthesia. For delivery, 50–80 μL of co-encapsulated LNP was injected into a single 29-gauge insulin syringe and administered intravenously (IV) through the posterior orbital sinus. Plasma was collected from the treated mice 6 days post-treatment. For plasma collection, treated mice were placed under isoflurane anesthesia, and approximately 150 μL of whole blood was collected retroorbitally. The whole blood was mixed with 10% volume of 3.2% sodium citrate, centrifuged at 15,000 g, 20°C for 15 minutes, and the plasma supernatant was collected. hFVIII antigen levels were measured using the Visualize® Factor VIII Antigen Plus Kit (Affinity Biologicals® Inc.).

[0505] The results of FVIII antigen expression are shown in Table 26. Table 26 TIFF2026530178000090.tif44170

[0506] As shown in Table 26, the LNP composition containing compound number 1 resulted in an increase of up to approximately 41% in FVIII antigen expression compared to the vehicle. Example 32 - Preparation and in vivo screening of LNPs of the present disclosure, including DNA.

[0507] The following are non-limiting examples that provide exemplary methods for formulating multiple multi-component LNP compositions containing exemplary compounds of formula (I) and DNA.

[0508] The LNP compositions of this disclosure, containing DNA encoding compound number 1 and firefly luciferase (Nature Technology Corporation), were prepared as described in Example 23. The LNP compositions are shown in Table 27. Table 27 TIFF2026530178000091.tif34170

[0509] Using the IVIS Lumina in vivo imaging system (Perkin-Elmer) according to the manufacturer's instructions, the location and extent of luciferase expression in treated and control mice were determined at 48 hours of DNA bioluminescence imaging (BLI) of anesthetized mice. Briefly, mice were anesthetized with isoflurane in oxygen and placed supine on a heated stage. Subsequently, D-luciferin (Perkin-Elmer number 122799) was administered intravenously to the mice, and BLI was performed. The results are shown in Table 28. Table 28 TIFF2026530178000092.tif42170

[0510] The results of this embodiment show that the LNP composition of this disclosure successfully delivered DNA in vivo, as shown by visual BLI analysis, primarily to liver cells, and the encoded protein was subsequently expressed by the cells.

[0511] Example 33 - LNP compositions containing tannic acid promote DNA delivery to liver cells in vivo. This experiment demonstrates the ability of the LNP composition of this disclosure, which contains tannic acid, to promote DNA delivery to liver cells in vivo.

[0512] In this experiment, each group of adult female BALB / C mice (n=3 / group) was injected via tail vein with a DNA nanoplasmid encoding the fluc gene operably bound to a constitutive CMV promoter. The DNA molecules were incorporated into the LNP compositions shown in Table 29, and the LNP compositions of this disclosure (0.5 mg / kg) were administered to the mice from each group. Table 29 TIFF2026530178000093.tif46170

[0513] The results of the BLI measurement (total flux [p / sec]) are shown in Table 30. Table 30 TIFF2026530178000094.tif42170

[0514] As shown in Table 30, the addition of tannic acid to the LNP composition resulted in an increase of up to 84 times in BLI compared to the LNP composition without tannic acid (C.1).

[0515] Example 34 - Preparation and in vivo screening of LNPs of the Disclosure containing mRNA or DNA An LNP composition of this disclosure, comprising one of compound numbers 1 to 22 and RNA encoding firefly luciferase (TriLink BioTechnologies) or DNA encoding firefly luciferase (Nature Technology Corporation), was prepared as described in Example 23. The LNP compositions are shown in Table 31. Table 31 TIFF2026530178000095.tif74170

[0516] Mice were treated with an LNP composition and subjected to BLI as described in Example 23. One group of mice was treated with a vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control. The results for RNA delivery are shown in Table 32, and the results for DNA delivery are shown in Table 33. Table 32 TIFF2026530178000096.tif63170Table 33 As demonstrated in this embodiment, the LNP composition of the present disclosure successfully delivered mRNA or DNA in vivo to primarily liver cells, as shown by visual BLI analysis, and the encoded transgene was subsequently expressed by the cells.

[0517] While the present invention has been described in relation to its specific embodiments, it will be understood that further modifications are possible, and this application is intended to co...

Claims

1. Compound of formula (I): or a salt thereof During the ceremony: Z is, or covalent bond; X ａ , X ｂ and X ｃ Each of them is independently NH or O; Y ａ , Y ｂ , Y ｃ , Y ｄ , Y ｅ and Y ｆ each independently is C or O; Each of r, s, and t is independently an integer in the range of 1 to 9; and R ２ａ , R ２ｂ , R ２ｃ , R ２ｄ , R ２ｅ and R ２ｆ Each of these is independent of C １ ~C １８ Alkyl, C ２ ~C １８ Alkenyl, -(cyclohexyl)-(C １ ~C １８ Alkyl),-(cyclohexyl)-(C ２ ~C １８ (Alkenyl) or -(cyclohexyl)-(CH ２ -O-CO)-(C １ ~C １８ Alkyl) and the above C １ ~C １８ Alkyl or C ２ ~C １８ An alkenyl is a compound or a salt thereof in which one or more cyclohexyl groups are optionally substituted.

2. Z is The compound according to claim 1.

3. Z is The compound according to claim 1.

4. X ａ , X ｂ and X ｃ The compound according to any one of claims 1 to 3, wherein each of them is NH.

5. X ａ , X ｂ and X ｃ The compound according to any one of claims 1 to 3, wherein each of the is O.

6. Y ａ , Y ｂ , Y ｃ , Y ｄ , Y ｅ and Y ｆ The compound according to any one of claims 1 to 5, wherein each of the is C.

7. Y ａ , Y ｂ , Y ｃ , Y ｄ , Y ｅ and Y ｆ The compound according to any one of claims 1 to 5, wherein each of the is O.

8. R ２ａ , R ２ｂ , R ２ｃ , R ２ｄ , R ２ｅ and R ２ｆ Each of them is C １ ~C １８ A compound according to any one of claims 1 to 7, wherein it is alkyl.

9. R ２ａ , R ２ｂ , R ２ｃ and R ２ｄ Each of these is a C substituted with one or more cyclohexyl groups. １ ~C １８ A compound according to any one of claims 1 to 8, wherein it is alkyl.

10. R ２ａ , R ２ｂ , R ２ｃ and R ２ｄ Each of them, The compound according to any one of claims 1 to 9.

11. R ２ａ , R ２ｂ , R ２ｃ and R ２ｄ Each of them, The compound according to any one of claims 1 to 9.

12. Z is X ａ and X ｂ The compound according to claim 1, wherein each of them is NH.

13. Z is X ａ and X ｂ The compound according to claim 1, wherein each of them is O.

14. Z is a covalent bond, and X ａ and X ｂ The compound according to claim 1, wherein each of them is O.

15. Z is X ａ and X ｂ The compound according to claim 1, wherein each of them is NH.

16. Y ａ , Y ｂ , Y ｃ and Y ｄ The compound according to any one of claims 12 to 15, wherein each of the is C.

17. Y ａ , Y ｂ , Y ｃ and Y ｄ The compound according to any one of claims 12 to 15, wherein each of the is O.

18. R ２ａ , R ２ｂ , R ２ｃ and R ２ｄ are each C ３ to C １８ alkyl, the compound according to any one of claims 12 to 17.

19. R ２ａ , R ２ｂ , R ２ｃ and R ２ｄ each is C substituted with one or more cyclohexyl groups ３ -C １８ alkyl, the compound according to any one of claims 12 to 18.

20. R ２ａ , R ２ｂ , R ２ｃ and R ２ｄ Each of them, The compound according to any one of claims 12 to 19.

21. R ２ａ , R ２ｂ , R ２ｃ and R ２ｄ Each of them, The compound according to any one of claims 12 to 19.

22. The compound according to any one of claims 1 to 21, wherein each of r and s is 3. A compound selected from

23. .

24. Compound of formula (II): or a salt thereof During the ceremony: G is, or And; Y ａ , Y ｂ , Y ｃ and Y ｄ Each of them is independently either C or O; Each of r and s is an integer in the range of 1 to 9, independently of the others; R １ is H or C1-C3 alkyl; and R ２ａ , R ２ｂ , R ２ｃ and R ２ｄ Each of these is independent of C １ ~C １８ Alkyl, C ２ ~C １８ Alkenyl, -(cyclohexyl)-(C １ ~C １８ Alkyl),-(cyclohexyl)-(C ２ ~C １８ (Alkenyl), or -(cyclohexyl)-(CH ２ -O-CO)-(C １ ~C １８ Alkyl) and the above C １ ~C １８ Alkyl or C ２ ~C １８ An alkenyl is a compound or a salt thereof in which one or more cyclohexyl groups are optionally substituted.

25. Y ａ , Y ｂ , Y ｃ and Y ｄ The compound according to claim 24, wherein each of them is C.

26. Y ａ , Y ｂ , Y ｃ and Y ｄ The compound according to claim 24, wherein each of them is O.

27. R １ The compound according to any one of claims 24 to 26, wherein is H.

28. R ２ａ , R ２ｂ , R ２ｃ and R ２ｄ Each of them is C １ ~C １８ The compound according to any one of claims 24 to 27, wherein it is alkyl.

29. R ２ａ , R ２ｂ , R ２ｃ and R ２ｄ Each of these is a C substituted with one or more cyclohexyl groups. １ ~C １８ The compound according to any one of claims 24 to 28, wherein it is alkyl.

30. R ２ａ , R ２ｂ , R ２ｃ and R ２ｄ Each of them, The compound according to any one of claims 24 to 29.

31. The compound according to any one of claims 24 to 30, wherein each of r and s is 3. A compound selected from

32. .

33. A composition comprising at least one lipid nanoparticle containing at least one compound of formula (I) as described in any one of claims 1 to 23.

34. The at least one lipid nanoparticle is The compound comprises at least one compound of formula (I) in a molar ratio of approximately 35%, 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 makes up approximately 56% of the molar ratio. DOPC at a molar ratio of approximately 7.5%, and Approximately 1.5% DMG-PEG2000 in molar ratio Furthermore, including, The composition according to claim 33, wherein the ratio of lipids to nucleic acids in at least one nanoparticle is about 50:1 (w / w).

35. The at least one lipid nanoparticle is The compound comprises at least one compound of formula (I) in a molar ratio of approximately 50%, 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 makes up approximately 38.5% of the molar ratio. DOPC at a molar ratio of approximately 10%, and Approximately 1.5% DMG-PEG2000 in molar ratio Furthermore, including, The composition according to claim 33, wherein the ratio of lipids to nucleic acids in the at least one nanoparticle is about 60:1 (w / w) or about 80:1 (w / w).

36. The at least one lipid nanoparticle is The compound comprises at least one compound of formula (I) in a molar ratio of approximately 50%, 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 makes up approximately 37.5% of the molar ratio. DOPC at a molar ratio of approximately 10%, and Approximately 2.5% DMG-PEG2000 in molar ratio Furthermore, including, The composition according to claim 33, wherein the ratio of lipids to nucleic acids in the at least one nanoparticle is about 40:1 (w / w).

37. The at least one lipid nanoparticle is The compound comprises at least one compound of formula (I) in a molar ratio of approximately 40%, 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 makes up approximately 52.5% of the molar ratio. DOPC at a molar ratio of approximately 5%, and Approximately 2.5% DMG-PEG2000 in molar ratio Furthermore, including, The composition according to claim 33, wherein the ratio of lipids to nucleic acids in at least one nanoparticle is about 50:1 (w / w).

38. The at least one lipid nanoparticle is The compound comprises at least one compound of formula (I) in a molar ratio of approximately 45%, 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 makes up approximately 45.5% of the molar ratio. DOPC at a molar ratio of approximately 7.5%, and Approximately 2% molar ratio of DMG-PEG2000 Furthermore, including, The composition according to claim 33, wherein the ratio of lipids to nucleic acids in at least one nanoparticle is about 50:1 (w / w).

39. The at least one lipid nanoparticle is The mixture contains at least one compound of formula (I) in a molar ratio of approximately 43.17%, 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 accounts for approximately 43.17% of the molar ratio. DOPC at a molar ratio of approximately 11.96%, and Approximately 1.7% DMG-PEG2000 in molar ratio Furthermore, including, The composition according to claim 33, wherein the ratio of lipids to nucleic acids in at least one nanoparticle is about 80:1 (w / w).

40. The at least one lipid nanoparticle is The compound comprises at least one compound of formula (I) in a molar ratio of approximately 45%, 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 makes up approximately 43% of the molar ratio. DOPC at a molar ratio of approximately 10%, and Approximately 2% molar ratio of DMG-PEG2000 Furthermore, including, The composition according to claim 33, wherein the ratio of lipids to nucleic acids in at least one nanoparticle is about 80:1 (w / w).

41. The at least one lipid nanoparticle is The compound comprises at least one compound of formula (I) in a molar ratio of approximately 45%, 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 makes up approximately 46% of the molar ratio. DOPC at a molar ratio of approximately 7.5%, and Approximately 1.5% DMG-PEG2000 in molar ratio Furthermore, including, The composition according to claim 33, wherein the ratio of lipids to nucleic acids in at least one nanoparticle is about 60:1 (w / w).

42. The at least one lipid nanoparticle is The compound comprises at least one compound of formula (I) in a molar ratio of approximately 50%, 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 makes up approximately 39% of the molar ratio. DOPC at a molar ratio of approximately 10%, and Approximately 1% molar ratio of DMG-PEG2000 Furthermore, including, The composition according to claim 33, wherein the ratio of lipids to nucleic acids in at least one nanoparticle is about 60:1 (w / w).

43. The at least one lipid nanoparticle is The compound comprises at least one compound of formula (I) in a molar ratio of approximately 50%, 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 makes up approximately 41.5% of the molar ratio. DOPC at a molar ratio of approximately 7.5%, and Approximately 1% molar ratio of DMG-PEG2000 Furthermore, including, The composition according to claim 33, wherein the ratio of lipids to nucleic acids in at least one nanoparticle is about 80:1 (w / w).

44. The at least one lipid nanoparticle is The compound comprises at least one compound of formula (I) in a molar ratio of approximately 40%, 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 makes up approximately 48.5% of the molar ratio. DOPC at a molar ratio of approximately 10%, and Approximately 1.5% DMG-PEG2000 in molar ratio Furthermore, including, The composition according to claim 33, wherein the ratio of lipids to nucleic acids in at least one nanoparticle is about 80:1 (w / w).

45. The at least one lipid nanoparticle is The compound comprises at least one compound of formula (I) in a molar ratio of approximately 35% to approximately 50%, 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, in molar ratio, is approximately 37.5% to 56%. DOPC in molar ratio of approximately 5% to approximately 12%, and DMG-PEG2000 in a molar ratio of approximately 1% to 2.5% Furthermore, including, The composition according to claim 33, wherein the ratio of lipids to nucleic acids in at least one nanoparticle is about 40:1 (w / w) to about 80:1 (w / w).

46. A composition comprising at least one lipid nanoparticle containing at least one compound of formula (II) as described in any one of claims 24 to 32.

47. The at least one lipid nanoparticle is The compound comprises at least one compound of formula (II) in a molar ratio of approximately 35%, 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 makes up approximately 59% of the molar ratio. DOPC at a molar ratio of approximately 5%, and Approximately 1% molar ratio of DMG-PEG2000 Furthermore, including, The composition according to claim 46, wherein the ratio of lipids to nucleic acids in at least one nanoparticle is about 60:1 (w / w).

48. The at least one lipid nanoparticle is The compound comprises at least one compound of formula (II) in a molar ratio of approximately 50%, 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 makes up approximately 41.5% of the molar ratio. DOPC at a molar ratio of approximately 7.5%, and Approximately 1% molar ratio of DMG-PEG2000 Furthermore, including, The composition according to claim 46, wherein the ratio of lipids to nucleic acids in at least one nanoparticle is about 60:1 (w / w).

49. The at least one lipid nanoparticle is The compound comprises at least one compound of formula (II) in a molar ratio of approximately 50%, 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 makes up approximately 39% of the molar ratio. DOPC at a molar ratio of approximately 10%, and Approximately 1% molar ratio of DMG-PEG2000 Furthermore, including, The composition according to claim 46, wherein the ratio of lipids to nucleic acids in at least one nanoparticle is about 50:1 (w / w).

50. The at least one lipid nanoparticle is The mixture contains at least one compound of formula (II) in a molar ratio of approximately 42.5%, 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 makes up approximately 51.5% of the molar ratio. DOPC at a molar ratio of approximately 5%, and Approximately 1% molar ratio of DMG-PEG2000 Furthermore, including, The composition according to claim 46, wherein the ratio of lipids to nucleic acids in at least one nanoparticle is about 40:1 (w / w).

51. The at least one lipid nanoparticle contains at least one compound of formula (II) in a molar ratio of about 35% to about 50%. 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, in molar ratio, is approximately 37% to 59%. DOPC in molar ratio of approximately 5% to approximately 10%, and DMG-PEG2000 in a molar ratio of approximately 1% to 3% Furthermore, including, The composition according to claim 46, wherein the ratio of lipids to nucleic acids in at least one nanoparticle is about 50:1 (w / w) to about 80:1 (w / w).

52. The composition according to any one of claims 33 to 51, wherein the RNA molecule is an mRNA molecule, and preferably the mRNA molecule further comprises 5'-CAP.

53. The composition according to any one of claims 33 to 51, wherein the at least one RNA molecule comprises a nucleic acid sequence encoding at least one transposase, and preferably the transposase is piggyBac™ (PB) transposase, piggyBac-like (PBL) transposase, Super piggyBac™ (SPB) transposase polypeptide, Sleeping Beauty transposase, Hyperactive Sleeping Beauty (SB100X) transposase, helitron transposase, Tol2 transposase, TcBaster transposase, or mutant TcBaster transposase.

54. The composition according to any one of claims 33 to 51, wherein the DNA molecule is a circular DNA molecule, a DoggyBone DNA molecule, a DNA plasmid, a DNA nanoplasmid, or a linearized DNA molecule.

55. The composition according to any one of claims 33 to 51, wherein the at least one DNA molecule comprises a nucleic acid sequence encoding at least one transposon.

56. The composition according to any one of claims 33 to 55, wherein the at least one nucleic acid molecule comprises a nucleic acid sequence encoding at least one therapeutic protein.

57. The composition according to any one of claims 33 to 56, 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.

58. A pharmaceutical composition comprising the composition according to any one of claims 33 to 57 and at least one pharmaceutically acceptable excipient or diluent.

59. 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 described in any one of claims 33 to 57.

60. A method for genetically modifying at least one cell, comprising contacting the at least one cell with at least one composition described in any one of claims 33 to 57.

61. The method according to claim 59 or 60, wherein the at least one cell is a liver cell.

62. The method according to claim 61, wherein the liver cells are hepatocytes, hepatic stellate cells, Kupffer cells, or hepatic sinusoidal endothelial cells.

63. At least one cell modified according to the method described in any one of claims 59 to 62.

64. A method for treating at least one disease or disorder in a subject that requires treatment of at least one disease or disorder, comprising administering at least one therapeutically effective amount of a composition according to any one of claims 33 to 57, a pharmaceutical composition according to claim 58, or at least one cell according to claim 63 to the subject.

65. The method according to claim 64, wherein the at least one disease or disorder is a liver disease or liver disorder.

66. The composition according to any one of claims 33 to 57, wherein the at least one RNA molecule comprises a nucleic acid sequence encoding a fusion protein, and the fusion protein comprises (i) an inactivated Cas9 (dCas9) protein or its inactivated nuclease domain, and (ii) a Clo051 protein or its nuclease domain.

67. The composition according to claim 66, wherein the composition further comprises at least one guide RNA molecule.

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