Lipidoid Compounds and Related Compositions and Uses

Abstract

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

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 480,815, filed January 20, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing The Sequence Listing XML associated with this application has been provided electronically in XML format and is incorporated herein by reference. The XML file containing the Sequence Listing XML is named "POTH-076_001WO_SeqList.xml." The XML file is 20,860 bytes and was created on January 18, 2024, and has been submitted electronically via the USPTO Patent Center.

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

[0004] There is a long-standing but unmet need in the art for compositions and methods for delivering nucleic acids to cells, as well as for genetically modifying cells in vivo, ex vivo, and in vitro. Widely accepted gene delivery and genetic modification techniques, such as the use of viral vectors, including AAV, can cause acute toxicity and adverse side effects in patients. The present disclosure provides improved compositions, methods, and kits for delivering nucleic acids to various types of cells in vivo, ex vivo, and in vitro. More specifically, the present disclosure provides improved lipid nanoparticle compositions and methods for using the same. These lipid nanoparticle compositions and methods enable the delivery of nucleic acids to cells with high efficiency and low toxicity. Therefore, the compositions and methods of the present disclosure have broad applicability to a variety of fields, including gene therapy. Furthermore, these lipid nanoparticles and methods enable targeted delivery of nucleic acids to the lungs and lung cells, rather than the liver, which is the target of most developed lipid nanoparticle compositions. Therefore, the compositions and methods of the present disclosure also have broad applicability for the development of treatments for a wide range of lung diseases. Summary of the Invention

[0005] In some embodiments, novel compounds are provided. In one embodiment, the novel compounds are compounds of formula (I): [ka] Formula (I) or a salt thereof, During the ceremony, A is, [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , [ka] ,or [ka] and each B independently: [ka] or [ka] where * indicates the bond to A and ** indicates the bond to C; Each C is independent, [ka] where: [ka] indicates a single or double bond; n is an integer from 2 to 6; a is an integer from 1 to 5; b is an integer from 1 to 5; Each R1 is independently C1-C 18 Alkyl or C2-C 18 is alkenyl; each R2 is independently H or methyl; Each R3 is independently H or methyl.

[0006] In some embodiments, novel lipid nanoparticles ("LNPs") are provided that comprise novel compounds. In one embodiment, the novel compounds are compounds of formula (I):

[0007] In some embodiments, a pharmaceutical composition is provided comprising a composition of the present disclosure and at least one pharmaceutically acceptable excipient or diluent.

[0008] In some embodiments, methods are provided for delivering at least one nucleic acid to at least one cell, comprising contacting the at least one cell with at least one composition of the present disclosure.

[0009] In some embodiments, methods of genetically modifying at least one cell are provided, comprising contacting the at least one cell with at least one composition of the present disclosure.

[0010] In some embodiments, methods are provided for treating at least one disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one composition of the present disclosure.

[0011] In some embodiments, methods are provided for delivering at least one nucleic acid to at least one cell, comprising contacting the at least one cell with at least one composition of the present disclosure.

[0012] In some aspects, cells modified according to the methods of the present disclosure are provided.

[0013] In some embodiments, provided are methods for preferentially delivering at least one composition of the present disclosure to the lungs or lung cells of a subject in need thereof, the method comprising administering at least one composition of the present disclosure to the subject.

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

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

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

[0017] [Figure 1] 1 shows bioluminescence images of the lungs, liver, and spleen of a mouse after administration of a composition of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present disclosure provides novel lipidoid compounds, novel lipid nanoparticle compositions (LNPs) containing novel lipidoid compounds, methods for preparing LNPs, and methods for using them. In a non-limiting example, the compositions and methods of the present disclosure can be used for gene delivery. In a non-limiting example, the compositions and methods of the present disclosure can be widely used to deliver nucleic acids to lung cells in vivo, ex vivo, or in vitro for the treatment of certain diseases and disorders, including but not limited to, lung disorders. In a non-limiting example, the compositions and methods of the present disclosure can be widely used to deliver nucleic acids to induce the expression of secreted therapeutic proteins.

[0019] Compositions of the Disclosure—Lipid Nanoparticles

[0020] The present disclosure provides a composition comprising at least one lipid nanoparticle comprising the compound of the present disclosure and at least one nucleic acid molecule.In some embodiments, lipid nanoparticle can further comprise at least one structured lipid.In some embodiments, lipid nanoparticle can further comprise at least one phospholipid.In some embodiments, lipid nanoparticle can further comprise at least one PEGylated lipid.

[0021] compound

[0022] In one aspect, the present disclosure provides a compound of formula (I): [ka] Formula (I) or a salt thereof, During the ceremony, A is, [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , [ka] ,or [ka] and; Each B is independently [ka] or [ka] where * indicates the bond to A and ** indicates the bond to C; Each C independently: [ka] where: [ka] indicates a single or double bond; n is an integer from 2 to 6; a is an integer from 1 to 5; b is an integer from 1 to 5; Each R1 is independently C1-C 18 Alkyl or C2-C 18 is alkenyl; each R2 is independently H or methyl; Each R3 is independently H or methyl.

[0023] In some embodiments, A is [ka] is.

[0024] In some embodiments, each B is [ka] where * indicates the bond to A and ** indicates the bond to C.

[0025] In some embodiments, each B is [ka] where * indicates the bond to A and ** indicates the bond to C.

[0026] In some embodiments, A is [ka] and each B is [ka] where * indicates the bond to A and ** indicates the bond to C.

[0027] In some embodiments, A is [ka] and each B is [ka] where * indicates the bond to A and ** indicates the bond to C.

[0028] In some embodiments, each C is [ka] where: [ka] represents a single or double bond. In some embodiments, each C is [ka] In some embodiments, each C is [ka] In some embodiments, each C is [ka] is.

[0029] In some embodiments, each R2 is H.

[0030] In some embodiments, each R2 is methyl.

[0031] In some embodiments, each R3 is H.

[0032] In some embodiments, each R3 is methyl.

[0033] In some embodiments, each R is C-C 18 In some embodiments, each R is alkenyl. [ka] In some embodiments, each R is [ka] is.

[0034] In some embodiments, each R is C-C 18 In some embodiments, each R is alkyl. [ka] is.

[0035] In some embodiments, each R3 is H and each R1 is C2-C 18 In some embodiments, each R is alkenyl. [ka] In some embodiments, each R is [ka] is.

[0036] In some embodiments, each R3 is methyl and each R1 is C2-C 18 In some embodiments, each R is alkenyl. [ka] is.

[0037] In some embodiments, each R3 is H and each R1 is C1-C 18 In some embodiments, each R is alkyl. [ka] is.

[0038] In some embodiments, each R2 is H, each R3 is H, and each R1 is C2-C 18 In some embodiments, each R is alkenyl. [ka] In some embodiments, each R is [ka] is.

[0039] In some embodiments, each R2 is methyl, each R3 is H, and each R1 is C2-C 18In some embodiments, each R is alkenyl. [ka] is.

[0040] In some embodiments, each R2 is H, each R3 is H, and each R1 is C1-C 18 In some embodiments, each R is alkyl. [ka] is.

[0041] In some embodiments, a is 2.

[0042] In some embodiments, b is 2.

[0043] In some embodiments, a is 2 and b is 2.

[0044] In some embodiments, n is 4.

[0045] In some embodiments, the compound of formula (I) is a compound selected from the following: [ka] , [ka] , [ka] , [ka] , [ka] , [ka] ,or [ka] .

[0046] It will be understood that the compounds of any one of the formulae disclosed herein, and any pharmaceutically acceptable salts thereof, include all isomeric forms, stereoisomers, mixtures of stereoisomers, and polymorphs of the compounds.

[0047] It is understood that the compounds disclosed herein may be presented without a specific configuration (e.g., without a specific stereochemistry). Such presentation is intended to encompass all available isomers, tautomers, positional isomers, and stereoisomers of the compound. In some embodiments, presentation of a compound herein without a specific configuration is intended to refer to each of the available isomers, tautomers, positional isomers, and stereoisomers of the compound, or any mixture thereof.

[0048] It should be understood that the compounds of any formula described herein include the compounds themselves, as well as their salts and solvates, if applicable.Salts can be formed, for example, between an anion and a positively charged group (e.g., amino) on the substituted compounds disclosed herein.Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and acetate (e.g., trifluoroacetate).

[0049] In any of the formulas described herein, when a "-" is used to indicate a link between two variables (e.g., AB), it is understood that the linkage can be one or more covalent bonds.

[0050] General Methods for Preparing Compounds of Formula (I) of the Present Disclosure

[0051] Compounds of formula (I) can 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.

[0052] General procedure for the synthesis of lipidoid (A) [ka]

[0053] Generally, the first step in the preparation of compounds of formula (I) of the present disclosure according to General Scheme A is the reaction of a suitable terpene, such as trans-β-farnesene, β-myrcene, or other suitable biorenewable terpene, with a suitable unsaturated carboxylic acid derivative, such as methyl acrylate, which acts as a dienophile to form a suitable Diels-Alder adduct. [ka]

[0054] Suitable Diels-Alder products for use in the methods herein include: [ka]

[0055] Diels-Alder reactions and Diels-Alder products, such as those used in the preparation of compounds of formula (I) of the present disclosure according to general Scheme A, are described in WO 2022 / 087175, the contents of which are incorporated by reference in their entirety.

[0056] General procedure for Diels-Alder cycloaddition:

[0057] Myrcene (7.2 g, 52.8 mmol, 1.0 equiv.) and methyl acrylate (5.0 g, 58.1 mmol, 1.1 equiv.) were placed in a 150 mL sealed tube. The tube was then capped and stirred at 130 °C for 16 h. The cooled reaction mixture was transferred to a round-bottom flask containing CHCl and evaporated. The crude residue was purified by silica gel flash column chromatography using 3% EtOAc / hexane as the eluent to give myrcene methyl ester (MME). This reaction produces an unequal mixture of 1,3- and 1,4-regioisomers that cannot be separated using conventional column chromatography methods. This mixture of regioisomers was used in subsequent reactions without isomer separation.

[0058] Suitable temperatures are generally between 30° C. and 150° C. Higher temperatures are generally associated with higher yields.

[0059] Following the Diels-Alder reaction, one optional step is hydrogenation, which can be performed as follows:

[0060] Hydrogenation:

[0061] In a round-bottom flask, compound MME was dissolved in a mixture of ethanol and CHCl (5:1). 10% Pd / C (15% w / w) was added in one portion to the solution, and the resulting dark suspension was stirred at room temperature under a hydrogen atmosphere for 20 hours. The reaction mixture was filtered through Celite and rinsed with CHCl (5x). The filtrate was evaporated to give the hydrogenated compound HMME. The resulting residue was carried on to the next step without further purification.

[0062] The compounds prepared by either the Diels-Alder reaction or hydrogenation can then be saponified to give My-COOH (using the product of the Diels-Alder reaction) or hydrogenated My-COOH (HMy-COOH) (using the product of the hydrogenation reaction).

[0063] Saponification:

[0064] In a round-bottom flask, MME or HMME (1.0 equiv.) was added in one portion to methanol (5x) and KOH (1.5 equiv.). The resulting suspension became clear after stirring at 50 °C for 20 h. The reaction was cooled, and water was added. The reaction mixture was extracted with diethyl ether (3x), and the aqueous layer was then acidified to pH 2-3 with 6 N HCl and extracted with ethyl acetate (4x). The combined ethyl acetate extracts were washed with brine; dried over Na2SO4; filtered, and evaporated.

[0065] The saponification reaction can be accomplished with other bases (e.g., NaOH or LiOH), in different solvents, and at lower temperatures with longer waiting times.

[0066] Following saponification, the compound My-COOH or HMy-COOH can be esterified to give the acrylamide compound 2M or hydrogenated 2M (2HM).

[0067] Esterification:

[0068] In a round-bottom flask, My-COOH or HMy-COOH (1.0 equiv.) was dissolved in dichloromethane (10x). EDC-HCl (1.5 equiv.) and DMAP (0.4 equiv.) were added, and the resulting solution was stirred at ambient temperature for 20 min. N-hydroxyethylacrylamide (1.2 equiv.) was added dropwise to the reaction mixture, and the reaction mixture was stirred at ambient temperature for 20 h. Brine (50 mL) was added, and the mixture was extracted with dichloromethane (4x). The combined organic extracts were dried over Na2SO4; filtered, and evaporated. The crude product was purified by silica gel flash column chromatography using a 30-40% ethyl acetate / hexane eluent.

[0069] The esterification reaction can be accomplished using other ester-forming catalysts such as DCC, HATU, or HBTU; or in other solvents such as DMF or NMP.

[0070] Finally, compounds of formula (I) of the present disclosure according to general Scheme A can be prepared from these acrylamide compounds by aza-Michael addition.

[0071] Aza Michael adds:

[0072] Amine 404 (1.0 equiv.) and either 2M or 2HM (5.0 equiv.) were combined in a scintillation vial. The vial was capped and the reaction mixture was stirred at 85 °C for 3 days. The cooled crude reaction product was purified by silica gel flash column chromatography using 5-10% MeOH / CHCl eluent.

[0073] Any suitable amine substrate can be used in the above general scheme A to generate the lipidoid compounds of formula (I) of the present disclosure. For example, the amines shown in the general procedure for the aza-Michael addition step below can be combined with the acrylamide compounds 2M or 2HM, respectively, to provide lipidoid compounds of the present invention. [ka]

[0074] General procedure for the synthesis of lipidoids (B) [ka]

[0075] Intermediate 3

[0076] 1 (6 g, 53.8 mmol) and 2 (7.34 g, 53.8 mmol) were heated in a sealed tube at 130 °C for 30 h. The reaction mixture was cooled to room temperature and loaded onto a column (EtOAc / hexanes). Intermediate 3 (6.15 g, 52%) was obtained as a clear oil. 1H NMR(500MHz,CDCl3)δ 5.34(m,1H),5.08(m,1H),4.11(m,2H),2.55-2.39(m,1H),2.13-2.01(m,3H), 1.99-1.77(m,5H),1.67(m,3H),1.59(m,3H),1.52-1.46(m,1H),1.23(t,J=7.1 Hz,3H),1.18(m,3H).

[0077] Intermediate 4

[0078] 1N NaOH (90 mL) was added to intermediate 3 (5.1 g, 20.37 mmol) in EtOH (90 mL). The resulting mixture was stirred at 75° C. overnight. 1N HCl was added to make the solution acidic (pH approx. 2), which was then extracted with ethyl acetate. The combined organic phases were washed with brine, dried over NaSO, and concentrated. Purification by column chromatography (ethyl acetate / hexane) afforded intermediate 4 (4.37 g, 96%) as a clear oil. 1 H NMR(499MHz,CDCl3)δ 5.40-5.31(m,1H),5.07(m,1H),2.52(m,1H),2.14-2.03(m,3H),2.02-1.84(m,5H),1.67(m,3H),1.65-1.50(m,4H),1.21-1.23(m,3H).

[0079] Intermediate 6

[0080] To 4 (250 mg, 1.12 mmol) in DCM (20 mL) was added 5 (142 mg, 1.23 mmol, 1.1 equiv), EDCI (236 mg, 1.23 mmol, 1.1 equiv), and DMAP (151 mg, 1.23 mmol, 1.1 equiv). The resulting mixture was stirred at room temperature overnight and washed with 1 N HCl, saturated NaHCO3, and brine. The combined organic phases were dried over Na2SO4 and concentrated. Purification by column chromatography (ethyl acetate / hexane) afforded intermediate 6 (276 mg, 77%) as a clear oil. 1H NMR(499MHz,CDCl3)δ 6.27(dd,J=17.0,1.3 Hz,1H),6.12-6.01(m,1H),5.66(dd,J=10.3,1.3 Hz,1H),5.35(m,1H),5.06(m,1H),4.30-4.15(m,2H),3.60(q,J=5.5 Hz,2H),2.40-2.52(m,1H),2.09-2.02(m,3H),2.00-1.80(m,5H),1.67(m,3H),1.63-1.47(m,4H),1.19-1.20(m,3H).

[0081] product 7

[0082] A mixture of N1-(3-aminopropyl)-N1-methylpropane-1,3-diamine (10.5 mg, 0.072 mmol) and 6 (139 mg, 0.43 mmol) was heated at 90 °C for 42 h. The reaction mixture was cooled to room temperature and loaded onto a column (MeOH / DCM). The product 7 (15 mg, 15%) was obtained as a clear gel. 1 H NMR(499MHz,CDCl3)δ 5.33(m,4H),5.06(m,4H),4.14(t,J=5.8 Hz,8H),3.48(q,J=5.8 Hz,8H),2.71(t,J=6.3 Hz,8H),2.52-2.43(m,8H),2.34(t,J=6.3 Hz,8H),2.25(br,3H),2.07-2.01(M,8H),2.00-1.79(m,24H),1.67(M,16H),1.60-1.57(m,20H),1.18-1.17(m,12H).MS actual value 1423.4[M+H] +、 Calculated value [C83H136N7O12 = 1423.0] [ka]

[0083] Intermediate 9

[0084] To 4 (1.8 g, 8.1 mmol) in DCM (100 mL) was added 8 (544 mg, 8.9 mmol, 1.1 equiv), EDCI (1.7 g, 8.9 mmol, 1.1 equiv), and DMAP (1.1 g, 8.9 mmol, 1.1 equiv). The resulting mixture was stirred at room temperature overnight and washed with 1 N HCl, saturated NaHCO3, and brine. The combined organic phases were dried over Na2SO4 and concentrated. Purification by column chromatography (ethyl acetate / hexane) afforded intermediate 9 (1.77 g, 82%) as a clear oil. 1 H NMR(499MHz,CDCl3)δ 6.28-6.21(m,1H),5.46-5.37(m,1H),5.07-5.03(m,1H),3.71-3.67(m,2H),3.49-3.32(m,2H),2.43-2.27(m,1H) ),2.12-2.04(m,3H),2.01-1.78(m,5H),1.68-1.67(m,3H),1.60-1.59(m,3H),1.58-1.45(m,1H),1.20(d,J=2.6 Hz,3H).

[0085] Intermediate 11

[0086] To 9 (563 mg, 2.12 mmol) in DCM (30 mL) at 0 °C was added TEA (236 mg, 2.33 mmol, 1.1 equiv) and 10 (211 mg, 2.33 mmol, 1.1 equiv) dropwise. The resulting mixture was stirred from 0 °C to room temperature overnight and washed with 1 N HCl, saturated NaHCO3, and brine. The combined organic phases were dried over Na2SO4 and concentrated. Purification by column chromatography (ethyl acetate / hexanes) afforded intermediate 11 (357 mg, 52%) as a clear oil. 1H NMR(499MHz,CDCl3)δ 6.42(dd,J=17.3,1.4 Hz,1H),6.16-6.09(m,2H),5.86(dd,J=10.4,1.3 Hz,1H),5.45-5.35(m,1H),5.08-5.03(m,1H),4.25(m,2H),3.61-3.47(m,2H),2.41-2.26(m,1H) ,2.12-2.02(m,3H),2.01-1.79(m,5H),1.68-1.66(m,3H),1.61-1.45(m,4H),1.17-1.18(m,3H).

[0087] product 12

[0088] A mixture of N1-(3-aminopropyl)-N1-methylpropane-1,3-diamine (10.5 mg, 0.072 mmol) and 11 (139 mg, 0.43 mmol) was heated at 90 °C for 42 h. The reaction mixture was cooled to room temperature and loaded onto a column (MeOH / DCM). The product 12 (23 mg, 23%) was obtained as a clear gel. δ 6.23(s,4H),5.43-5.35(m,4H),5.07(m,4H),4.15(t,J=5.5 Hz,8H),3.53-3.44(m,8H),2.75(t,J=7.1 Hz,8H),2.44(t,J=7.1 Hz,12H),2.39-2.25(m,4H),2.17(br,3H),2.10-2.03(m,10H),2.00-1.93( m,16H),1.91-1.84(m,6H),1.68(m,12H),1.62-1.48(m,24H),1.17(d,J=4.6 Hz,12H).MS actual value 1424.3[M+H] +、 Calculated value [C83H136N7O12 = 1423.0]

[0089] General procedure for the synthesis of lipidoids (C) [ka]

[0090] Intermediate 14

[0091] β-Myrcene (30 g, 0.22 mol) and methyl acrylate (1.1 equiv., 20.86 g) were mixed in a round 250 ml pressure flask, sealed with a screw cap, and the reaction mixture was heated to 130 °C for 48 h. After cooling the reaction mixture, the residue was suspended in NaOH (15%, 2 equiv.) in MeOH / HO (1 / 1) and warmed to 50 °C for 3–4 h until the starting material was consumed, as monitored by TLC. MeOH was removed under reduced pressure, and the remaining residue was extracted with EtOAc and washed three times with 1 N NaOH. The combined aqueous layers were neutralized to pH 1–2 with 1 N HCl, followed by extraction three times with EtOAc and washing with brine. The crude acid intermediate 14 was obtained as a slightly brown oil, which was used in the next step without purification.

[0092] Intermediate 15

[0093] To a solution of intermediate 14 (300 mg, 1.44 mmol) and N-OH succinimide (1.2 equiv., 200 mg), EDCl (1.5 equiv., 413 mg) and DIEA (2.0 equiv., 488 μl) were added at room temperature. The resulting reaction mixture was stirred overnight at room temperature until most of the starting material was consumed. The reaction mixture was quenched with saturated NaHCO3 and extracted with DCM, then washed and dried. The crude NHS ester was treated with 1-Me ethanol (108 mg, 1.0 equiv.) in dry DMF (3 ml), followed by the addition of DIEA (0.36 ml, 1.5 equiv.). The resulting mixture was stirred overnight at room temperature to afford the desired amide intermediate 15 (135 mg, 35% yield) after purification by silica gel column (Hex / EtOAc). 1 H NMR(500MHz,CDCl3)of 15:δ5.45-5.44(m(m,1H),5.15-5.08(m,1H),3.80-3.77(m,2H),3.59-3.52(m,2H),3.12(s,2.2H),3.98(s,0.8H),2.72-2.69(m,1H ),2.30-2.25(m,1H),2.12-2.03(m,5H),1.97-1.96(m,2H),1.87-1.80(m,1H),1.77-1.73(m,1H),1.66(s,3H),1.62(s,3H).MS:m / z 266.2(M+H).

[0094] Intermediate 16

[0095] To a solution of intermediate 15 (105 mg, 0.396 mmol) and DIEA (80 μL, 1.2 equiv.) in dry DCM (10 ml), chloride (51 μL, 1.6 equiv.) was added in an ice bath. The reaction mixture was stirred from 0° C. to room temperature for 20 hours. The reaction mixture was quenched with 1N HCl (2 ml), extracted with DCM, and then purified by silica gel column to give intermediate 16 (88 mg) in 70% yield. 1 H NMR(300MHz,CDCl3)of 16:δ 6.43-6.39(m,1H),6.15-6.09(m,1H),5.89-5.84(m,1H),5.41-5.42(m,1 H),5.15-5.08(m,1H),4.34-4.29(m,2H),3.71-3.64(m,2H),3.11(s,1.94 H),3.98(s,1.06H),2.69-2.67(m,1H),2.30-2.27(m,1H),2.12-2.03(m,5 H),1.97-1.94(m,2H),1.82-1.75(m,1H),1.71(s,3H),1.60(s,3H).MS:m / z 342(M+Na).

[0096] product 19

[0097] The final product 19 was prepared by aza-Michael addition according to general procedure A.

[0098] 1 H NMR(500MHz,CDCl3)of 19:δ 5.42-5.43(m,8H),5.08-5.11(m,8H),4.23-4.17(m,8H),3.71-3.58(m,8H),3.13-.91(m,12H),2.71-2.65(m,12H),2.45-2.38(m,12H) ),2.35-2.25(m,8H),2.27-2.15(m,3H),2.14-2.01(m,16H),2.02-193(m,6H),1.8-1.67(m,12H),1.68(s,12H),1.60(s,12H).MS:m / z 1423(M+1).

[0099] Intermediate 17

[0100] To a solution of N-methylaminoethanol (2.47 g, 33 mmol) in dry THF (20 ml) was added acryloyl chloride (2.93 ml, 1.1 equiv.) dropwise at 0 °C, and the resulting reaction mixture was stirred from 0 °C to room temperature for 3 h. The reaction mixture was quenched with saturated NHCO and extracted with EtOAc to give intermediate 17 (0.65 g, 15% yield) after purification by silica gel column. 1 H NMR(300MHz,CDCl3)of 17:δ 6.71-6.56(m,1H),6.36-6.28(m,1H),5.75-5.64(m,1H),3.8-3.61(m,2H),3.54-3.52(m,2H),3.15(s,2H),3.03(s,1H).MS:m / z 130.2(M+1).

[0101] Intermediate 18

[0102] Intermediate 18 was prepared according to the procedure for preparing intermediate 16.

[0103] 1 H NMR(500MHz,CDCl3)of 18:δ 6.64-6.55(m,1H),6.37-6.31(m,1H),5.72-5.69(m,1H),5.40-5.37(m,1H),5.10-5.08(m,1H),4.29-4.21(m,2H),3.72-3 .64(m,2H),3.14-3.04(m,3H),2.69-2.67(m,1H),2.50-2.48(m,1H),2.22-1.93(m,10H),1.71(s,3H),1.60(s,3H).MS:m / z 342(M+Na).

[0104] product 20

[0105] The final product 20 was prepared by aza-Michael addition according to general procedure A.

[0106] 1H NMR(500MHz,CDCl3)of 20:δ 5.32-5.37(m,8H),5.09-5.06(m,8H),4.21-4.20(m,8H),3.61-3.58(m,8H),3.07-2.95(m,12H),2.84-2.7 9(m,8H),2.55-2.45(m,16H),2.30-2.14(m,15H),2.08-1.93(m,28H),1.66(s,12H),1.61(s,12H).MS:m / z 1423(M+1).

[0107] Lipid nanoparticles of the present disclosure

[0108] The present disclosure provides lipid nanoparticles (LNPs) comprising one or more compounds of formula (I). In addition to one or more compounds of formula (I), the LNPs of the present disclosure can include one or more additional LNP components, as described below.

[0109] In some embodiments, LNPs of the disclosure can comprise, on a molar basis, 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, the at least one compound is at least one compound of formula (I) described herein. In some embodiments, the at least one compound of the present disclosure is a mixture of two or more compounds of formula (I).

[0110] In some embodiments, the LNPs of the present disclosure can comprise, on a molar basis, 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% of at least one compound of the present disclosure. In some embodiments, the at least one compound is at least one compound of Formula (I) described herein. In some embodiments, at least one compound of the present disclosure is a mixture of two or more compounds of formula (I).

[0111] structural lipids

[0112] In some embodiments, the LNPs can 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% on a molar basis of at least one structural lipid.

[0113] In some embodiments, the LNPs can further comprise 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% of at least one structural lipid on a molar basis.

[0114] In some embodiments, the structured lipids can be steroids. In some embodiments, the structured lipids can be sterols. In some embodiments, the structured lipids can comprise cholesterol. In some embodiments, the structured lipids can comprise ergosterol. In some embodiments, the structured lipids can be phytosterols.

[0115] In some embodiments, the at least one structured lipid is a mixture of two structured lipids.

[0116] phospholipids

[0117] In some embodiments, the LNPs can 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 on a molar basis.

[0118] In some embodiments, the LNPs can further comprise 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% of at least one phospholipid on a molar basis.

[0119] As used herein, the term "phospholipid" is used in its broadest sense to refer to any amphipathic molecule containing a polar (hydrophilic) head group containing phosphate and two hydrophobic fatty acid chains. In some embodiments, the phospholipid can comprise dioleoylphosphatidylethanolamine (DOPE). In some embodiments, the phospholipid can comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments, the phospholipid can comprise 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some embodiments, the phospholipid is DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPA-NA (1,2-dierucoyl-sn-glycero-3-phosphate (sodium salt)), DEPC (1,2-dierucoyl-sn-glycero-3-phosphocholine), DEPE (1,2-dierucoyl-sn-glycero-3-phosphoethanolamine), DEPG-NA ... 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-dimyristoyl-sn-glycero-3-phosphate (sodium salt)), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DMPG-NA (1,2-dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol) (sodium salt)), DMPG-NH4 (1,2-Dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol)(ammonium salt)), DMPG-NH4 / NA (1,2-Dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium / ammonium salt)), DMPS-NA (1,2-Dimyristoyl-sn-glycero-3-phosphoserine(sodium salt)), DOPA-NA (1,2-Dioleoyl-sn-glycero-3-phosphate(sodium salt)), DOPC (1,2-Dioleoyl-sn-glycero-3-phosphate(sodium salt)), 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-distearoyl 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), 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.

[0120] In some embodiments, at least one phospholipid is a mixture of two structured lipids.

[0121] PEGylated lipids

[0122] In some embodiments, the LNPs can further comprise at least about 0.25%, or at least about 0.5%, or at least about 0.75%, or at least about 1%, or at least about 1.25%, or at least about 1.5%, or at least about 1.75%, or at least about 2%, or at least about 2.25%, or at least about 2.5%, or at least about 2.75%, or at least about 3%, or at least about 3.25%, or at least about 3.5%, or at least about 3.75%, or at least about 4%, or at least about 4.25%, or at least about 4.5%, or at least about 4.75%, or at least about 5%, or at least about 7.5%, or at least about 10% PEGylated lipid on a molar basis.

[0123] In some embodiments, the LNPs can further comprise about 0.25%, or about 0.5%, or about 0.75%, or about 1%, or about 1.25%, or about 1.5%, or about 1.75%, or about 2%, or about 2.25%, or about 2.5%, or about 2.75%, or about 3%, or about 3.25%, or about 3.5%, or about 3.75%, or about 4%, or about 4.25%, or about 4.5%, or about 4.75%, or about 5%, or about 7.5%, or about 10% PEGylated lipid on a molar basis.

[0124] As used herein, the term "PEGylated lipid" refers to any lipid to which at least one polyethylene glycol molecule has been modified (e.g., covalently attached). In some embodiments, the PEGylated lipid can comprise 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (hereinafter referred to as DMG-PEG2000).

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

[0126] Exemplary LNP Compositions

[0127] The following are exemplary LNP compositions of the present disclosure comprising at least one compound of Formula (I), at least one structured lipid, at least one PEGylated lipid, and at least one phospholipid.

[0128] In some embodiments, lipid nanoparticles containing at least one nucleic acid can comprise about 40.75% by molar of at least one compound of Formula (I), about 51.75% by molar of at least one structural lipid, about 5% by molar of at least one phospholipid, and about 2.5% by molar of at least one PEGylated lipid. In some embodiments, lipid nanoparticles containing at least one nucleic acid can comprise about 30.75% to about 50.75% by molar of at least one compound of Formula (I), about 41.75% to about 61.75% by molar of at least one structural lipid, about 0.1% to about 15% by molar of at least one phospholipid, and about 0.1% to about 12.5% ​​by molar of at least one PEGylated lipid. In some embodiments, lipid nanoparticles comprising at least one nucleic acid can comprise from about 35.75% to about 45.75% by molar of at least one compound of Formula (I), from about 46.75% to about 56.75% by molar of at least one structural lipid, from about 1% to about 10% by molar of at least one phospholipid, and from about 1% to about 7.5% by molar of at least one PEGylated lipid.

[0129] Table A shows additional exemplary LNP compositions of the present disclosure.

[0130] [table] TIFF2026503551000065.tif115170

[0131] In some embodiments, including the LNP compositions described in Table A, the compound of Formula (I) included in the LNP composition is one of Compound Nos. 1-7.

[0132] In some embodiments, including the LNP compositions described in Table A, the structural lipid can be cholesterol.

[0133] In some embodiments, including the LNP compositions described in Table A, the phospholipid is DOPE.

[0134] In some embodiments, including the LNP compositions described in Table A, the phospholipid is DSPC.

[0135] In some embodiments, including the LNP compositions described in Table A, the phospholipid is DOPC.

[0136] In some embodiments, including the LNP compositions described in Table A, the phospholipid is DPPC.

[0137] In some embodiments of the preceding LNPs, including the LNP compositions set forth in Tables 1A-1C, the PEGylated lipid is DMG-PEG2000.

[0138] In some embodiments, including the LNP compositions described in Table A, the structural lipid is cholesterol, the phospholipid is DOPE, and the PEGylated lipid is DMG-PEG2000.

[0139] In some embodiments, including the LNP compositions described in Table A, the structural lipid is cholesterol, the phospholipid is DOPC, and the PEGylated lipid is DMG-PEG2000.

[0140] In some embodiments, including the LNP compositions described in Table A, the structural lipid is cholesterol, the phospholipid is DSPC, and the PEGylated lipid is DMG-PEG2000.

[0141] In some embodiments, including the LNP compositions described in Table A, the structural lipid is cholesterol, the phospholipid is DPPC, and the PEGylated lipid is DMG-PEG2000.

[0142] nucleic acid molecule

[0143] In some embodiments, the lipid nanoparticles of the present disclosure, including those shown in Tables 1A-1C, can further comprise at least one nucleic acid. In some embodiments, the lipid nanoparticles can comprise multiple nucleic acid molecules. In some embodiments, at least one nucleic acid or multiple nucleic acid molecules can be formulated into the lipid nanoparticles.

[0144] Thus, the lipid nanoparticles can comprise at least one nucleic acid, at least one compound of the present disclosure, at least one structural lipid, at least one phospholipid, and at least one PEGylated lipid. In some embodiments, the lipid nanoparticles can further comprise at least one targeting ligand.

[0145] In some embodiments, at least one nucleic acid is a DNA molecule. In one embodiment, at least one DNA molecule is a DoggyBone DNA molecule. In some embodiments, at least one DNA molecule is a DNA nanoplasmid.

[0146] At least one nucleic acid is an RNA molecule. In some embodiments, the RNA molecule is an mRNA molecule. In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, all of the cytidine residues in the mRNA molecule can be 5-methylcytidine.

[0147] In some embodiments, at least one RNA molecule is a guide RNA (gRNA) molecule.

[0148] In some embodiments, at least one nucleic acid can include both an mRNA molecule and a guide RNA (gRNA) molecule. That is, the LNPs of the present disclosure can include both an mRNA molecule and a gRNA molecule. In some embodiments in which 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 can be a Cas-CLOVER protein. In some embodiments, the gRNA molecule can target the psk9 gene.

[0149] In some embodiments in which the LNP comprises both mRNA and gRNA molecules, the ratio of mRNA:gRNA can be about 1:2, or about 1:3, or about 1:4, or about 1:5, or about 1:6, or about 1:7, or about 1:8, or about 1:9, or about 1:10, or about 1:1, or about 2:1, or about 3:1, or about 4:1, or about 5:1, or about 6:1, or about 7:1, or about 8:1, or about 9:1, or about 10:1.

[0150] In some embodiments, the at least one nucleic acid can comprise at least one RNA molecule and at least one DNA molecule, i.e., the LNPs of the present disclosure can comprise both RNA and DNA molecules.

[0151] In some embodiments, the LNPs of the present disclosure can include both RNA molecules and DNA molecules, where the RNA molecules include at least one nucleic acid sequence encoding a transposase, and the DNA molecules include at least one nucleic acid sequence comprising a transposon. In some embodiments, the transposase can be any of the transposases described herein. In some embodiments, the transposon can be a transposon comprising at least one nucleic acid sequence encoding a therapeutic protein to be expressed in the lungs of a subject.

[0152] In some embodiments in which the LNPs of the present disclosure comprise both RNA (e.g., mRNA) and DNA, the ratio of RNA to DNA (RNA:DNA) in the LNP can be about 1:2, or about 1:3, or about 1:4, or about 1:1, or about 2:1, or about 3:1, or about 4:1, or about 5:1, or about 6:1, or about 7:1, or about 8:1, or about 9:1, or about 10:1.

[0153] In some embodiments, the lipid nanoparticles can comprise lipids and nucleic acids in a specified ratio (weight / weight).

[0154] In some embodiments, lipid nanoparticles comprising at least one nucleic acid have a molecular weight of about 5:1 to about 15:1, or about 10:1 to about 20:1, or about 15:1 to about 25:1, or about 20:1 to about 30:1, or about 25:1 to about 35:1, or about 30:1 to about 40:1, or about 35:1 to about 45:1, or about 40:1 to about 50:1, or about 45:1 to about 55:1, or about 50:1 to about 60:1, or about 55:1 to about 65:1, or about 60:1 to about 70:1, or about 65:1 to about 75:1, or about 70:1 to about 80:1, or about 75:1 to about 85:1. The lipids and nucleic acids can comprise a lipid:nucleic acid (weight / weight) ratio of about 80:1 to about 90:1, or about 85:1 to about 95:1, or about 90:1 to about 100:1, or about 95:1 to about 105:1, or about 100:1 to about 110:1, or about 105:1 to about 115:1, or about 110:1 to about 120:1, or about 115:1 to about 125:1, or about 120:1 to about 130:1, or about 125:1 to about 135:1, or about 130:1 to about 140:1, or about 135:1 to about 145:1, or about 140:1 to about 150:1.

[0155] In some embodiments, lipid nanoparticles comprising at least one nucleic acid have a lipid to lipid ratio of about 5:1, or about 10:1, or about 15:1, or about 20:1, or about 25:1, or about 30:1, or about 35:1, or about 40:1, or about 45:1, or about 50:1, or about 55:1, or about 60:1, or about 65:1, or about 70:1, or about 75:1, or about 80:1 , or about 85:1, or about 90:1, or about 95:1, or about 100:1, or about 105:1, or about 110:1, or about 115:1, or about 120:1, or about 125:1, or about 130:1, or about 135:1, or about 140:1, or about 145:1, or about 150:1 lipid:nucleic acid (weight / weight).

[0156] In some embodiments, lipid nanoparticles comprising at least one nucleic acid can comprise lipid and nucleic acid at a lipid:nucleic acid (weight / weight) ratio of about 10:1, or about 25:1, or about 40:1. In some embodiments, lipid nanoparticles comprising at least one nucleic acid can comprise lipid and nucleic acid at a lipid:nucleic acid (weight / weight) ratio of about 20:1, or about 40:1, or about 60:1, or about 80:1, or about 120:1.

[0157] In some embodiments, including the LNPs shown in Table A, the lipid to nucleic acid ratio in the nanoparticles can be about 30:1 to about 50:1 (w / w), or about 35:1 to about 45:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 40:1 (w / w).

[0158] In some embodiments, including the LNPs shown in Table A, the lipid to nucleic acid ratio in the nanoparticles can be about 40:1 to about 60:1 (w / w), or about 45:1 to about 55:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 50:1 (w / w).

[0159] In some embodiments, including the LNPs shown in Table A, the lipid to nucleic acid ratio in the nanoparticles can be about 50:1 to about 70:1 (w / w), or about 55:1 to about 65:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 60:1 (w / w).

[0160] In some embodiments, including the LNPs shown in Table A, the ratio of lipid to nucleic acid in the nanoparticles can be about 70:1 to about 90:1 (w / w), or about 75:1 to about 85:1 (w / w). In some embodiments, the ratio of lipid to nucleic acid in the nanoparticles can be about 80:1 (w / w).

[0161] In some embodiments, including the LNPs shown in Table A, the ratio of lipid to nucleic acid in the nanoparticles can be about 90:1 to about 110:1 (w / w), or about 95:1 to about 105:1 (w / w). In some embodiments, the ratio of lipid to nucleic acid in the nanoparticles can be about 100:1 (w / w).

[0162] In some embodiments, including the LNPs shown in Table A, the lipid to nucleic acid ratio in the nanoparticles can be about 110:1 to about 130:1 (w / w), or about 115:1 to about 125:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 120:1 (w / w).

[0163] In some embodiments in which the LNPs of the present disclosure comprise both RNA (e.g., mRNA) and DNA, the ratio of RNA to DNA (RNA:DNA) in the LNPs can be 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.

[0164] In some embodiments in which the LNPs of the present disclosure comprise mRNA, gRNA, and DNA, the ratio of mRNA to gRNA to DNA (mRNA:gRNA:DNA) can be about 1:1:1, about 2:1:1, about 3:1:1, about 4:1:1, or about 5:1:1.

[0165] In some embodiments in which the LNPs of the present disclosure comprise mRNA, gRNA, and DNA, the ratio of gRNA to mRNA to DNA (gRNA:mRNA:DNA) can be about 1:1:1, about 2:1:1, about 3:1:1, about 4:1:1, or about 5:1:1.

[0166] In some embodiments in which the LNPs of the present disclosure comprise mRNA, gRNA, and DNA, the ratio of DNA to gRNA to mRNA (DNA:gRNA:mRNA) can be about 1:1:1, about 2:1:1, about 3:1:1, about 4:1:1, or about 5:1:1.

[0167] Further features of the nucleic acid molecules of the present disclosure are provided herein.

[0168] Exemplary LNPs of the Disclosure

[0169] The following are exemplary LNPs of the present disclosure:

[0170] In some embodiments, lipid nanoparticles are provided comprising about 40.75% by molar of at least one compound of Formula (I), about 51.75% by molar of at least one structural lipid, about 5% by molar of at least one phospholipid, and about 2.5% by molar of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, and the at least one nucleic acid comprises at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 30.75% to about 50.75% by molar of at least one compound of Formula (I), about 41.75% to about 61.75% by molar of at least one structural lipid, about 0.1% to about 15% by molar of at least one phospholipid, and about 0.1% to about 12.5% ​​by molar of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, and the at least one nucleic acid comprises at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 35.75% to about 45.75% by molar of at least one compound of Formula (I), about 46.75% to about 56.75% by molar of at least one structural lipid, about 1% to about 10% by molar of at least one phospholipid, and about 1% to about 7.5% by molar of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, and the at least one nucleic acid comprises at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 50:1 to about 70:1 (w / w), or about 55:1 to about 65:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 60:1 (w / w).

[0171] In some embodiments, the nucleic acid molecule is a DNA molecule. Thus, the present disclosure provides lipid nanoparticles comprising about 40.75% by molar of at least one compound of Formula (I), about 51.75% by molar of at least one structural lipid, about 5% by molar of at least one phospholipid, and about 2.5% by molar of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, and the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 30.75% to about 50.75% by molar of at least one compound of Formula (I), about 41.75% to about 61.75% by molar of at least one structural lipid, about 0.1% to about 15% by molar of at least one phospholipid, and about 0.1% to about 12.5% ​​by molar of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, and the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 35.75% to about 45.75% by molar of at least one compound of Formula (I), about 46.75% to about 56.75% by molar of at least one structural lipid, about 1% to about 10% by molar of at least one phospholipid, and about 1% to about 7.5% by molar of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, and the at least one nucleic acid comprises at least one DNA molecule. In 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 at least one nucleic acid further comprises at least one RNA molecule. In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 10:1 to about 30:1 (w / w), or about 15:1 to about 25:1 (w / w). In some embodiments, the ratio of lipid to nucleic acid in the nanoparticles can be about 20:1 (w / w).

[0172] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid can comprise at least one nucleic acid molecule, wherein the at least one nucleic acid molecule is at least one RNA molecule and at least one DNA molecule.

[0173] In some embodiments, lipid nanoparticles are provided comprising about 40.75% by molar of at least one compound of Formula (I), about 51.75% by molar of at least one structural lipid, about 5% by molar of at least one phospholipid, and about 2.5% by molar of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one RNA molecule and at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 30.75% to about 50.75% by molar of at least one compound of Formula (I), about 41.75% to about 61.75% by molar of at least one structural lipid, about 0.1% to about 15% by molar of at least one phospholipid, and about 0.1% to about 12.5% ​​by molar of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one RNA molecule and at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 35.75% to about 45.75% by molar of at least one compound of Formula (I), about 46.75% to about 56.75% by molar of at least one structural lipid, about 1% to about 10% by molar of at least one phospholipid, and about 1% to about 7.5% by molar of at least one PEGylated lipid, wherein the lipid nanoparticles comprise at least one nucleic acid, the at least one nucleic acid comprising at least one RNA molecule and 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 mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 10:1 to about 30:1 (w / w), or about 15:1 to about 25:1 (w / w). In some embodiments, the ratio of lipid to nucleic acid in the nanoparticles can be about 20:1 (w / w). In some embodiments, the at least one nucleic acid comprises at least one RNA molecule and at least one DNA molecule in a ratio of 1:2.

[0174] Pharmaceutical Compositions of the Present Disclosure

[0175] In some embodiments, the present disclosure provides a pharmaceutical composition comprising at least one lipid nanoparticle of the present disclosure.In some embodiments, the present disclosure provides a pharmaceutical composition comprising at least one first nanoparticle of the present disclosure and at least one second nanoparticle of the present disclosure, wherein at least one first nanoparticle comprises at least one nucleic acid molecule encoding at least one transposase, and at least one second nanoparticle comprises at least one nucleic acid molecule encoding at least one transposon.In some embodiments, the at least one nucleic acid molecule encoding at least one transposase can be an RNA molecule (e.g., an mRNA molecule), and the at least one nucleic acid molecule encoding at least one transposon can be a DNA molecule (e.g., a DoggyBone DNA molecule or a DNA nanoplasmid).

[0176] In some embodiments, the present disclosure provides a composition comprising at least one cell contacted with at least one nanoparticle of the present disclosure. In some embodiments, the present disclosure provides a composition comprising at least one cell genetically modified using at least one nanoparticle of the present disclosure. In some embodiments, the present disclosure provides a composition comprising at least one cell genetically modified using any of the methods of the present disclosure.

[0177] In some embodiments, the present disclosure provides a pharmaceutical composition comprising at least one cell contacted with at least one nanoparticle of the present disclosure. In some embodiments, the present disclosure provides a pharmaceutical composition comprising at least one cell genetically modified using at least one nanoparticle of the present disclosure. In some embodiments, the present disclosure provides a pharmaceutical composition comprising at least one cell genetically modified using any of the methods of the present disclosure.

[0178] Methods of the present disclosure

[0179] The present disclosure provides a method for delivering at least one nucleic acid to at least one cell, comprising contacting the at least one cell with at least one composition of the present disclosure.The present disclosure provides a method for delivering at least one nucleic acid to at least one cell, comprising contacting the at least one cell with at least one nanoparticle of the present disclosure.

[0180] In all of the methods, compositions, and kits of the present disclosure, at least one cell can be a lung cell, which can include, but is not limited to, endothelial cells, epithelial cells, and leukocytes.

[0181] In some aspects of any of the methods of the present disclosure, the cells can be in vivo, ex vivo, or in vitro. In some aspects, any of the methods of the present disclosure can be applied in vivo, ex vivo, or in vitro.

[0182] The present disclosure provides a method for genetically modifying at least one cell, comprising contacting the at least one cell with at least one composition of the present disclosure.The present disclosure provides a method for genetically modifying at least one cell, comprising contacting the at least one cell with at least one nanoparticle of the present disclosure.

[0183] In some embodiments, genetically modifying a cell can include delivering at least one exogenous nucleic acid to the cell such that the cell expresses at least one protein that the cell does not otherwise normally express, or such that the cell expresses at least one protein at a level higher than the level at which the cell would otherwise normally express the at least one protein, or such that the cell expresses at least one protein at a level lower than the level at which the cell would otherwise normally express the at least one protein. In some embodiments, genetically modifying a cell can include delivering at least one exogenous nucleic acid to the cell such that the at least one exogenous nucleic acid is integrated into the genome of the at least one cell.

[0184] In some embodiments, the methods of the present disclosure can produce a plurality of cells, wherein at least about 1%, or at least about 2%, or at least about 3%, or at least about 4%, or at least about 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% of the cells of the plurality of cells express at least one protein encoded by at least one nucleic acid delivered to the plurality of cells via the nanoparticles of the present disclosure.

[0185] The present disclosure provides methods for treating at least one disease in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one composition of the present disclosure comprising at least one nucleic acid encoding a therapeutic protein.

[0186] The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering a therapeutically effective amount of at least one nanoparticle of the present disclosure comprising at least one nucleic acid encoding a therapeutic protein.

[0187] The present disclosure provides methods of treating at least one disease in a subject, comprising administering a therapeutically effective amount of cells, wherein the cells are in contact with at least one nanoparticle of the present disclosure, the nanoparticle comprising at least one nucleic acid encoding a therapeutic protein.The present disclosure provides methods of treating at least one disease in a subject, comprising administering a therapeutically effective amount of cells, the cells being genetically modified using the compositions and / or methods of the present disclosure.

[0188] 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 pulmonary disease or pulmonary disorder. In some embodiments, the at least one disease may be cystic fibrosis.

[0189] Thus, the present disclosure provides a method of treating a pulmonary disease or disorder in a subject in need thereof, comprising administering to the subject at least one composition comprising at least one lipid nanoparticle of the present disclosure.

[0190] Thus, the present disclosure provides a method of treating cystic fibrosis in a subject in need thereof, comprising administering to the subject at least one composition comprising at least one lipid nanoparticle of the present disclosure.

[0191] The present disclosure provides a method for preferential delivery of a composition to lungs or lung cells in a subject in need thereof, comprising administering at least one composition comprising at least one lipid nanoparticle of the present disclosure to the subject, thereby providing greater expression or activity of the at least one composition in the subject's lungs or lung cells compared to that achieved in non-pulmonary organs or non-pulmonary cells of the subject.

[0192] nucleic acid molecule

[0193] In some embodiments, the nucleic acid molecule can be an RNA molecule.Therefore, in some embodiments, the lipid nanoparticle can comprise at least one RNA molecule.At least one RNA molecule can be encapsulated in the lipid nanoparticle.In some embodiments, the RNA molecule can be an mRNA molecule.In some embodiments, the lipid nanoparticle can comprise at least one mRNA molecule.The mRNA molecule can be encapsulated in the lipid nanoparticle.

[0194] In some embodiments, the nucleic acid molecule can be a synthetic nucleic acid molecule. In some embodiments, the nucleic acid molecule can be a non-naturally occurring nucleic acid molecule. In some embodiments, the non-naturally occurring nucleic acid molecule can comprise at least one non-naturally occurring nucleotide. The at least one non-naturally occurring nucleotide can be any non-naturally occurring nucleotide known in the art. In some embodiments, the nucleic acid molecule can be a modified nucleic acid molecule. In some embodiments, the modified nucleic acid molecule can comprise at least one modified nucleotide. The at least one modified nucleotide can be any modified nucleic acid known in the art.

[0195] In some embodiments, mRNA molecules can be capped using any method and / or capping moiety known in the art. mRNA molecules can be capped with an m7G(5')ppp(5')G moiety. The m7G(5')ppp(5')G moiety is also referred to herein as "Cap0." mRNA molecules can be capped with a CleanCap® moiety. The CleanCap® moiety can comprise an m7G(5')ppp(5')(2'OMeA) (CleanCap® AG) moiety. The CleanCap® moiety can comprise an m7G(5')ppp(5')(2'OMeG) (CleanCap® GG) moiety. mRNA molecules can be capped with an anti-reverse cap analog (ARCA®) moiety. The ARCA® moiety can comprise an m7(3'-O-methyl)G(5')ppp(5')G moiety. The mRNA molecule can be capped with a CleanCap® 3'OMe moiety (CleanCap® + ARCA®).

[0196] In some embodiments, the mRNA molecule can include at least one modified nucleic acid.

[0197] At least one modified nucleic acid can contain 5-methoxyuridine (5moU). In some embodiments, at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, at least about 90%, or at least about 95%, or at least about 99% of the uridine bases in the mRNA molecule are 5-methoxyuridine bases. In some embodiments, all of the uridine bases in the mRNA molecule are 5-methoxyuridine bases. Without wishing to be bound by theory, 5-methoxyuridine can 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).

[0198] In some embodiments, the mRNA molecule can include at least one modified nucleic acid.

[0199] At least one modified nucleic acid is N1-methylpseudouridine (me 1Ψ). In some embodiments, at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, at least about 90%, or at least about 95%, or at least about 99% of the uridine bases in the mRNA molecule are N1-methylpseudouridine bases. In some embodiments, all of the uridine bases in the mRNA molecule are N1-methylpseudouridine bases. Without wishing to be bound by theory, N1-methylpseudouridine can improve protein expression (see Li et al., Bioconjugate Chem. 2016, 27, 3, 849-853).

[0200] In some embodiments, the mRNA molecule can include at least one modified nucleic acid.

[0201] At least one modified nucleic acid can contain pseudouridine (Ψ). In some embodiments, at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, at least about 90%, or at least about 95%, or at least about 99% of the uridine bases in the mRNA molecule are pseudouridine bases. In some embodiments, all of the uridine bases in the mRNA molecule are pseudouridine bases. Without wishing to be bound by theory, pseudouridine can 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).

[0202] In some embodiments, the mRNA molecule can include at least one modified nucleic acid.

[0203] At least one modified nucleic acid can contain 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%, at least about 90%, or at least about 95%, or at least about 99% of the cytidine bases in mRNA are 5-MeC bases.In some embodiments, all of the cytidine bases in mRNA molecules are 5-MeC bases.

[0204] In some embodiments, the nucleic acid molecule can comprise a DNA molecule. Thus, in some embodiments, the lipid nanoparticle can comprise a DNA molecule. In some embodiments, the DNA molecule can be a circular DNA molecule, such as, but not limited to, a DNA plasmid or a DNA nanoplasmid. Thus, in some embodiments, the lipid nanoparticle can comprise a circular DNA molecule. In some embodiments, the lipid nanoparticle can comprise a doggybone DNA molecule. In some embodiments, the lipid nanoparticle can comprise a DNA plasmid. In some embodiments, the lipid nanoparticle can comprise a DNA nanoplasmid. In some embodiments, the DNA molecule can be a linearized DNA molecule, such as, but not limited to, a linearized DNA plasmid or a linearized DNA nanoplasmid.

[0205] The DNA plasmid or DNA nanoplasmid is at least about 0.25 kb, or at least about 0.5 kb, or at least about 0.75 kb, or at least about 1.0 kb, or at least about 1.25 kb, or at least about 1.5 kb, or at least about 1.75 kb, or at least about 2.0 kb, or at least about 2.25 kb, or at least about 2.5 kb, or at least about 2.75 kb, or at least about 3.0 kb, or at least about 3.25 kb, or at least about 3.5 kb, or at least or at least about 3.75 kb, or at least about 4.0 kb, or at least about 4.25 kb, or at least about 4.5 kb, or at least about 4.75 kb, or at least about 5.0 kb, or at least about 5.25 kb, or at least about 5.5 kb, or at least about 5.75 kb, or at least about 6.0 kb, or at least about 6.25 kb, or at least about 6.5 kb, or at least about 6.75 kb, or at least about 7.0 kb, or at least about 7.25 kb, or at least about 7.5 kb, or at least about 7.75 kb, or at least about 8.0 kb, or at least about 8.25 kb, or at least about 8.5 kb, or at least about 8.75 kb, or at least about 9.0 kb, or at least about 9.25 kb, or at least about 9.5 kb, or at least about 9.75 kb, or at least about 10.0 kb, or at least about 10.25 kb, or at least about 10.5 kb, or at least about 10.75 kb, or at least about 11.0 kb, or at least about 11.25 kb, or at least It may be about 11.5 kb, or at least about 11.75 kb, or at least about 12 kb, or at least about 12.25 kb, or at least about 12.5 kb, or at least about 12.75 kb, or at least about 13.0 kb, or at least about 13.25 kb, or at least about 13.5 kb, or at least about 13.75 kb, or at least about 14.0 kb, or at least about 14.25 kb, or at least about 14.5 kb, or at least about 14.75 kb or at least about 15.0 kb in length.

[0206] In some embodiments, the nucleic acid molecule formulated in the lipid nanoparticles of the present disclosure can comprise at least one transgene sequence. In some embodiments, the transgene sequence can comprise a nucleotide sequence encoding at least one therapeutic protein. In some embodiments, the transgene sequence can comprise a nucleotide sequence encoding at least one transposase. In some embodiments, the transgene sequence can comprise a nucleotide sequence encoding at least one transposon. In some embodiments, the transposon can comprise a nucleotide sequence encoding at least one therapeutic protein. In some embodiments, the transposon can comprise a nucleotide sequence encoding at least one therapeutic protein and at least one protomer sequence, and the at least one therapeutic protein is operably linked to at least one promoter sequence.

[0207] In some aspects, the lipid nanoparticles of the present disclosure can be produced using a microfluidic mixing platform, hi some aspects, the microfluidic mixing platform can be a non-turbulent microfluidic mixing platform.

[0208] In some embodiments, the microfluidic mixing platform can produce the lipid nanoparticles of the present invention by combining a miscible solvent phase containing the lipid components of the nanoparticles with an aqueous phase containing the lipid nanoparticle cargo (e.g., nucleic acid, DNA, mRNA, etc.) using a microfluidic device. In some embodiments, the miscible solvent phase and the aqueous phase are mixed in the microfluidic device under laminar flow conditions that do not allow the two phases to mix immediately. When the two phases move under laminar flow in the microfluidic channel, the fine features in the channel allow for controlled, homogeneous mixing, which can produce the lipid nanoparticles of the present disclosure.

[0209] 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).

[0210] In some embodiments, the lipid nanoparticles of the present disclosure can be produced using a microfluidic mixing platform that mixes at a rate of at least about 2.5 ml / min, or at least about 5 ml / min, or at least about 7.5 ml / min, or at least about 10 ml / min, or at least about 12.5 ml / min, or at least about 15 ml / min, or at least about 17.5 ml / min, or at least about 20 ml / min, or at least about 22.5 ml / min, or at least about 25 ml / min, or at least about 27.5 ml / min, or at least about 30 ml / min.

[0211] In some embodiments, the lipid nanoparticles of the present disclosure can be produced using a microfluidic mixing platform that mixes a miscible solvent phase and an aqueous phase in a solvent:water (v:v) 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.

[0212] piggyBac ITR sequences

[0213] In some embodiments, the nucleic acid can comprise a piggyBac ITR sequence. In some embodiments, the nucleic acid can comprise a first piggyBac ITR sequence and a second piggyBac ITR sequence.

[0214] In some embodiments, the piggyBac ITR sequences can include any piggyBac ITR sequences known in the art.

[0215] In some embodiments of the methods of the present disclosure, the piggyBac ITR sequences, such as the first piggyBac ITR sequence and / or the second piggyBac ITR sequence in an AAV piggyBac transposon, can comprise, consist essentially of, or consist of Sleeping Beauty transposon ITRs, Helraiser transposon ITRs, Tol2 transposon ITRs, TcBuster transposon ITRs, or any combination thereof.

[0216] Transposition System

[0217] In some embodiments, the nucleic acid can comprise a transposon or nanotransposon comprising a first nucleic acid sequence comprising: (a) a first inverted terminal repeat (ITR) or sequence encoding the first ITR, (b) a second ITR or sequence encoding the second ITR, and (c) an intra-ITR sequence or intra-ITR coding sequence, wherein the intra-ITR sequence comprises a transposon sequence or a transposon-coding sequence.

[0218] In some embodiments, the nucleic acid can comprise a transposon or nanotransposon comprising a first nucleic acid sequence comprising: (a) a first inverted terminal repeat (ITR) or sequence encoding the first ITR, (b) a second ITR or sequence encoding the second ITR, and (c) an intra-ITR sequence or intra-ITR coding sequence, where the intra-ITR sequence comprises a transposon sequence or a transposon-encoding sequence, and a second nucleic acid sequence comprising an inter-ITR sequence or inter-ITR coding sequence, where the inter-ITR sequence is 700 nucleotides in length or less.

[0219] The transposon or nanotransposon of the present disclosure can be a piggyBac™ (PB) transposon. In some embodiments where the transposon is a PB transposon, the transposase is a piggyBac™ (PB) transposase, a piggyBac-like (PBL) transposase, or a Super piggyBac™ (SPB) transposase. Preferably, the sequence encoding the SPB transposase is an mRNA sequence.

[0220] Non-limiting examples of PB transposons and PB, PBL and SPB transposases are described in detail in U.S. Patent No. 6,218,182; U.S. Patent No. 6,962,810; U.S. Patent No. 8,399,643 and PCT Publication No. WO 2010 / 099296.

[0221] PB, PBL, and SPB transposases recognize transposon-specific inverted terminal repeats (ITRs) at the ends of the transposon and insert the contents between the ITRs into the sequence 5'-TTAT-3' (TTAT target sequence) or the sequence 5'-TTAA-3' (TTAA target sequence) within the chromosomal site. The target sequences of the PB or PBL transposons are 5'-CTAA-3', 5'-TTAG-3', 5'-ATAA-3', 5'-TCAA-3', 5'AGTT-3', 5'-ATTA-3', 5'-GTTA-3', 5'-TTGA-3', 5'-TTTA-3', 5'-TTAC-3', 5'-ACTA-3', 5'-AGGG-3', 5'-CTAG-3', 5'-TGAA-3', 5'-AGGT-3', 5'-ATCA-3', 5'-CTCC-3', 5'-TAAA-3', 5'-TCTC-3', 5'TGAA-3', 5'-A The PB or PBL transposon system can include or consist of the following ITRs: AAT-3', 5'-AATC-3', 5'-ACAA-3', 5'-ACAT-3', 5'-ACTC-3', 5'-AGTG-3', 5'-ATAG-3', 5'-CAAA-3', 5'-CACA-3', 5'-CATA-3', 5'-CCAG-3', 5'-CCCA-3', 5'-CGTA-3', 5'-GTCC-3', 5'-TAAG-3', 5'-TCTA-3', 5'-TGAG-3', 5'-TGTT-3', 5'-TTCA-3', 5'-TTCT-3', and 5'-TTTT-3'. The PB or PBL transposon system does not limit the payload of the gene of interest that can be included between the ITRs.

[0222] 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. In preferred embodiments, the PB transposase comprises or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:1.

[0223] The PB or PBL transposase can comprise or consist of an amino acid sequence having an amino acid substitution at two or more, three or more, or each of positions 30, 165, 282, and / or 538 of the sequence of SEQ ID NO: 1. The transposase can be an SPB transposase comprising or consisting of the amino acid sequence of SEQ ID NO: 1, wherein the amino acid substitution at position 30 can be a substitution of valine (V) for isoleucine (I), the amino acid substitution at position 165 can be a substitution of serine (S) for glycine (G), the amino acid substitution at position 282 can be a substitution of valine (V) for methionine (M), and the amino acid substitution at position 538 can be a substitution of lysine (K) for asparagine (N). In preferred embodiments, the SPB transposase comprises or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:2.

[0224] In certain embodiments, where the transposase comprises the above-described mutations at positions 30, 165, 282, and / or 538, the PB, PBL, and SPB transposases can further comprise amino acid substitutions at one or more of 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, 421, 436, 456, 470, 486, 503, 552, 570, and 591 of the sequence of SEQ ID NO:1 or SEQ ID NO:2, as described in more detail in PCT Publication Nos. WO 2019 / 173636 and PCT / US2019 / 049816.

[0225] In preferred embodiments, the PB transposase comprises or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:3.

[0226] The PB or PBL transposase can comprise or consist of an amino acid sequence having an amino acid substitution at two or more, three or more, or each of positions 29, 164, 281, and / or 537 of the sequence of SEQ ID NO: 3. The transposase can be an SPB transposase comprising or consisting of the amino acid sequence of SEQ ID NO: 3, wherein the amino acid substitution at position 29 can be a substitution of valine (V) for isoleucine (I), the amino acid substitution at position 164 can be a substitution of serine (S) for glycine (G), the amino acid substitution at position 281 can be a substitution of valine (V) for methionine (M), and the amino acid substitution at position 537 can be a substitution of lysine (K) for asparagine (N). In preferred embodiments, the SPB transposase comprises or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:4.

[0227] In certain embodiments where the transposase comprises the above-described mutations at positions 29, 164, 281 and / or 537, the PB, PBL and SPB transposases can further comprise an amino acid substitution at one or more of 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, 339, 420, 435, 455, 469, 485, 502, 551, 569 and 590 of the sequence of SEQ ID NO:3 or SEQ ID NO:4, as described in more detail in PCT Publication Nos. WO 2019 / 173636 and PCT / US2019 / 049816.

[0228] The PB, PBL, or SPB transposase can be isolated or derived from an insect, vertebrate, crustacean, or urochordate, as described in more detail in PCT Publication Nos. WO 2019 / 173636 and PCT / US2019 / 049816. In a preferred embodiment, the PB, PBL, or SPB transposase is isolated or derived from the insect Trichoplusia ni (GenBank Accession No. AAA87375) or Bombyx mori (GenBank Accession No. BAD11135).

[0229] A hyperactive PB or PBL transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In a preferred embodiment, the hyperactive PB or PBL transposase is isolated or derived from Bombyx mori or Xenopus tropicalis. Examples of hyperactive PB or PBL transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810; U.S. Patent No. 8,399,643; and International Publication No. WO 2019 / 173636. A list of hyperactive amino acid substitutions is disclosed in U.S. Patent No. 10,041,077. The transposon or nanotransposon of the present disclosure may be a Sleeping Beauty transposon. In some embodiments, when the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase (eg, as disclosed in US Pat. No. 9,228,180) or a hyperactive Sleeping Beauty (SB100X) transposase.

[0230] The transposon or nanotransposon of the present disclosure can be a Helraiser transposon. Exemplary Helraiser transposons include Helibat1. In some embodiments, when the transposon is a Helraiser transposon, the transposase is a Helitron transposase (for example, as disclosed in WO2019 / 173636).

[0231] The transposon or nanotransposon of the present disclosure can be a Tol2 transposon. In some embodiments, when the transposon is a Tol2 transposon, the transposase is a Tol2 transposase (for example, as disclosed in WO2019 / 173636).

[0232] The transposon or nanotransposon of the present disclosure can be a TcBuster transposon. In some embodiments, when the transposon is a TcBuster transposon, the transposase is a TcBuster transposase or a hyperactive TcBuster transposase (e.g., as disclosed in International Publication No. 2019 / 173636). The TcBuster transposase can comprise or consist of a naturally occurring or non-naturally occurring amino acid sequence. The polynucleotide encoding the TcBuster transposase can comprise or consist of a naturally occurring or non-naturally occurring nucleic acid sequence.

[0233] In some embodiments, the mutant TcBuster transposase comprises one or more sequence mutations when compared to the wild-type TcBuster transposase, which is described in more detail in PCT Publication Nos. WO 2019 / 173636 and PCT / US2019 / 049816.

[0234] The cell delivery compositions (e.g., transposons) disclosed herein can include nucleic acid molecules encoding therapeutic proteins or therapeutic agents. Examples of therapeutic proteins include those disclosed in PCT Publication Nos. WO 2019 / 173636 and PCT / US2019 / 049816.

[0235] Gene editing system

[0236] The present disclosure provides a gene editing composition and / or a cell containing the gene editing composition. The gene editing composition can include nanoparticles containing a nucleic acid, where the nucleic acid includes 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 can include a DNA sequence, an RNA sequence, or a combination thereof. The nuclease or its nuclease domain can include one or more of a CRISPR / Cas protein, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), and an endonuclease.

[0237] The nuclease or its nuclease domain can comprise a nuclease-inactivated Cas (dCas) protein and an endonuclease. The endonuclease can comprise a Clo051 nuclease or its nuclease domain. The gene editing composition can comprise a fusion protein. The fusion protein can comprise a nuclease-inactivated Cas9 (dCas9) protein and a Clo051 nuclease or a Clo051 nuclease domain. In some embodiments, the fusion protein can further comprise at least one nuclear localization signal (NLS). In some embodiments, the fusion protein can further comprise at least two NLSs. The gene editing composition can further comprise a guide sequence. The guide sequence can comprise an RNA sequence.

[0238] The transgene can comprise a nucleic acid sequence encoding a mini-Cas9 (Cas9) operably linked to an effector. The present disclosure provides fusion proteins comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a mini-Cas9 (Cas9). The mini-Cas9 constructs of the present disclosure can comprise an effector comprising a type IIS endonuclease.

[0239] The transgene can include a nucleic acid sequence encoding an inactivated small Cas9 (dSaCas9) operably linked to an effector. The transgene can include a nucleic acid sequence encoding a fusion protein comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a small inactivated Cas9 (dSaCas9). The small inactivated Cas9 (dSaCas9) construct of the present disclosure can include an effector comprising a type IIS endonuclease.

[0240] The transgene can include a nucleic acid sequence encoding an inactivated Cas9 (dCas9) operably linked to an effector. The transgene can include a nucleic acid sequence encoding a fusion protein comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule, wherein the effector comprises an inactivated Cas9 (dCas9). The inactivated Cas9 (dCas9) construct of the present disclosure can include an effector comprising a type IIS endonuclease.

[0241] The dCas9 can be isolated or derived from Streptococcus pyogenes. The dCas9 can include dCas9 with substitutions at amino acid positions 10 and 840 that inactivate the catalytic site. In some embodiments, these substitutions are D10A and H840A.

[0242] The cell containing the gene editing composition can express the gene editing composition stably or transiently.Preferably, the gene editing composition is expressed transiently.The guide RNA can comprise a sequence that is complementary to the target sequence in the genomic DNA sequence.The target sequence in the genomic DNA sequence can be the target sequence in the safe harbor site of the genomic DNA sequence.

[0243] Gene editing compositions comprising Cas-CLOVER and methods of using these compositions for gene editing are described in detail in U.S. Patent Application Publication Nos. 2017 / 0107541, 2017 / 0114149, 2018 / 0187185 and U.S. Patent No. 10,415,024. In some embodiments, the Cas-CLOVER protein can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:5.

[0244] Thus, the present disclosure provides any of the lipid nanoparticle compositions described herein, wherein the lipid nanoparticles comprise at least one genome editing composition, and the at least one genome editing composition comprises: a) a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises (i) an inactivated Cas9 (dCas9) protein or its inactivated nuclease domain, (ii) a Clo051 protein or its nuclease domain; and b) at least one gRNA molecule. In some embodiments, the fusion protein can further comprise at least one NLS. In some embodiments, the at least one genome editing composition can comprise at least two gRNA molecules.

[0245] Formulations, Dosages and Modes of Administration

[0246] The present disclosure provides formulations, dosages and methods for administering the compositions described herein.

[0247] The disclosed compositions and pharmaceutical compositions can further comprise any suitable auxiliary agent, for example, but not limited to, at least one of a diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant, etc. Pharmaceutically acceptable auxiliary agents are preferred. Non-limiting examples of such sterile solutions and methods for their preparation are well known in the art, for example, but not 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 mode of administration, solubility, and / or stability of the composition can be routinely selected and are well known in the art or as described herein.

[0248] For example, the disclosed LNP compositions of the present invention can further comprise a diluent. In some compositions, the diluent can be phosphate buffered saline ("PBS").

[0249] Non-limiting examples of pharmaceutical excipients and additives suitable for use include proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derivatized sugars, such as alditols, aldonic acids, and esterified sugars; and polysaccharides or sugar polymers), which can be present alone or in combination and comprise 1 to 99.99% (by weight or volume), alone or in combination. Non-limiting examples of protein excipients include serum albumins, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acids / protein components that can also function in buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine.

[0250] The composition can also contain a buffer or pH adjuster. Typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts, such as citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid salts; Tris, tromethamine hydrochloride, or phosphate buffer. Preferred buffers are organic acid salts such as citrate. In some embodiments, the buffer can contain sucrose.

[0251] Many known and developed modes can be used to administer the therapeutically effective amount of the compositions or pharmaceutical compositions disclosed herein.Non-limiting examples of administration modes include bolus, buccal, injection, intra-articular, intrabronchial, intraperitoneal, intravesical, intrachondral, intracavity, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intralesional, intramuscular, intramyocardial, intranasal, intraocular, intraosseous, intraskeletal, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intratumoral, intravenous, intravesical, oral, parenteral, rectal, sublingual, subcutaneous, transdermal or intravaginal means.

[0252] The compositions of the present disclosure may be used for parenteral (subcutaneous, intramuscular, or intravenous) or any other administration, particularly in the form of a liquid solution or suspension; for vaginal or rectal administration, particularly, but not limited to, in semi-solid forms such as creams and suppositories; for buccal or sublingual administration, such as, but not limited to, in the form of tablets or capsules; or intranasally, for example, but not limited to, in the form of powders, nasal sprays, or aerosols or specific medications; or transdermally, for example, but not limited to, in gels, ointments, lotions, suspensions, or patch delivery systems using chemical enhancers such as dimethyl sulfoxide to modify skin structure or to increase drug concentration in transdermal patches (Junginger, et al. In "Drug Permeation Enhancement"; Hsieh, D.S., Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994,), or to create temporary transport pathways, such as electroporation, or to increase the mobility of charged drugs through the skin, such as iontophoresis, or the application of an electric field, such as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (the above publications and patents are incorporated herein by reference in their entireties).

[0253] For parenteral administration, any composition disclosed herein can be formulated as a solution, suspension, emulsion, granules, powder, or lyophilized powder, either combined with a pharmaceutically acceptable parenteral vehicle or provided separately. Parenteral formulations can contain common excipients such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalene, etc. Aqueous or oily suspensions for injection can be prepared using known methods by using appropriate emulsifiers or wetting agents and suspending agents. Injectable preparations can be suspensions in non-toxic parenterally administrable diluents, such as aqueous solutions, sterile injectable solutions, or solvents. Usable vehicles or solvents include water, Ringer's solution, isotonic saline, etc.; sterile fixed oils can be used as common solvents or suspending solvents. For these purposes, any type of fixed oil and fatty acid can be used, including natural, synthetic, or semisynthetic fatty oils or fatty acids; natural, synthetic, or semisynthetic mono-, di-, or triglycerides. Parenteral administration is known in the art and includes, but is not limited to, conventional injection means, gas-pressurized needleless injection devices such as those described in U.S. Pat. No. 5,851,198, and laser perforation devices such as those described in U.S. Pat. No. 5,839,446.

[0254] For pulmonary administration, the compositions or pharmaceutical compositions described herein are preferably delivered in particle sizes effective to reach the lower respiratory tract of the lungs or paranasal sinuses. The compositions or pharmaceutical compositions can be delivered by any of a variety of inhalation or nasal devices known in the art for administering therapeutic agents by inhalation. These devices, which can deposit aerosolized formulations into a patient's paranasal sinuses or alveoli, include metered-dose inhalers, nebulizers (e.g., jet nebulizers, ultrasonic nebulizers), dry powder generators, sprayers, and the like. All such devices can employ formulations suitable for administration of the compositions or pharmaceutical compositions described herein in aerosols. Such aerosols can be composed of either solutions (both aqueous and non-aqueous) or solid particles. In metered-dose inhalers (MDIs), the propellant, the compositions or pharmaceutical compositions described herein, and any excipients or other additives are contained in a canister as a mixture with a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol. A more detailed description of pulmonary administration, formulations and related devices is disclosed in PCT Publication No. WO 2019 / 049816.

[0255] For absorption through mucosal surfaces, the composition comprises an emulsion containing a plurality of submicron particles, a mucoadhesive polymer, a bioactive peptide, and an aqueous continuous phase, which promotes absorption through mucosal surfaces by achieving mucoadhesion of the emulsion particles (U.S. Patent No. 5,514,670). Mucosal surfaces suitable for application of the emulsions of the present disclosure include corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, gastric, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, such as suppositories, can contain excipients such as polyalkylene glycols, petrolatum, cocoa butter, etc. Formulations for intranasal administration can be solid and contain excipients such as lactose, or can be aqueous or oily solutions of nasal drops. For buccal administration, excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, etc. (U.S. Patent No. 5,849,695). A more detailed description of mucosal administration and formulations is disclosed in PCT Publication No. WO 2019 / 049816.

[0256] For transdermal administration, the compositions or pharmaceutical compositions disclosed herein are encapsulated in a delivery device such as liposomes or polymer nanoparticles, microparticles, microcapsules, or microspheres (collectively referred to as microparticles unless otherwise specified).Several suitable devices are known, including microparticles made of polyhydroxy acids such as polylactic acid, polyglycolic acid and its copolymers, polyorthoesters, polyanhydrides, and polyphosphazenes, as well as synthetic polymers such as collagen, polyamino acids, natural polymers such as 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 PCT Publication No. WO 2019 / 049816.

[0257] It may be desirable to deliver the disclosed compounds to a subject over an extended period of time, for example, from one week to one year from a single administration. A variety of sustained release, depot, or implant dosage forms are available.

[0258] Suitable dosages are well known in the art.See, for example, Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, California (2000); Nursing 2001 Handbook of Drugs, 21st Edition, Springhouse Corp., Springhouse, Pa., 2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc., Upper Saddle River, NJ. Preferred doses can optionally include about 0.1-99 and / or 100-500 mg / kg / administration, or any range, value, or fraction thereof, or can achieve a serum concentration of about 0.1-5000 μg / ml, or any range, value, or fraction thereof, per single or multiple administration. Preferred dosage ranges for the compositions or pharmaceutical compositions disclosed herein are from about 1 mg / kg up to about 3, about 6, or about 12 mg / kg of subject body weight.

[0259] Alternatively, the dosage administered may vary depending on known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the age, health, and weight of the recipient; the nature and extent of the symptoms, type of concurrent treatment, frequency of treatment, and the desired effect.

[0260] As a non-limiting example, human or animal treatment can be provided as a single or periodic dosage of a composition or pharmaceutical composition disclosed herein of about 0.1 to 100 mg / kg per day, or any range, value, or fraction thereof, on at least day 1 of days 1 to 40, or alternatively or additionally, at least once from 1 to 52 weeks, or alternatively or additionally, at least once from 1 to 20 years, or any combination thereof, using single, infusion, or repeated doses.

[0261] In embodiments where the composition administered to a subject in need thereof is a modified cell disclosed herein, the cells are administered in an amount of about 1 x 10 3 ~1x10 15 cells; 1x10 3 ~1x10 15 cells, approximately 1x10 4 ~1x10 12 cells; approx. 1x10 5 ~1x10 10 cells; approx. 1x10 6 ~1x10 9 cells; approx. 1x10 6 ~1x10 8 cells; approx. 1x10 6 ~1x10 7 cells; or approximately 1x10 6 ~25x10 6 In one embodiment, the cells are administered in amounts of about 5x10 6 ~25x10 6 It is administered in cells.

[0262] A more detailed description of the pharmaceutically acceptable excipients, formulations, dosages and methods of administration of the disclosed compositions and pharmaceutical compositions is disclosed in PCT Publication No. WO 2019 / 04981.

[0263] The present disclosure provides for the use of the disclosed compositions and pharmaceutical compositions to treat 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 the cell, tissue, organ, animal, or subject. In one aspect, the subject is a mammal. Preferably, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0264] The present disclosure provides a method for regulating or treating at least one malignant disease or disorder in a cell, tissue, organ, animal, or subject. Non-limiting examples of malignant diseases or disorders include cancer, lung diseases or disorders, and cystic fibrosis.

[0265] Any of the methods can include administering an effective amount of any of the compositions or pharmaceutical compositions disclosed herein to a cell, tissue, organ, animal, or subject in need of such modulation, treatment, or therapy. Such methods can optionally further include co-administration or combination therapy for treating such a disease or disorder, and the administration of any of the compositions or pharmaceutical compositions disclosed herein can further include the administration of at least one chemotherapeutic agent (e.g., alkylating agent, mitotic inhibitor, radiopharmaceutical) before, simultaneously with, and / or after.

[0266] In some embodiments, the subject does not develop graft versus host (GvH) and / or host versus graft (HvG) after administration. In one embodiment, administration is systemic. Systemic administration can be by any means known in the art and described in detail herein. Preferably, systemic administration is by intravenous injection or infusion. In one embodiment, administration is local. Local administration can be by any means known in the art and described in detail herein. Preferably, local administration is by intratumoral, intraspinal, intraventricular, intraocular, or intraosseous injection or infusion.

[0267] In some embodiments, the therapeutically effective dose is a single dose. In some embodiments, the single dose is one of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of doses therebetween that are produced simultaneously. In some embodiments, when the composition is autologous or allogeneic cells, the dose is sufficient to allow the cells to engraft and / or persist for a sufficient time to treat the disease or disorder.

[0268] In some embodiments of the treatment methods described herein, treatment can be modified or terminated. Specifically, in embodiments in which the composition used for treatment includes an inducible pro-apoptotic polypeptide, apoptosis can be selectively induced in cells by contacting the cells with an inducer. Treatment can be modified or terminated, for example, in response to signs of recovery or a decrease in disease severity / progression, signs of disease remission / cessation, and / or the occurrence of adverse events. In some embodiments, the method includes administering an inhibitor of the inducer to inhibit the modification of the cell therapy, thereby restoring the function and / or effectiveness of the cell therapy (e.g., when signs or symptoms of the disease reappear or the increase in severity and / or adverse events resolve).

[0269] Nucleic acid construction

[0270] The isolated nucleic acids of the present disclosure can be produced using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and / or (d) combinations thereof, as are known in the art.

[0271] The nucleic acid can advantageously contain sequences in addition to the polynucleotide of the present disclosure. For example, a multicloning site containing one or more endonuclease restriction sites can be inserted into the nucleic acid to facilitate isolation of the polynucleotide. Also, a translatable sequence can be inserted to facilitate isolation of the translated polynucleotide of the present disclosure. For example, a hexa-histidine marker sequence provides a convenient means for purifying the protein of the present disclosure. The nucleic acid of the present disclosure, excluding the coding sequence, is optionally a vector, adapter, or linker for cloning and / or expressing the polynucleotide of the present disclosure.

[0272] Additional sequences can be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to aid in the isolation of polynucleotides, or to improve the introduction of polynucleotides into cells. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra; or Sambrook, supra.)

[0273] Recombinant methods for constructing nucleic acids

[0274] The isolated nucleic acid compositions of the present disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methodologies known to those skilled in the art. In some embodiments, oligonucleotide probes that selectively hybridize to the polynucleotides of the present disclosure under stringent conditions are used to identify the desired sequence in a cDNA or genomic DNA library. The isolation of RNA and the construction of cDNA and genomic libraries are well known to those skilled in the art. (See, for example, Ausubel, supra; or Sambrook, supra).

[0275] Nucleic Acid Screening and Isolation Methods

[0276] cDNA or genomic libraries can be screened using probes based on the sequences of the polynucleotides disclosed herein. The probes can hybridize to genomic DNA or cDNA sequences and be used to isolate homologous genes within the same or different organisms. Those skilled in the art will understand that varying degrees of hybridization stringency can be used in the assay. Either the hybridization or wash medium can be stringent. The more stringent the hybridization conditions, the greater the degree of complementarity between the probe and target must be for duplex formation to occur. The degree of stringency can be controlled by one or more of temperature, ionic strength, pH, and the presence of a partially denaturing solvent such as formamide. For example, hybridization stringency can be conveniently altered by changing the polarity of the reaction solution, e.g., by manipulating the formamide concentration within the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding will vary depending on the stringency of the hybridization medium and / or wash medium. The degree of complementarity is optimally 100%, or 70-100%, or any range or value therein. However, it should be understood that minor sequence variations in the probes and primers can be compensated for by reducing the stringency of the hybridization and / or wash medium.

[0277] Methods for amplifying RNA or DNA are well known in the art and can be used in accordance with the present disclosure without undue experimentation, based on the teachings and guidance presented herein.

[0278] Known methods of DNA or RNA amplification include polymerase chain reaction (PCR) and related amplification processes (e.g., U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159, and 4,965,188 to Mullis et al.; 4,795,699 and 4,921,794 to Tabor et al.; 5,142,033 to Innis; 5,122,464 to Wilson et al.; 5,091,310 to Innis; 5,142,033 to Gyllensten et al.). Nos. 4,066,584 by Gelfand et al.; 4,889,818 by Silver et al.; 4,994,370 by Biswas; 4,766,067 by Biswas; and 4,656,134 by Ringold), as well as RNA-mediated amplification (U.S. Pat. No. 5,130,238 by Malek et al., under the trade name NASBA), which uses antisense RNA against a target sequence as a template for double-stranded DNA synthesis, the entire contents of which are incorporated herein by reference. (See, e.g., Ausubel, supra; or Sambrook, supra).

[0279] For example, polymerase chain reaction (PCR) technology can be used to amplify the sequences of the polynucleotides and related genes of the present disclosure directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods can also be useful, for example, for cloning nucleic acid sequences encoding expressed proteins, for preparing nucleic acids to be used as probes to detect the presence of desired mRNA in a sample, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to guide those skilled in the art through in vitro amplification methods can be found in Berger (supra), Sambrook (supra), and Ausubel (supra), as well as Mullis 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). Commercially available kits for genomic PCR amplification are known in the art. See, for example, the Advantage-GC Genomic PCR Kit (Clontech). Additionally, for example, T4 gene 32 protein (Boehringer Mannheim) can be used to improve yields of long PCR products.

[0280] Synthetic methods for constructing nucleic acids

[0281] The isolated nucleic acids of the present disclosure can also be prepared by direct chemical synthesis using known methods (see, for example, Ausubel et al., supra). Chemical synthesis generally produces a single-stranded oligonucleotide, which can be converted into double-stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. While chemical synthesis of DNA may be limited to sequences of about 100 bases or more, those skilled in the art will recognize that longer sequences can be obtained by ligating shorter sequences.

[0282] Recombinant Expression Cassette

[0283] The present disclosure further provides a recombinant expression cassette comprising the nucleic acid of the present disclosure. The nucleic acid sequence of the present disclosure can be used to construct a recombinant expression cassette that can be introduced into at least one desired host cell. The recombinant expression cassette typically comprises a polynucleotide of the present disclosure operably linked to a transcription initiation regulatory sequence that directs the transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct the expression of the nucleic acid of the present disclosure.

[0284] In some embodiments, isolated nucleic acids that act as promoters, enhancers, or other elements can be introduced into appropriate locations (upstream, downstream, or within an intron) of non-heterologous polynucleotides of the present disclosure to up- or down-regulate expression of the polynucleotides of the present disclosure. For example, endogenous promoters can be altered in vivo or in vitro by mutation, deletion, and / or substitution.

[0285] Expression vectors and host cells

[0286] The present disclosure also relates to vectors comprising the isolated nucleic acid molecules of the present disclosure, and host cells genetically engineered with recombinant vectors, as are well known in the art. See, e.g., Sambrook et al., supra; Ausubel et al., supra, each of which is incorporated herein by reference in its entirety.

[0287] Polynucleotide can optionally be linked to the vector that contains the selectable marker for propagation in host.Generally, plasmid vector is introduced into precipitate such as calcium phosphate precipitate or into the complex with charged lipid.If vector is virus, it can be packaged in vitro using suitable packaging cell line, and then transduced into host cell.

[0288] The DNA insert should be operably linked to a suitable promoter. The expression construct will also contain sites for transcription initiation, termination, and a ribosome binding site for translation within the transcribed region. The coding portion of the mature transcript expressed by the construct preferably contains a translation initiation codon (e.g., UAA, UGA, or UAG) appropriately positioned at the beginning and at the end of the mRNA to be translated, with UAA and UAG being preferred for mammalian or eukaryotic cell expression.

[0289] Expression vectors will preferably, but optionally, include at least one selectable marker, such as, but not limited to, ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / geneticin (neo gene), DHFR (encoding dihydrofolate reductase and conferring resistance to methotrexate), mycophenolic acid or glutamine synthetase (GS, U.S. Patent Nos. 5,122,464, 5,770,359; 5,827,739), blasticidin (bsd gene), eukaryotic cell culture resistance genes, as well as genes encoding ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / geneticin (neo gene), DHFR (encoding dihydrofolate reductase and conferring resistance to methotrexate), mycophenolic acid or glutamine synthetase (GS, U.S. Patent Nos. 5,122,464, 5,770,359; 5,827,739), blasticidin (bsd gene), and eukaryotic cell culture resistance genes. Examples of suitable host cell resistance genes include those for resistance to erythromycin (bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline for culturing in E. coli and other bacteria or prokaryotes (the above patents are incorporated herein by reference in their entireties). Appropriate culture media and conditions for the above host cells are known in the art. Suitable vectors will be readily apparent to those skilled in the art. Introduction of vector constructs into host cells can be 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 (supra), Chapters 1-4 and 16-18; Ausubel (supra), Chapters 1, 9, 13, 15, and 16.

[0290] The expression vector will preferably, but optionally, include at least one selectable cell surface marker for isolating cells modified by the disclosed compositions and methods. Selectable cell surface markers of the present disclosure include surface proteins, glycoproteins, or proteins that distinguish cells or cell subsets from other defined cell subsets. Preferably, the selectable cell surface marker distinguishes cells modified by the disclosed compositions or methods from cells not modified by the disclosed compositions or methods. Examples of such cell surface markers include, but are not limited to, "cluster of designation" or "classification determinant" proteins (often abbreviated as "CD"), such as truncated or full-length forms of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. Cell surface markers include the suicide gene marker RQR8 (Philip B et al. Blood. 2014 Aug 21;124(8):1277-87).

[0291] The expression vector will preferably, but optionally, include at least one selectable drug resistance marker for isolating cells modified by the disclosed compositions and methods. 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.

[0292] Those of skill in the art are familiar with the numerous expression systems available for expression of nucleic acid molecules encoding the proteins of the present disclosure.

[0293] definition

[0294] As used throughout this disclosure, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes a plurality of such methods, reference to "a dose" includes a reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0295] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend to some extent on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations. Alternatively, "about" can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When a particular value is described in this application and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range for the particular value.

[0296] In the chemical formulas shown herein, the marking [ka] indicates the point at which the functional group is attached to another part of the molecule. Definitions of certain functional groups and chemical terms are described in more detail below.

[0297] Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, racemic mixtures thereof, and other mixtures thereof, as being within the scope of the present invention. Additional asymmetric carbon atoms may be present in a substituent, such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in the present invention.

[0298] Isomeric mixtures containing any of a variety of isomer ratios can be utilized in accordance with the present invention. For example, when combining only two isomers, mixtures containing isomer ratios of 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 are all contemplated by the present invention. Those skilled in the art will readily understand that similar ratios are contemplated for more complex isomer mixtures.

[0299] For example, if a particular enantiomer of a compound of the invention is desired, it may be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary, the resulting diastereomeric mixture separated, and the auxiliary group cleaved to yield the pure desired enantiomer. Alternatively, if the molecule contains a basic functional group such as amino or an acidic functional group such as carboxyl, diastereomeric salts are formed with an appropriate optically active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, followed by recovery of the pure enantiomers.

[0300] 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 that a specific functional moiety, such as O, S, or N, is temporarily blocked so that a reaction can be selectively carried out at another reactive site of a multifunctional compound. In certain embodiments, the protecting group reacts selectively in good yield to give a protected substrate that is stable for the anticipated reaction; the protecting group must be selectively removable in good yield with a readily available, preferably non-toxic, reagent that does not attack other functional groups; the protecting group forms an easily separable derivative (more preferably, without generating a new stereocenter); and the protecting group has minimal additional functional groups to avoid further reactive sites. As detailed herein, oxygen, sulfur, nitrogen, and carbon protecting groups may be utilized.

[0301] The term "aliphatic," as used herein, includes both saturated and unsaturated straight-chain (i.e., unbranched), branched-chain, acyclic, cyclic, or polycyclic aliphatic hydrocarbons, optionally substituted with one or more functional groups. As understood by those skilled in the art, "aliphatic," as used herein, is intended to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term "alkyl" includes straight-chain, branched, and cyclic alkyl groups. A similar rule applies to other generic terms, such as "alkenyl" and "alkynyl." Furthermore, as used herein, terms such as "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.

[0302] In certain embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-18 aliphatic carbon atoms. In certain embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-15 aliphatic carbon atoms. In certain other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-10 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-6 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-4 carbon atoms. Thus, exemplary aliphatic groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, -CH-cyclopropyl, vinyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, -CH-cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, cyclopentyl, -CH-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, -CH-cyclohexyl moieties, and the like, which may also bear one or more substituents. Alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

[0303] The term "alkyl" as used herein refers to a saturated straight or branched chain aliphatic group having 1 to 18 carbon atoms, and thus "alkyl" includes C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C2 10 , C 11 and C 12Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, and dodecyl.

[0304] The term "alkenyl" refers to an unsaturated straight-chain or, where applicable, branched-chain aliphatic group having 2 to 18 carbon atoms and one or more carbon-carbon double bonds. Thus, "alkenyl" includes C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, ​​C47, C48, C49, C50, C51, C52, C 10 , C 11 and C 12 Alkenyl groups include, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.

[0305] The term "alkynyl" refers to an unsaturated straight-chain or, where applicable, branched-chain aliphatic group having 2 to 18 carbon atoms and one or more carbon-carbon triple bonds. Thus, "alkynyl" includes C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, ​​C47, C48, C49, C50, C51, C52, C 10 , C 11 and C 12 Representative alkynyl groups include ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

[0306] As used herein, the term "aryl" group refers to an optionally substituted C-C group containing 1 to 3 aromatic rings. 14 It is an aromatic moiety. Thus, "aryl" refers to C6, C7, C8, C9, C 10 , C 11 , C 12 C 13 , and C 14 Exemplary aryl groups include C-C 10 Particular aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl and fluorenyl.

[0307] As used herein, the term "cycloalkyl" includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons. Thus, "cycloalkyl" includes C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26 10 , C 11 and C 12 Representative cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0308] As used herein, the term "halo" or "halogen" refers to fluoro, chloro, bromo, and iodo.

[0309] It will be understood that the compounds of any one of the formulae disclosed herein, and any pharmaceutically acceptable salts thereof, include all isomeric forms, stereoisomers, mixtures of stereoisomers, and polymorphs of the compounds.

[0310] The term "independently selected" is used herein to indicate that the R groups can be the same or different.

[0311] As used herein, the term "substituted," whether preceded by the term "optionally" or not, and "substituent," refers to the ability to change one functional group to another, provided that the valence of all atoms is maintained, as recognized by those skilled in the art. When more than one position in any given structure can be substituted with more than one substituent selected from a particular group, the substituents can be the same or different at all positions. Substituents can also be further substituted (e.g., an aryl group substituent can have another substituent, such as another aryl group further substituted with fluorine at one or more positions).

[0312] The present disclosure provides isolated or substantially purified polynucleotide or protein compositions. An "isolated" or "purified" polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an "isolated" polynucleotide is free of sequences that naturally flank the polynucleotide in the genomic DNA of the organism from which the polynucleotide is derived (i.e., sequences located at the 5' and 3' ends of the polynucleotide) (optimally, protein-coding sequences). For example, in various embodiments, an isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. Proteins that are substantially free of cellular material include preparations of proteins having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating proteins. When a protein of the present disclosure or a biologically active portion thereof is recombinantly produced, optimally, the culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-target protein chemicals.

[0313] The present disclosure provides fragments and variants of the disclosed DNA sequences and proteins encoded by these DNA sequences. As used throughout this disclosure, the term "fragment" refers to a portion of a DNA sequence or a portion of an amino acid sequence, and thus the protein encoded thereby. A fragment of a DNA sequence, including a coding sequence, may encode a protein fragment that retains the biological activity of the native protein, thus retaining the DNA recognition or binding activity to the target DNA sequence described herein. Alternatively, a fragment of a DNA sequence useful as a hybridization probe generally does not encode a protein that retains biological activity or does not retain promoter activity. Thus, a fragment of a DNA sequence can range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide of the present disclosure.

[0314] The nucleic acids or proteins of the present disclosure can be constructed by a modular approach, involving preassembling monomeric and / or repeating units in a target vector that can then be assembled into a final destination vector. Polypeptides of the present disclosure can include repeating monomers of the present disclosure and can be constructed by a modular approach by preassembling repeating units in a target vector that can then be assembled into a final destination vector. The present disclosure provides polypeptides produced by this method as well as nucleic acid sequences encoding these polypeptides. The present disclosure provides host organisms and cells containing nucleic acid sequences encoding polypeptides produced by this modular approach.

[0315] The term "antibody" is used in the broadest sense and specifically encompasses single monoclonal antibodies (including agonist and antagonist antibodies) and antibody compositions with polyepitopic specificity. It is also within the scope of the present specification to use natural or synthetic analogs, mutants, variants, alleles, homologs, and orthologs of the antibodies of the present specification (collectively referred to herein as "analogs"). Thus, according to one aspect of the present specification, the term "antibody of the present specification" in its broadest sense also encompasses such analogs. Generally, in such analogs, one or more amino acid residues may be substituted, deleted, and / or added compared to the antibodies of the present specification as defined herein.

[0316] The term "comprising" is intended to mean that compositions and methods include the recited elements, but do not exclude other elements. "Consisting essentially of," when used to define compositions and methods, shall mean excluding other elements that are essential to the combination when used for its intended purpose. Thus, a composition consisting essentially of the elements defined herein does not exclude trace amounts of contaminants or inert carriers. "Consisting of" shall mean excluding more than trace amounts of other components and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0317] 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 may include splicing of the mRNA in a eukaryotic cell.

[0318] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. A gene product can be a direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, microRNA, structural RNA, or any other type of RNA) or a protein produced by translation of mRNA. Gene products also include RNAs that are modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins that are modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0319] "Modulation" or "regulation" of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression.

[0320] The term "operatively linked" or its equivalents (e.g., "linked operatively") means that two or more molecules are positioned relative to each other so that they can interact to affect a function ascribed to one or both molecules or a combination thereof.

[0321] Non-covalently linked components and methods for making and using non-covalently linked components are disclosed. Various components can take a variety of different forms as described herein. For example, non-covalently linked (i.e., operably linked) proteins can be used to allow temporary interactions, which avoids one or more problems in the art. The ability of non-covalently linked components, such as proteins, to associate and dissociate allows for functional association only or primarily under circumstances where such association is required for the desired activity. The association can be of sufficient duration to allow for the desired effect.

[0322] Disclosed is a method for targeting a protein to a specific locus within the genome of an organism. The method can include providing a DNA localization component and providing an effector molecule, wherein the DNA localization component and the effector molecule can be operably linked via a non-covalent bond.

[0323] A "target site" or "target sequence" is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist.

[0324] The term "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked to each other. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid can also encompass the complementary strand of the depicted single strand. The nucleic acids of the present disclosure also encompass substantially identical nucleic acids and their complements that retain the same structure or encode the same protein.

[0325] A probe of the present disclosure can comprise a single-stranded nucleic acid capable of hybridizing to a target sequence under stringent hybridization conditions. Thus, a nucleic acid of the present disclosure can refer to a probe that hybridizes under stringent hybridization conditions.

[0326] The nucleic acids of the present disclosure may be single-stranded or double-stranded. The nucleic acids of the present disclosure may contain double-stranded sequences even if the majority of the molecule is single-stranded. The nucleic acids of the present disclosure may contain single-stranded sequences even if the majority of the molecule is double-stranded. The nucleic acids of the present disclosure may include genomic DNA, cDNA, RNA, or hybrids thereof. The nucleic acids of the present disclosure may contain a combination of deoxyribonucleotides and ribonucleotides. The nucleic acids of the present disclosure may contain a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. The nucleic acids of the present disclosure may be synthesized to contain unnatural amino acid modifications. The nucleic acids of the present disclosure may be obtained by chemical synthesis or recombinant methods.

[0327] The nucleic acids of the present disclosure may not be naturally occurring in their entirety or in any part thereof. The nucleic acids of the present disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not occur in nature, making the entire nucleic acid sequence non-naturally occurring. The nucleic acids of the present disclosure may contain one or more duplicated sequences, inverted sequences, or repeated sequences, resulting in sequences that do not occur in nature, making the entire nucleic acid sequence non-naturally occurring. The nucleic acids of the present disclosure may contain non-naturally occurring modified nucleotides, artificial nucleotides, or synthetic nucleotides, making the entire nucleic acid sequence non-naturally occurring.

[0328] Given the redundancy of the genetic code, multiple nucleotide sequences may encode any particular protein, and all such nucleotide sequences are contemplated herein.

[0329] As used throughout this disclosure, the term "operably linked" refers to the expression of a gene under the control of a promoter to which it is spatially connected. 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 it is derived. Variation in the distance between the promoter and the gene can be accommodated without loss of promoter function.

[0330] As used throughout this disclosure, the term "promoter" refers to a synthetic or naturally occurring molecule that can confer, activate, or enhance expression of a nucleic acid in a cell. A promoter can contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter can also contain distal enhancer or repressor elements, which can be located as far as thousands of base pairs from the start site of transcription. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters can regulate the expression of gene components constitutively or differentially with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, EF-1 alpha promoter, CAG promoter, SV40 early promoter or SV40 late promoter, and CMV IE promoter.

[0331] As used throughout this disclosure, the term "substantially complementary" refers to a first sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids, or where the two sequences hybridize under stringent hybridization conditions.

[0332] As used throughout this disclosure, the term "substantially identical" refers to first and second sequences that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or with respect to nucleic acids, where the first sequence is substantially complementary to the complement of the second sequence.

[0333] As used throughout this disclosure, the term "variant," when used to describe a nucleic acid, refers to: (i) a portion or fragment of a referenced nucleotide sequence; (ii) a complement of the referenced nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to the referenced nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, its complement, or a sequence substantially identical thereto.

[0334] As used throughout this disclosure, the term "vector" refers to a nucleic acid sequence containing a replication origin. A vector can be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector, preferably a DNA plasmid. A vector can contain a combination of amino acids and DNA sequences, RNA sequences, or both DNA and RNA sequences.

[0335] As used throughout this disclosure, the term "variant," when used to describe a peptide or polypeptide, refers to a peptide or polypeptide that differs in amino acid sequence by amino acid insertions, deletions, or conservative substitutions, but retains at least one biological activity. A variant can also refer to a protein having an amino acid sequence substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity.

[0336] Conservative amino acid substitutions, i.e., replacing an amino acid with a different amino acid with similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are recognized in the art as typically resulting in minor changes. These minor changes can be identified, in part, by considering the hydropathic index of the amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on consideration of its hydrophobicity and charge. Amino acids with similar hydropathic indices may be substituted and still retain protein function. In one embodiment, amino acids with hydropathic indices of ±2 are substituted. Amino acid hydrophilicity can also be used to identify substitutions that result in proteins that retain biological function. Considering the hydrophilicity of amino acids in the context of a peptide allows for calculation of the peptide's maximum local average hydrophilicity, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101, incorporated herein by reference in its entirety.

[0337] Substitution of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, such as immunogenicity. Substitutions can be made with amino acids that have hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with this observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids, particularly their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0338] As used herein, "conservative" amino acid substitutions can be defined as shown in Tables 1, 2, or 3 below. In some embodiments, fusion polypeptides and / or nucleic acids encoding such fusion polypeptides contain conservative substitutions introduced by modifying the polynucleotide encoding the polypeptide of the present disclosure. Amino acids can be classified according to their physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is the substitution of one amino acid for another amino acid with similar properties. Exemplary conservative substitutions are shown in Table 1.

[0339] [Table 1]

[0340] Alternatively, conserved amino acids can be grouped as shown in Table 2, as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY, NY (1975), pp. 71-77).

[0341] [Table 2]

[0342] Alternatively, exemplary conservative substitutions are shown in Table 3.

[0343] [Table 3]

[0344] It should be understood that the polypeptides of the present disclosure are intended to include polypeptides possessing one or more insertions, deletions, or substitutions of amino acid residues, or any combination thereof, as well as modifications other than insertions, deletions, or substitutions of amino acid residues. A polypeptide or nucleic acid of the present disclosure may contain one or more conservative substitutions.

[0345] As used throughout this disclosure, the term "two or more" of the foregoing amino acid substitutions refers to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more of the listed amino acid substitutions. The term "two or more" can refer to 2, 3, 4, or 5 of the listed amino acid substitutions.

[0346] The polypeptides and proteins of the present disclosure may not be naturally occurring in their entirety or in any part thereof.The polypeptides and proteins of the present disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not occur in nature, making the entire amino acid sequence non-naturally occurring.The polypeptides and proteins of the present disclosure may contain one or more overlapping sequences, inverted sequences, or repeat sequences, resulting in sequences that do not occur in nature, making the entire amino acid sequence non-naturally occurring.The polypeptides and proteins of the present disclosure may contain non-naturally occurring modified amino acids, artificial amino acids, or synthetic amino acids, making the entire amino acid sequence non-naturally occurring.

[0347] As used throughout this disclosure, "sequence identity" can be determined by using a standalone executable BLAST engine program (bl2seq) to blast two sequences, which is available from the National Center for Biotechnology Information (NCBI) ftp site using default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; incorporated herein by reference in its entirety). The term "identical" or "identity," when used in the context of two or more nucleic acid or polypeptide sequences, refers to a specific percentage of residues that are the same across a specific region of each sequence. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences across a specified region, determining the number of positions where identical residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are of different lengths or the alignment produces one or more staggered ends and a particular 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 determined manually or by using a computer alignment algorithm such as BLAST or BLAST 2.0.

[0348] As used throughout this disclosure, the term "endogenous" refers to a nucleic acid or protein sequence that is naturally associated with the target gene or host cell into which it is introduced.

[0349] As used throughout this disclosure, the term "exogenous" refers to a nucleic acid or protein sequence that is not naturally associated with the target gene or the host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid (e.g., a DNA sequence) or a naturally occurring nucleic acid sequence located in a non-naturally occurring genomic location.

[0350] The present disclosure provides a method for introducing a polynucleotide construct containing a DNA sequence into a host cell. "Introduction" refers to presenting the polynucleotide construct to the cell so that the construct gains access to the interior of the host cell. The method of the present disclosure does not rely on a specific method for introducing the polynucleotide construct into the host cell, but only requires that the polynucleotide construct gain access to the interior of one cell of the host. Methods for introducing polynucleotide constructs into bacteria, plants, fungi, and animals are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0351] Example

[0352] For the examples below, compound numbers are assigned to compounds of formula (I) of the present disclosure according to the following: [table] TIFF2026503551000070.tif187170

[0353] Example 1 - Preparation of Compound No. 1

[0354] Compound No. 1 was prepared according to general scheme A. The crude product was purified by silica gel flash column chromatography using 5-8% MeOH / CH2Cl2. Light brown oil, 32 mg, yield: 31%; MS found 1383.1 [M+H] +、 Calculated value [C79H143N7O12 = 1382.08].

[0355] Example 2 - Preparation of Compound No. 2 Compound No. 2 was prepared according to general scheme A. The crude product was purified by silica gel flash column chromatography using 8% MeOH / CH2Cl2. Light brown oil, 0.76 g, yield: 37%; 1H NMR(499MHz,CDCl3)δ 5.43-5.34(m,4H),5.13-5.03(m,4H),4.20-4.08(m,8H),3.58-3.42(m,8H),3.16-3.00(m,4H),2.74-1.79(m,71H),1.69-1.56(m,28H).

[0356] Example 3 - Preparation of Compound No. 3

[0357] Compound No. 3 was prepared according to general scheme B2. The crude product was purified by silica gel flash column chromatography using 8% MeOH / CH2Cl2. δ 6.23(s,4H),5.43-5.35(m,4H),5.07(m,4H),4.15(t,J=5.5 Hz,8H),3.53-3.44(m,8H),2.75(t,J=7.1 Hz,8H),2.44(t,J=7.1 Hz,12H),2.39-2.25(m,4H),2.17(br,3H),2.10-2.03(m,10H),2.00-1.93( m,16H),1.91-1.84(m,6H),1.68(m,12H),1.62-1.48(m,24H),1.17(d,J=4.6 Hz,12H).MS actual value 1424.3[M+H] +、 Calculated value [C83H136N7O12 = 1423.0]

[0358] Example 4 - Preparation of Compound No. 4

[0359] Compound No. 4 was prepared according to general scheme B1. The crude product was purified by silica gel flash column chromatography using 8% MeOH / CH2Cl2. 1H NMR(499MHz,CDCl3)δ 5.33(m,4H),5.06(m,4H),4.14(t,J=5.8 Hz,8H),3.48(q,J=5.8 Hz,8H),2.71(t,J=6.3 Hz,8H),2.52-2.43(m,8H),2.34(t,J=6.3 Hz,8H),2.25(br,3H),2.07-2.01(M,8H),2.00-1.79(m,24H),1.67(M,16H),1.60-1.57(m,20H),1.18-1.17(m,12H).MS actual value 1423.4[M+H] +、 Calculated value [C83H136N7O12 = 1423.0]

[0360] Example 5 - Preparation of Compound No. 5

[0361] Compound No. 5 was prepared according to general scheme A. The crude product was purified by silica gel flash column chromatography using 30-50% MeOH / CH2Cl2 as eluent. Brown oil, 138 mg, yield=61%; 1 H NMR(499MHz,CDCl3)δ 7.36-7.29(m,4H),5.43-5.34(m,4H),5.13-5.04(m,8H),4.22-4.10(m,8H),3.56-3.42(m,8H),2.75- 2.66(m,8H),2.59-2.43(m,8H),2.39-2.31(m,8H),2.25-1.91(m,59H),1.70-1.56(m,44H).Mass (ESI):C 99 H 160 N7O 12 Calculated value: 1639, measured value: 1640.

[0362] Example 6 - Preparation of Compound No. 6

[0363] Compound No. 6 was prepared according to general Scheme C. 1H NMR(500MHz,CDCl3):δ 5.42-5.43(m,8H),5.08-5.11(m,8H),4.23-4.17(m,8H),3.71-3.58(m,8H),3.13-.91(m,12H),2.71-2.65(m,12H),2.45-2.38(m,12H) ),2.35-2.25(m,8H),2.27-2.15(m,3H),2.14-2.01(m,16H),2.02-193(m,6H),1.8-1.67(m,12H),1.68(s,12H),1.60(s,12H).MS:m / z 1423(M+1).

[0364] Example 7 - Preparation of Compound No. 7

[0365] Compound No. 7 was prepared according to general Scheme C. 1 H NMR(500MHz,CDCl3):δ 5.32-5.37(m,8H),5.09-5.06(m,8H),4.21-4.20(m,8H),3.61-3.58(m,8H),3.07-2.95(m,12H),2.84-2.7 9(m,8H),2.55-2.45(m,16H),2.30-2.14(m,15H),2.08-1.93(m,28H),1.66(s,12H),1.61(s,12H).MS:m / z 1423(M+1).

[0366] Example 8 - LNPs of the present disclosure deliver RNA with high specificity to the lung in vivo

[0367] The following are non-limiting examples that provide exemplary methods for formulating multiple multi-component LNP compositions comprising an exemplary compound of Formula (I) and mRNA.

[0368] A. Preparation

[0369] To formulate LNPs, Compound No. 2, the phospholipid DOPC, the structural lipid cholesterol (Chol), and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (DMG-PEG2000; Avanti Polar Lipids, Alabaster, Alabama, USA) were combined to prepare the LNP composition.

[0370] Individual 25 mg / ml stock solutions were prepared by solubilizing lipids in 200-proof HPLC-grade ethanol, and the stock solutions were stored at -80 °C until formulation. At the time of formulation, the lipid stock solutions were briefly equilibrated to room temperature and then placed on a hot plate maintained at a temperature range of 50-55 °C. The hot lipid stock solutions were then combined to obtain the desired final molar percentages. The LNP compositions are shown in Table 4.

[0371] [Table 4]

[0372] A 1 mg / ml solution of 5'-CleanCap-fLuciferase mRNA (TriLink Biotech) to be incorporated into the LNPs was added to 150 mM sodium acetate buffer (pH 5.2) to form a stock solution and kept on ice. The lipid phase was mixed with the aqueous mRNA phase in a microfluidic chip using a NanoAssemblr® instrument (Precision Nanosystems, Vancouver, BC, Canada) according to the manufacturer's instructions to form an LNP composition containing the encapsulated mRNA. The NanoAssemblr process parameters for mRNA encapsulation are shown in Table 5.

[0373] [Table 5]

[0374] The resulting mRNA LNP composition was then transferred to a Repligen Float-A-Lyzer dialysis device (Spectrum Chemical Mfg. Corp., CA, USA) with a molecular weight cutoff (MWCO) of 8-10 kDa and processed by dialysis against phosphate-buffered saline (PBS) (dialysate:dialysis buffer volume at least 1:200 v / v), pH 7.4, at 4 °C overnight (or alternatively, at least 4 h at room temperature) to remove 25% ethanol and achieve complete buffer exchange. In some experiments, the LNP was further concentrated with an Amicon® Ultra-4 centrifugal filter unit, MWCO-30 kDa (Millipore Sigma, USA), and spun at approximately 4100 × g in an ultracentrifuge. The mRNA LNP was then stored at 4 °C until further use.

[0375] B. In Vivo Screening

[0376] Adult BALB / C mice (n=3) were administered 0.5 mg / kg of total RNA formulated into the LNP compositions listed in Table 4. One group of mice was treated with vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control.

[0377] The location and extent of luciferase expression in treated and control mice was determined 4 hours after RNA delivery by bioluminescence imaging (BLI) of anesthetized mice using an IVIS Lumina in vivo imaging system (Perkin-Elmer) according to the manufacturer's instructions. Briefly, mice were anesthetized using isoflurane in oxygen and placed in a supine position on a heated stage. Mice were then administered D-luciferin (Perkin-Elmer #122799) IP, and BLI was performed on the lungs, liver, and spleen. BLI images are shown in Figure 1, and quantitative results are shown in Table 6.

[0378] [Table 6]

[0379] As shown in Table 6 and Figure 1, the LNP compositions of the present disclosure successfully delivered RNA to lung cells, and the encoded transgene was expressed in the lung cells.

[0380] Example 9 - LNPs of the present disclosure deliver DNA with high specificity to the lung in vivo

[0381] The following are non-limiting examples that provide exemplary methods for formulating multiple multi-component LNP compositions comprising exemplary compounds of Formula (I) and DNA.

[0382] Adult BALB / C mice (n = 3) were administered either (1) LNPs (Nature Technology Corporation) encapsulating a firefly luciferase transposon or (2) a single co-encapsulated LNP encapsulating both the firefly luciferase transposon and SPB. Mice treated with LNPs encapsulating only the luciferase transposon received 0.33 mg / kg or 0.5 mg / kg of LNPs. Mice treated with co-encapsulated LNPs received 0.5 mg / kg or 0.75 mg / kg of co-encapsulated LNPs encapsulating mRNA and DNA at a 1:2 mRNA:DNA ratio. One group of mice was treated with vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control.

[0383] The LNPs encapsulating the firefly luciferase transposon were LNPs of the present disclosure containing nanoplasmid DNA (SEQ ID NO: 6 or SEQ ID NO: 7) containing a transposon, where the transposon contained an expression cassette containing a first piggyBac inverted terminal repeat (right ITR), a nucleic acid sequence encoding firefly luciferase, and a second piggyBac inverted terminal repeat (left ITR). The LNPs encapsulating the firefly luciferase transposon contained Compound No. 2, DOPC, cholesterol, and DMG-PEG2000 in the molar ratios shown in Table 7.

[0384] The single co-encapsulated LNP encapsulating both the firefly luciferase transposon and SPB was an LNP of the present disclosure containing Nanoplasmid DNA of SEQ ID NO: 6 or SEQ ID NO: 7, mRNA encoding active SPB, and Compound No. 2, DOPC, cholesterol, and DMG-PEG2000 in the molar ratios shown in Table 7. All cytidine residues in the mRNA were 5-methylcytidine (5-MeC).

[0385] [Table 7]

[0386] The location and extent of luciferase expression in treated and control mice was determined 7 days after injection for DNA delivery by bioluminescence imaging (BLI) of anesthetized mice using an IVIS Lumina in vivo imaging system (Perkin-Elmer) according to the manufacturer's instructions. Briefly, mice were anesthetized using isoflurane in oxygen and placed in a supine position on a heated stage. Mice were then administered D-luciferin (Perkin-Elmer #122799) IP, and BLI was performed on the lungs, liver, and spleen. The BLI results on the lungs are shown in Table 8 for the low-dose treatment (0.33 mg / kg for luciferase transposon alone and 0.5 mg / kg for co-encapsulation treatment) and the high-dose treatment (0.5 mg / kg for luciferase transposon alone and 0.75 mg / kg for co-encapsulation treatment).

[0387] [Table 8]

[0388] As shown in Table 8, co-encapsulated LNPs (SPB mRNA + FLuc DNA at a 1:2 ratio) were significantly more potent than LNPs encapsulating only FLuc DNA at an equivalent DNA dose, indicating that SPB-facilitated translocation may occur. This example demonstrates that the LNP composition of the present disclosure successfully delivered a two-component DNA / RNA system to lung cells, and the desired transgene was stably integrated into the genome rather than episomes.

[0389] While the invention has been described in relation to particular embodiments thereof, it will be understood that the invention is capable of further modifications, and this application is generally intended to cover any variations, uses, or adaptations of the invention which are in accordance with the principles of the invention and which are within known or customary practice in the art to which this invention pertains and which are applicable to the essential features hereinabove described and which comply with the scope of the appended claims, including departures from the present disclosure.

Claims

1. Compounds of formula (I): 【Chemistry 1】 Formula (I) And, During the ceremony, A is, 【Chemistry 2】 、 【Transformation 3】 、 【Chemistry 4】 、 【Transformation 5】 、 【Transformation 6】 、 【Transformation 7】 、 【Transformation 8】 ,or 【Chemistry 9】 and Each B is independently 【Chemistry 10】 or 【Chemistry 11】 where * indicates the bond to A and ** indicates the bond to C; Each C is independently 【Chemistry 12】 wherein: 【Chemistry 13】 represents a single or double bond; n is an integer from 2 to 6; a is an integer from 1 to 5; b is an integer from 1 to 5; Each R １ are independently １ -C １８ Alkyl or C ２ -C １８ alkenyl; Each R ２ is independently H or methyl; Each R ３ is independently H or methyl, or a salt thereof.

2. A is, 【Chemistry 14】 2. The compound of claim 1, wherein:

3. Each B, 【Chemistry 15】 2. The compound of claim 1, wherein * indicates a bond to A and ** indicates a bond to C.

4. Each B, 【Chemistry 16】 2. The compound of claim 1, wherein * indicates a bond to A and ** indicates a bond to C.

5. A is, 【Chemistry 17】 and each B is [Chemistry 18] 2. The compound of claim 1, wherein * indicates a bond to A and ** indicates a bond to C.

6. A is, 【Chemistry 19】 and each B is 【Chemistry 20】 2. The compound of claim 1, wherein * indicates a bond to A and ** indicates a bond to C.

7. Each R ２ The compound according to any one of claims 2 to 6, wherein is H.

8. Each R ２ The compound according to any one of claims 2 to 6, wherein is methyl.

9. Each R ３ The compound according to any one of claims 2 to 8, wherein is H.

10. Each R ３ The compound according to any one of claims 2 to 7, wherein is methyl.

11. Each R １ is C ２ -C １８ The compound according to any one of claims 2 to 10, which is alkenyl.

12. Each R １ but 【Chemistry 21】 12. The compound of claim 11, wherein:

13. Each R １ but 【Chemistry 22】 12. The compound of claim 11, wherein:

14. Each R １ is C １ -C １８ The compound of any one of claims 2 to 7 or 9, which is alkyl.

15. Each R １ but 【Chemistry 23】 15. The compound of claim 14, wherein:

16. Each R ３ is H, and each R １ is C ２ -C １８ The compound of any one of claims 2 to 8, which is alkenyl.

17. Each R ３ is H, and each R １ is C １ -C １８ The compound of any one of claims 2 to 7, which is alkyl.

18. Each R ２ is H, and each R ３ is H, and each R １ is C ２ -C １８ The compound of any one of claims 2 to 6, which is alkenyl.

19. Each R ２ is H, and each R ３ is H, and each R １ is C １ -C １８ The compound according to any one of claims 2 to 6, which is alkyl.

20. Each R １ but 【Chemistry 24】 19. The compound of claim 16 or claim 18, wherein

21. Each R １ but 【Chemistry 25】 19. The compound of claim 16 or claim 18, wherein

22. Each R １ but 【Chemistry 26】 20. The compound of claim 17 or claim 19, wherein:

23. The compound according to any one of claims 2 to 22, wherein a is 2.

24. The compound of any one of claims 2 to 22, wherein b is 2.

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

26. The compound of any one of claims 2 to 25, wherein n is 4.

27. A compound selected from: 【Chemistry 27】 、 【Chemistry 28】 、 【Chemistry 29】 、 【Transformation 30】 、 【Chemistry 31】 、 【Chemistry 32】 ,or 【Transformation 33】 。

28. A composition comprising at least one lipid nanoparticle comprising at least one compound of formula (I) according to any one of claims 1 to 27.

29. the at least one lipid nanoparticle comprises about 40.75% on a molar basis of the at least one compound of formula (I); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle further comprises: approximately 51.75% cholesterol on a molar basis, about 5% DOPC on a molar basis, and About 2.5% DMG-PEG2000 on a molar basis; and The lipid to nucleic acid ratio in the at least one nanoparticle is about 120:1, about 60:1, or about 20:1 (w / w).

29. The composition of claim 28, wherein

30. the at least one lipid nanoparticle comprises about 40.75% on a molar basis of the at least one compound of formula (I); the at least one nucleic acid molecule comprises at least one RNA molecule; The at least one lipid nanoparticle further comprises: approximately 51.75% cholesterol on a molar basis, about 5% DOPC on a molar basis, and About 2.5% DMG-PEG2000 on a molar basis; and The lipid to nucleic acid ratio in the at least one nanoparticle is about 120:1, about 60:1, or about 20:1 (w / w).

29. The composition of claim 28, wherein

31. the at least one lipid nanoparticle comprises about 40.75% on a molar basis of the at least one compound of formula (I); the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule; The at least one lipid nanoparticle further comprises: approximately 51.75% cholesterol on a molar basis, about 5% DOPC on a molar basis, and Contains about 3% DMG-PEG2000 on a molar basis; The lipid to nucleic acid ratio in the at least one nanoparticle is about 120:1, about 60:1, or about 20:1 (w / w).

29. The composition of claim 28, wherein

32. the at least one lipid nanoparticle comprises about 40.75% on a molar basis of the at least one compound of formula (I); the at least one nucleic acid molecule comprises at least one RNA molecule; The at least one lipid nanoparticle further comprises: approximately 51.75% cholesterol on a molar basis, about 5% DOPC on a molar basis, and Contains about 3% DMG-PEG2000 on a molar basis; The lipid to nucleic acid ratio in the at least one nanoparticle is about 120:1, about 60:1, or about 20:1 (w / w).

29. The composition of claim 28, wherein

33. The composition of any one of claims 28 to 32, wherein the RNA molecule is an mRNA molecule, preferably the mRNA molecule further comprises 5'-CAP.

34. 34. The composition of any one of claims 28 to 33, wherein the at least one RNA molecule comprises a nucleic acid sequence encoding at least one transposase, preferably wherein the transposase is a piggyBac™ (PB) transposase, a piggyBac-like (PBL) transposase, a Super piggyBac™ (SPB) transposase polypeptide, a Sleeping Beauty transposase, a hyperactive Sleeping Beauty (SB100X) transposase, a Helitron transposase, a Tol2 transposase, a TcBuster transposase, or a mutant TcBuster transposase.

35. The composition of any one of claims 28 to 34, wherein the DNA molecule is a circular DNA molecule, a DoggyBone DNA molecule, a DNA plasmid, a DNA nanoplasmid, or a linearized DNA molecule, preferably the DNA molecule is a DoggyBone DNA molecule or a DNA nanoplasmid.

36. The composition of any one of claims 28 to 35, wherein the at least one DNA molecule comprises a nucleic acid sequence encoding at least one transposon.

37. The composition of any one of claims 28 to 36, wherein the at least one nucleic acid molecule comprises a nucleic acid sequence encoding at least one therapeutic protein.

38. 38. The composition of any one of claims 28 to 37, 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.

39. A pharmaceutical composition comprising the composition of any one of claims 28 to 38 and at least one pharmaceutically acceptable excipient or diluent.

40. 40. A method for delivering at least one nucleic acid to at least one cell, the method comprising contacting said at least one cell with at least one composition according to any one of claims 28 to 39.

41. 40. A method for genetically modifying at least one cell, comprising contacting said at least one cell with at least one composition according to any one of claims 28 to 39.

42. 42. The method of claim 40 or 41, wherein the at least one cell is a lung cell.

43. At least one cell modified according to the method of any one of claims 40 to 42.

44. 44. A method of treating at least one disease or disorder in a subject in need thereof, comprising administering to said subject at least one therapeutically effective amount of the composition of any one of claims 28 to 39 or at least one cell of claim 43.

45. 45. The method of claim 44, wherein the at least one disease or disorder is a pulmonary disease or disorder.

46. 46. ​​The method of claim 45, wherein the pulmonary disease or disorder is cystic fibrosis.

47. 40. A method of preferential delivery of a composition to the lungs or lung cells in a subject in need thereof, comprising administering a composition according to any one of claims 28 to 39, thereby providing a greater amount, expression or activity of said composition in said lungs or lung cells of said subject compared to that achieved in a non-pulmonary organ or non-pulmonary cell of said subject.

Images