Lipidoid compounds and related compositions and uses

Abstract

Lipidoid compounds, compositions containing such compounds, methods of preparing these compounds, and the use of these compositions in gene delivery. The compositions and methods of the present disclosure can be used to deliver a nucleic acid to liver cells, in vivo, ex vivo or in vitro, for the treatment of certain diseases and disorders, including, but not limited to liver disorders. The compositions and methods of the present disclosure can be broadly used to deliver a nucleic acid to induce the expression of a secreted therapeutic protein.

Description

LIPIDOID COMPOUNDS AND RELATED COMPOSITIONS AND USESSEQUENCE LISTING

[0001] The Sequence Listing XML associated with this application is provided electronically in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing XML is “POTH-077_001WO_SeqList.xml”. The XML file is 33676 bytes, created on October 29, 2024, and is being submitted electronically via USPTO Patent Center.FIELD OF THE INVENTION

[0002] The present invention relates generally to lipidoid compounds, compositions containing such compounds, methods of preparing these compounds, and the use of these compositions in gene delivery.BACKGROUND OF THE INVENTION

[0003] There has been a long-felt but unmet need in the art for compositions and methods for delivering nucleic acids to cells and for genetically modifying cells in vivo, ex vivo and in vitro. Widely accepted gene delivery and genetic modification techniques, such as the use of viral vectors, including AAVs, can cause acute toxicity and harmful side-effects in patients. The present disclosure provides improved compositions, methods and kits for the delivery of 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 of using the same. These lipid nanoparticle compositions and methods allow for the delivery of nucleic acids to cells with high efficiency and low toxicity. Thus, the compositions and methods of the present disclosure have wide applicability to a diverse number of fields, including gene therapy. Further, these lipid nanoparticles and methods allow for the targeted delivery of nucleic acids to the lungs and lung cells, instead of to the liver which is the target of most developed lipid nanoparticle compositions. Thus, the compositions and methods of the present disclosure also have wide applicability to the development of treatments for a broad spectrum of pulmonary diseases.SUMMARY OF THE INVENTION

[0004] In some aspects, provided are novel compounds. In one aspect, the novel compound is a compound of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII) or Formula (IX):Formula (IX)or a salt thereof,wherein:each R2 is independently: (i) C5-C20 alkyl with optionally one or more alkenyl or (ii)each R4 is independently:Ry each Rs is independently: (i) C5-C20 alkyl with optionally one or more alkenyl, (ii)■v, or(iii)each Re is independently:each R7 is independently:each Rs is independently: (i) C5-C20 alkyl with optionally one or more alkenyl, (ii)each R9 is independently: (i) C5-C20 alkyl with optionally one or more alkenyl or (ii)5 •each R11 is independently: (i) C5-C20 alkyl with optionally one or more alkenyl or (ii)each R13 is independently: (i) C5-C20 alkyl with optionally one or more alkenyl, (ii)-(C5-C20 alkyl)(iii)n is an integer ranging from 0 to 6;p is 0, 1 or 2;each Ryis independently: C5-C20 alkyl with optionally one or more cyclohexyl; andeach Rzis independently:

[0005] In some aspects, provided are novel lipid nanoparticles (“LNPs”) comprising a novel compound. In one aspect, the novel compound is a compound of Formula (I). In another aspect, the novel compound is a compound of Formula (II). In another aspect, the novel compound is a compound of Formula (III). In another aspect, the novel compound is a compound of Formula (IV). In another aspect, the novel compound is a compound of Formula (V). In another aspect, the novel compound is a compound of Formula (VI). In another aspect, the novel compound is a compound of Formula (VII). In another aspect, the novel compound is a compound of Formula (VIII). In another aspect, the novel compound is a compound of Formula (IX).

[0006] In some aspects, a lipid nanoparticle comprises at least one compound of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII) and / or Formula (IX), at least one phospholipid, at least one structural lipid, and at least one PEGylated lipid.

[0007] In some aspects, the LNP may further comprise at least one nucleic acid molecule.

[0008] In some aspects, the at least one nucleic acid molecule is an RNA molecule.

[0009] In some aspects, the at least one nucleic acid molecule is a DNA molecule.

[0010] In some aspects, the ratio of total lipid to nucleic acid in the at least one nanoparticle is from about 40: 1 to about 100: 1 (w / w). The total lipid is the sum of the masses of the four lipid components- cationic lipid, structural lipid, phospholipid, and pegylated lipid.

[0011] In some aspects, provided are pharmaceutical compositions, comprising a composition of the present disclosure and at least one pharmaceutically-acceptable excipient or diluent.

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

[0013] In some aspects, provided are methods of genetically modifying at least one cell comprising contacting the at least one cell with at least one composition of the present disclosure.

[0014] In some aspects, provided are methods of treating at least one disease or disorder in a subject in need thereof comprising administering to the subject at least one therapeutically effective amount of at least one composition of the present disclosure.

[0015] In some aspects, provided are methods of 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.

[0016] In some aspects, provided are cells modified according to methods of the present disclosure.

[0017] Any of the aspects and / or embodiments described herein can be combined with any other aspect and / or embodiment described herein.

[0018] 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. In the Specification, the singular forms also include the plural unless the context clearly dictates otherwise; as examples, 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 element. Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply 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.”

[0019] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present 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. The references cited herein are not admitted to be prior art to the claimed invention. In the case of conflict, the present Specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the disclosure will be apparent from the following detailed description and claim.DETAILED DESCRIPTION OF THE INVENTION

[0020] The present disclosure provides novel lipidoid compounds, novel lipid nanoparticle compositions (LNPs) comprising the novel lipidoid compounds, methods for preparing the LNPs, and methods for using same. In a non-limiting example, the compositions and methods of the present limiting disclosure can be used for gene delivery. In a non-limiting example, the compositions and methods of the present disclosure can be broadly used to deliver a nucleic acid to liver cells, in vivo, ex vivo or in vitro, for the treatment of certain diseases and disorders, including, but not limited to liver disorders. In a non-limiting example, the compositions and methods of the present disclosure can be broadly used to deliver a nucleic acid to induce the expression of a secreted therapeutic protein.

[0021] Compounds of the Present Disclosure

[0022] In one aspect, the present disclosure provides compounds of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII) or Formula (IX):Formula (IX)or a salt thereof,wherein:each R2 is independently: (i) C5-C20 alkyl with optionally one or more alkenyl or (ii)each R3 is independently:each R4 is independently:each Rs is independently: (i) C5-C20 alkyl with optionally one or more alkenyleach Rs is independently: (i) C5-C20 alkyl with optionally one or more alkenyl, (ii)each R9 is independently: (i) C5-C20 alkyl with optionally one or more alkenyl or (ii)Rs;each Rio is independently: (i) -H or (ii);each R11 is independently: (i) C5-C20 alkyl with optionally one or more alkenyl or (ii)each R13 is independently: (i) C5-C20 alkyl with optionally one or more alkenyl, (ii)-(C5-C20 alkyl)n is an integer ranging from 0 to 6;p is 0, 1 or 2;each Ryis independently: C5-C20 alkyl with optionally one or more cyclohexyl; andeach Rz is independently:

[0025] In some aspects, each R2 is independently O

[0026] In some aspects, each R3 is independently C5-C20 alkyl.

[0027] In some aspects, each R3 is independently O

[0028] In some aspects, n is 0.

[0029] In some aspects, n is 1.

[0030] In some aspects, the compound of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII) and / or Formula (IX) is a compound selected from:

[0031] It will be understood that the compounds of any one of the Formulas disclosed herein and any pharmaceutically acceptable salts thereof, comprise stereoisomers, mixtures of stereoisomers, polymorphs of all isomeric forms of said compounds.

[0032] It will be understood that while compounds disclosed herein may be presented without specified configuration (e.g., without specified stereochemistry). Such presentation intends to encompass all available isomers, tautomers, regioisomers, and stereoisomers of the compound. In some embodiments, the presentation of a compound herein without specified configuration intends to refer to each of the available isomers, tautomers, regioisomers, and stereoisomers of the compound, or any mixture thereof.

[0033] It is to be understood that the compounds of any Formula described herein include the compounds themselves, as well as their salts, and their solvates, if applicable. A salt, for example, can be formed between an anion and a positively charged group (e.g., amino) on a substituted compound 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).

[0034] It will be understood that in any of the formulae described herein, when ais used to indicate linkage between two variables (e.g., A-B), the linkage could be one or more covalent bonds.

[0035] General Methods for the Preparation of Compounds of the Present Disclosure

[0036] Compounds of the present disclosure 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.

[0037] General Procedure for Synthesis of Lipidoids (A)General Scheme A

[0038] Intermediate 5

[0039] 3.27 g of beta-octyl glucoside (11.2 mmol) was suspended in dry acetonitrile (100 ml) followed by addition of benzyl dimethyl acetal (2.2 g, 1.3 eq) and racemic camphorsulfonic acid (520 mg, 0.2 eq) at room temperature. The resulting suspension was warmed to 50°C for 12 hrs until most of the starting material was used up as monitored by TLC. After cooling down to room temperature, the reaction mixture was quenched with 0.5 ml DIEA and filtered to obtain a white solid filtrate, which was washed with cold acetonitrile. The mother liquid was stripped under vacuum and followed by silica gel column purification. A combined total of 3.2 g intermediate 2 was obtained after drying under high vacuum (75% yield).

[0040] 2.5 g of 4,6-benzylidene beta-octyl glucoside (6.6 mmol) was dissolved in dry dichloromethane (DCM, 80 ml) followed by addition of 4-dimethylaminopyridine (DMAP, 1.93 g, 1.2 eq per OH) at room temperature. To the resulting solution was added lauroyl chloride (3.7 ml, 1.2 eq per OH) dropwise via syringe at 0°C in 30 mins. The resulting reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with 50 ml DCM followed by addition of cold saturated NaHCO3(100 ml). After stirring at room temperature for 30 minutes, the reaction mixture was extracted with DCM twice and washed with NaHCO3and brine to get crude product which was applied to a silica gel column to yield desired intermediate 3 (2.85 g, 58% yield).

[0041] Intermediate 3 (2.0 g, 2.7 mmol) was suspended in a solvent mixture of DCM / MeOH (20 ml, 1 / 1) followed by addition of racemic camphorsulfonic acid (31 mg, 0.05 eq). The reaction mixture was stirred at room temperature for 24 hrs until most of the starting material was converted into product. The reaction mixture was neutralized by tri ethyl amine (60 pl) and stripped to dryness. The desired intermediate 4 (1.43 g, 81% yield) was obtained after silica gel column purification.

[0042] Intermediate 4 (1.5 g, 2.29 mmol) was dissolved in dry DCM (50 ml) followed by addition of DIEA (0.58 ml, 1.5 eq). To the solution of DCM was added 1.6 eq acryloyl chloride 294 pl dropwise at 0°C via syringe. The resulting reaction mixture was stirred from 0°C to room temperature for 3-4 hrs until most of the starting material was used up as monitored by TLC analysis. After the reaction was quenched with 1 N HC1, the mixture was extracted with DCM to provide crude product, which was purified by silica gel column to give desired acylate 5 (976 mg, 60% yield).1HNMR(500 MHz, CDCl3): δ6.49-6.46 (d, 1H, J=15Hz), 6.21-6.15 (d, J=10 Hz), 6.17-6.15 (d, J=10 Hz), 5.91-5.89 (1H, J=10 Hz), 5.06-5.02 (1H, t, J=10 Hz), 4.96-4.93 (1H, dd, 3. J=5Hz, 10 Hz), 4.59-4.56 (lH,dd, J=5 Hz, 15 Hz), 4.46 (1H, dd, J=10Hz), 4.42-4.39 (1H, dd, J=5Hz, 15Hz), 3.87-3.82 (m, 1H), 3.61-3.54 (m, 2H), 3.47-3.42 (m, 1H), 3.01 (1H, d, J= 5 Hz), 1.54 (m, 8H), 1.35-1.19 (m, 40H), 0.89-0.86 (m, 9H). MS: m / z 733.2 (M+Na).

[0043] 6 -acetate (2’-aminoalkyl)-2,3-Acylate Octyl Glucoside 6

[0044] To a solution of acrylate 5 (25 mg, 35 pmol) in dry acetonitrile (0.5 ml) was added a secondary amine (1.05 eq) at room temperature in a screwed vial, and the resulting reaction was heated to 55°C overnight. After removing the solvent under vacuum, the desired product 6 was obtained as crude product, which was analyzed by LCMS to determine if further purification by silica gel column was necessary.

[0045] Any suitable secondary amine can be used in General Scheme A above to generate lipidoid compounds of the present disclosure. For example, the amines shown below can be combined with acrylate 5 to provide lipidoid compounds of the invention:

[0046] General Procedure for Synthesis of Lipidoids (B)General Scheme B

[0047] a, a -Trehalose dihydrate (1 eq) was dehydrated by boiling in absolute ethanol (0.4 M) under reflux for 30 min. The dry residue was then suspended in dry A', A'-dimethylformamide(DMF, 0.4 M), and dimethyl acetal (2 eq.) was added, together with 10-camphorsulfonic acid (CSA, 0.05 eq). The mixture was heated (60°C) for 1 h on a rotary evaporator with a light vacuum to eliminate the methanol. More DMA (0.25 eq) and CSA (0.01 eq) were then added, and the mixture was attached again to a rotary evaporator. At the end of the reaction, DMF was evaporated. The mixture was stirred overnight in an aqueous sodium hydrogen carbonate solution (5%) to give crystalline diacetal 8. The product was recrystallized by dissolution in boiling ethanol, addition of hot water, and slow cooling. Finally, the white crystals of 8 were filtered, washed with water and ether petroleum and dried (84%).

[0048] Diacetal 8 (1.55 g, 2.99 mmol) was dissolved in dry DCM (30 ml) followed by addition of DMAP (1.82 g, 1.25 eq per OH) at room temperature. To the resulting solution was added lauroyl chloride (3.4 ml, 1.25 eq per OH) dropwise via syringe at 0°C in 30 mins. The resulting reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with 50 ml DCM and followed by addition of cold saturated NaHCOa (100 ml). After stirring at room temperature for 30 mins, the reaction mixture was extracted with DCM twice and washed with NaHCOa and brine to get crude product which was applied to a silica gel column to desired product 9 (2.68 g, 72% yield).

[0049] Product 9 (2.68 g, 2.15 mmol) was suspended in a solvent mixture of DCM (6mL) / MeOH (12 mb) followed by addition of racemic camphorsulfonic acid (50 mg, 0.1 eq). The reaction mixture was stirred at room temperature for 24 hrs until most of the starting material was converted into product. The reaction mixture was neutralized by triethyl amine (60 pl) and stripped to dryness. The desired product 10 (1.64 g, 71% yield) was obtained after silica gel column purification.

[0050] Product 10 (760 mg, 0.71 mmol) was dissolved in dry DCM (10 ml) followed by addition of DIEA (404 mg, 4.4 eq). To the solution of DCM was added 2.2 eq acryloyl chloride (141 mg) dropwise at 0°C via syringe. The resulting reaction mixture was stirred from 0°C to room temperature overnight. After quenching the reaction with 1 N HC1 and extraction with DCM, the crude product was purified by silica gel column to give desired acylate Ila (162 mg, 20%) and lib (385 mg, 48% yield).

[0051] COMPOUND NOS. 55-58 were obtained from acylate 11a or lib following the addition of secondary amine in accordance with General Scheme A.

[0052] General Procedure for Synthesis of Lipidoids (C)General Scheme C

[0053] Synthesis of 5’-OTf-2,3-Di-Lauryl Octyl Glucoside 13

[0054] To a solution of octyl glycoside diol 12 (2.1 g, 3.2 mmol) in dry DCM (20 ml) and Py(0.5 ml) was added triflic anhydride (0.86 ml, 1.6 eq) dropwise at -78°C. The resulting reactionmixture was stirred from -78 °C to -30°C for 1 hr until most starting material was used up asmonitored by TLC. The reaction mixture was diluted with 50 ml cold DCM followed byquenching with 1 N HC1 30 ml at 0°C. The organic phase was sequentially washed with 1 N HC1solution twice, then saturated NaHCO3 to get crude product 13, which was applied to the nextstep without further purification.

[0055] Synthesis of 5’-N3-5’-Deoxyl-2,3-Di-Laurayl Octyl Glucoside 14

[0056] To a solution of intermediate 13 (253 mg, 0.321 mmol) in dry DMF (2 ml) was addedsodium azide (209 mg, 10.0 eq) at room temperature and the resulting reaction mixture wasstirred for 24 hours. The reaction mixture was extracted with EtOAc and sat. Nal ICO3 followedby column purification to give the desired azide compound 14(185 mg, 85%).

[0057] Synthesis of Acrylamide 16

[0058] A suspension of Pd-C (18 mg) and azide 14(180 mg) in EtOAc / MeOH (1 / 2) 2 ml was stirred in the presence of an H2 balloon for 24 hrs until the starting material was used up as monitored by TLC. After filtration and concentration, the residue was applied to the next step without further purification.

[0059] To a dry DCM solution of 5’-NH2-5’-deoxyl Glucoside 15 (150 mg, 0.23 mmol) and DIEA (3.0eq) was added acryl chloride (1.8eq) in an ice bath. The resulting reaction mixture was stirred at room temperature to give the desired product 16 after standard work and chromatography purification.

[0060] Synthesis of Bromo Intermediate 18

[0061] To a solution of 4-bromo propanoic acid (0.64 g, 3.85 mmol) in 10 ml dry DCM was added EDC1 (0.89 g, 1.2 eq) and DIEA (0.98 ml, 1.5 eq) at room temperature. The resulting mixture was stirred at room temperature for 20 mins followed by addition of compound 12 (2.1 g, 3.2 mmol) in dry DCM at 0°C. The reaction mixture was stirred to room temperature overnight. The reaction was quenched with saturated NaHCO3 and extracted with DCM followed by brine and drying over anhydrous Na2SO4. After purification, the desired intermediate 18 was obtained as colorless oil (1.87 g, 73% yield).

[0062] Synthesis of Product 19

[0063] Intermediate 18 (50 mg, 62.1 pmol) and an HC1 salt of a secondary amine (16 mg, 1.5 eq) were mixed in 0.5 ml dry THF followed by addition of DIEA (32 pF, 3.0 eq). The resulting reaction mixture was stirred at 60°C for 24 hrs. After stripping the solvent, the residue was purified by silica gel column to give desired product 19 (14 mg, 39% yield).

[0064] Products 17 and 20 were obtained from the addition of a secondary amine in a similar procedure used to obtain product 19.

[0065] Synthesis of Product 24

[0066] Following the syntheses of intermediates 21 and 23 in accordance with similar procedures shown in General Schemes A and C, product 24 was obtained in a similar procedure used to obtain product 20.

[0067] Any suitable secondary amine can be used in General Scheme C above to generate lipidoid compounds of the present disclosure. For example, the amines shown below can becombined with the triflate, acrylamide, or bromo intermediates to provide lipidoid compounds of the invention:

[0068] General Procedure for Synthesis ofLipidoids (D)General Scheme D

[0069] To a solution of 4.3 (2.8 g, 0.0192 mmol) and DTEA (7 ml, 2.0 eq) in dry DCM (70 ml) was added acyl chloride 4.4 (5.3 g, 0.0288 mmol) dropwise via a dropping funnel in an ice bath. The resulting reaction mixture was stirred at room temperature overnight. After quenching the reaction mixture with sat. NaHCO3, the reaction mixture was extracted with DCM to get crude product. The desired product 4.5 was obtained as a white solid (4.4 g, 78% yield).!H NMR (500 MHz, CDCl3) 84.20 (d, J= 7.1 Hz, 2H), 4.00 (dd, J= 12.1, 4.0 Hz, 2H), 3.73 (dd, J= 12.0, 5.6 Hz, 2H), 3.46 (t, J= 6.4 Hz, 2H), 2.52 (t, J= 7.2 Hz, 2H), 2.21 - 2.14 (m, 2H), 1.98 (dtdd, J = 7.1, 5.6, 4.4, 1.6 Hz, 1H), 1.48 - 1.35 (m, 6H).

[0070] The isopropylidene protected bromide 4.5 (3.1g) was treated with IR120 (2 g) in MeOH (20 ml) at room temperature for 24 hours to afford diol 4.6 (1.98 g, 74% yield) after silica gelchromatography column purification.1H NMR (500 MHz, CDCI3) 84.27 (d, J= 6.3 Hz, 2H), 3.80 (dd, J = 11.1, 4.8 Hz, 2H), 3.75 (dd, J= 11.1, 5.5 Hz, 2H), 3.47 (t, J= 6.4 Hz, 2H), 2.54 (t, J= 7.2 Hz, 2H), 2.18 (p, J = 6.8 Hz, 2H), 2.05 (p, J= 5.7 Hz, 1H).

[0071] Following the protocol to obtain 4.5, the desired DLin-C4 bromide 4.7 was obtained in 65% yield as an oil.JH NMR (500 MHz, CDCl3) 85.4-5.30 (m, 8H), 4.16-4.13 (t, 4H, J=10Hz), 4.14 (dd, J=10 Hz, 2H), 3.46 (t, J=5 Hz, 2H), 2.77 (t, J=5Hz, 4H), 2.52 (t, J=5Hz, 2H), 2.4 (m, 2H), 2.31 (t, J=10 hz, 4H), 2.17 (m, 2H), 2.07-2.03 (m, 8 H), 1.63-1.60 (m, 4H), 1.4-1.26 (m, 28 H), 0.90-0.87 (m, 6H). MS found 800.2 [M+H]+’calcd for [C44H75BrO6=778.5]

[0072] Synthesis of COMPOUND NO, 59

[0073] To a dry small vial with intermediate 4.7 (120 mg, 0.153 mmol) and CS2CO3 (55 mg, 1.1 eq) in a dry mixture of solvents THF / ACN (1 / 1 ratio, 2 ml) was added SAI 7 HC1 salt (40 mg, 1.5 eq) and the resulting reaction mixture was heated to 50 °C overnight with stirring. The reaction mixture was diluted with DCM (10 ml) followed by filtration and extraction with sat. NaHCO3to get crude product, which was applied to silica gel chromatography to afford desired product COMPOUND NO. 59 (44 mg, 35% yield).

[0074] General Procedure for Synthesis of Lipidoids (E)General Scheme E

[0075] Standard Synthetic Procedure of 4.2

[0076] To 1.0 eq dimethyl malonate in dry THF was added 2.2 eq 60% NaH at 0°C and stirred at room temperature for 30 mins followed by addition of alkyl bromide at 0°C. The resultingreaction mixture was stirred from room temperature to 50°C until most of the mono alkylated product had disappeared by TLC analysis. After workup with sat NH4CI, the combined organic layer was concentrated and purified to get desired product 4.2B.!H NMR (500 MHz, CDCl3) 5 3.74 (s, 6H), 1.97 - 1.81 (m, 4H), 1.43 - 1.22 (m, 28H), 1.15 (dq, J= 15.5, 6.5 Hz, 4H), 0.91 (t, J = 7.0 Hz, 6H).

[0077] Standard Synthetic Procedure of 4.3

[0078] 4.2 was dissolved in a mixture of solvents EtOH / H2O (1 / 1) and treated with KOH (8.0 eq) at 40 °C overnight to get saponified diacid, which was re-dissolved in decane and heated to170°C for 24hrs until the diacid was converted to monoacid 4.3. 4.3B 'HNMR (500 MHz, CDCl3) 8 1.98 - 1.88 (m, 4H), 1.58 (ddt, J= 14.1, 9.8, 5.1 Hz, 1H), 1.35 - 1.15 (m, 32H), 0.87 (t, J = 6.9 Hz, 6H).

[0079] Synthesis of 4.8C

[0080] Ester 4.8C was prepared from diol 4.6 with acid 4.3C by following a similar protocol used to synthesize 20 from 18 in General Scheme C.1H NMR (500 MHz, CDCl3) 64.14 (dd, J = 8.1, 6.0 Hz, 6H), 3.59 (t, J= 6.3 Hz, 1H), 3.46 (t, J= 6.4 Hz, 2H), 2.52 (t, J= 7.2 Hz, 2H), 2.40 (p, J= 6.0 Hz, 1H), 2.33 (tt, J= 8.5, 5.4 Hz, 2H), 2.24 - 2.14 (m, 2H), 1.59 (d, J= 4.2 Hz, 3H), 1.44 (dq, J= 14.8, 7.7 Hz, 6H), 1.25 (s, 64H), 0.88 (t, J= 7.0 Hz, 12H).

[0081] Synthesis of COMPOUND NO. 63

[0082] COMPOUND NO. 63 was obtained from 4.8 following a similar protocol to Scheme D.

[0083] Synthesis of 4.11

[0084] A mixture of 4.3 (6.32g, 2 eq) and sodium hydroxide (powder, 5g, 1 eq) was heated at 40°C for 30 min. Bu4NBr (2.27g, 0.2 eq) was added followed by 4.9 (5g, 34.2 mmol, 1 eq). The resulting mixture was heated at 40°C over about 65 hours, then cooled to room temperature and the solid was filtered off. Purification by column chromatography (ethyl acetate / hexane) afforded intermediate 4.11 (4.4 g, 68%) as a clear oil. 'H NMR (500 MHz, CDCl3) 64.00 - 3.93 (m, 2H), 3.78 - 3.72 (m, 3H), 3.56 (qd, J= 9.3, 6.8 Hz, 2H), 3.42 - 3.32 (m, 1H), 3.13 (ddt, J = 5.7, 4.2, 2.8 Hz, 1H), 2.84 - 2.74 (m, 1H), 2.63 - 2.55 (m, 1H), 1.97 (pt, J = 6.5, 4.2 Hz, 1H), 1.40 (dd,,7 = 14.8, 3.2 Hz, 6H).

[0085] Synthesis of 4.12

[0086] To 4.11 (622 mg, 3 mmol, 1 eq) in methanol (10 mL) was added Amberlite IR120H (470 mg). The mixture was stirred at room temperature overnight. The solid was filtered off and the filtrate was concentrated. Purification by column chromatography (methanol / DCM) afforded intermediate 4.12 (206 mg, 42%) as a clear oil.1H NMR (500 MHz, CDCl3) 53.85 - 3.75 (m, 5H), 3.71 (dd, <7= 9.3, 5.6 Hz, 1H), 3.65 (dd,<7= 9.3, 5.8 Hz, 1H), 3.42 (dd, J= 11.7, 5.7 Hz, 1H), 3.15 (ddt, J= 5.6, 4.2, 2.7 Hz, 1H), 2.81 (dd, J = 5.0, 4.1 Hz, 1H), 2.63 (dd, J = 5.0, 2.7 Hz, 1H), 2.23 (td, J= 5.5, 1.5 Hz, 2H), 2.03 (hept, J = 5.4 Hz, 1H).

[0087] Synthesis of 4.13

[0088] To 4.12 (204 mg, 1 eq) in DCM (10 mL) was added DMAP (338 mg, 2.2 eq), Linoleic Acid (777 mg, 2.2 eq) and EDCI (530 mg, 2.2 eq). The resulting mixture was stirred at roomtemperature overnight, then washed with IN HC1, saturated NaHCO3 and brine. The combined organic phase was dried over Na2SC>4 and concentrated. Purification by column chromatography (ethyl acetate / hexane) afforded 4.13 (163 mg, 19%). 'HNMR (500 MHz, CDCl3) 85.45 - 5.27 (m, 8H), 4.23 - 4.05 (m, 4H), 3.74 (dd, J= 11.6, 2.8 Hz, 1H), 3.57 (dd, J= 9.4, 6.0 Hz, 1H), 3.50 (dd, J= 9.5, 6.0 Hz, 1H), 3.35 (dd, J= 11.6, 5.9 Hz, 1H), 3.12 (ddt, J= 5.7, 4.2, 2.8 Hz, 1H), 2.81 - 2.74 (m, 4H), 2.59 (dd, J= 5.0, 2.7 Hz, 1H), 2.39 - 2.26 (m, 5H), 2.09 - 1.99 (m, 8H), 1.61 (t, J= 7.5 Hz, 4H), 1.41 - 1.22 (m, 29H), 0.94 - 0.84 (m, 6H).

[0089] Synthesis of 4.14

[0090] In a 250 mb round bottom flask, trans-4'-pentyl-(l,l'-bicyclohexyl)-4-carboxylic acid (1.33 g, 2.2 eq), EDC (1.24 g, 3.0 eq) and DMAP are combined in dichloromethane (20 mL). The solution was stirred for 15 minutes at room temperature before adding diol (350 mg, 1.0 eq). The resulting solution was left stirring for 20 h at room temperature under nitrogen atmosphere. Water (50 mL) was added and stirring continued for 10 minutes. Both layers were separated, then the aqueous layer was extracted with dichloromethane (3 x 50 mL). Combined organic extracts were washed with brine (50 mL), dried over Na2SC>4, filtered, and evaporated. The crude was purified by flash silica gel chromatography with 10-20% EtOAc / hexane eluants. Off-white solid, 0.96 g (yield 65%); 'H NMR (499 MHz, CDCl3) 84.18 - 4.04 (m, 4H), 3.72 (dd, J = 11.6, 2.9 Hz, 1H), 3.59 - 3.45 (m, 2H), 3.36 (dd, J = 11.5, 5.7 Hz, 1H), 3.15 - 3.08 (m, 1H), 2.78 (dd, J = 5.0, 4.1 Hz, 1H), 2.59 (dd, J = 5.0, 2.7 Hz, 1H), 2.31 (hept, J = 6.1 Hz, 1H), 2.20 (tt, J = 12.2, 3.5 Hz, 2H), 2.02 - 1.93 (m, 4H), 1.82 - 1.64 (m, 12H), 1.44 - 0.77 (m, 44H). MS (ES): m / z calcd for C43H74O6Na (M+Na), 709.54; found, 709.4.

[0091] Synthesis of COMPOUND NO, 67

[0092] To 4.13 (97mg, 0.14 mmol, 1 eq) in ACN (2mL) was added compound SA17 (25 mg, 1 eq) and CS2CO3 (46 mg, 1 eq). The mixture was stirred at 70°C overnight. The solid was filtered off and the filtrate was concentrated. Purification by column chromatography (methanol / DCM) afforded desired product COMPOUND NO. 67 (56 mg, 48%). 'H NMR (500 MHz, CDCl3) 8 5.46 - 5.25 (m, 8H), 4.21 -4.04 (m, 4H), 3.84 (ddt, J= 9.7, 5.4, 4.2 Hz, 1H), 3.51 (d, J= 6.1 Hz, 2H), 3.42 (h, J= 5.6 Hz, 2H), 2.77 (t, J= 6.4 Hz, 4H), 2.56 (s, 2H), 2.41 - 2.25 (m, 8H), 2.09 -2.01 (m, 8H), 1.66- 1.54 (m, 8H), 1.47 (td, J= 7.3, 4.0 Hz, 5H), 1.43 - 1.22 (m, 32H), 0.94 -0.83 (m, 6H). MS found 826.6 [M+H calcd for [C52H91NO6=825.7]

[0093] Synthesis of COMPOUND NO. 70

[0094] In a 20 mL scintillation vial, 8-azaspiro[4.5]decane hydrochloride (SAI 7) (50 mg, 1.0 eq), 4.14 (235 mg, 1.2 eq) and cesium carbonate (205 mg, 2.2 eq) were combined in anhydrous acetonitrile. The suspension was stirred for 20 h at 90°C. The reaction was cooled and filtered. The filtrate was evaporated, water (50 mL) added and the aqueous layer was extracted with di chloromethane (4 x 50 mL). Combined organic extracts were washed with brine (50 mL), dried over Na2SO4, filtered, and evaporated. The crude was purified by flash silica gel chromatography with 5-6% MeOH / dichloromethane eluants. Off-white solid, 193 mg (yield 72%); MS (ES): m / z calcd for C52H9iO6NNa (M+Na), 848.67; found, 848.6.

[0095] General Procedure for Synthesis of Lipidoids (F)General Scheme F

[0096] Synthesis of Intermediate 5.12

[0097] To a solution of Gallic methyl ester (1.68 g, 9.13 mmol) in 40l dry DMF was added 1-iodo dodecane (12.2 g, 4.12 mmol) and potassium carbonate (7.69 g, 55.7 mmol) at room temperature. The resulting suspension was heated to 120°C in a sealed tube for 48 hrs, rotor-evaporated to dryness under vacuum followed by extraction with water and EtOAC. The crude product 5.1 (5.3 g) was carried out to the next step without further purification. 5.1 (1g) was treated with a mixture of solvents EtOH / Et2O (1 / 2) in the presence of excess KOH (5 eq per crude product) at 110°C for 24 hrs in a sealed tube. After working up with 1 HC1 and EtOAC,the cruder product was purified by silica gel to desired product 5.12 (450 mg). 'H NMR (500 MHz, CDCl3) 87.3 l(s, 2H), 4.05-4.01 (m, 6H), 1.83-1.72 (m, 6H), 1.50-1.31(m, 6H), 1.30-1.28 (m, 54H), 0.88 (t, J=5Hz, 9 H). MS found 697.4 [M+Na]+’ calcd for [C43H78NO5=674.6],

[0098] Synthesis of Intermediate 5.13

[0099] SAI 7 was heated with bromo propyl diol in dry acetonitrile in the presence of K2CO3 to 60°C for 24 hrs to get desired SA17-Propyl Diol 5.13 in 35% yield after purification by silica gel chromatography.

[0100] Syntheses of COMPOUND NOS. 60 and 61

[0101] By following a similar protocol used to prepare intermediate 4.8 in General Scheme E, diol 5.13 was reacted with an excess of acid 5.12 in the presence of EDCI / DMAP in dry DCM at room temperature to provide the desired COMPOUND NOS. 60 and 61.

[0102] Synthesis of 5.2

[0103] A mixture of 4,6 benzylidene glucose (1.33g, 4.96 mmol), trans-4-pentyl-cyclohexane-l-carboxylic acid (3.5 g, 17.86 mmol) in DCM (20 ml) and ACN (1 ml) was treated with DMAP (2.2 g, 1786 mmol) and EDCI (3.41 g, 1786 mmol) sequentially. The resulting reaction mixture was stirred at room temperature overnight to offer the desired product Benzylidene 5.2 (2.0 g, 50% yield) as an alpha and beta mixture after standard workup and silica gel column purification.

[0104] Synthesis of 5.3A

[0105] To a solution of 5.2 (1.0 g, 1.23 mmol) and 2.5 ml mixture of solvents DCM / MeOH (1 / 1) was added CSA (13 mg) at room temperature. The resulting reaction mixture was stirred at room temperature for 24 hrs until most of the starting material disappeared as viewed by TLC. After neutralizing with TEA, the resulting reaction mixture was concentrated and applied to a silica gel chromatography to afford diol alpha isomer 5.3A (265 mg, 30% yield) and 5.3B (310 mg, 35%).

[0106] Synthesis of 5.4A

[0107] To a solution of 5.3A in dry DCM was added EDCI and DMAP and the resulting reaction solution was stirred for 30 mins followed by addition of compound 5.2, then the resulting reaction mixture was stirred at room temperature for 24 hours. After workup and drying, the desired compound 5.4 was obtained in 35% yield. 86.27 (d, J=5.0 Hz, 1H), 5.30 (t, J=5 Hz, 1H), 5.06 (dd, 1H, J=2.5, 5.0 Hz, 1H), 4.46 (dd, J-2.5, 5.0Hz, 1H), 4.33-4.32 (m, 1H), 3.95-3.93 (m, 1H), 3.62-3.58 (m, 1H), 3.48-3.46 (t, J=10 Hz, 2H), 2.98 (m, 1H), 2.59-2.56 (t, J=10 Hz, 2H),2.32-2.21 (m, 3H), 2.19 (m, J=10 Hz, 2H), 2.16-1.76 (m, 12H), 1.42-1.14 (m, 33H), 0.95-0.86 (m, 15H). MS found 891.8 [M+Na]+- calcd for [C46H77BrO10=868.5]

[0108] Synthesis of COMPOUND NO, 68

[0109] Following a similar procedure for preparing COMPOUND NO. 67, COMPOUND NO.68 was obtained in a thick colorless gel like molecule in 56% yield.JH NMR (500 MHz, DMSO)86.11 (d, J=5.0 Hz, 1H), 5.72 (d, J=10Hz, 1H), 5.21 (t, J=5.0Hz, 1H), 4.83 (dd, 1H, J=2.5, 5.0Hz, 1H), 4.31(m, 1H), 4.10-4.08 (m, 1H), 3.89-3.85 (m, 1H), 3.58-3.53 (m, 1H), 2.39-2.17 (m,11H), 1.99-1.64 (m, 14H), 1.38-1.13 (m, 45H), 0.99-0.83 (m, 15H). MS found 927.8 [M+H]calcd for [C55H93NO10=927.7]

[0110] Synthesis of COMPOUND NO. 69

[0111] Following a similar procedure for preparing COMPOUND NO. 67, COMPOUND NO.69 was obtained in thick colorless gel like molecule in 56% yield.1H NMR (500 MHz, CDCl3) δ5.71 (d, J=10 Hz, 1H), 5.10-5.08 (m, 2H), 4.52-4.50 (m, 1H), 4.27-4.25 (m, 1H), 3.64 (m, 2H),2.43-2.12(m, 11H), 1.95-1.79 (m, 14H), 1.59-1.11 (m, 45H), 0.94-0.83 (m, 15H). MS found927.8 [M+H]+’ calcd for [C55H93NO10=927.7],

[0112] General Procedure for Synthesis of Lipidoids (G)General Scheme G

[0113] Synthesis of 5.5 A: To cyclohexyl C5 (91.2 mg, 2.0 eq) in dry DCM (2 ml), EDC (71.4mg, 2.0 eq) was added. The reaction mixture was stirred at room temperature for 10 mins,followed by addition of compound 5.4A (200 mg, 1.0 eq) and stirring at room temperature for16h. After the reaction was complete, the resulting mixture was washed with IN HC1, saturated NaHCO3, and brine. The combined organic phase was dried over Na2SO4 andconcentrated. Purification by column chromatography (10% ethyl acetate / hexane) afforded intermediate 5.5A (226 mg, 93% yield).

[0114] Synthesis of COMPOUND NO. 77: To a mixture of 5.5A (226 mg, 1.0 eq) and SA17 (43mg, 1.1 eq) in ACN: THF (1:1), CS2CO3 (77 mg, 1.1 eq) and catalytic amount of KI was addedand the reaction mixture stirred at 50°C for 16 h. After the reaction was complete, the resultingmixture was filtered and concentrated in vacuo. Purification by column chromatography (10% MeOH: DCM) afforded target COMPOUND NO. 77 (156 mg, 65.5 % yield). ’H NMR (499 MHz, CDCl3) 86.29 (d, J= 3.1 Hz, 1H), 5.49 (t, J= 9.9 Hz, 1H), 5.13 - 5.05 (m, 2H), 4.28 (dd, J= 12.5, 4.4 Hz, 1H), 4.08 - 3.99 (m, 2H), 2.86 (s, 3H), 2.45 (t, J= 6.7 Hz, 2H), 2.34 (tt, J = 12.3, 3.6 Hz, 1H), 2.25 -2.07 (m, 5H), 2.03 - 1.95 (m, 3H), 1.87 - 1.76 (m, 15H), 1.65 - 1.61 (m, 4H), 1.49 - 1.39 (m, 6H), 1.26 (qd, J= 13.7, 8.2 Hz, 36H), 1.16 (d, J= 7.9 Hz, 11H), 0.88 (q, J= 7.2 Hz, 20H). MS found 1108.7 [M+H]+- calcd for [ C67H113NO11= 1107.8314],

[0115] General Procedure for Synthesis of Lipidoids (H)General Scheme H

[0116] Synthesis of mono-allyl pentaerythritol (6.1): Pentaerythritol (20g, 2.5 eq) was dissolved in NaOH solution (2.82g NaOH in 60 ml water). Allyl bromide (5.0 ml, 1.0 eq) was slowly added to the reaction mixture while stirring over 2h. The reaction was stirred at 70°C for 16 h. After the reaction was done, the reaction mixture was filtered and concentrated. The crude mixture was purified by column chromatography to get intermediate 6.1 (2.8g, 11.2% yield).

[0117] Synthesis of intermediate 6.3: To lauric acid (3.4g, 6.0 eq) in dry DCM (2 ml), EDC (2.64g, 6.0 eq) was added. The reaction mixture was stirred at room temperature for 10 mins, followed by addition of compound 6.1 (500 mg, 1.0 eq) and stirring at room temperature for 16h. After the reaction was complete, the resulting mixture was washed with IN HC1, saturatedNaHCO3, and brine. The combined organic phase was dried over Na2SO4 andconcentrated. Purification by column chromatography (10% ethyl acetate / hexane) afforded intermediate 6.3 (1.5g, 73% yield).

[0118] Synthesis of intermediate 6.4: Intermediate 6.3 (800 mg, 1.0 eq) was dissolved in anhydrous DCM (5 ml) and kept under ice bath at 0°C for 30 mins. While the reaction mixture was cooling down, meta-chloroperoxybenzoic acid (mCPBA, 381 mg, 2.0 eq) was added in portions over 20 mins. Once the addition was complete, the reaction mixture was stirred at room temperature for 20h, then washed with saturated NaHCO3. The combined organic phase was dried over Na2SO4 and concentrated. Purification by column chromatography (20% ethylacetate / hexane) afforded intermediate 6.4 (663 mg, 81% yield).

[0119] Synthesis of COMPOUND NO. 78: To mixture of 6.4 (400 mg, 1.0 eq) and SAI 7 (115 mg, 1.2 eq) in ACN: THF (1: 1), CS2CO3 (211 mg, 1.1 eq) was added and the reaction mixture stirred at 60°C for 16 h. After the reaction was complete, resulting mixture was filtered and concentrated in vacuo. Purification by column chromatography (10% MeOH: DCM) afforded target COMPOUND NO. 78 (158 mg, 40% yield). 'H NMR (499 MHz, CDCI3) 85.30 (s, 1H), 4.10 (s, 6H), 3.83 - 3.78 (m, 1H), 3.47 (s, 2H), 3.44 - 3.36 (m, 2H), 2.54 (s, 2H), 2.33 - 2.27 (m, 9H), 1.59 (dt, J = 7.5, 3.8 Hz, 9H), 1.50 - 1.42 (m, 4H), 1.41 - 1.37 (m, 4H), 1.31 - 1.22 (m, 50H), 0.87 (t, J= 7.0 Hz, 9H). MS found 879 [M+H] - calcd for [ C53H99NO8= 877.7371],

[0120] General Procedure for Synthesis of Lipidoids (I)General Scheme I

[0121] Synthesis of intermediate 7.1: Intermediate 6.1 (2.0 g, 1.0 eq) was dissolved in anhydrous DCM (10 mL) and kept under ice bath at 0°C for 30 mins. While the reaction mixture was cooling down, mCPBA (4.2g, 2.0 eq) was added in portions over 20 mins. Once the addition was complete, the reaction mixture was stirred at room temperature for 20h, then washed with saturated NaHCO3. The combined organic phase was dried over Na2SO4 andconcentrated. Purification by column chromatography (10% MeOH / DCM) afforded intermediate 7.1 (1.5g, 68% yield).

[0122] Synthesis of intermediate 7.3: Intermediate 7.1 (282 mg, 1.0 eq) was dissolved in anhydrous DCM (1.0 ml) and kept under an ice bath. When the mixture cooled down, triethylamine (1.22 ml, 6.0 eq) was added and the resulting mixture was stirred at 0°C for 60 mins. After generation of anion, oleoyl chloride 7.2 (3.39 ml, 6.0 eq) was added as a 1:1 mixture with THF in a dropwise manner for 30 mins and the reaction mixture was stirred at room temperature for 16h. After the completion of reaction as monitored by TLC, the reaction mixture was extracted with water, dried over Na2SO4 and concentrated. Purification by column chromatography (ethylacetate / hexane) afforded intermediate 7.3 (588 mg, 43% yield).

[0123] Synthesis of COMPOUND NO. 81: To a mixture of intermediate 7.3 (500 mg, 1.0 eq) and SA17 (117 mg, 1.2 eq) in ACN: THF (1:1), CS2CO3 (214 mg, 1.1 eq) was added and the reaction mixture stirred at 60°C for 16 h. After the reaction was complete, the resulting mixture was filtered and concentrated in vacuo. Purification by column chromatography (10% MeOH: DCM) afforded target COMPOUND NO. 81 (145 mg, 26% yield). 'H NMR (500 MHz, CDCl3) / : 5.30-5.37 (m, 6H), 4.10 (s, 6H), 3.79-3.84 (m, 1H), 3.46 (s, 2H), 3.36-3.43 (m, 2H), 2.56 (br, 1H, OH), 2.33-2.37 (m, 3H), 2.29 (t, 6H, J = 7.9Hz), 2.00 (dd, J = 5.75, 6.85 Hz, 11H), 1.57-1.62 (m, 10H), 1.45-1.49 (m, 4H), 1.39 (t, 4H, J = 7.3Hz), 1.24-1.35 (m, 63H), 0.86-0.89 (m, 9H). MS found 1125[M+H]+- calcd for [ C71H129NO8= 1123.9718],

[0124] General Procedure for Synthesis of Lipidoids (J)General Scheme J

[0125] Synthesis of HC12E-acid 8.3: To a solution of HC12E alcohol (8.1) (1.5g, l. Oeq) in DCM (10 ml), DMAP (1.12g, 1.2 eq) was added followed by addition of succinic anhydride (0.994g, 1.3eq). The resulting reaction mixture was stirred at room temperature for 16h. After completion of the reaction, the reaction mixture was extracted with DCM, dried over Na2SO4 and concentrated. Purification by column chromatography (EtOAc / hexane) afforded intermediate 8.3 (798 mg, 37% yield).

[0126] Synthesis of intermediate SA17-3OE1 (8.4): To a mixture of intermediate 7.1 (300 mg, 1.0 eq) and SA17 (333 mg, 1.2 eq) in ACN: THF (1:1), CS2CO3 (609 mg, 1.1 eq) was added and the reaction mixture stirred at 60°C for 16 h. After the reaction was complete, the resulting mixture was filtered and concentrated in vacuo to get intermediate SA17-3OH 8.4 (675 mg) which was taken to next steps without further purification.

[0127] Synthesis of COMPOUND NO. 83: To HC12E-acid 8.3 (391 mg, 4.0 eq) in anhydrous THF (2 ml) was added EDC (187 mg, 4.0 eq) and DMAP (147 mg, 4.0 eq). The reaction mixture was stirred at room temperature for 10 mins, followed by addition of compound intermediate SA17-3OH 8.4 (100 mg, 1.0 eq). The reaction mixture was stirred at room temperature for 16h. After the reaction was complete, the resulting mixture was washed with IN HC1, saturated NaHCO3, and brine. The combined organic phase was dried over Na2SO4 andconcentrated. Purification by column chromatography (5% MeOH / DCM) afforded target COMPOUND NO. 83 (52 mg, 10% yield). H NMR (499 MHz, MeOD) 85.44 - 5.32 (m, 2H),4.14 (d, J= 23.8 Hz, 6H), 3.90 (d, J= 6.6 Hz, 6H), 3.67 - 3.39 (m, 9H), 3.28 - 3.10 (m, 3H), 2.64 (s, 12H), 2.08 - 1.93 (m, 5H), 1.89 - 1.38 (m, 38H), 1.34 - 1.18 (m, 11H), 1.01 (dd, J = 22.9, 11.4 Hz, 9H), 0.96 - 0.88 (m, 18H). MS found 1251.2 [M+H] - calcd for [ C74H123NO14= 1249.8944],

[0128] General Procedure for Synthesis of Lipidoids (K)General Scheme K

[0129] Synthesis of bis-allyl protected mannitol (9.2): To compound 9.1 (2.0g, 1.0 eq) in anhydrous DMF at 0°C, NaH (806 mg, 2.2 eq) was added. The reaction mixture was stirred at 0°C for 1 hour for complete anion generation. To the mixture, allyl bromide (1.649 mL, 2.5 eq) was slowly added at 0°C. Once the addition was complete, the reaction was brought to room temperature and stirred for 4 hours until the reaction was complete (TLC control). After completion of the reaction, the reaction was quenched with water and the solution extracted with hexane. The reaction mixture was dried over anhydrous sodium sulfate and concentrated in vacuum to obtain intermediate 9.2 (3.0g). Intermediate 9.2 was taken to the next step without further purification.

[0130] Synthesis of 3, 4-bis(allyloxy)hexane-l, 2,5, 6-tetraol (9.3): Intermediate 9.2 (1.0g, 1.0 eq) was dissolved in MeOH (5.0 ml) and Amberlite IR 120H (3.0g, 3.0 eq) was added to the reaction mixture. The resulting mixture was heated at 60°C and stirred for 16 h. Once the reaction wascomplete (monitored by TLC), the reaction mixture was filtered, and eluent was concentrated in vacuum. The crude mixture was loaded on a silica column and purified by 10% MeOH: DCM to get pure compound 9.3 (462 mg, 38% yield).

[0131] Synthesis of intermediate 9.5: Trans cyclohexyl C5 (2.645g, 7.0 eq) was dissolved in anhydrous DCM (10 ml). To this mixture, EDC (2.07g, 7.0 eq) and DMAP (1.629g, 7.0 eq) were added. The reaction mixture was stirred for 10 mins at room temperature. After 10 mins, intermediate 9.3 (500 mg, 1.0 eq) was added and the reaction mixture was stirred for 16h at room temperature under an argon balloon. After the reaction was complete, the resulting mixture was washed with IN HC1, saturated NaHCO3 and brine. The combined organic phase was dried over Na2SO4 and concentrated. Purification by column chromatography (10% ethyl acetate / hexane) afforded compound 9.5 (1.19g, 63% yield).

[0132] Synthesis of bis-epoxy intermediate 9.6: Intermediate 9.5 (584 mg, 1.0 eq) was dissolved in anhydrous DCM (10 ml) and kept under an ice bath at 0°C for 30 mins. While the reaction mixture was cooling down, mCPBA (492 mg, 5.0 eq) was added in portions over 20 mins. Once the addition was complete, the reaction mixture was stirred at room temperature for 20h, then washed with saturated NaHCO3. The combined organic phase was dried over Na2SO4 and concentrated. Purification by column chromatography (20% ethyl acetate / hexane) afforded compound 9.6 (174 mg, 28% yield).

[0133] Synthesis of COMPOUND NO. 85: To a mixture of 9.6 (260 mg, 1.0 eq) and SA17 (105 mg, 2.3 eq) in ACN: THF (1: 1), CS2CO3 (2.2 eq) was added and the reaction mixture was stirred at 60°C for 16 h. After the reaction was complete, the resulting mixture was filtered and concentrated in vacuo. Purification by column chromatography (10% MeOH: DCM) afforded target COMPOUND NO. 85 (110 mg, 31% yield). 'HNMR (499 MHz, CDCI3) 85.22 - 5.15 (m, 2H), 4.62 (dtd, J= 24.8, 12.3, 2.6 Hz, 2H), 4.21 (dt, J= 12.0, 5.9 Hz, 2H), 3.89 (s, 2H), 3.64 - 3.58 (m, 4H), 3.52 - 3.45 (m, 2H), 2.62 (s, 3H), 2.43 (s, 6H), 2.20 (ddt, J= 15.7, 12.2, 3.4 Hz, 4H), 1.93 (d, J= 13.6 Hz, 8H), 1.80 (d, J= 12.2 Hz, 8H), 1.62- 1.57 (m, 8H), 1.51 (s, 7H), 1.43 - 1.32 (m, 17H), 1.31 - 1.21 (m, 27H), 1.20 - 1.13 (m, 12H), 0.88 (t, J= 7.1 Hz, 22H). MS found 1293.9 [M+H]+calcd for [ C78H136N2O12= 1293.0093],

[0134] General Procedure for Synthesis of Lipidoids (L)General Scheme L

[0135] Intermediate 9.2 (2.3g, 1.0 eq) was dissolved in anhydrous DCM (10 ml) and kept under an ice bath at 0°C for 30 mins. While the reaction mixture was cooling down, mCPBA (492 mg, 5.0 eq) was added in portions over 20 mins. Once addition was complete, the reaction mixture was stirred at room temperature for 20h under Argon, then washed with saturated NaHCO3. The combined organic phase was dried over Na2SO4 and concentrated. Purification by column chromatography (10% MeOH: DCM) afforded intermediate 10.1 (1.314g, 53%).

[0136] Synthesis of intermediate 10.3a: To intermediate 10.1 (400 mg, 1.0 eq) in anhydrous THF (10 ml), tri ethylamine (1.109g, 8.0eq) was added at 0°C. The reaction mixture was stirred at 0°C for 2 hours followed by addition of oleoyl chloride (3.275 g, 8.0 eq) as a 1:1 mixture with THF in a dropwise manner. The reaction mixture was stirred at room temperature for 16h, then diluted with DCM and extracted with water. The combined organic layers were dried over anhydrous sodium sulfate and concentrated in vacuum. Purification with 15% ethylacetate: hexane afforded compound 10.3a (391 mg, 22%).

[0137] Synthesis of COMPOUND NO. 88: To a mixture of intermediate 10.3a (390 mg, 1.0 eq) and SA17 (154 mg, 3.0 eq) in ACN: THF (1:1), CS2CO3 (281 mg, 3.0 eq) was added and the reaction mixture stirred at 60°C for 16 h. After the reaction was complete, the resulting mixture was filtered and concentrated in vacuo. Purification by column chromatography (10% MeOH: DCM) afforded COMPOUND NO. 88 (150 mg, 11% yield). 'H NMR (499 MHz,CDCl3) 8 'HNMR (499 MHz, CDCI3) 85.38 - 5.20 (m, 10H), 4.62 (ddd, J= 24.4, 12.3, 2.7 Hz,2H), 4.27 -4.18 (m, 2H), 3.85 (s, 2H), 3.71 - 3.49 (m, 5H), 2.62 - 2.21 (m, 18H), 2.00 (q, J =6.3 Hz, 16H), 1.58 (p, J= 3.6 Hz, 15H), 1.47 (s, 8H), 1.39 (t, J= 5.5 Hz, 7H), 1.35 - 1.23 (m,87H), 0.89 -0.86 (m, 12H). MS found 1630 [M+H]+- calcd for [ C102H184N2O12= 1629.3849],

[0138] General Procedure for Synthesis of Lipidoids (M)General Scheme M

[0139] Synthesis of intermediate 11.2: To compound 11.1 (5.0g, 1.0 eq) in anhydrous DMF at0°C, NaH (2.0 g, 2.2 eq) was added. The reaction mixture was stirred at 0°C for 1 hour forcomplete anion generation. To the mixture, benzyl bromide (5.6 mL, 2.5 eq) was slowly added at0°C. Once the addition was complete, the reaction was brought to room temperature and stirredfor 4 hours until the reaction was complete (TLC control). After completion of the reaction, the reaction was quenched with water and the solution was extracted with hexane. The reactionmixture was dried over anhydrous sodium sulfate and concentrated in vacuum to obtainintermediate 11.2 (9.2g). Intermediate 11.2 was taken to the next step without further purification.

[0140] Synthesis of intermediate 11.3: Intermediate 11.2 (9.2 g, 1.0 eq) was dissolved in MeOH (30.0 ml) and Amberlite IR 120H (27.0 g, 3.0 eq) was added to the reaction mixture. The resulting mixture was heated at 60°C and stirred for 16 h. Once the reaction was complete (monitored by TLC), the reaction mixture was filtered and the eluent was concentrated in vacuum. The crude mixture was loaded on a silica column and purified by 10% MeOH: DCM to get pure product 11.3 (4.7 g, 62.6%).

[0141] Synthesis of intermediate 11.4: Trans cyclohexyl C5 (3.3g, 6.0 eq) was dissolved in anhydrous DCM (10 ml). To this mixture, EDC (2.56g, 6.0 eq) and DMAP (1.34g, 4.0 eq) were added. The reaction mixture was stirred for 10 mins at room temperature. After 10 mins, intermediate 11.3 (1.0g, 1.0 eq) was added and the reaction mixture stirred for 16h at room temperature under Argon balloon. After the reaction was complete, the resulting mixture was washed with IN HC1, saturated NaHCO3, and brine. The combined organic phase was dried over Na2SO4 and concentrated. Purification by column chromatography (10% ethyl acetate / hexane) afforded compound 11.4b (1.6g, 57.5%).

[0142] Synthesis of intermediate 11.5: Intermediate 11.4b (1.6g, 1.0 eq) was dissolved in an anhydrous THF:ethanol (1:1) mixture, followed by addition of 20% Pd / C (528 mg). The resulting mixture was stirred under Argon for 48 hours. Upon completion of the reaction (as monitored by TLC), the reaction mixture was filtered through a celite bed, then concentrated in vacuum. Purification by column chromatography (0-5% A: B (A = ethylacetate, B = 1:1 (DCM: Hexane)) provided desired compound 11.5 (452 mg, 34.7%).

[0143] Synthesis of intermediate 11.6: Bromohexanoic acid (226 mg, 1.5 eq) was dissolved in anhydrous DCM (2 ml). To this mixture, EDC (232 mg, 1.5 eq) and DMAP (85 mg, 0.5 eq) were added. The reaction mixture was stirred for 10 mins at room temperature. After 10 mins, intermediate 11.5 (700 mg, 1.0 eq) was added and the reaction mixture stirred for 16h at room temperature under an Argon balloon. After the reaction was complete, the resulting mixture was washed with IN HC1, saturated NaHCO3, and brine. The combined organic phase was dried over Na2SO4 and concentrated. Purification by column chromatography (10% ethyl acetate / hexane) afforded compound 11.6bc (195 mg, 23.3%) and 11.7 (195 mg, 20%).

[0144] Synthesis of Compound NO. 90: To a mixture of 11.6bc (184 mg, 1.0 eq) and SAI 7 (33.4 mg, 1.1 eq) in ACN: THF (1:1), CS2CO3 (61 mg, 1.1 eq) along with a catalytic amount of KI was added and the reaction mixture was stirred at 50°C for 16 h. After the reaction was complete, the resulting mixture was filtered and concentrated in vacuo. Purification by column chromatography (10% MeOH: DCM) afforded target COMPOUND NO. 90 (78 mg, 40.2% yield). 'H NMR (499 MHz, CDCl3) 55.27 (ddd, J = 8.4, 5.8, 2.7 Hz, 1H), 5.17 (dd, J= 8.1, 1.5 Hz, 1H), 4.82 (dt, J = 10.0, 3.0 Hz, 1H), 4.43 - 4.27 (m, 3H), 4.11 (dd, J= 12.5, 5.9 Hz, 1H), 3.80 (d, J= 10.0 Hz, 1H), 2.40 - 2.14 (m, 7H), 2.01 - 1.89 (m, 9H), 1.80 (d, J= 14.0 Hz, 8H), 1.68 - 1.54 (m, 11H), 1.48 - 1.11 (m, 56H), 0.88 (t, J= 7.0 Hz, 20H). MS found 1138.8 [M+H] calcd for [ C69H119NO11 =1137.8783],

[0145] LNP COMPONENTS

[0146] The present disclosure provides lipid nanoparticles (LNPs) comprising one or more compounds of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII) and / or Formula (IX). In addition to the one or more compounds of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII) and / or Formula (IX), the LNPs of the present disclosure can comprise one or more additional LNP components, as described below.

[0147] In some aspects, a lipid nanoparticle comprises at least one compound of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII) and / or Formula (IX), at least one phospholipid, at least one structural lipid, and at least one PEGylated lipid.

[0148] In some aspects, the LNP may further comprise at least one nucleic acid molecule.

[0149] In some aspects, the at least one nucleic acid molecule is an RNA molecule.

[0150] In some aspects, the at least one nucleic acid molecule is a DNA molecule.

[0151] In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle is from about 40:1 to about 100: 1 (w / w).

[0152] In some aspects, the lipid nanoparticle retains at least 80% activity after storage at 4°C for six months.

[0153] In some aspects, an LNP of the present disclosure can 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 leastabout 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 present disclosure by moles. In some aspects, the at least one compound is at least one compound of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII) and / or Formula (IX), as described herein. In some aspects, the at least one compound of the present disclosure is a mixture of two or more compounds of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII) and / or Formula (IX).

[0154] Structural Lipid

[0155] In some aspects, an LNP 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 structural lipid by moles.

[0156] In some aspects, a structural lipid can be a steroid. In some aspects, a structural lipid can be a sterol. In some aspects, a structural lipid can comprise cholesterol. In some aspects, a structural lipid can comprise ergosterol. In some aspects, a structural lipid can be a phytosterol.

[0157] In some aspects, the at least one structural lipid is a mixture of two structural lipids.

[0158] Phospholipid

[0159] In some aspects, an LNP 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 atleast 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 by moles.

[0160] As used herein, the term “phospholipid” is used in its broadest sent to refer to any amphiphilic molecule that comprises a polar (hydrophilic) headgroup comprising phosphate and two hydrophobic fatty acid chains. In some aspects, a phospholipid can comprisedi oleoylphosphatidylethanolamine (DOPE). In some aspects, a phospholipid can comprise 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC). In some aspects, a phospholipid can comprise l,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some aspects, a phospholipid can comprise DPPC (l,2-Dipalmitoyl-sn-glycero-3-phosphocholine). In some aspects, a phospholipid can comprise DDPC (l,2-Didecanoyl-sn-glycero-3 -phosphocholine), DEPA-NA (l,2-Dierucoyl-sn-glycero-3-phosphate (Sodium Salt)), DEPC (l,2-Dierucoyl-sn-glycero-3-phosphocholine), DEPE (l,2-Dierucoyl-sn-glycero-3-phosphoethanolamine), DEPG-NA (1,2-Dierucoyl-sn-glycero-3[Phospho-rac-(l -glycerol) (Sodium Salt)), DLOPC (1,2-Dilinoleoyl-sn-glycero-3-phosphocholine), DLPA-NA (l,2-Dilauroyl-sn-glycero-3-phosphate (Sodium Salt)), DLPC (l,2-Dilauroyl-sn-glycero-3 -phosphocholine), DLPE (l,2-Dilauroyl-sn-glycero-3-phosphoethanolamine), DLPG-NA (l,2-Dilauroyl-sn-glycero-3[Phospho-rac-(l -glycerol) (Sodium Salt)), DLPG-NH4 (l,2-Dilauroyl-sn-glycero-3[Phospho-rac-(l-glycerol) (Ammonium Salt)), DLPS-NA (l,2-Dilauroyl-sn-glycero-3 -phosphoserine (Sodium Salt)), DMPA-NA (1,2-Dimyristoyl-sn-glycero-3 -phosphate (Sodium Salt)), DMPC (l,2-Dimyristoyl-sn-glycero-3-phosphocholine), DMPE (l,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine), DMPG-NA (l,2-Dimyristoyl-sn-glycero-3[Phospho-rac-(l -glycerol) (Sodium Salt)), DMPG-NH4 (1,2-Dimyristoyl-sn-glycero-3[Phospho-rac-(l -glycerol) (Ammonium Salt)), DMPG-NH4 / NA (1,2-Dimyristoyl-sn-glycero-3[Phospho-rac-(l -glycerol) (Sodium / Ammonium Salt)), DMPS-NA (l,2-Dimyristoyl-sn-glycero-3-phosphoserine (Sodium Salt)), DOPA-NA (1,2-Dioleoyl-sn-glycero-3-phosphate (Sodium Salt)), DOPC (l,2-Dioleoyl-sn-glycero-3-phosphocholine), DOPE ( 1,2-Dioleoyl-sn-glycero-3 -phosphoethanolamine), DOPG-NA ( 1,2-Dioleoyl-sn-glycero-3 [Phospho-rac-(l -glycerol) (Sodium Salt)), DOPS-NA (l,2-Dioleoyl-sn-glycero-3-phosphoserine (Sodium Salt)), DPPA-NA (l,2-Dipalmitoyl-sn-glycero-3 -phosphate (Sodium Salt)), DPPE (l,2-Dipalmitoyl-sn-glycero-3 -phosphoethanolamine), DPPG-NA (1,2-Dipalmitoyl-sn-glycero-3[Phospho-rac-(l -glycerol) (Sodium Salt)), DPPG-NH4 (1,2-Dipalmitoyl-sn-glycero-3[Phospho-rac-(l -glycerol) (Ammonium Salt)), DPPS-NA (1,2-Dipalmitoyl-sn-glycero-3-phosphoserine (Sodium Salt)), DSPA-NA (1,2-Distearoyl-sn-glycero-3-phosphate (Sodium Salt)), DSPC (l,2-Distearoyl-sn-glycero-3-phosphocholine), DSPE (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine), DSPG-NA (1,2-Distearoyl-sn-glycero-3 [Phospho-rac-(l -glycerol) (Sodium Salt)), DSPG-NH4 (l,2-Distearoyl-sn-glycero-3[Phospho-rac-(l -glycerol) (Ammonium Salt)), DSPS-NA (l,2-Distearoyl-sn-glycero-3-phosphoserine (Sodium Salt)), EPC (Egg-PC), HEPC (Hydrogenated Egg PC), HSPC (Hydrogenated Soy PC), LYSOPC MYRISTIC (l-Myristoyl-sn-glycero-3-phosphocholine), LYSOPC PALMITIC (l-Palmitoyl-sn-glycero-3-phosphocholine), LYSOPC STEARIC (l-Stearoyl-sn-glycero-3-phosphocholine), Milk Sphingomyelin (MPPC; l-Myristoyl-2-palmitoyl-sn-glycero 3-phosphocholine), MSPC (l-Myristoyl-2-stearoyl-sn-glycero-3-phosphocholine), PMPC (1-Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine), POPC (l-Palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine), POPE (l-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), POPG-NA (l-Palmitoyl-2-oleoyl-sn-glycero-3[Phospho-rac-(l -glycerol)] (Sodium Salt)), PSPC (1-Palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine), SMPC (l-Stearoyl-2-myristoyl-sn-glycero-3-phosphocholine), SOPC (l-Stearoyl-2-oleoyl-sn-glycero-3 -phosphocholine), SPPC (1-Stearoyl-2-palmitoyl-sn-glycero-3 -phosphocholine), or any combination thereof.

[0161] PEGylated Lipid

[0162] In some aspects, an LNP can further comprise at least about 0.25%, or at least about 0.5%, or at least about 0.75%, or at least about 1.0%, or at least about 2.5%, or at least about 5%, or at least about 7.5%, or at least about 10% PEGylated lipid by moles.

[0163] As used herein, the term “PEGylated lipid” is used to refer to any lipid that is modified (e.g. covalently linked to) at least one polyethylene glycol molecule. In some aspects, a PEGylated lipid can comprise l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, hereafter referred to as DMG-PEG2000 or PEG-DMG.

[0164] In some aspects, the at least one PEGylated lipid is a mixture of two PEGylated lipids.

[0165] Nucleic Acid Molecules

[0166] In some aspects, a lipid nanoparticle of the present disclosure can further comprise at least one nucleic acid. In some aspects, a lipid nanoparticle can comprise a plurality of nucleic acid molecules. In some aspects, the at least one nucleic acid or the plurality of nucleic acid molecules can be formulated in a lipid nanoparticle.

[0167] Accordingly, a lipid nanoparticle 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.

[0168] In some aspects, the at least one nucleic acid is a DNA molecule. In one aspect, the at least one DNA molecule is a DoggyBone® DNA molecule. In some aspects, the at least one DNA molecule is a DNA nanoplasmid.

[0169] In some aspects, the at least one nucleic acid is an RNA molecule. In some aspects, the RNA molecule is an mRNA molecule. In some aspects, the mRNA molecule further comprises a 5 ’-CAP. In some aspects, all of the cytidine residues in an mRNA molecule can be 5-methylcytidine.

[0170] In some aspects, the at least one RNA molecule is a guide RNA (gRNA) molecule.

[0171] In some aspects, an at least one nucleic acid can comprise both mRNA molecules and guide RNA (gRNA) molecules. That is, the LNPs of the present disclosure can comprise both mRNA molecules and gRNA molecules. In some aspects wherein the LNPs comprise both mRNA molecules and gRNA molecules, the mRNA molecules comprise at least one nucleic acid sequence that encodes a fusion protein, wherein the fusion protein comprises: (i) an inactivated Cas9 (dCas9) protein or an inactivated nuclease domain thereof; and (ii) a Clo051 protein or a nuclease domain thereof, and wherein the gRNA molecules encode guide RNA sequence targeting one or more specific genomic loci. In some aspects, the fusion protein can be a Cas-CLOVER protein. In some aspects, the gRNA molecules can target the psk9 gene. In some aspects, the gRNA molecules can target the KLKB1 gene.

[0172] In some aspects wherein the LNPs comprise both mRNA molecules and gRNA molecules, the ratio of mRNA:gRNA can be about 1:2, or about 1:3, or aboutl: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:l, or about 4:1, or about 5:l, or about6:l, or about 7:1, orabout 8:l, or about 9:1 or about 10:1.

[0173] In some aspects, an at least one nucleic acid can comprise at least one RNA molecule and at least one DNA molecule. That is, the LNPs of the present disclosure can comprise both RNA molecules and DNA molecules.

[0174] In some aspects, the LNPs of the present disclosure can comprise both RNA molecules and DNA molecules, wherein the RNA molecules comprise at least one nucleic acid sequencethat encodes a transposase and wherein the DNA molecules comprise at least one nucleic acid sequence that comprises a transposon. In some aspects, the transposase can be any of the transposases described herein. In some aspects, the transposon can be a transposon comprising at least one nucleic acid sequence encoding a FVIII polypeptide. In some aspects, the transposon can be a transposon comprising at least one nucleic acid sequence encoding a human propionyl-CoA carboxylase subunit alpha (PCCA) polypeptide.

[0175] In some aspects wherein 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 1:2, or about 1:3, oraboutl:4, or about 1:1, orabout 2:l, or about3:l, or about 4:1, orabout 5:l, or about 6:1, or about 7:1, or about 8:1, or about 9:1 or about 10:1.

[0176] In some aspects, a lipid nanoparticle can comprise lipid and nucleic acid at a specified ratio (weight / weight).

[0177] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise lipid and nucleic acid at a ratio of about 5: 1 to about 15: 1, or about 10: 1 to about 20: 1, or about 15:1 to about 25:1, or about 20:1 to about 30:1, or about 25:1 to about 35:1 or about 30:1 to about 40: 1, or about 35:1 to about 45:1, or about 40:1 to about 50: 1, or about 45:1 to about 55:1, or about 50: 1 to about 60: 1, or about 55: 1 to about 65:1, or about 60: 1 to about 70: 1, or about 65:1 to about 75:1, or about 70:1 to about 80:1, or about 75:1 to about 85:1, or 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, lipid:nucleic acid, weight / weight.

[0178] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise lipid and nucleic acid at a 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.

[0179] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise lipid and nucleic acid at a ratio of about 10:1, or about 25: 1, or about 40: 1, lipidmucleic acid, weight / weight.

[0180] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise lipid and nucleic acid at a ratio of about 20: 1, or about 40: 1, or about 60: 1, or about 80: 1, or about 120:1 lipidmucleic acid, weight / weight.

[0181] In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 30: 1 to about 50:1 (w / w), or about 35:1 to about 45:1 (w / w). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 40: 1 (w / w).

[0182] In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 40: 1 to about 60:1 (w / w), or about 45:1 to about 55:1 (w / w). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 50: 1 (w / w).

[0183] In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 50: 1 to about 70:1 (w / w), or about 55:1 to about 65:1 (w / w). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 60:1 (w / w).

[0184] In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 70: 1 to about 90:1 (w / w), or about 75:1 to about 85:1 (w / w). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 80:1 (w / w).

[0185] In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 90: 1 to about 110:1 (w / w), or about 95:1 to about 105:1 (w / w). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 100:1 (w / w).

[0186] In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 110: 1 to about 130: 1 (w / w), or about 115:1 to about 125: 1 (w / w). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 120:1 (w / w).

[0187] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise a compound of the present disclosure and nucleic acid at a specified ratio (weight / weight).

[0188] In some aspects, a lipid nanoparticle comprising a compound of the present disclosure and at least one nucleic acid can comprise a compound and nucleic acid at a ratio of about 0.1:1, or about 0.15:1, or about 0.2:1, or about 0.25:1, or about 0.3:1, or about 0.35:1, or about 0.4:1, or about 0.45:1, or about 0.5:1, or about 1:1, or about 1.5:1, or about 2:1, or about 2.5:1, or about 3:1, or about 3.5:1, or about 4:1, or about 4.5:1, or about 5:1, or about 5.5:1, or about 6:1, orabout 6.5:1, or about 7:1, or about 7.5:1, or about 8:1, or about 8.5:1, or about 9:1, or about 9.5:1, or about 10:1, or about 10.5:1, or about 11:1, or about 11.5:1, or about 12:1, or about 12.5:1, or about 13:1, or about 13.5:1, or about 14:1, or about 14.5:1, or about 15:1, or about 15.5:1, or about 16:1, or about 16.5:1, or about 17:1, or about 17.5:1, or about 18:1, or about 18.5:1, or about 19:1, or about 19.5: 1, or about 20: 1 compound: nucleic acid. In some aspects, the at least one nucleic acid can comprise DNA.

[0189] In some aspects, a lipid nanoparticle can comprise a compound of the present disclosure and lipid at a specified ratio (weight / weight).

[0190] In some aspects, a lipid nanoparticle can comprise a compound of the present disclosure and lipid at a ratio of about 0.08:1, 0.1:1, or about 0.17:1, or about 0.2:1 or about 0.25:1, or about 0.3:1 compound:lipid, weight / weight.

[0191] Further characteristics of the nucleic acid molecules of the present disclosure are provided herein.

[0192] Polyphenol Additives

[0193] In some aspects, a lipid nanoparticle of the present disclosure comprises at least one polyphenol (also referred to herein as a “polyphenol additive”).

[0194] Accordingly, a lipid nanoparticle can comprise at least one nucleic acid, at least one compound of the present disclosure, at least one structural lipid, at least one phospholipid, at least one PEGylated lipid, and at least one polyphenol. In some aspects, the at least one nucleic acid comprises at least one DNA molecule.

[0195] As used herein, the term “polyphenol” is used to refer to any compound that has at least two phenol subunits, wherein a phenol is an aromatic ring, as defined herein, that has at least one hydroxyl substituent. Polyphenols include compounds that have at least two phenol subunits, for example flavonoids, catechins, anthocyanins, stilbenes and ellagic acid. Polyphenols also include compounds that have at least three phenol subunits, for example proanthocyanins, tannins and punicalagin.

[0196] In some aspects, the at least one polyphenol is selected from: malvidin 3-o-(6"-p-coumaroyl- glucoside), delphinidin 3-o-(6"-acetyl-galactoside), cyanidin 3-o-(6"-acetyl-galactoside), cyanidin 3-o-galactoside, cyanidin 3-o-glucoside, cyanidin 3-o-rutinoside, cyanidin 3-o- sophoroside, pelargonidin 3-o-glucoside, cyanidin 3-o-(6"-malonyl-glucoside), peonidin, peonidin 3-o-glucoside, peonidin 3-o-rutinoside, pelargonidin 3-o-rutinoside, pelargonidin,cyanidin, malvidin 3,5-o-diglucoside, cyanidin 3-o-glucosyl-rutinoside, pelargonidin 3-o-sophoroside, pelargonidin 3-o-glucosyl-rutinoside, cyanidin 3-o-(6"-succinyl-glucoside), pelargonidin 3-o-(6"-succinyl-glucoside), delphinidin 3-o-galactoside, delphinidin 3-o-glucoside, delphinidin 3-o-arabinoside, petunidin 3-o-galactoside, cyanidin 3-o-arabinoside, petunidin 3-o-glucoside, peonidin 3-o-galactoside, petuni din 3-o-arabinoside, malvidin 3-o-glucoside, malvidin 3-o-arabinoside, delphinidin 3-o-(6"-acetyl-glucoside), petunidin 3-o-(6"-acetyl-galactoside), peonidin 3-o-(6"-acetyl-galactoside), cyanidin 3-o-(6"-acetyl-ghicoside), malvidin 3-o-(6"-acetyl- galactoside), petunidin 3-o-(6"-acetyl-glucoside), malvidin 3-o-(6"-acetyl-glucoside), peonidin 3- o-(6"-acetyl-glucoside), pelargonidin 3-o-arabinoside, delphinidin 3-o-rutinoside, cyanidin 3-o- sambubioside, pelargonidin 3-o-(6"-malonyl-glucoside), peonidin 3-o-(6"-p-coumaroyl- glucoside), cyanidin 3-o-xyloside, malvidin 3-o-galactoside, peonidin 3-o-arabinoside, petunidin 3-o-rutinoside, delphinidin 3-o-xyloside, petunidin 3-o-(6"-p-coumaroyl-glucoside), pelargonidin 3-o-galactoside, pelargonidin 3-o-sambubioside, delphinidin 3-o-sambubioside, cyanidin 3-o- xylosyl-rutinoside, vitisin a, delphinidin 3-o-(6"-p-coumaroyl-glucoside), pigment a, cyanidin 3- o-(6"-p-coumaroyl-glucoside), cyanidin 3-o-sambubioside 5-o-glucoside, cyanidin 3-o-(6"- caffeoyl-glucoside), cyanidin 3,5-o-diglucoside, pinotin a, delphinidin 3,5-o-diglucoside, pelargonidin 3,5-o-diglucoside, malvidin 3-o-(6"-caffeoyl-glucoside), cyanidin 3-o-(6"-dioxalyl- glucoside), delphinidin 3-o-glucosyl-glucoside, cyanidin 3-o-(6"-malonyl-3 "-glucosyl- glucoside), delphinidin 3-o-feruloyl-glucoside, petunidin 3,5-o-diglucoside, petunidin 3-o- rhamnoside, cyanidin 3-o-diglucoside-5-o-glucoside, peonidin 3-o-diglucoside-5-o-glucoside, peonidin 3-o-(2-o-(6-o-(e)-caffeoyl-d-glucosyl)-d-glucoside)-5-o-d-glucoside, peonidin 3-o- sophoroside, peonidin 3-o-sambubioside, peonidin 3-o-sambubioside-5-o-glucoside, peonidin 3- o-xyloside, 4'-o-methylcyanidin 3-o-d-glucoside, cyanidin 3-o-(3",6"-o-dimalonyl-glucoside), 4- o-methyldelphinidin 3-o-d-glucoside, isopeonidin 3-o-arabinoside, isopeonidin 3-o-galactoside, isopeonidin 3-o-glucoside, isopeonidin 3-o-rutinoside, isopeonidin 3-o-sambubioside, isopeonidin 3-o-xyloside, cyanidin 3-o-(2-o-(6-o-(e)-caffeoyl-d glucoside)-d-glucoside)-5-o-d- glucoside, 4'-o-methyldelphinidin 3-o-rutinoside, butein, xanthohumol, phloretin, phloridzin, phloretin 2'-o-xylosyl-glucoside, 3-hydroxyphloretin 2'-o-xylosyl-glucoside, 3-hydroxyphloretin 2'-o-glucoside, phloretin 2'-o-glucuronide, dihydroquercetin 3-o-rhamnoside, dihydroquercetin, dihydromyricetin 3-o-rhamnoside, (+)-catechin, (-)-epi catechin, (+)-gallocatechin, (-)- epigallocatechin, (-)-epicatechin 3-o-gallate, (-)-epigallocatechin 3-o-gallate, theaflavin, theaflavin 3-o-gallate, theaflavin 3'-o-gallate, theaflavin 3,3'-o-digallate, (+)-gallocatechin 3-o- gallate, (+)-catechin 3-o-gallate, procyanidin dimer bl, procyanidin dimer b2, procyanidin dimer b3, procyanidin dimer b4, procyanidin dimer b5, procyanidin dimer b7, prodelphinidin dimer b3, procyanidin trimer cl, procyanidin trimer eec, procyanidin trimer t2, procyanidin trimer c2, prodelphinidin trimer gc-gc-c, prodelphinidin trimer gc-c-c, prodelphinidin trimer c-gc-c, (-)- epicatechin-(2a-7)(4a-8)-epicatechin 3-o-galactoside, cinnamtannin a2, (+)-catechin 3-o-glucose, 3'-o-methylepicatechin, 4'-o-methyl-(-)-epicatechin 3'-o-glucuronide, epicatechin 3'-o- glucuronide, 3'-o-methylcatechin, 4',4"-o-dimethylepigallocatechin 3-o-gallate, 4'-o- methylepigallocatechin, 4"-o-methylepigallocatechin 3-o-gallate, 4'-o-methylepicatechin, epigallocatechin 3-o-gallate-7-o-glucoside-4"-o-glucuronide, (-)-epigallocatechin 3-o- glucuronide, 3'-o-methyl-(-)-epicatechin 7-o-glucuronide, epicatechin 7-o-glucuronide, (-)- epigallocatechin 3'-o-glucuronide, (-)-epigallocatechin 7-o-glucuronide, 4'-o-methyl-(-)- epigallocatechin 3'-o-glucuronide, 4'-o-methyl-(-)-epigallocatechin 7-o-glucuronide, naringenin, eriodictyol, hesperetin, eriocitrin, hesperidin, naringin, narirutin, neoeriocitrin, neohesperidin, poncirin, didymin, narirutin 4'-o-glucoside, naringin 4'-o-glucoside, naringin 6'-malonate, isosakuranetin, naringenin 7-o-glucoside, pinocembrin, 8-prenylnaringenin, 6-prenylnaringenin, 6-geranylnaringenin, isoxanthohumol, eriodictyol 7-o-glucoside, sakuranetin, hesperetin 3'-o- glucuronide, hesperetin 7-o-glucuronide, hesperetin 3'-sulfate, homoeriodictyol, naringenin 4'-o- glucuronide, naringenin 5-o-glucuronide, naringenin 7-o-glucuronide, hesperetin 3', 7-o- diglucuronide, hesperetin 5,7-o-diglucuronide, apigenin, luteolin, diosmin, isorhoifolin, neodiosmin, rhoifolin, sinensetin, nobiletin, tangeretin, luteolin 7-o-diglucuronide, chrysin, luteolin 7-o-rutinoside, tetramethylscutellarein, luteolin 7-o-glucoside, apigenin 7-o-glucoside, apigenin 6,8-di-c-glucoside, apigenin 6,8-c-arabinoside-c-glucoside, apigenin 6,8-c-galactoside-c-arabinoside, luteolin 7-o-glucuronide, apigenin 7-o-glucuronide, luteolin 7-o-malonyl-glucoside, luteolin 6-c-glucoside, luteolin 7-o-(2-apiosyl-glucoside), luteolin 7-o-(2-apiosyl-6-malonyl)-glucoside, apigenin 7-o-apiosyl-glucoside, 7,3',4'-trihydroxyflavone, 7,4'-dihydroxyflavone, geraldone, baicalein, apigenin 6-c-glucoside, hispidulin, cirsimaritin, 5,6-dihydroxy-7,8,3',4'-tetramethoxyflavone, pebrellin, gardenin b, nepetin, jaceosidin, cirsilineol, eupatorin, 6-hydroxyluteolin, 6-hydroxyluteolin 7-o-rhamnoside, scutellarein, apigenin 7-o-(6"-malonyl-apiosyl-glucoside), chrysoeriol 7-o-apiosyl-glucoside, chrysoeriol 7-o-(6"-malonyl-apiosyl-glucoside), chrysoeriol 7-o-glucoside, chrysoeriol 7-o-(6"-malonyl-glucoside), apigenin7-o-diglucuronide, rhoifolin 4'-o-glucoside, kaempferol, quercetin, quercetin 3-o-galactoside, quercetin 3-o-glucoside, quercetin 3-o-xyloside, quercetin 3-o-rhamnoside, quercetin 3-o-rutinoside, quercetin 3-o-sophoroside, quercetin 3-o-arabinoside, quercetin 3-o-xylosyl-glucuronide, isorhamnetin 3-o-glucoside 7-o-rhamnoside, isorhamnetin 3-o-rutinoside, kaempferol 3-o-glucuronide, isorhamnetin 7-o-rhamnoside, quercetin 3, 4'-o-di lucoside, myricetin 3-o-rutinoside, myricetin, morin, kaempferide, myricetin 3-o-galactoside, myricetin 3-o-glucoside, quercetin 3-o-glucosyl-xyloside, quercetin 3-o-acetyl-rhamnoside, kaempferol 3-o-galactoside, galangin, isorhamnetin, kaempferol 3-o-glucoside, kaempferol 3-o-rutinoside, kaempferol 3-o-glucosyl-rhamnosyl-galactoside, kaempferol 3-o-glucosyl-rhamnosyl-glucoside, quercetin 3-o-glucosyl-rhamnosyl-galactoside, quercetin 3-o-glucosyl-rhamnosyl-glucoside, rhamnetin, isorhamnetin 3-o-glucoside, myricetin 3-o-rhamnoside, quercetin 3-o-rhamnosyl-galactoside, quercetin 3-o-glucuronide, isorhamnetin 3-o-glucuronide, myricetin 3-o-arabinoside, quercetin 7,4'-o-diglucoside, quercetin 4'-o-glucoside, isorhamnetin 4'-o-glucoside, 3,7-dimethylquercetin, kaempferol 3-o-sophoroside, kaempferol 3,7-o-diglucoside, kaempferol 3-o-sophoroside 7-o-glucoside, quercetin 3-o-(6"-malonyl-glucoside), kaempferol 3-o-(6"-malonyl-glucoside), kaempferol 3-o-rhamnoside, quercetin 3-o-(6"-malonyl-glucoside) 7-o-glucoside, patuletin 3-o-glucosyl-(l->6)-[apiosyl(l->2)]-glucoside, spinacetin 3-o-glucosyl-(l->6)- [apiosyl(l->2)]-glucoside, patuletin 3-o-(2"-feruloylglucosyl)(l->6)-[apiosyl(l->2)]-glucoside, spinacetin 3-o-(2"-p-coumaroylglucosyl)(l->6)-[apiosyl(l->2)]-glucoside, spinacetin 3-o-(2"-feruloylglucosyl)( 1 ->6)-[apiosyl( 1 ->2)]-glucoside, spinacetin 3 -o-glucosyl-( 1 ->6)-glucoside, jaceidin 4’-o-glucuronide, 5,3',4'-trihydroxy-3-methoxy-6:7-methylenedioxyflavone 4'-o-glucuronide, 5,4'-dihydroxy-3,3'-dimethoxy-6:7-methylenedioxyflavone 4'-o-glucuronide, kaempferol 3-o-xylosyl-glucoside, kaempferol 3-o-acetyl-glucoside, quercetin 3-o-xylosyl-rutinoside, kaempferol 3-o-xylosyl-rutinoside, kaempferol 7-o-glucoside, kaempferol 3-o-galactoside 7-o-rhamnoside, kaempferol 3-o-(6"-acetyl-galactoside) 7-o-rhamnoside, quercetin 3-o-galactoside 7-o-rhamnoside, quercetin 3-o-(6"-acetyl-galactoside) 7-o-rhamnoside, kaempferol 3-o-(2"-rhamnosyl-galactoside) 7-o-rhamnoside, kaempferol 3-o-(2"-rhamnosyl-6"-acetyl-galactoside) 7-o-rhamnoside, 6,8-dihydroxykaempferol, isorhamnetin 3-o-galactoside, quercetin 3 -o-rhamnosyl-rhamnosyl -glucoside, kaempferol 3-o-rhamnosyl-rhamnosyl-glucoside, methylgalangin, kaempferol 3,7,4'-o-triglucoside, 3-methoxynobiletin, 3-methoxysinensetin, quercetin 3'-o-glucuronide, quercetin 3'-sulfate, quercetin 4'-o-glucuronide,isorhamnetin 4'-o- glucuronide, daidzein, formononetin, genistein, biochanin a, glycitein, glycitin, 6"-o- acetyldaidzin, 6"-o-malonylgenistin, daidzin, genistin, 6"-o-acetylgenistin, 6"-o-acetylglycitin, 6"-o-malonyldaidzin, 6"-o-malonylglycitin, 2',7-dihydroxy-4',5'-di methoxy isoflavone, 2- dehydro-o-desmethylangolensin, 2'-hydroxyformononetin, 3',4',7-trihydroxyisoflavan, 3', 4', 7- trihydroxyisoflavanone, 3'-hydroxydaidzein, 3'-hydroxy-o-desmethylangolensin, 4', 6, 7- trihydroxyisoflavanone, 4',7-dihydroxy-3'-methoxyisoflavan, 4', 7-dihydroxy-6- m ethoxy isoflavan, 4'-o-methylequol, 5,6,7,3',4'-pentahydroxyisoflavone, 5, 6,7,4'-tetrahydroxyisoflavone, 5,7,8,3',4'-pentahydroxyisoflavone, 5,7,8,4'-tetrahydroxyisoflavone, 5'-hydroxy-o-desmethylangolensin, 5'-methoxy-o-desmethylangolensin, 6, 7, 3', 4'-tetrahydroxyisoflavone, 6,7,4'-trihydroxyisoflavone, 6'-hydroxyangolensin, 6'-hydroxy-o-desmethylangolensin, 7,8,3',4'-tetrahydroxyisoflavone, 7,8,4'-trihydroxyisoflavone, angolensin, calycosin, daidzein 4'-o-glucuronide, daidzein 7-o-glucuronide, dihydrobiochanin a, dihydrodaidzein, dihydrodaidzein 7-o-glucuronide, dihydroformononetin, dihydrogeni stein, dihydroglycitein, equol, formononetin 7-o-glucuronide, genistein 4',7-o-diglucuronide, genistein 4'-o-glucuronide, genistein 5-o-glucuronide, genistein 7-o-glucuronide, glycitein 4'-o-glucuronide, glycitein 7-o-glucuronide, koparin, o-desmethylangolensin, orobol, prunetin, pseudobaptigenin, puerarin, daidzin 4'-o-glucuronide, irisolidone 7-o-glucuronide, tectorigenin 7-sulfate, tectorigenin 4'-sulfate, irisolidone, tectorigenin, tectoridin, 5,7-dihydroxy-8,4'-dimethoxyisoflavone, isotectorigenin, equol 7-o-glucuronide, equol 4'-o-glucuronide, 3', 4', 5,7-tetrahydroxyisoflavanone, 3'-o-methylequol, 6-o-methylequol, 3 '-hydroxy genistein, 6-hydroxydihydrodaidzein, 3 '-hydroxy equol, cis-4-hydroxyequol, 4'-methoxy-2',3,7-trihydroxyisoflavanone, irilone, vestitone, sativanone, butin, 3'-hydroxymelanettin, melanettin, stevenin, violanone, isoliquiritigenin, dalbergin, 3'-o-methylviolanone, 8-hydroxydihydrodaidzein, secoisolariciresinol, matairesinol, lariciresinol, pinoresinol, syringaresinol, isolariciresinol, arctigenin, trachelogenin, medioresinol, 1-acetoxypinoresinol, sesamin, sesamolin, sesamolinol, sesaminol, sesamol, 7-hydroxymatairesinol, isohydroxymatairesinol, secoisolariciresinol-sesquilignan, cyclolariciresinol, 7-oxomatairesinol, todolactol a, conidendrin, 7-hydroxysecoisolariciresinol, nortrachelogenin, lariciresinol-sesquilignan, anhydro-secoisolariciresinol, dimethylmatairesinol, episesamin, episesaminol, enterodiol, enterolactone, sesaminol 2-o-triglucoside, schisandrin, gomisin d, schisandrol b, tigloylgomicin h, schisanhenol, schisantherin a, gomisin m2, deoxyschisandrin, schisandrin b,schi sandrin c, 2-hydroxyenterodiol, 4-hydroxyenterodiol, 6-hydroxyenterodiol, 2-hydroxyenterolactone, 4-hydroxy enterolactone, 6-hydroxyenterolactone, 2'-hydroxyenterolactone, 4'-hydroxyenterolactone, 6'-hydroxyenterolactone, 5-hydroxy enterolactone, 7-hydroxyenterolactone, 4-ethylbenzoic acid, glycine, 1,3,5-trimethoxybenzene, vanilloylglycine, 4-vinylguaiacol, 4-ethylguaiacol, 4-vinylsyringol, 5-heneicosenylresorcinol, 5 -heneicosylresorcinol, 5-heptadecylresorcinol, 5-nonadecenylresorcinol, 5-nonadecylresorcinol, 5-pentacosenylresorcinol, 5-pentacosylresorcinol, 5-pentadecylresorcinol, 5-tricosenylresorcinol, 5-tricosylresorcinol, 3-methylcatechol, 4-methylcatechol, 4-ethylcatechol, 4-vinylphenol, 4-ethylphenol, curcumin, demethoxycurcumin, bisdem ethoxy curcumin, bergapten, psoralen, xanthotoxin, isopimpinellin, syringaldehyde, protocatechuic aldehyde, vanillin, 4-hydroxybenzaldehyde, gallic aldehyde, p-anisaldehyde, vanillin 4-sulfate, 3 -meth oxy acetophenone, 2,3 -dihydroxy- 1-guaiacylpropanone, paeonol, 2.4- dihydroxyacetophenone 5-sulfate, 2-hydroxy-4-methoxyacetophenone 5-sulfate, resacetophenone, norathyriol, ferul aldehyde, sinapaldehyde, coumarin, mellein, scopoletin, esculetin, esculin, umbelliferone, 4-hydroxycoumarin, urolithin a 3,8-o-diglucuronide, urolithin a, urolithin b, urolithin b 3-o-glucuronide, urolithin c, 2-methoxy-5-prop-l-enylphenol, anethole, eugenol, acetyl eugenol, [6]-gingerol, estragole, guaiacol, jugl one, 1,4-naphtoquinone, camosic acid, rosmanol, carnosol, epirosmanol, rosmadial, thymol, carvacrol, tyrosol, hydroxytyrosol, 3.4-dhpea-ac, p-hpea-ac, oleuropein, demethyloleuropein, 3,4-dhpea-ea, ligstroside, 3,4-dhpea-eda, hydroxytyrosol 4-o-glucoside, oleoside dimethylester, oleoside 11 -methylester, p-hpea-eda, p-hpea-ea, ol europein-aglycone, ligstroside-aglycone, tyrosol 4-sulfate, coumestrol, catechol, pyrogallol, phlorin, phenol, arbutin, 3,4-dihydroxyphenylglycol, lithospermic acid, salvianolic acid b, salvianolic acid c, salvianolic acid d, salvianolic acid g, isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate, ellagic acid glucoside, protocatechuic acid, gallic acid, vanillic acid, gentisic acid, ellagic acid, 4-hydroxybenzoic acid, syringic acid, 5-o-galloylquinic acid, ellagic acid arabinoside, ellagic acid acetyl-xyloside, ellagic acid acetyl-arabinoside, benzoic acid, 2-hydroxy benzoic acid, 3 -hydroxybenzoic acid, 2,3 -dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid 4-o-glucoside, protocatechuic acid 4-o-glucoside, gallic acid 4-o-glucoside, 3,5-dihydroxybenzoic acid, 2,6-dihydroxybenzoic acid, gallic acid 3-o-gallate, gallic acid ethyl ester, valoneic acid dilactone, galloyl glucose, lambertianin c, sanguiin h-6, punicalagin, gallagic acid, 3 -o-m ethylgallic acid, 4-o-m ethylgallic acid, 3,4-o-dimethylgallic acid, punicalin, 4-hydroxyhippuric acid, 3 -hydroxy hippuric acid, 2-hydroxyhippuric acid, hippuric acid, paeoniflorin, vanillic acid 4-sulfate, p-coumaric acid, 5-p-coumaroylquinic acid, 4- p-coumaroylquinic acid, caffeic acid, feruloyl glucose, ferulic acid, caffeoyl tartaric acid, rosmarinic acid, o-coumaric acid, m-coumaric acid, sinapic acid, p-coumaroyl glucose, p- coumaroylquinic acid, 3-caffeoylquinic acid, verbascoside, 4-caffeoylquinic acid, p-coumaroyl tartaric acid, 2,5-di-s-glutathionyl caftaric acid, feruloyl tartaric acid, caffeic acid ethyl ester, cinnamoyl glucose, 5-caffeoylquinic acid, 3 -p-coumaroylquinic acid, 2-s-glutathionyl caftaric acid, 5-feruloylquinic acid, 4-feruloylquinic acid, 3-feruloylquinic acid, 5-sinapoylquinic acid, 4- sinapoylquinic acid, 3-sinapoylquinic acid, 3,5-dicaffeoylquinic acid, isoferulic acid, caffeoyl glucose, p-coumaric acid 4-o-glucoside, caffeic acid 4-o-glucoside, ferulic acid 4-o-glucoside, p- coumaroyl tartaric acid glucosidic ester, p-coumaric acid ethyl ester, hydroxy caffeic acid, chicoric acid, 5-5'-dehydrodiferulic acid, 5-8'-dehydrodiferulic acid, 1,2-disinapoylgentiobiose, l-sinapoyl-2-feruloylgentiobiose, 1,2-diferuloylgentiobiose, 1,2,2'-trisinapoylgentiobiose, 1,2'- disinapoyl-2-feruloylgentiobiose, l-sinapoyl-2,2'-diferuloylgentiobiose, 1,2,2'- triferuloylgentiobiose, 8-o-4'-dehydrodiferulic acid, 5-8'-benzofuran dehydrodiferulic acid, 3,4- dicaffeoylquinic acid, 3,4-diferuloylquinic acid, 3,5-diferuloylquinic acid, 1,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, avenanthramide 2p, avenanthramide 2c, avenanthramide 2f, p- coumaroyl malic acid, p-coumaroyl glycolic acid, cinnamic acid, caffeoyl aspartic acid, p- coumaroyl tyrosine, sinapine, avenanthramide k, 24-methylcholestanol ferulate, 24- methylcholesterol ferulate, 24-methyllathosterol ferulate, stigmastanol ferulate, sitosterol ferulate, schottenol ferulate, 24-methylenecholestanol ferulate, 3-o-methylrosmarinic acid, feruloylglycine, isoferulic acid 3-o-glucuronide, isoferulic acid 3-sulfate, ferulic acid 4-sulfate, ferulic acid 4-o-glucuronide, caffeic acid 4-sulfate, caffeic acid 3-sulfate, feruloyl cl- glucuronide, isoferuloyl cl -glucuronide, caffeic acid 3-o-glucuronide, caffeic acid 4-o- glucuronide, caffeoyl cl -glucuronide, 1,5-diferuloylquinic acid, l-caffeoyl-5-feruloylquinic acid, l-feruloyl-5-caffeoylquinic acid, 3,4-dihydroxyphenylacetic acid, 4-hydroxyphenylacetic acid, homovanillic acid, homoveratric acid, methoxyphenylacetic acid, 3 -hydroxyphenylacetic acid, 2- hydroxyphenylacetic acid, phenacetylglycine, phenylacetic acid, 4-hydroxymandelic acid, 2- hydroxy-2 -phenylacetic acid, homovanillic acid 4-sulfate, dihydro-p-coumaric acid, di hydrocaffeic acid, 3 -hydroxy-3 -(3 -hydroxyphenyl)propionic acid, 3 -(3, 4-dihydroxyphenyl)-2- methoxypropionic acid, 3 -hydroxyphenylpropionic acid, dihydroferulicacid 4-sulfate, di hydrocaffeic acid 3-o-glucuronide, di hydrocaffeic acid 3 -sulfate, dihydroferulic acid, dihydroferulic acid 4-o-glucuronide, dihydrosinapic acid, dihydroferuloylglycine, danshensu, 3- methoxy -4-hydroxyphenyllactic acid, 3,4-dihydroxyphenyllactic acid methyl ester, hydroxydanshensu, 3 -phenylpropionic acid, 3 -hydroxy-4-m ethoxyphenyllactic acid, 4-hydroxyphenyl-2-propionic acid, 5-(3'-methoxy-4'-hydroxyphenyl)-y-valerolactone, 4-hydroxy-(3',4'-dihydroxyphenyl)valeric acid, 5-(3',4'-dihydroxyphenyl)-valeric acid, 5-(3',4',-dihydroxyphenyl)-y-valerolactone, 5-(3', 4', 5'-trihydroxyphenyl)-y -valerolactone, 5-(3',5'-dihydroxyphenyl)-y-valerolactone, 3 -hydroxyphenylvaleric acid, 5-(3',5'-dihydroxyphenyl)-y-valerolactone 3-o-glucuronide, trans-resveratrol, piceatannol, e-viniferin, pterostilbene, d-viniferin, pallidol, piceatannol 3-o-glucoside, pinosylvin, resveratrol 5-o-glucoside, resveratrol, resveratrol 3-o-glucoside, 3,4,5,4'-tetramethoxystilbene, 3'-hydroxy-3, 4,5,4'-tetramethoxystilbene, 4'-hydroxy-3,4,5-trimethoxystilbene, 4-hydroxy-3,5,4'-trimethoxystilbene, cis-resveratrol 3-o-glucuronide, cis-resveratrol 3-sulfate, cis-resveratrol 4'-o-glucuronide, cisresveratrol 4'-sulfate, resveratrol 3-sulfate, trans-resveratrol 3,5-disulfate, trans-resveratrol 3,4'- di sulfate, trans-resveratrol 3-o-glucuronide, trans-resveratrol 3-sulfate, trans-resveratrol 4'-o- glucuronide, trans-resveratrol 4'-sulfate, dihydroresveratrol, tannic acid, or any combination thereof.

[0197] In some aspects, the at least one polyphenol is a polyphenol additive of the following structure:

[0198] In some aspects, a lipid nanoparticle can comprise a polyphenol and nucleic acid at a specified ratio (weight / weight).

[0199] In some aspects, a lipid nanoparticle comprising a polyphenol and at least one nucleic acid can comprise a polyphenol and nucleic acid at a ratio of about 0.1: 1, or about 0.15: 1, orabout 0.2:1, or about 0.25:1, or about 0.3:1, or about 0.35:1, or about 0.4:1, or about 0.45:1, or about 0.5:1, or about 1:1, or about 1.5:1, or about 2:1, or about 2.5:1, or about 3:1, or about 3.5:1, or about 4:1, or about 4.5:1, or about 5:1, or about 5.5:1, or about 6:1, or about 6.5:1, or about 7:1, or about 7.5:1, or about 8:1, or about 8.5:1, or about 9:1, or about 9.5:1, or about 10:1, or about 10.5:1, or about 11:1, or about 11.5:1, or about 12:1, or about 12.5:1, or about 13:1, or about 13.5:1, or about 14:1, or about 14.5:1, or about 15:1, or about 15.5:1, or about 16:1, or about 16.5:1, or about 17:1, or about 17.5:1, or about 18:1, or about 18.5:1, or about 19:1, or about 19.5:1, or about 20:1 polyphenol:nucleic acid. In some aspects, the at least one nucleic acid can comprise DNA.

[0200] In some aspects, a lipid nanoparticle can comprise a polyphenol and lipid at a specified ratio (weight / weight).

[0201] In some aspects, a lipid nanoparticle can comprise a polyphenol and lipid at a ratio of about 0.08:1, 0.1:1, or about 0.15:1, or about 0.2:1 or about 0.25:1, or about 0.3:1 additivelipid (weight / weight).

[0202] Targeting Ligand

[0203] In some aspects, an LNP can further comprise at least one targeting ligand.

[0204] Accordingly, a lipid nanoparticle 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, and at least one targeting ligand.

[0205] In some aspects, an LNP of the present disclosure can further comprise at least about 0.05%, or at least about 0.1%, or at least about 0.15%, or at least about 0.2%, or at least about 0.25%, or at least about 0.3%, or at least about 0.35%, or at least about 0.4%, or at least about 0.45%, or at least about 0.5%, or at least about 0.55%, or at least about 0.6%, or at least about 0.65%, or at least about 0.7%, or at least about 0.75%, or at least about 0.8%, or at least about 0.85%, or at least about 0.9%, or at least about 0.95%, or at least about 1.0%, or at least about 1.1%, or at least about 1.2%, or at least about 1.3%, or at least about 1.4%, or at least about 1.5%, or at least about 1.6%, or at least about 1.7%, or at least about 1.8%, or at least about 1.9%, or at least about 2.0% of at least one targeting ligand by moles.

[0206] A targeting ligand may be any ligand that provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand.

[0207] In some aspects, a composition comprising a targeting lipid is well -tolerated and provides an adequate therapeutic index, such that patient treatment with an effective dose of the composition is associated with an improved toxicity and / or risk profile to the patient, compared to patient treatment with an effective dose of a composition that does not comprise a targeting ligand.

[0208] In some aspects, a targeting ligand provides an enhanced affinity for the liver or liver cells, such as hepatocytes. A non-limiting example of a targeting ligand with enhanced affinity for the liver or liver cells is GalNac (n-acetyl-galactosamine). Thus, in some embodiments, the invention provides LNP compositions comprising a targeting ligand comprising GalNac.

[0209] In some aspects, a targeting ligand comprising GalNac can be a pegylated GalNac molecule. In some aspects, a pegylated GalNac molecule can be Tri-GalNac-PEG2000-DSPE (referred to herein as “GalNac-PEG”), and which structure is shown below:some aspects, the present disclosure provides LNPs comprising GalNac-PEG.

[0210] In some aspects, a targeting ligand can also include targeting groups, for example a group of tissue targeting agents. A non-limiting example of a targeting group can be multivalent GalNac molecule. Thus, in some embodiments, the invention provides LNP compositions comprising a targeting ligand comprising multivalent GalNac. A non-limiting example of a multivalent GalNac molecule is GalNac-PEG.

[0211] In some aspects, a targeting ligand can comprise DSPE (1, 2-Distearoyl-sn-glycero-3- phosphoethanolamine). Thus, in some embodiments, the invention provides LNP compositions comprising a targeting ligand comprising DSPE. In some aspects, the DSPE can be pegylated. In some aspects, a targeting ligand comprising DSPE can be l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], also referred to herein as DSPE- PEG2000 or DSPE-PEG, and whose structure is shown below:

[0212] Exemplary LNPs of the Present Disclosure

[0213] The following are exemplary LNPs of the present disclosure.

[0214] In some aspects, a lipid nanoparticle is provided comprising about 50% of at least one compound of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII) and / or Formula (IX) by moles, about 38.5% of at least one structural lipid by moles, about 10% of at least one phospholipid by moles, about 1.5% of at least one PEGylated lipid by moles, and at least one nucleic acid molecule. In some aspects, the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 40: 1 (w / w), about 50:1 (w / w), or about 80:1 (w / w).

[0215] In some aspects, a lipid nanoparticle is provided comprising about 54% of at least one compound of Formula (1), Formula (11), Formula (111), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII) and / or Formula (IX) by moles, about 35% of at least one structural lipid by moles, about 10% of at least one phospholipid by moles, about 1% of at least one PEGylated lipid by moles, and at least one nucleic acid molecule. In some aspects, the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 40: 1 (w / w) or about 100:1 (w / w).

[0216] In some aspects, a lipid nanoparticle is provided comprising about 50% of COMPOUND NO. 37 by moles, about 38.5% of at least one structural lipid by moles, about 10% of at least one phospholipid by moles, about 1.5% of at least one PEGylated lipid by moles, at least one DNA molecule, and pentagalloylglucose. In some aspects, the ratio of lipid to DNA in the nanoparticle can be about 80: 1 (w / w). In some aspects of the preceding LNPs, the lipid nanoparticle comprises pentagalloylglucose in a ratio of pentagalloylglucose to DNA of about 2.5:1, or about 5:1, or about 10:1.

[0217] In some aspects, a lipid nanoparticle is provided comprising about 50% of at least one compound of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII) and / or Formula (IX) by moles, about 38.5% of at least onestructural lipid by moles, about 10% of at least one phospholipid by moles, about 1.5% of at least one PEGylated lipid by moles, at least one nucleic acid molecule, and at least one polyphenol ofthe following structure:. in some aspects, the at least one nucleic acid molecule comprises at least one DNA molecule. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 40: 1 (w / w), about 50: 1 (w / w), or about 80: 1 (w / w). In some aspects, the ratio of polyphenol additive to total lipid in the nanoparticle can be about 0.1, about 0.15, or about 0.2.

[0218] Pharmaceutical Compositions of the Present Disclosure

[0219] In some aspects, the present disclosure provides a pharmaceutical composition comprising at least one lipid nanoparticle of the present disclosure. In some aspects, 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 the at least one first nanoparticle comprises at least one nucleic acid molecule encoding at least one transposase, wherein the at least one second nanoparticle comprises at least one nucleic acid molecule encoding at least one transposon. In some aspects, the at least one nucleic acid molecule encoding at least one transposase can be an RNA molecule (e.g. 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).

[0220] In some aspects, the present disclosure provides a composition comprising at least one cell that has been contacted by at least one nanoparticle of the present disclosure. In some aspects, the present disclosure provides a composition comprising at least one cell that has been genetically modified using at least one nanoparticle of the present disclosure. In some aspects, the present disclosure provides a composition comprising at least one cell that has been genetically modified using any method of the present disclosure.

[0221] In some aspects, the present disclosure provides a pharmaceutical composition comprising at least one cell that has been contacted by at least one nanoparticle of the present disclosure. In some aspects, the present disclosure provides a pharmaceutical composition comprising at least one cell that has been genetically modified using at least one nanoparticle of the present disclosure. In some aspects, the present disclosure provides a pharmaceutical composition comprising at least one cell that has been genetically modified using any method of the present disclosure.Methods of the Present Disclosure

[0222] The present disclosure provides a method of delivering at least one nucleic acid to at least one target cell comprising contacting the at least one cell with at least one composition of the present disclosure. The present disclosure provides a method of delivering at least one nucleic acid to at least one target cell comprising contacting the at least one target cell with at least one nanoparticle of the present disclosure.

[0223] In one aspect, the target cell may be selected from a group consisting of immune cells, stem cells, epithelial cells, and liver cells.

[0224] In all methods, compositions and kits of the present disclosure, at least one cell can be a liver cell. A liver cell can include, but is not limited to, a hepatocyte, a hepatic stellate cell, Kupffer cell or a liver sinusoidal endothelial cell.

[0225] In some aspects of any methods of the present disclosure, a cell 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.

[0226] The present disclosure provides a method of 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 of genetically modifying at least one cell comprising contacting the at least one cell with at least one nanoparticle of the present disclosure.

[0227] In some aspects, genetically modifying a cell can comprise delivering at least one exogenous nucleic acid to the cell such that the cell expresses at least one protein that the cell otherwise would not normally express, or such that the at least one cell expresses at least one protein at a level that is higher than the level that the cell would otherwise normally express the at least one protein, or such that the cell expresses at least one protein at a level that is lower thanthe level that the cell would otherwise normally express. In some aspects, genetically modifying a cell can comprise delivering at least one exogenous nucleic to the cell such that at least one exogenous nucleic acid is integrated into the genome of the at least one cell.

[0228] In some aspects, the methods of the present disclosure can yield 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 cell in the plurality express at least one protein that was encoded in at least one nucleic acid that was delivered to the plurality of cells via a nanoparticle of the present disclosure.

[0229] The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering to the subject at least one therapeutically effective amount of at least one composition of the present disclosure comprising at least one nucleic acid encoding a therapeutic protein.

[0230] 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.

[0231] The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering a therapeutically effective amount of cells, wherein the cells have been contacted by at least one nanoparticle of the present disclosure comprising at least one nucleic acid encoding a therapeutic protein. The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering a therapeutically effective amount of cells, wherein the cells have been genetically modified using the compositions and / or methods of the present disclosure.

[0232] The disclosure provides methods for the treatment of a disease or disorder in a cell, tissue, organ, animal, or subject, comprising administering or contacting the cell, tissue, organ, animal, or subject with a therapeutic effective amount of a composition disclosed herein. In one aspect, the subject is a mammal. Preferably, the subject is human. The terms “subject” and “patient” are used interchangeably herein.

[0233] The disclosure provides methods of treating at least one disease or disorder in a subject, comprising administering to the subject at least one therapeutically effective amount of at least one composition disclosed herein comprising at least one nucleic acid encoding a therapeutic protein.

[0234] Without wishing to be bound by theory, it is hypothesized that the LNP compositions of the present disclosure target liver cells more effectively than other cells, thus reducing off-target effects associated with other delivery compositions.

[0235] In some embodiments, the LNP compositions provided herein that comprise a targeting ligand result in less cytokine release than the same LNP composition not comprising the targeting ligand. Cytokine release may be measured using any suitable method known in the art or described herein. For example, cytokine levels may be determined in the blood of a subject receiving the LNP composition comprising the targeting ligand using enzyme-linked immunosorbent assays (ELISAs). The cytokine levels may then be compared to pre-treatment baseline levels.

[0236] The disclosure provides a method for modulating or treating at least one malignant disease or disorder in a cell, tissue, organ, animal or subject. In some aspects, the at least one disease can be a genetic disorder. In some aspects, the at least one disease can be a malignant disease, including, but not limited to, cancer. In some aspects, the at least one disease can be Hemophilia A or Hemophilia B. In some aspects, the at least one disease can be a metabolic liver disorder (MLD). In some aspects, the at least one disease can be a urea cycle disorder (UCD). An MLD and / or UCD can include, but is not limited to, N-Acetylglutamate Synthetase (NAGS) Deficiency, Carbamoylphosphate Synthetase I Deficiency (CPSI Deficiency), Ornithine Transcarbamylase (OTC) Deficiency, Argininosuccinate Synthetase Deficiency (ASSD) (Citrullinemia I), Citrin Deficiency (Citrullinemia II), Argininosuccinate Lyase Deficiency (Argininosuccinic Aciduria), Arginase Deficiency (Hyperargininemia), Ornithine Translocase Deficiency (HHH Syndrome), methylmalonic acidemia (MMA) or any combination thereof.

[0237] Methods of the disclosure may be used to treat a disease or disorder by use of a therapeutic transgene encoding for an exogenous nucleic acid sequence or exogenous amino acid sequence. In such methods, the transgene is delivered to a target cell to replace or repair a mutated gene. Diseases that may be treated with such methods are generally caused by a mutation in a gene that results in no protein being expressed or non-functional proteins beingexpressed. Examples of therapeutic transgenes that can be delivered using the compositions disclosed herein include: Beta-Thalassemia (HBB T87Q, BCL11A shRNA, IGF2BP1), Sickle Cell Disease (HBB T87Q, BCL11A shRNA, IGF2BP1), Hemophilia A (Factor VIII), Hemophilia B (Factor IX), X-linked Severe Combined Immunodeficiency (Interleukin 2 receptor gamma (IL2RG)), Hypophosphatasia (Tissue Non-specific Alkaline Phosphatase (TNAP)), Osteopetrosis (TCIRG1), Glycogen Storage Disease Type II (Pompe Disease) (Alpha Glucosidase (GAA)), Alpha-Galactosidase A Deficiency (Fabry disease) (Alpha-galactosidase A (GLA)), Mucopolysaccharidosis Type I (MPS I) (Alpha-L-iduronidase (IDUA)), Mucopolysaccharidosis Type II (MPS II) (Iduronate 2-sulfatase (IDS)), Mucopolysaccharidosis Type IIIA (MPS IIIA) (sulfoglycosamine-sulfohydrolase (SGSH)), Mucopolysaccharidosis Type IIIB (MPS IIIB) (N-alpha-acetylglucosaminidase (NAGLU)), Mucopolysaccharidosis Type IV A (MPS IVA) (Morquio) (N-acetylgalactosamine-6-sulfate sulfatase (GALNS)), Mucopolysaccharidosis Type IV B (MPS IVB) Beta-galactosidase (GLB1 (Beta-galactosidase (GLB1)), Cholesteryl Ester Storage Disease (CESD) (Lysosomal acid lipase (LIPA)), Cystinosis (Cystinosin lysosomal cystine transporter (CTNS)), X-linked chronic granulomatous disease (X-CGD) (CYBB), Wiskott-Aldrich Syndrome (WAS) (WAS), X-linked Adrenoleukodystrophy (X-ALD) (ABCD1), Metachromatic leukopdystrophy (MLD) (ARSA), Phenylketonuria (PAH), Methylmalonic academia (MMUT), Propionic Acidemia (PCCA, PCCB), Retinitis Pigmentosa (RPE65), Usher Syndrome (MY07A), and Gaucher Disease (GBA).

[0238] Methods of the present disclosure can optionally further comprise co-administration or combination therapy for treating such diseases or disorders, wherein the administering of any composition or pharmaceutical composition disclosed herein, further comprises administering, before concurrently, and / or after, at least one chemotherapeutic agent (e.g., an alkylating agent, a mitotic inhibitor, a radiopharmaceutical).

[0239] The present disclosure also provides methods of preparing a lipid nanoparticle composition, comprising mixing a compound of Formula (I), Formula (II), Formula (III), Formula (IV) or Formula (V), a phospholipid, and a structural lipid under conditions allowing the formation of a stable lipid nanoparticle.Nucleic Acid Molecules

[0240] In some aspects, a nucleic acid molecule can be a synthetic nucleic acid molecule. In some aspects, a nucleic acid molecule can be a non-naturally occurring nucleic acid molecule. Insome aspects, a 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 aspects, a nucleic acid molecule can be a modified nucleic acid molecule. In some aspects, a 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.

[0241] In some aspects, an mRNA molecule can be capped using any method and / or capping moiety known in the art. An mRNA molecule can be capped with m7G(5’)ppp(5’)G moiety. A m7G(5’)ppp(5’)G moiety is also referred to herein as a “CapO”. An mRNA molecule can be capped with a CleanCap® moiety. A CleanCap® moiety can comprise a m7G(5')ppp(5')(2'OMeA) (CleanCap® AG) moiety. A CleanCap® moiety can comprise a m7G(5')ppp(5')(2'OMeG) (CleanCap® GG) moiety. An mRNA molecule can be capped with an anti-reverse cap analog (ARCA®) moiety. An ARCA® moiety can comprise a m7(3’-O-methyl)G(5’)ppp(5’)G moiety. An mRNA molecule can be capped with a CleanCap® 3’OMe moiety (CleanCap®+ARCA®).

[0242] In some aspects, an mRNA molecule can comprise at least one modified nucleic acid.

[0243] The at least one modified nucleic acid can comprise 5-methoxyuridine (5moU). In some aspects, 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 an mRNA molecule are 5-methoxyuridine bases. In some aspects, all of the uridine bases in an 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).

[0244] In some aspects, an mRNA molecule can comprise at least one modified nucleic acid.

[0245] The at least one modified nucleic acid can comprise / Vi-methylpseudouridine (me1'P). In some aspects, 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%, orat 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 an mRNA Ai-methylpseudouridine bases. In some aspects, all of the uridine bases in an mRNA molecule are Ai-methylpseudouridine bases. Without wishing to be bound by theory, Ai-methylpseudouridine can improve protein expression (see Li et al., Bioconjugate Chem. 2016, 27, 3, 849-853).

[0246] In some aspects, an mRNA molecule can comprise at least one modified nucleic acid.

[0247] The at least one modified nucleic acid can comprise pseudouridine ( ). In some aspects, 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 an mRNA pseudouridine bases. In some aspects, all of the uridine bases in an 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).

[0248] In some aspects, an mRNA molecule can comprise at least one modified nucleic acid.

[0249] The at least one modified nucleic acid can comprise 5-methylcytidine (5-MeC). In some aspects, 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 an mRNA 5-MeC bases. In some aspects, all of the cytidine bases in an mRNA molecule are 5-MeC bases.

[0250] In some aspects, a nucleic acid molecule can comprise a DNA molecule. Thus, in some aspects, a lipid nanoparticle can comprise a DNA molecule. In some aspects, the DNA molecule can be a circular DNA molecule, such as, but not limited to, a DNA plasmid or DNA nanoplasmid. Thus, in some aspects, a lipid nanoparticle can comprise a circular DNA molecule. In some aspects, a lipid nanoparticle can comprise a Doggybone DNA molecule. In someaspects, a lipid nanoparticle can comprise a DNA plasmid. In some aspects, a lipid nanoparticle can comprise a DNA nanoplasmid. In some aspects, a DNA molecule can be a linearized DNA molecule, such as, but not limited to, a linearized DNA plasmid or a linearized DNA nanoplasmid.

[0251] A DNA plasmid or DNA nanoplasmid can comprise can be 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 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 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.

[0252] In some aspects, a nucleic acid molecule formulated in a lipid nanoparticle of the present disclosure can comprise at least one transgene sequence. In some aspects, a transgene sequence can comprise a nucleotide sequence encoding at least one therapeutic protein. In some aspects, a transgene sequence can comprise a nucleotide sequence encoding at least one transposase. In some aspects, a transgene sequence can comprise a nucleotide sequence encoding at least one transposon. In some aspects, a transposon can comprise a nucleotide sequence encoding at least one therapeutic protein. In some aspects, a transposon can comprise a nucleotide sequence encoding at least one therapeutic protein and at least one protomer sequence, wherein the at least one therapeutic protein is operatively linked to the at least one promoter sequence.

[0253] In some aspects, the lipid nanoparticles of the present disclosure can be produced using a microfluidic-mixing platform. In some aspects, the microfluidic-mixing platform can be a non-turbulent microfluidic mixing platform.

[0254] In some aspects, a microfluidic-mixing platform can produce the lipid nanoparticles of the present invention by combining a miscible solvent phase comprising the lipid components of the nanoparticle and an aqueous phase comprising the lipid nanoparticle cargo (e.g. nucleic acid, DNA, mRNA, etc.) using a microfluidic device. In some aspects, the miscible solvent phase and the aqueous phase are mixed in the microfluidic device under laminar flow conditions that do not allow for immediate mixing of the two phases. As the two phases move under laminar flow in a microfluidic channel, microscopic features in the channel can allow for controlled, homogenous mixing to produce the lipid nanoparticles of the present disclosure.

[0255] In some aspects, the microfluidic-mixing platform can include, but are not limited to the NanoAssemblr® Spark (Precision NanoSystems), the NanoAssemblr® Ignite™ (Precision NanoSystems), the NanoAssemblr® Benchtop (Precision NanoSystems), the NanoAssemblr® Blaze (Precision NanoSystems) or the NanoAssemblr® GMP System (Precision NanoSystems).

[0256] In some aspects, the lipid nanoparticles of the present disclosure can be produced using a microfluidic-mixing platform, wherein the microfluidic mixing platform 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.

[0257] In some aspects, the lipid nanoparticles of the present disclosure can be produced using a microfluidic-mixing platform, wherein the microfluidic mixing platform mixes a miscible solvent phase and an aqueous phase at a ratio of about 10:1, or about 9: 1, or about 8:1, or about 7:1, or about 6:1, or about 5:1, or about 4:1, or about 3:1, or about 2:1, or about 1:1, or about 1:2, or about 1:3, or about 1:4, or about 1:5, or about 1:6, or about 1:7, or about 1:8, or about 1:9, or about 1:10, solvent: aqueous, v:v.piggyBac® ITR sequences

[0258] In some aspects, a nucleic acid can comprise a piggyBac® ITR sequence. In some aspects, a nucleic acid can comprise a first piggyBac® ITR sequence and a second piggyBac® ITR sequence.

[0259] In some aspects, a piggyBac® ITR sequence can comprise any piggyBac® TTR sequence known in the art.

[0260] In some aspects of the methods of the present disclosure, a piggyBac® ITR sequence, such as a first piggyBac® ITR sequence and / or a second piggyBac® ITR sequence in an AAV piggyBac® transposon can comprise, consist essentially of, or consist of a Sleeping Beauty transposon ITR, a Helraiser transposon ITR, a Tol2 transposon ITR, a TcBuster transposon ITR or any combination thereof.Transposition systems

[0261] In some aspects, a nucleic acid can comprise a transposon or a nanotransposon comprising: a first nucleic acid sequence comprising: (a) a first inverted terminal repeat (ITR) or a sequence encoding a first ITR, (b) a second ITR or a sequence encoding a second ITR, and (c) an intra-ITR sequence or a sequence encoding an intra-ITR, wherein the intra-ITR sequence comprises a transposon sequence or a sequence encoding a transposon.

[0262] In some aspects, a nucleic acid can comprise a transposon or a nanotransposon comprising: a first nucleic acid sequence comprising: (a) a first inverted terminal repeat (ITR) or a sequence encoding a first ITR, (b) a second ITR or a sequence encoding a second ITR, and (c) an intra-ITR sequence or a sequence encoding an intra-ITR, wherein the intra-ITR sequence comprises a transposon sequence or a sequence encoding a transposon, and a second nucleic acid sequence comprising an inter-ITR sequence or a sequence encoding an inter-ITR, wherein the length of the inter-ITR sequence is equal to or less than 700 nucleotides.

[0263] The transposon or nanotransposon of the present disclosure can be a piggyBac® (PB) transposon. In some aspects when 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.

[0264] 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.

[0265] The PB, PBL and SPB transposases recognize transposon-specific inverted terminal repeat sequences (ITRs) on the ends of the transposon, and inserts the contents between the ITRs at the sequence 5’-TTAT-3’ within a chromosomal site (a TTAT target sequence) or at the sequence 5’-TTAA-3’ within a chromosomal site (a TTAA target sequence). The target sequenceof the PB or PBL transposon can comprise or consist of 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’-AAAT-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 has no payload limit for the genes of interest that can be included between the ITRs.

[0266] Exemplary amino acid sequences for one or more PB, PBL and SPB transposases are disclosed in U. S. Patent No. 6,218,185; U. S. Patent No. 6,962,810 and U. S. Patent No.8,399,643, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein. In some embodiments, the PB transposase comprises or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 1. In some embodiments, the PB transposases comprises the amino acid sequence of SEQ ID NO: 1.

[0267] The PB or PBL transposase can comprise or consist of an amino acid sequence having an amino acid substitution at two or more, at three or more or at each of positions 30, 165, 282, and / or 538 of the sequence of SEQ ID NO: 1. The transposase can be a SPB transposase that comprises or consists of the amino acid sequence of the sequence of SEQ ID NO: 1 wherein the amino acid substitution at position 30 can be a substitution of a valine (V) for an isoleucine (I), the amino acid substitution at position 165 can be a substitution of a serine (S) for a glycine (G), the amino acid substitution at position 282 can be a substitution of a valine (V) for a methionine (M), and the amino acid substitution at position 538 can be a substitution of a lysine (K) for an asparagine (N). In some embodiments, the SPB transposase comprises or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 2. In some embodiments, the SPB transposase comprises the amino sequence set forth in SEQ ID NO: 2.

[0268] In certain aspects wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and / or 538, the PB, PBL and SPB transposases can further comprise an amino acid substitution 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 are described in more detail in PCT Publications No. WO 2019 / 173636 and No. WO 2020 / 051374, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein.

[0269] In some embodiments, the PB transposase comprises or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 3. In some embodiments, the PB transposase comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0270] The PB or PBL transposase can comprise or consist of an amino acid sequence having an amino acid substitution at two or more, at three or more or at each of positions 29, 164, 281, and / or 537 of the sequence of SEQ ID NO: 3. The transposase can be a SPB transposase that comprises or consists of the amino acid sequence of the sequence of SEQ ID NO: 3 wherein the amino acid substitution at position 29 can be a substitution of a valine (V) for an isoleucine (I), the amino acid substitution at position 164 can be a substitution of a serine (S) for a glycine (G), the amino acid substitution at position 281 can be a substitution of a valine (V) for a methionine (M), and the amino acid substitution at position 537 can be a substitution of a lysine (K) for an asparagine (N). In some embodiments, the SPB transposase comprises or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 4. In some embodiments, the SPB transposase comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0271] In certain aspects wherein 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 are described in more detail in PCT Publication No. WO 2019 / 173636 and No. WO 2020 / 051374, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein.

[0272] The PB, PBL or SPB transposases can be isolated or derived from an insect, vertebrate, crustacean or urochordate as described in more detail in PCT Publication No. WO 2019 / 173636and PCT / US2019 / 049816. In preferred aspects, the PB, PBL or SPB transposases is isolated or derived from the insect Trichoplusia ni (GenBank Accession No. AAA87375) or Bonibyx mori (GenBank Accession No. BAD 11135).

[0273] A hyperactive PB or PBL transposase is a transposase that is more active than the endogenous transposase from which it is derived. In a preferred aspect, a 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 WO 2019 / 173636, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein. A list of hyperactive amino acid substitutions is disclosed in U. S. Patent No. 10,041,077, which is incorporated herein by reference in its entirety for examples of amino acid substitutions that may be introduced into the transposases described herein. A transposon or nanotransposon of the present disclosure can be a Sleeping Beauty transposon. In some aspects, when the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase (for example as disclosed in U. S. Patent No. 9,228,180, which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein) or a hyperactive Sleeping Beauty (SB100X) transposase.

[0274] In some aspects, the PB or PBL transposase is integration deficient. An integration deficient PB or PBL transposase is a transposase that can excise its corresponding transposon, but that integrates the excised transposon at a lower frequency than a corresponding wild type transposase. Examples of integration deficient PB or PBL transposases are disclosed in U. S. Patent No. 6,218,185; U. S. Patent No. 6,962,810, U. S. Patent No. 8,399,643 and WO 2019 / 173636, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein. A list of integration deficient amino acid substitutions is disclosed in US patent No. 10,041,077, which is incorporated herein by reference in its entirety for examples of amino acid substitutions that may be introduced into transposases described herein.

[0275] In some aspects, the PB or PBL transposase is fused to a nuclear localization signal. Examples of PB or PBL transposases fused to a nuclear localization signal are disclosed in U. S. Patent No. 6,218,185; U. S. Patent No. 6,962,810, U. S. Patent No. 8,399,643 and WO2019 / 173636, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein.

[0276] A transposon or nanotransposon of the present disclosure can be a Sleeping Beauty transposon. In some aspects, when the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase (for example as disclosed in U. S. Patent No.9,228,180, which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein) or a hyperactive Sleeping Beauty (SB100X) transposase.

[0277] A transposon or nanotransposon of the present disclosure can be a Helraiser transposon. An exemplary Helraiser transposon includes Helibatl. In some aspects, when the transposon is a Helraiser transposon, the transposase is a Helitron transposase (for example, as disclosed in WO 2019 / 173636, which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein).

[0278] A transposon or nanotransposon of the present disclosure can be a Tol2 transposon. In some aspects, when the transposon is a Tol2 transposon, the transposase is a Tol2 transposase (for example, as disclosed in WO 2019 / 173636).

[0279] A transposon or nanotransposon of the present disclosure can be a TcBuster transposon. In some aspects, when the transposon is a TcBuster transposon, the transposase is a TcBuster transposase or a hyperactive TcBuster transposase (for example, as disclosed in WO 2019 / 173636, which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein). The TcBuster transposase can comprise or consist of a naturally occurring amino acid sequence or a non-naturally occurring amino acid sequence. The polynucleotide encoding a TcBuster transposase can comprise or consist of a naturally occurring nucleic acid sequence or a non-naturally occurring nucleic acid sequence.

[0280] In some aspects, a mutant TcBuster transposase comprises one or more sequence variations when compared to a wild type TcBuster transposase as described in more detail in PCT Publications No. WO 2019 / 173636 and No. WO 2020 / 051374, each of which isincorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein.

[0281] The cell delivery compositions (e.g., transposons) disclosed herein can comprise a nucleic acid molecule encoding a therapeutic protein or therapeutic agent. Examples of therapeutic proteins include those disclosed in PCT Publications No. WO 2019 / 173636 and No. WO 2020 / 051374, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein.

[0282] In some aspects, a therapeutic protein can comprise a FVIII polypeptide. An exemplary nanoplasmid encoding an FVIII polypeptide is provided in SEQ ID NO: 9. Accordingly, a nucleic acid formulated in a nanoparticle of the present disclosure can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 9.

[0283] In some aspects, a therapeutic protein can comprise a propionyl-CoA carboxylase subunit alpha (PCCA) polypeptide. An exemplary transposon encoding a PCCA polypeptide is provided in SEQ ID NO: 10. Accordingly, a nucleic acid formulated in a nanoparticle of the present disclosure can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 10.Gene editing systems

[0284] The present disclosure provides a gene editing composition and / or a cell comprising the gene editing composition. The gene editing composition can comprise a nanoparticle comprising a nucleic acid, wherein the nucleic acid comprises a sequence encoding a DNA binding domain and a sequence encoding a nuclease protein or a nuclease domain thereof. The sequence encoding a nuclease protein or the sequence encoding a nuclease domain thereof can comprise a DNA sequence, an RNA sequence, or a combination thereof. The nuclease or the nuclease domain thereof can comprise one or more of a CRISPR / Cas protein, a Transcription Activator-Like Effector Nuclease (TALEN), a Zinc Finger Nuclease (ZFN), and an endonuclease.

[0285] The nuclease or the nuclease domain thereof can comprise a nuclease-inactivated Cas (dCas) protein and an endonuclease. The endonuclease can comprise a Clo051 nuclease or a nuclease domain thereof. The gene editing composition can comprise a fusion protein. Thefusion protein can comprise a nucl ease-inactivated Cas9 (dCas9) protein and a Clo051 nuclease or a Clo051 nuclease domain. In some aspects, the fusion protein can further comprise at least one nuclear localization signal (NLS). In some aspects, 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.

[0286] A transgene can comprise a nucleic sequence encoding a small, Cas9 (Cas9) operatively-linked to an effector. The disclosure provides a fusion protein comprising, consisting essentially of or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a small, Cas9 (Cas9). A small Cas9 construct of the disclosure can comprise an effector comprising a type IIS endonuclease.

[0287] A transgene can comprise a nucleic sequence encoding an inactivated, small, Cas9 (dSaCas9) operatively-linked to an effector. A transgene can comprise a nucleic 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). A small, inactivated Cas9 (dSaCas9) construct of the disclosure can comprise an effector comprising a type IIS endonuclease.

[0288] A transgene can comprise a nucleic sequence encoding an inactivated Cas9 (dCas9) operatively-linked to an effector. A transgene can comprise a nucleic 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). An inactivated Cas9 (dCas9) construct of the disclosure can comprise an effector comprising a type IIS endonuclease.

[0289] The dCas9 can be isolated or derived from Streptoccocus pyogenes. The dCas9 can comprise a dCas9 with substitutions at amino acid positions 10 and 840, which inactivate the catalytic site. In some aspects, these substitutions are D10A and H840A.

[0290] A cell comprising the gene editing composition can express the gene editing composition stably or transiently. Preferably, the gene editing composition is expressed transiently. The guide RNA can comprise a sequence complementary to a target sequence within a genomic DNA sequence. The target sequence within a genomic DNA sequence can be a target sequence within a safe harbor site of a genomic DNA sequence.

[0291] Gene editing compositions, including Cas-CLOVER, and methods of using these compositions for gene editing are described in detail in U. S. Patent Publication Nos.2017 / 0107541, 2017 / 0114149, 2018 / 0187185 and U. S. Patent No. 10,415,024, each of which is incorporated herein by reference in its entirety for examples of gene editing systems that may be used in conjunction with the compositions and methods described herein. In some aspects, a 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: 11. In some embodiments, the Cas-CLOVER protein comprises the amino acid sequence set forth in SEQ ID NO: 11.

[0292] Accordingly, the present disclosure provides any of the lipid nanoparticle compositions described herein, wherein the lipid nanoparticle comprises at least one genomic editing composition, wherein the at least one genomic 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 an inactivated nuclease domain thereof, (ii) a Clo051 protein or a nuclease domain thereof; and b) at least one gRNA molecule. In some aspects, the fusion protein can further comprise at least one NLS. In some aspects, the at least one genomic editing composition can comprise at least two species of gRNA molecules.

[0293] An exemplary nucleic acid sequence encoding a fusion protein is presented in SEQ ID NO: 5. Accordingly, a nucleic acid molecule formulated in a lipid nanoparticle of the present disclosure can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 5.

[0294] Exemplary gRNA sequences are presented in SEQ ID NOs: 6 and 7. Accordingly, gRNA molecules formulated in a lipid nanoparticle of the present disclosure can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 6 or SEQ ID NO: 7.Formulations, Dosages and Modes of Administration

[0295] The present disclosure provides formulations, dosages and methods for administration of the compositions described herein.

[0296] The disclosed compositions and pharmaceutical compositions can further comprise at least one of any suitable auxiliary, such as, but not limited to, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like. Pharmaceutically acceptable auxiliaries are preferred. Non-limiting examples of, and methods of preparing such sterile solutions are well known in the art, such as, but limited to, Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990 and in the “Physician's Desk Reference”, 52nd ed., Medical Economics (Montvale, N. J.) 1998.Pharmaceutically acceptable carriers can be routinely selected that are suitable for the mode of administration, solubility and / or stability of the composition as well known in the art or as described herein.

[0297] 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”).

[0298] Non-limiting examples of pharmaceutical excipients and additives suitable for use include proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars, such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Non-limiting examples of protein excipients include serum albumin, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like.Representative amino acid / protein components, which can also function in a 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.

[0299] The compositions can also include a buffer or a pH-adjusting agent; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts, such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers. Preferred buffers are organic acid salts, such as citrate. In some aspects, the buffer can include sucrose.

[0300] Many known and developed modes can be used for administering therapeutically effective amounts of the compositions or pharmaceutical compositions disclosed herein. Nonlimiting examples of modes of administration include bolus, buccal, infusion, intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intralesional, intramuscular, intramyocardial, intranasal, intraocular, intraosseous, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intratumoral, intravenous, intravesical, oral, parenteral, rectal, sublingual, subcutaneous, transdermal or vaginal means.

[0301] A composition of the disclosure can be prepared for use for parenteral (subcutaneous, intramuscular or intravenous) or any other administration particularly in the form of liquid solutions or suspensions; for use in vaginal or rectal administration particularly in semisolid forms, such as, but not limited to, creams and suppositories; for buccal, or sublingual administration, such as, but not limited to, in the form of tablets or capsules; or intranasally, such as, but not limited to, the form of powders, nasal drops or aerosols or certain agents; or transdermally, such as not limited to a gel, ointment, lotion, suspension or patch delivery system with chemical enhancers such as dimethyl sulfoxide to either modify the skin structure or to increase the drug concentration in the transdermal patch (Junginger, et al. In “Drug Permeation Enhancement;” Hsieh, D. S., Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994,), or applications of electric fields to create transient transport pathways, such as electroporation, or to increase the mobility of charged drugs through the skin, such as iontophoresis, or application of ultrasound, such as sonophoresis (U. S. Pat. Nos. 4,309,989 and 4,767,402) (the above publications and patents being entirely incorporated herein by reference).

[0302] For parenteral administration, any composition disclosed herein can be formulated as a solution, suspension, emulsion, particle, powder, or lyophilized powder in association, or separately provided, with a pharmaceutically acceptable parenteral vehicle. Formulations for parenteral administration can contain as common excipients sterile water or saline, polyalkylene glycols, such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. Aqueous or oily suspensions for injection can be prepared by using an appropriate emulsifier or humidifier and a suspending agent, according to known methods. Agents for injection can be a non-toxic, non-orally administrable diluting agent, such as aqueous solution, a sterile injectable solution or suspension in a solvent. As the usable vehicle or solvent, water, Ringer's solution, isotonic saline, etc. are allowed; as an ordinary solvent or suspending solvent,sterile involatile oil can be used. For these purposes, any kind of involatile oil and fatty acid can be used, including natural or synthetic or semisynthetic fatty oils or fatty acids; natural or synthetic or semisynthtetic mono- or di- or tri-glycerides. Parental administration is known in the art and includes, but is not limited to, conventional means of injections, a gas pressured needleless injection device as described in U. S. Pat. No. 5,851,198, and a laser perforator device as described in U. S. Pat. No. 5,839,446, each of which is incorporated herein by reference in its entirety for examples of injection devices that may be used in conjunction with the compositions and methods described herein.

[0303] For pulmonary administration, preferably, a composition or pharmaceutical composition described herein is delivered in a particle size effective for reaching the lower airways of the lung or sinuses. The composition or pharmaceutical composition can be delivered by any of a variety of inhalation or nasal devices known in the art for administration of a therapeutic agent by inhalation. These devices capable of depositing aerosolized formulations in the sinus cavity or alveoli of a patient include metered dose inhalers, nebulizers (e.g., jet nebulizer, ultrasonic nebulizer), dry powder generators, sprayers, and the like. All such devices can use formulations suitable for the administration for the dispensing of a composition or pharmaceutical composition described herein in an aerosol. Such aerosols can be comprised of either solutions (both aqueous and non-aqueous) or solid particles. In a metered dose inhaler (MDI), a propellant, a composition or pharmaceutical composition described herein, and any excipients or other additives are contained in a canister as a mixture including 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, which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein.

[0304] For absorption through mucosal surfaces, compositions include an emulsion comprising a plurality of submicron particles, a mucoadhesive macromolecule, a bioactive peptide, and an aqueous continuous phase, which promotes absorption through mucosal surfaces by achieving mucoadhesion of the emulsion particles (see, e.g., U. S. Pat. No. 5,514,670, which is incorporated herein by reference in its entirety for examples). Mucous surfaces suitable for application of the emulsions of the disclosure can include corneal, conjunctival, buccal, sublingual, nasal, vaginal,pulmonary, stomachic, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, e.g., suppositories, can contain as excipients, for example, polyalkyleneglycols, vaseline, cocoa butter, and the like. Formulations for intranasal administration can be solid and contain as excipients, for example, lactose or can be aqueous or oily solutions of nasal drops. For buccal administration, excipients include sugars, calcium stearate, magnesium stearate, pregelinatined starch, and the like (see, e.g., U. S. Pat. No.5,849,695, which is incorporated herein by reference in its entirety for examples). A more detailed description of mucosal administration and formulations is disclosed in PCT Publication No. WO 2019 / 049816, each of which is incorporated herein by reference in its entirety for examples of formulations that may be used in conjunction with the compositions and methods described herein.

[0305] For transdermal administration, a composition or pharmaceutical composition disclosed herein is encapsulated in a delivery device, such as a liposome or polymeric nanoparticles, microparticle, microcapsule, or microspheres (referred to collectively as microparticles unless otherwise stated). A number of suitable devices are known, including microparticles made of synthetic polymers, such as polyhydroxy acids, such as polylactic acid, polyglycolic acid and copolymers thereof, polyorthoesters, polyanhydrides, and polyphosphazenes, and natural polymers, such as collagen, polyamino acids, albumin and other proteins, alginate and other polysaccharides, and combinations thereof (see, e.g., U. S. Pat. No. 5,814,599, each of which is incorporated herein by reference in its entirety for examples). A more detailed description of transdermal administration, formulations and suitable devices is disclosed in PCT Publication No. WO 2019 / 049816, which is incorporated herein by reference in its entirety for examples of formulations and devices that may be used in conjunction with the compositions and methods described herein.

[0306] It can be desirable to deliver the disclosed compounds to the subject over prolonged periods of time, for example, for periods of one week to one year from a single administration. Various slow release, depot or implant dosage forms can be utilized.

[0307] Suitable dosages are well known in the art. See, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000); Nursing 2001 Handbook of Drugs, 21st edition, Springhouse Corp., Springhouse, Pa.,2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, N. J. Preferred doses can optionally include about 0.1-99 and / or 100-500 mg / kg / administration, or any range, value or fraction thereof, or to achieve a serum concentration of about 0.1-5000 pg / ml serum concentration per single or multiple administration, or any range, value or fraction thereof. A preferred dosage range for the compositions or pharmaceutical compositions disclosed herein is from about 1 mg / kg, up to about 3, about 6 or about 12 mg / kg of body weight of the subject.

[0308] Alternatively, the dosage administered can vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired.

[0309] As a non-limiting example, treatment of humans or animals can be provided as a onetime or periodic dosage of the compositions or pharmaceutical compositions disclosed herein about 0.1 to 100 mg / kg or any range, value or fraction thereof per day, on at least one of day 1-40, or, alternatively or additionally, at least one of week 1-52, or, alternatively or additionally, at least one of 1-20 years, or any combination thereof, using single, infusion or repeated doses.

[0310] In aspects where the compositions to be administered to a subject in need thereof are modified cells as disclosed herein, the cells can be administered between about IxlO3and IxlO15cells; IxlO3and IxlO15cells, about IxlO4and IxlO12cells; about IxlO5and IxlO10cells; about IxlO6and IxlO9cells; about IxlO6and IxlO8cells; about IxlO6and IxlO7cells; or about IxlO6and 25xl06cells. In an aspect the cells are administered between about 5xl06and 25xl06cells.

[0311] A more detailed description of 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, which is incorporated herein by reference in its entirety for examples of transposases that may be used in conjunction with the compositions and methods described herein.

[0312] The disclosure provides the use of a disclosed composition or pharmaceutical composition for the treatment of a disease or disorder in a cell, tissue, organ, animal, or subject, as known in the art or as described herein, using the disclosed compositions and pharmaceutical compositions, e.g., administering or contacting the cell, tissue, organ, animal, or subject with a therapeutic effective amount of the composition or pharmaceutical composition. In an aspect, thesubject is a mammal. Preferably, the subject is human. The terms “subject” and “patient” are used interchangeably herein.

[0313] The disclosure provides a method for modulating or treating at least one malignant disease or disorder in a cell, tissue, organ, animal or subject. Non-limiting examples of a malignant disease or disorder include cancer and liver diseases or disorders.

[0314] Any method can comprise administering an effective amount of any composition or pharmaceutical composition disclosed herein to a cell, tissue, organ, animal or subject in need of such modulation, treatment or therapy. Such a method can optionally further comprise coadministration or combination therapy for treating such diseases or disorders, wherein the administering of any composition or pharmaceutical composition disclosed herein, further comprises administering, before concurrently, and / or after, at least one chemotherapeutic agent (e.g., an alkylating agent, an a mitotic inhibitor, a radiopharmaceutical).

[0315] In some aspects, the subject does not develop graft vs. host (GvH) and / or host vs. graft (HvG) following administration. In an aspect, the administration is systemic. Systemic administration can be any means known in the art and described in detail herein. Preferably, systemic administration is by an intravenous injection or an intravenous infusion. In an aspect, the administration is local. Local administration can be any means known in the art and described in detail herein. Preferably, local administration is by intra-tumoral injection or infusion, intraspinal injection or infusion, intracerebroventricular injection or infusion, intraocular injection or infusion, or intraosseous injection or infusion.

[0316] In some aspects, the therapeutically effective dose is a single dose. In some aspects, 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 in between that are manufactured simultaneously. In some aspects, where the composition is autologous cells or allogeneic cells, the dose is an amount sufficient for the cells to engraft and / or persist for a sufficient time to treat the disease or disorder.

[0317] In some aspects of the methods of treatment described herein, the treatment can be modified or terminated. Specifically, in aspects where the composition used for treatment comprises an inducible proapoptotic polypeptide, apoptosis may be selectively induced in the cell by contacting the cell with an induction agent. A treatment may be modified or terminated in response to, for example, a sign of recovery or a sign of decreasing diseaseseverity / progression, a sign of disease remission / cessation, and / or the occurrence of an adverse event. In some aspects, the method comprises the step of administering an inhibitor of the induction agent to inhibit modification of the cell therapy, thereby restoring the function and / or efficacy of the cell therapy (for example, when a sign or symptom of the disease reappear or increase in severity and / or an adverse event is resolved).Construction of Nucleic Acids

[0318] The isolated nucleic acids of the disclosure can be made using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and / or (d) combinations thereof, as well-known in the art.

[0319] The nucleic acids can conveniently comprise sequences in addition to a polynucleotide of the present disclosure. For example, a multi-cloning site comprising one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in isolation of the polynucleotide. Also, translatable sequences can be inserted to aid in the isolation of the translated polynucleotide of the disclosure. For example, a hexa-histidine marker sequence provides a convenient means to purify the proteins of the disclosure. The nucleic acid of the disclosure, excluding the coding sequence, is optionally a vector, adapter, or linker for cloning and / or expression of a polynucleotide of the disclosure.

[0320] Additional sequences can be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. Use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra, or Sambrook, supra).Recombinant Methods for Constructing Nucleic Acids

[0321] The isolated nucleic acid compositions of this 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 of skill in the art. In some aspects, oligonucleotide probes that selectively hybridize, under stringent conditions, to the polynucleotides of the present disclosure are used to identify the desired sequence in a cDNA or genomic DNA library. The isolation of RNA, and construction of cDNA and genomic libraries are well known to those of ordinary skill in the art. (See, e.g., Ausubel, supra, or Sambrook, supra).Nucleic Acid Screening and Isolation Methods

[0322] A cDNA or genomic library can be screened using a probe based upon the sequence of a polynucleotide of the disclosure. Probes can be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. Those of skill in the art will appreciate that various degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target 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, the stringency of hybridization is conveniently varied by changing the polarity of the reactant solution through, for example, manipulation of the concentration of formamide within the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding will vary in accordance with the stringency of the hybridization medium and / or wash medium. The degree of complementarity will optimally be 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.

[0323] Methods of amplification of RNA or DNA are well known in the art and can be used according to the disclosure without undue experimentation, based on the teaching and guidance presented herein.

[0324] Known methods of DNA or RNA amplification include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (see, e.g., U. S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 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; 5,066,584 to Gyllensten, et al; 4,889,818 to Gelfand, et al; 4,994,370 to Silver, et al; 4,766,067 to Biswas; 4,656,134 to Ringold) and RNA mediated amplification that uses anti-sense RNA to the target sequence as a template for double-stranded DNA synthesis (U. S. Pat. No. 5,130,238 to Malek, et al, with the tradename NASBA), the entire contents of which references are incorporated herein by reference. (See, e.g., Ausubel, supra, or Sambrook, supra.)

[0325] For instance, polymerase chain reaction (PCR) technology can be used to amplify the sequences of polynucleotides of the disclosure and related genes directly from genomic DNA orcDNA libraries. PCR and other in vitro amplification methods can also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to direct persons of skill through in vitro amplification methods are found in Berger, supra, Sambrook, supra, and Ausubel, supra, as well as Mullis, et al., U. S. Pat. No. 4,683,202 (1987); and Innis, et al., PCR Protocols A Guide to Methods and Applications, Eds., Academic Press Inc., San Diego, Calif. (1990). Commercially available kits for genomic PCR amplification are known in the art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech). Additionally, e.g., the T4 gene 32 protein (Boehringer Mannheim) can be used to improve yield of long PCR products.Synthetic Methods for Constructing Nucleic Acids

[0326] The isolated nucleic acids of the disclosure can also be prepared by direct chemical synthesis by known methods (see, e.g., 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. One of skill in the art will recognize that while chemical synthesis of DNA can be limited to sequences of about 100 or more bases, longer sequences can be obtained by the ligation of shorter sequences.Recombinant Expression Cassettes

[0327] The disclosure further provides recombinant expression cassettes comprising a nucleic acid of the disclosure. A nucleic acid sequence of the disclosure can be used to construct a recombinant expression cassette that can be introduced into at least one desired host cell. A recombinant expression cassette will typically comprise a polynucleotide of the disclosure operably linked to transcriptional initiation regulatory sequences that will direct the transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the disclosure.

[0328] In some aspects, isolated nucleic acids that serve as promoter, enhancer, or other elements can be introduced in the appropriate position (upstream, downstream or in the intron) of a non-heterologous form of a polynucleotide of the disclosure so as to up or down regulateexpression of a polynucleotide of the disclosure. For example, endogenous promoters can be altered in vivo or in vitro by mutation, deletion and / or substitution.Expression Vectors and Host Cells

[0329] The disclosure also relates to vectors that include isolated nucleic acid molecules of the disclosure and host cells that are genetically engineered with the recombinant vectors, as is well known in the art. See, e.g., Sambrook, et al., supra, Ausubel, et al., supra, each entirely incorporated herein by reference.

[0330] The polynucleotides can optionally be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

[0331] The DNA insert should be operatively linked to an appropriate promoter. The expression constructs will further contain sites fortranscription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (e.g., UAA, UGA or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG preferred for mammalian or eukaryotic cell expression.

[0332] Expression vectors will preferably but optionally include at least one selectable marker. Such markers include, e.g., but are not limited to, ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / Geneticin neo gene), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Methotrexate), mycophenolic acid, or glutamine synthetase (GS, U. S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739), blasticidin (bsd gene), resistance genes for eukaryotic cell culture as well as ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / Geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline resistance genes for culturing in E. coli and other bacteria or prokaryotics (the above patents are entirely incorporated hereby by reference). Appropriate culture mediums and conditions for the above-described host cells are known in the art. Suitable vectors will be readily apparent to the skilled artisan. Introduction of a vector construct into a host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other known methods. Suchmethods are described in the art, such as Sambrook, supra Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, 16.

[0333] Expression vectors will preferably but optionally include at least one selectable cell surface marker for isolation of cells modified by the compositions and methods of the disclosure. Selectable cell surface markers of the disclosure comprise surface proteins, glycoproteins, or group of proteins that distinguish a cell or subset of cells from another defined subset of cells. Preferably the selectable cell surface marker distinguishes those cells modified by a composition or method of the disclosure from those cells that are not modified by a composition or method of the disclosure. Such cell surface markers include, e.g., but are not limited to, “cluster of designation” or “classification determinant” proteins (often abbreviated as “CD”) such as a truncated or full length form of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. Cell surface markers further include the suicide gene marker RQR8 (Philip B et al. Blood. 2014 Aug 21; 124(8): 1277-87).

[0334] Expression vectors will preferably but optionally include at least one selectable drug resistance marker for isolation of cells modified by the compositions and methods of the disclosure. Selectable drug resistance markers of the disclosure may comprise wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[0335] Those of ordinary skill in the art are knowledgeable in the numerous expression systems available for expression of a nucleic acid molecule encoding a protein of the disclosure.Definitions

[0336] As used throughout the disclosure, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0337] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 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 meanwithin an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0338] In the chemical formulas shown herein, the marking indicates the position where a functional group bonds to another portion of a molecule. Definitions of specific functional groups and chemical terms are described in more detail below.

[0339] Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cisand trans-i somers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the 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 this invention.

[0340] Isomeric mixtures containing any of a variety of isomer ratios may be utilized in accordance with the present invention. For example, where only two isomers are combined, mixtures containing 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios are all contemplated by the present invention. Those of ordinary skill in the art will readily appreciate that analogous ratios are contemplated for more complex isomer mixtures.

[0341] If, for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where 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, and subsequent recovery of the pure enantiomers.

[0342] One of ordinary skill in the art will appreciate that the synthetic methods, as described herein, utilize a variety of protecting groups. By the term "protecting group," as used herein, it is meant that a particular functional moiety, e.g., O, S, or N, is temporarily blocked so that a reaction can be carried out selectively at another reactive site in a multifunctional compound. In certain embodiments, a protecting group reacts selectively in good yield to give a protectedsubstrate that is stable to the projected reactions; the protecting group should be selectively removable in good yield by readily available, preferably non-toxic reagents that do not attack the other functional groups; the protecting group forms an easily separable derivative (more preferably without the generation of new stereogenic centers); and the protecting group has a minimum of additional functionality to avoid further sites of reaction. As detailed herein, oxygen, sulfur, nitrogen, and carbon protecting groups may be utilized.

[0343] The term "aliphatic," as used herein, includes both saturated and unsaturated, straight chain (i.e., unbranched), branched, acyclic, cyclic, or polycyclic aliphatic hydrocarbons, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, "aliphatic" is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term "alkyl" includes straight, branched and cyclic alkyl groups. An analogous convention applies to other generic terms such as "alkenyl," "alkynyl," and the like. Furthermore, as used herein, the terms "alkyl," "alkenyl," "alkynyl," and the like encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, "lower alkyl" is used to indicate those alkyl groups (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) having 1-6 carbon atoms.

[0344] 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 6-18 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 yet 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 yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-4 carbon atoms. Illustrative aliphatic groups thus include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, — CH2-cyclopropyl, vinyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, — CFb-cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, cyclopentyl, — CFb-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, — CFh-cyclohexyl moieties and the like, which again, may bear one or moresubstituents. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, l-methyl-2-buten-l-yl, and the like. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

[0345] The term "alkyl" as used herein refers to saturated, straight- (e.g., unbranched) or branched-chain aliphatic groups having from 1 to 18 carbon atoms. As such, "alkyl" encompasses Ci, C2, C3, C4, Cs, Ce, C7, Cs, C9, C10, C11 and C12 groups. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, and dodecyl.

[0346] The term “alkylene” refers to a divalent alkyl radical. Any of the above-mentioned monovalent alkyl groups may be an alkylene by abstraction of a second hydrogen atom from the alkyl. As herein defined, alkylene may also be a Ci-Cis alkylene. An alkylene may further be a C1-C12 alkylene. Typical alkylene groups include, but are not limited to, -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH2CH(CH3)-, -CH2C(CH3)2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and the like.

[0347] The term "alkenyl" refers to an unsaturated straight or, when applicable, branched chain aliphatic group with one or more carbon-carbon double bonds, having from 2 to 18 carbon atoms. As such, "alkenyl" encompasses C2, C3, C4, Cs, Ce, C7, Cs, C9, C10, C11 and C12 groups. Alkenyl groups include, for example, ethenyl, propenyl, butenyl, l-methyl-2-buten-l-yl, and the like.

[0348] The term "alkynyl" refers to an unsaturated straight or, when applicable, branched chain aliphatic group with one or more carbon-carbon triple bonds, having from 2 to 18 carbon atoms. As such, "alkynyl" encompasses C2, C3, C4, C5, Ce, C7, Cs, C9, C10, C11 and C12 groups.Representative alkynyl groups include ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

[0349] As used herein, the term "aryl" group is a Ce - C14 aromatic moiety comprising one to three aromatic rings, which is optionally substituted. As such, "aryl" includes Ce, C7, Cs, C9, C10, C11, C12 Ci3, and C14 cyclic hydrocarbon groups. An exemplary aryl group is a Ce-Cio aryl group. Particular aryl groups include, without limitation, phenyl, naphthyl, anthracenyl, and fluorenyl.

[0350] As used herein, the term "cycloalkyl" as employed herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons. As such, "cycloalkyl" includes C3, C4, C5, Ce, C7, Cs, C9, C10, C11 and C12 cyclic hydrocarbon groups. Representative cycloalkylgroups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0351] As used herein, the term “hydroxyalkyl” refers to -alkyl-OH or an alkyl chain substituted with at least one -OH.

[0352] As used herein, the term “halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

[0353] It will be understood that the compounds of any one of the Formulae disclosed herein and any pharmaceutically acceptable salts thereof, comprise stereoisomers, mixtures of stereoisomers, polymorphs of all isomeric forms of said compounds.

[0354] The term "independently selected" is used herein to indicate that the R groups can be identical or different.

[0355] The term "substituted," whether preceded by the term "optionally" or not, and "substituent," as used herein, refer to the ability, as appreciated by one skilled in this art, to change one functional group for another functional group provided that the valency of all atoms is maintained. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. The substituents may also be further substituted (e.g., an aryl group substituent may have another substituent off it, such as another aryl group, which is further substituted with fluorine at one or more positions).

[0356] The 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 (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various aspects, the 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 sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free ofcellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the protein of the disclosure or biologically active portion thereof is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

[0357] The disclosure provides fragments and variants of the disclosed DNA sequences and proteins encoded by these DNA sequences. As used throughout the disclosure, the term "fragment" refers to a portion of the DNA sequence or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a DNA sequence comprising coding sequences may encode protein fragments that retain biological activity of the native protein and hence DNA recognition or binding activity to a target DNA sequence as herein described. Alternatively, fragments of a DNA sequence that are useful as hybridization probes generally do not encode proteins that retain biological activity or do not retain promoter activity. Thus, fragments of a DNA sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide of the disclosure.

[0358] Nucleic acids or proteins of the disclosure can be constructed by a modular approach including preassembling monomer units and / or repeat units in target vectors that can subsequently be assembled into a final destination vector. Polypeptides of the disclosure may comprise repeat monomers of the disclosure and can be constructed by a modular approach by preassembling repeat units in target vectors that can subsequently be assembled into a final destination vector. The disclosure provides polypeptide produced by this method as well nucleic acid sequences encoding these polypeptides. The disclosure provides host organisms and cells comprising nucleic acid sequences encoding polypeptides produced this modular approach.

[0359] The term "antibody" is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies) and antibody compositions with polyepitopic specificity. It is also within the scope hereof to use natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (herein collectively referred to as “analogs”) of the antibodies hereof as defined herein. Thus, according to an aspect hereof, the term “antibody hereof’ in its broadest sense also covers such analogs. Generally, in such analogs, one or more amino acid residues may have been replaced, deleted and / or added, compared to the antibodies hereof as defined herein.

[0360] The term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination when used for the intended purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants or inert carriers."Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps. Aspects defined by each of these transition terms are within the scope of this disclosure.

[0361] As used herein, "expression" refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0362] “ Gene expression” refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, micro RNA, structural RNA or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.

[0363] ‘ ‘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.

[0364] The term “operatively linked” or its equivalents (e.g., “linked operatively”) means two or more molecules are positioned with respect to each other such that they are capable of interacting to affect a function attributable to one or both molecules or a combination thereof.

[0365] Non-covalently linked components and methods of making and using non-covalently linked components, are disclosed. The various components may take a variety of different forms as described herein. For example, non-covalently linked (i.e., operatively linked) proteins may be used to allow temporary interactions that avoid one or more problems in the art. The ability of non-covalently linked components, such as proteins, to associate and dissociate enables afunctional association only or primarily under circumstances where such association is needed for the desired activity. The linkage may be of duration sufficient to allow the desired effect.

[0366] A method for directing proteins to a specific locus in a genome of an organism is disclosed. The method may comprise the steps of providing a DNA localization component and providing an effector molecule, wherein the DNA localization component and the effector molecule are capable of operatively linking via a non-covalent linkage.

[0367] 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.

[0368] The terms "nucleic acid" or "oligonucleotide" or "polynucleotide" refer to at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid may also encompass the complementary strand of a depicted single strand. A nucleic acid of the disclosure also encompasses substantially identical nucleic acids and complements thereof that retain the same structure or encode for the same protein.

[0369] Probes of the disclosure may comprise a single stranded nucleic acid that can hybridize to a target sequence under stringent hybridization conditions. Thus, nucleic acids of the disclosure may refer to a probe that hybridizes under stringent hybridization conditions.

[0370] Nucleic acids of the disclosure may be single- or double-stranded. Nucleic acids of the disclosure may contain double-stranded sequences even when the majority of the molecule is single-stranded. Nucleic acids of the disclosure may contain single- stranded sequences even when the majority of the molecule is double-stranded. Nucleic acids of the disclosure may include genomic DNA, cDNA, RNA, or a hybrid thereof. Nucleic acids of the disclosure may contain combinations of deoxyribo- and ribo-nucleotides. Nucleic acids of the disclosure may contain combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids of the disclosure may be synthesized to comprise non-natural amino acid modifications. Nucleic acids of the disclosure may be obtained by chemical synthesis methods or by recombinant methods.

[0371] Nucleic acids of the disclosure, either their entire sequence, or any portion thereof, may be non-naturally occurring. Nucleic acids of the disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not naturally-occur, rendering the entire nucleic acidsequence non-naturally occurring. Nucleic acids of the disclosure may contain one or more duplicated, inverted or repeated sequences, the resultant sequence of which does not naturally-occur, rendering the entire nucleic acid sequence non-naturally occurring. Nucleic acids of the disclosure may contain modified, artificial, or synthetic nucleotides that do not naturally-occur, rendering the entire nucleic acid sequence non-naturally occurring.

[0372] Given the redundancy in the genetic code, a plurality of nucleotide sequences may encode any particular protein. All such nucleotides sequences are contemplated herein.

[0373] As used throughout the disclosure, the term "operably linked" refers to the expression of a gene that is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5' (upstream) or 3' (downstream) of a gene under its control. The distance between a promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. Variation in the distance between a promoter and a gene can be accommodated without loss of promoter function.

[0374] As used throughout the disclosure, the term "promoter" refers to a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter can comprise one or more specific transcriptional regulatory sequences to further enhance expression and / or to alter the spatial expression and / or temporal expression of same. A promoter can also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter can regulate the expression of a gene component constitutively or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. 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 the CMV IE promoter.

[0375] As used throughout the disclosure, the term “substantially complementary" refers to a first sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99%identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

[0376] As used throughout the disclosure, the term "substantially identical" refers to a first and second sequence 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, if the first sequence is substantially complementary to the complement of the second sequence.

[0377] As used throughout the disclosure, the term "variant" when used to describe a nucleic acid, refers to (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.

[0378] As used throughout the disclosure, the term "vector" refers to a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector, and preferably, is a DNA plasmid. A vector may comprise a combination of an amino acid with a DNA sequence, an RNA sequence, or both a DNA and an RNA sequence.

[0379] As used throughout the 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 the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variant can also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity.

[0380] A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes canbe identified, in part, by considering the hydropathic index of amino acids, 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 a consideration of its hydrophobicity and charge. Amino acids of similar hydropathic indexes can be substituted and still retain protein function. In an aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U. S. Patent No. 4,554,101, incorporated fully herein by reference.

[0381] Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity. Substitutions can be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.

[0382] As used herein, “conservative” amino acid substitutions may be defined as set out in Tables 1, 2, or 3 below. In some aspects, fusion polypeptides and / or nucleic acids encoding such fusion polypeptides include conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the disclosure. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table 1.

[0383] Table 1 - Conservative Substitutions I

[0384] Alternately, conservative amino acids can be grouped as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY, N. Y. (1975), pp. 71-77) as set forth in Table 2.

[0386] Alternately, exemplary conservative substitutions are set out in Table 3.Table 3 - Conservative Substitutions III

[0387] It should be understood that the polypeptides of the disclosure are intended to include polypeptides bearing one or more insertions, deletions, or substitutions, or any combination thereof, of amino acid residues as well as modifications other than insertions, deletions, or substitutions of amino acid residues. Polypeptides or nucleic acids of the disclosure may contain one or more conservative substitution.

[0388] As used throughout the disclosure, the term “more than one” of the aforementioned 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 recited amino acid substitutions. The term “more than one” may refer to 2, 3, 4, or 5 of the recited amino acid substitutions.

[0389] Polypeptides and proteins of the disclosure, either their entire sequence, or any portion thereof, may be non-naturally occurring. Polypeptides and proteins of the disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not naturally-occur, rendering the entire amino acid sequence non-naturally occurring. Polypeptides and proteins of the disclosure may contain one or more duplicated, inverted or repeated sequences, the resultant sequence of which does not naturally-occur, rendering the entire amino acid sequence non-naturally occurring. Polypeptides and proteins of the disclosure may contain modified, artificial,or synthetic amino acids that do not naturally-occur, rendering the entire amino acid sequence non-naturally occurring.

[0390] As used throughout the disclosure, “sequence identity” may be determined by using the stand-alone executable BLAST engine program for blasting two sequences (bl2seq), which can be retrieved from the National Center for Biotechnology Information (NCBI) ftp site, using the default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; which is incorporated herein by reference in its entirety). The terms "identical" or "identity" when used in the context of two or more nucleic acids or polypeptide sequences, refer to a specified percentage of residues that are the same over a specified region of each of the sequences. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield 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 yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0391] As used throughout the disclosure, the term "endogenous" refers to nucleic acid or protein sequence naturally associated with a target gene or a host cell into which it is introduced.

[0392] As used throughout the disclosure, the term "exogenous" refers to nucleic acid or protein sequence not naturally associated with a target gene or a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid, e.g., DNA sequence, or naturally occurring nucleic acid sequence located in a non-naturally occurring genome location.

[0393] The disclosure provides methods of introducing a polynucleotide construct comprising a DNA sequence into a host cell. By "introducing" is intended presenting to the cell the polynucleotide construct in such a manner that the construct gains access to the interior of the host cell. The methods of the disclosure do not depend on a particular method for introducing a polynucleotide construct into a host cell, only that the polynucleotide construct gains access tothe interior of one cell of the host. Methods for introducing polynucleotide constructs into bacteria, plants, fungi and animals are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0394] EXAMPLES

[0395] For the following Examples, Compound Numbers are assigned to compounds of the present disclosure according to the following:

[0396] Example 1 — Preparation of COMPOUND NO. 1

[0397] COMPOUND NO. 1 was prepared in accordance with the General Scheme (A). 'H NMR (500 MHz, CDCl3): 55.08-5.04 (t, 1H, J=10Hz), 4.92-4.89 (dd, 1H, 5 Hz, 10 Hz), 4.51-4.48 (dd, J=5Hz, 10 Hz), 4.45 (d, 1H, J=10 Hz), 4.38-4.35 (m, 1H), 3.86-3.83 (m, 1H), 3.70 (t,1H, J=10 Hz), 3.56-3.53 (tn, 1H), 3.46-3.41 (m, 1H), 3.04-2.84 (m, 2H), 2.87-2.84 (tn, 2H), 2.74-2.70 (m, 6H), 2.68-2.65 (m, 4H), 2.39-2.10 (m, 6H), 1.57-1.53 (m, 4H), 1.41-1.16 (m, 44H), 0.89-0.86 (m, 9H). MS found 830.3 [M+H] - calcd for [C46H87NO7S=829.0]

[0398] Example 2 — Preparation of COMPOUND NO. 2

[0399] COMPOUND NO. 2 was prepared in accordance with the General Scheme (A).1H NMR (500 MHz, CDCl3): 55.09-5.05 (t, 1H, J=10Hz), 4.93-4.90 (dd, 1H, J=5 Hz, 10 Hz), 4.58-4.55 (dd, J=5Hz, 10 Hz), 4.45 (d, 1H, J=10 Hz), 4.25-4.22 (m, 1H), 3.86-3.84 (m, 1H), 3.71 (t, 1H, J=10Hz), 3.50-3.48 (m, 1H), 3.44-3.42 (m, 1H), 2.82-2.76 (m, 1H), 2.63-2.49 (m, 3H), 2.42-2.34 (m, 3H), 2.29-2.19 (m,14H), 1.69-1.53 (m, 9H), 1.34-1.21 (m, 42H), 0.89-0.86 (m, 9H). MS found 827.3 [M+H]+- calcd for [C47H9ON209=826.0]

[0400] Example 3 — Preparation of COMPOUND NO. 3

[0401] COMPOUND NO. 3 was prepared in accordance with the General Scheme (A).1H NMR (500 MHz, CDCI3): 55.10-5.06 (t, 1H, J=10Hz), 4.96-4.91 (dd, 1H, J=5 Hz, 10 Hz), 4.80-4.77 (dd, J=5Hz, 10 Hz), 4.45 (d, 1H, J=10 Hz), 4.49-4.44 (m, 1H), 4.15-4.13 (m, 1H), 3.86-3.82 (m, 1H), 3.73-3.69 (t, 1H, J=10Hz), 3.58-3.52 (m, 1H), 3.46-3.04 (m, 1H), 2.88-2.82 (m, 2H), 2.72-2.55 (m, 2H), 2.42-2.24 (m, 10H), 1.59-1.52 (m, 12H), 1.34-1.21 (m, 40H), 0.89-0.86 (m, 9H). MS found 796.4 [M+HJ - calcd for [C46H85NO9=795.0]

[0402] Example 4 — Preparation of COMPOUND NO. 4

[0403] COMPOUND NO. 4 was prepared in accordance with the General Scheme (A).1H NMR (500 MHz, CDCl3): 85.08-5.04 (t, 1H, J=10Hz), 4.93-4.89 (dd, 1H, J=5 Hz, 10 Hz), 4.45 (d, 1H, J-10 Hz), 4.38-4.29 (m, 2H), 4.49-4.44 (m, 1H), 4.15-4.13 (m, 1H), 3.86-3.82 (m, 1H), 3.77-3.75 (t, 1H, J=10Hz), 3.48-3.40 (m, 2H), 2.83-2.72 (m, 1H), 2.71-2.67 (m, 1H), 2.54-2.35 (m, 8H), 2.32-2.18 (m, 18H), 1.71-1.45 (m, 8H), 1.34-1.15 (m, 42H), 0.89-0.86 (m, 9H). MS found 898.4 [M+H]+- calcd for [C5iH99N3O9=897.0]

[0404] Example 5 — Preparation of COMPOUND NO. 5

[0405] COMPOUND NO. 5 was prepared in accordance with the General Scheme (A).1H NMR (500 MHz, CDCl3): 88.29-828 (d, 2H, J=5Hz), 6.66-6.65 (d, 2H, J-5Hz), 5.07-5.03 (t, 1H, J=10Hz), 4.95-4.92 (dd, 1H, J=5 Hz, 10 Hz), 4.56-4.53 (dd, 1H, J=5Hz, 10 Hz), 4.44 (d, 1H, J=10 Hz), 4.41-4.37 (m, 2H), 3.88-3.82 (m, 4H), 3.67-3.62 (m, 1H), 3.62-3.55(m, 2H), 3.45-3.36 (m, 2H), 3.29-3.26 (m, 4H), 2.79-2.52 (m, 3H), 2.38-2.25 (m, 4H), 1.64-1.45 (m, 6H), 1.34-1.15 (m, 44H), 0.89-0.86 (m, 9H). MS found 875.5 [M]+> calcd for [C5OH87N2OIO=875.0]

[0406] Example 6 — Preparation of COMPOUND NO. 6

[0407] COMPOUND NO. 6 was prepared in accordance with the General Scheme (A).JH NMR (500 MHz, CDCl3): 85.07-5.04 (t, 1H, J=10Hz), 4.96-4.89 (dd, 1H, J=5 Hz, 10 Hz), 4.58-4.53 (dd, 1H, J=5Hz, 10 Hz), 4.44 (d, 1H, J= 10 Hz), 4.31-4.18 (m, 1H), 3.88-3.82 (m, 2H), 3.71-3.62 (m,3H), 3.65-3.38(m, 3H), 2.75-2.52 (m, 10H), 2.28-2.15 (m, 6H), 1.64-1.45 (m, 6H), 1.34-1.15 (m, 44H), 0.89-0.86 (m, 15H). MS found 883.4 [M+H]+> calcd for [C5oH94N2Oio=882]

[0408] Example 7 — Preparation of COMPOUND NO. 7

[0409] COMPOUND NO. 7 was prepared in accordance with the General Scheme (A).1H NMR (500 MHz, CDCl3): 85.09-5.06 (t, 1H, J=10Hz), 4.93-4.90 (dd, 1H, J=5 Hz, 10 Hz), 4.85-4.82 (dd, 1H, J=5Hz, 10 Hz), 4.45 (d, 1H, J=10 Hz), 4.09-4.06 (m, 1H), 3.85-3.82 (m, 1H), 3.66 (t, 1H, J=10Hz), 3.54-3.52 (m,lH), 3.45-3.43(m, 1H), 2.98-2.96 (m, 1H), 2.79-2.77 (d, 1H, J=10Hz), 2.67-2.43 (m, 4H), 2.43-2.45 (d, 1H, J=10Hz), 2.29-2.24 (m, 4H), 1.98-1.78 (m, 8H), 1.59-1.52 (m, 6H), 1.29-1.24 (m, 44H), 0.89-0.86 (m, 9H). MS found 822.4 [M+H]+- calcd for [C48H87NO9=821]

[0410] Example 8 — Preparation of COMPOUND NO. 8

[0411] COMPOUND NO. 8 was prepared in accordance with the General Scheme (A). ’H NMR (500 MHz, CDCl3): 65.09-5.06 (t, 1H, J=10Hz), 4.94-4.90 (dd, 1H, J=5 Hz, 10 Hz), 4.72-4.69 (dd, 1H, J=5Hz, 10 Hz), 4.44 (d, 1H, J=10 Hz), 4.16-4.13 (m, 1H), 3.88-3.81 (m, 1H), 3.71-3.62 (m,3H), 3.51-3.41(m, 2H), 2.85-2.79 (m, 1H), 2.63-2.45 (m, 4 H), 2.58-2.49 (m, 5H), 2.30-2.25 (m, 3H), 1.64-1.45 (m, 12H), 1.34-1.15 (m, 44H), 1.04-0.99 (t, 3H, J=10Hz), 0.89-0.86 (m, 9H). MS found 869.2 [M+H]+- calcd for [C49H92N20IO=868]

[0412] Example 9 — Preparation of COMPOUND NO. 9

[0413] COMPOUND NO. 9 was prepared in accordance with the General Scheme (A). 'H NMR (500 MHz, CDCl3): 85.15-5.06 (m, 2H), 4.94-4.90 (dd, 1H, J=5 Hz, 10 Hz), 4.86-4.83 (dd, 1H, J=5Hz, 10 Hz), 4.44 (d, 1H, J=10 Hz), 4.35-4.34 (m, 1H), 4.11-4.08- (m, 1H), 3.84-3.82 (m,lH), 3.70-3.65 (m, 1H), 3.55-3.51(m, 2H), 3.46-3.42 (m, 1H), 2.75-2.72 (m, 1H), 2.65-2.58 (m, 2 H), 2.53-2.51 (m, 1H), 2.38-2.25 (m, 4H), 1.64-1.45 (m, 8H), 1.34-1.15 (m, 44H), 0.89-0.86 (m, 9H). MS found 756.6 [M+HJ - calcd for [C43H8INO9=755]

[0414] Example 10 — Preparation of COMPOUND NO. 10

[0415] COMPOUND NO. 10 was prepared in accordance with the General Scheme (A). 'H NMR (500 MHz, CDCl3): 85.01-5.05 (t, J=10 Hz, 1H), 4.93 (t, J=10 Hz, 1H), 4.52-4.50 (dd, 1H,J=5Hz, 10Hz), 4.44 (d, 1H, J=10 Hz), 4.35-4.32 (tn, 1H), 3.87-3.82 (m, 1H), 3.70 (t, 4H, J=5Hz), 3.63 (t, 1H, t=10Hz), 3.52-3.49 (m,lH), 3.46-3.41 (tn, 1H), 2.85-2.82 (m, 2H), 2.71-2.65 (m, 4 H), 2.59-2.56 (m, 2H), 2.48-2.45 (m, 4H), 2.32-2.24 (m, 4H), 1.64-1.45 (m, 6H), 1.34-1.15 (m, 44H), 0.89-0.86 (m, 9H). MS found 858.3 [M+H] calcd for [C47H87NOIOS=857]

[0416] Example 11 — Preparation of COMPOUND NO. 11

[0417] COMPOUND NO. 11 was prepared in accordance with the General Scheme (A). ' H NMR (500 MHz, CDCl3): 85.06-5.02 (t, J=10 Hz, 1H), 4.95-4.91 (t, J=10 Hz, 1H), 4.56-4.53 (dd, 1H, J=5Hz, 10Hz), 4.44 (d, 1H, J=10 Hz), 4.29-4.26 (m, 1H), 3.87-3.81 (m, 1H), 3.61 (t, 1H, t=10Hz), 3.52-3.49 (m,lH), 3.45-3.41 (m, 1H), 2.44-2.38 (m, 2H), 2.33-2.24 (m, 5 H), 2.20 (s, 6H), 1.88-1.74(m, 2H), 1.64-1.45 (m, 6H), 1.34-1.15 (m, 44H), 0.89-0.86 (m, 9H). MS found 770.5 [M+H] calcd for [C44H83NO9=809]

[0418] Example 12 — Preparation of COMPOUND NO. 12

[0419] COMPOUND NO. 12 was prepared in accordance with the General Scheme (A).JH NMR (500 MHz, CDCl3): 85.09-5.05 (t, J=10 Hz, 1H), 4.93-4.90 (t, J=10 Hz, 1H), 4.80-4.78 (m, 1H), 4.72-4.67 (m, 4H), 4.44 (d, 1H, J=10 Hz), 4.17-4.13 (m, 1H), 3.85-3.83 (m, 1H), 3.63 (t, 1H, J=10Hz), 3.53-3.52 (m,lH), 3.45-3.34 (m, 3H), 2.84-2.78 (m, 1H), 2.61-2.58 (m, 1 H), 2.47-2.24 (m, 6H), 1.8 (br, 1H), 1.59-1.54(m, 6H), 1.34-1.15 (m, 44H), 0.89-0.86 (m, 9H). MS found 810.5 [M+H] - calcd for [C46H83NOio=809]

[0420] Example 13 — Preparation of COMPOUND NO. 13

[0421] COMPOUND NO. 13 was prepared in accordance with the General Scheme (A). 'H NMR (500 MHz, CDCl3): 85.08-5.04 (t, J=10 Hz, 1H), 4.92-4.89 (t, >10 Hz, 1H), 4.72-4.69 (dd, 1H, J=5Hz, 10Hz), 4.72-4.56 (m, 4H), 4.44 (d, 1H, J=10 Hz), 4.18-4.15 (m, 1H), 3.87-3.81 (m, 1H), 3.63 (t, 1H, t=10Hz), 3.53-3.52 (m,lH), 3.45-3.42 (m, 1H), 2.99-2.90 (m, 2H), 2.64-2.53 (m, 4 H), 2.32-2.25 (m, 3H), 2.13-2.11 (m, 1H), 1.88-1.74(m, 1H), 1.59-1.54 (m, 8H), 1.34-1.15 (m, 44H), 0.89-0.86 (m, 9H). MS found 824.6 [M+H]+’ calcd for [C47H8SNOIO=823]

[0422] Example 14 — Preparation of COMPOUND NO. 14

[0423] COMPOUND NO. 14 was prepared in accordance with the General Scheme (A). 'H NMR (500 MHz, CDCl3): 85.11-5.07 (t, J=10 HZ, 1H), 4.95-4.91 (t, J=10 Hz, 1H), 4.85-4.82 (dd, 1H, J=5Hz, 10Hz), 4.44 (d, 1H, J=10 Hz), 4.12-4.09 (m, 1H), 3.87-3.82 (m, 1H), 3.70 (t, 1H, t=10Hz), 3.55-3.52 (m,lH), 3.46-3.42 (m, 1H), 3.03-2.98(m, 1H), 2.85-2.81 (m, 1H), 2.72-2.45 (tn, 5 H), 2.27-2.24 (tn, 4H), 1.81-1.77(m, 2H), 1.56-1.55 (tn, 6H), 1.34-1.15 (m, 44H), 0.89-0.86 (m, 9H), 0.55-0.48 (m, 4H). MS found 808.4 [M+H]+’ calcd for [C47Hs5NO9=807]

[0424] Example 15 — Preparation of COMPOUND NO. 15

[0425] COMPOUND NO. 15 was prepared in accordance with the General Scheme (A).JH NMR (500 MHz, CDCl3): 85.10-5.06 (t, J=10 Hz, 1H), 4.94-4.90 (t, J=10 Hz, 1H), 4.79-4.76 (dd, 1H, J=5Hz, 10Hz), 4.45 (d, 1H, J=10 Hz), 4.14-4.11 (m, 1H), 3.87-3.82 (m, 1H), 3.70 (t, 1H, t=10Hz), 3.55-3.52 (m,lH), 3.46-3.42 (m, 1H), 2.90-2.88(m, 1H), 2.63-2.51 (m, 6H), 2.72-2.45 (m, 4 H), 1.61-1.56(m, 14H), 1.41-1.39 (m, 4H), 1.34-1.15 (m, 44H), 0.89-0.86 (m, 9H). MS found 850.6 [M+H] ‘ calcd for [C5oH9iN09=849].

[0426] Example 16 — Preparation of COMPOUND NO. 16

[0427] COMPOUND NO. 16 was prepared in accordance with the General Scheme (A). 'H NMR (500 MHz, CDCl3): 85.12-5.08 (t, J=10 Hz, 1H), 4.93-4.90 (t, J=10 Hz, 1H), 4.80-4.76 (dd, 1H, J=5Hz, 10Hz), 4.46 (d, 1H, J=10 Hz), 4.14-4.11 (m, 1H), 3.87-3.82 (m, 1H), 3.74 (t, 1H, t=10Hz), 3.67 (br, 1H), 3.54-3.45 (m,lH), 3.44-3.42 (m, 1H), 3.08-2.88(m, 5H), 2.56-2.54 (m, 1H), 2.47-2.42 (m, 1 H), 2.31-2.24(m, 4H), 2.6-2.14(s, 6H), 1.58-1.53 (m, 6H), 1.34-1.15 (m, 44H), 0.89-0.86 (m, 9H). MS found 811.5 [M+H]+- calcd for [C46Hs6N2O9=810].

[0428] Example 17 — Preparation of COMPOUND NO, 17

[0429] COMPOUND NO. 17 was prepared in accordance with the General Scheme (A). 'H NMR (500 MHz, CDCl3) 85.08 (t, J= 9.6 Hz, 1H), 4.92 (dd, J= 9.8, 7.9 Hz, 1H), 4.77 (dd, J= 12.1, 4.1 Hz, 1H), 4.45 (d,. / = 8.0 Hz, 1H), 4.28 (d,,7= 7.6 Hz, 1H), 4.13 (dd,. / = 12.0, 3.6 Hz, 1H), 3.84 (dt, J= 9.6, 6.5 Hz, 1H), 3.74 (t, J= 9.5 Hz, 1H), 3.57 (dt, J= 9.6, 3.8 Hz, 1H), 3.44 (dt, J= 9.6, 6.9 Hz, 1H), 3.09 - 2.95 (m, 2H), 2.85 (d, J= 10.4 Hz, 1H), 2.66 - 2.50 (m, 3H), 2.41 (dd, J= 10.4, 5.5 Hz, 1H), 2.37 - 2.19 (m, 4H), 2.11 (q, J= 8.6 Hz, 1H), 1.70 (dtd, J= 13.6, 8.0, 2.6 Hz, 2H), 1.66 - 1.47 (m, 6H), 1.34 - 1.17 (m, 42H), 0.88 (td, J= 7.0, 1.2 Hz, 9H). MS: m / z 798.3 (M+Na).

[0430] Example 18 — Preparation of COMPOUND NO. 18

[0431] COMPOUND NO. 18 was prepared in accordance with the General Scheme (A). 'H NMR (500 MHz, CDCl3) 85.08 (t, J = 9.6 Hz, 1H), 4.95 - 4.82 (m, 2H), 4.45 (d, J = 8.0 Hz, 1H), 4.07 (dd, J= 12.2, 2.2 Hz, 1H), 3.87 (ddt, J= 12.8, 9.7, 5.9 Hz, 2H), 3.70 (t, J= 9.6 Hz, 1H), 3.50 (ddd, J= 9.7, 3.5, 2.2 Hz, 1H), 3.44 (dt, J= 9.7, 6.9 Hz, 1H), 3.24 (s, 3H), 3.03 - 2.90 (m, 1H), 2.87 - 2.71 (m, 2H), 2.71 - 2.58 (m, 2H), 2.58 -2.47 (m, 2H), 2.41 (td, J= 8.5, 6.1 Hz,1H), 2.34-2.18 (m, 4H), 2.05 (dtd, J = 13.7, 8.0, 5.8 Hz, 1H), 1.83 (dddd, J= 14.0, 8.4, 6.3, 2.5 Hz, 1H), 1.65 - 1.51 (m, 6H), 1.26 (d, J= 7.2 Hz, 42H), 0.92 - 0.83 (m, 9H). MS: m / z 812.3 (M+Na).

[0432] Example 19 — Preparation of COMPOUND NO. 19

[0433] COMPOUND NO. 19 was prepared in accordance with the General Scheme (A). 'H NMR (500 MHz, CDCl3) 55.07 (t, J= 9.5 Hz, 1H), 4.92 (dd, J= 9.8, 8.0 Hz, 1H), 4.76 (dd, J= 12.2, 3.8 Hz, 1H), 4.44 (d, J= 7.9 Hz, 1H), 4.11 (dd, J= 12.2, 2.3 Hz, 1H), 3.95 - 3.80 (m, 2H), 3.68 (t, J= 9.5 Hz, 1H), 3.52 (ddd, J= 9.7, 3.8, 2.3 Hz, 1H), 3.44 (dt, J= 9.6, 6.9 Hz, 1H), 3.24 (s, 3H), 2.92 (ddd, J= 11.7, 8.4, 6.0 Hz, 1H), 2.82 - 2.42 (m, 7H), 2.33 - 2.22 (m, 4H), 2.03 (dq, J= 14.2, 7.4 Hz, 1H), 1.78 (dddd, J= 13.5, 8.1, 5.6, 2.8 Hz, 1H), 1.61 - 1.52 (m, 6H), 1.26 (d, J = 6.6 Hz, 42H), 0.88 (td, J= 7.0, 1.6 Hz, 9H). MS: m / z 812.2 (M+Na).

[0434] Example 20 — Preparation of COMPOUND NO. 20

[0435] COMPOUND NO. 20 was prepared in accordance with the General Scheme (A). ’H NMR (500 MHz, CDCl3) 85.07 (td, J= 9.6, 2.2 Hz, 1H), 4.91 (dt, J= 9.7, 7.7 Hz, 1H), 4.72 (ddd, J= 12.2, 4.0, 2.5 Hz, 1H), 4.45 (dd, J= 7.9, 1.7 Hz, 1H), 4.19 (dt, J= 12.2, 3.0 Hz, 1H), 3.84 (dt, J= 9.6, 6.4 Hz, 1H), 3.66 (td, J= 9.5, 6.6 Hz, 1H), 3.57 - 3.48 (m, 1H), 3.44 (ddd, J = 10.0, 7.2, 6.1 Hz, 1H), 2.99 - 2.80 (m, 2H), 2.80 - 2.75 (m, 1H), 2.74 - 2.64 (m, 2H), 2.63 - 2.49 (m, 3H), 2.26 (dtd, J= 10.1, 6.8, 2.9 Hz, 4H), 2.13 (tt, J= 13.2, 6.8 Hz, 1H), 1.87 - 1.73 (m, 1H), 1.26 (d, J= 7 A Hz, 38H), 0.91 - 0.83 (m, 9H). MS: m / z 844.3 (M+Na).

[0436] Example 21 — Preparation of COMPOUND NO. 21

[0437] COMPOUND NO. 21 was prepared in accordance with the General Scheme (A).JH NMR (500 MHz, CDCl3) 85.05 (t, J= 9.5 Hz, 1H), 4.91 (dd, J= 9.8, 7.9 Hz, 1H), 4.66 (dd, J= 12.2, 3.9 Hz, 1H), 4.45 (dd, J= 8.0, 5.6 Hz, 1H), 4.24 (dd, J= 12.2, 2.5 Hz, 1H), 3.84 (dt, J = 9.6, 6.4 Hz, 1H), 3.62 (td, J= 9.5, 4.1 Hz, 1H), 3.51 (ddd, J= 9.7, 3.9, 2.5 Hz, 1H), 3.44 (dtd, J = 9.8, 6.8, 3.3 Hz, 1H), 3.36 (d, J= 4.9 Hz, 1H), 3.04 - 2.95 (m, 1H), 2.95 - 2.84 (m, 2H), 2.83 - 2.69 (m, 2H), 2.63 - 2.48 (m, 2H), 2.38 - 2.20 (m, 6H), 1.26 (d, J= 6.9 Hz, 36H), 0.88 (t, J= 6.8 Hz, 9H). MS: m / z 818.3 (M+Na).

[0438] Example 22 — Preparation of COMPOUND NO, 22

[0439] COMPOUND NO. 22 was prepared in accordance with the General Scheme (A).JH NMR (500 MHz, CDCl3) 85.24 - 5.09 (m, 1H), 5.06 (t, J= 9.6 Hz, 1H), 4.90 (dd, J= 9.7, 8.0 Hz, 1H), 4.76 (dd, J= 12.2, 3.8 Hz, 1H), 4.44 (d, J= 7.9 Hz, 1H), 4.16 (dd, J= 12.3, 2.4 Hz,1H), 3.85 (dt, J= 9.1, 6.5 Hz, 1H), 3.66 (t, J= 9.5 Hz, 1H), 3.50 (ddd, J= 9.1, 3.9, 2.4 Hz, 1H), 3.44 (dt, J= 9.6, 6.8 Hz, 1H), 3.01 - 2.83 (m, 3H), 2.83 - 2.70 (m, 2H), 2.67 - 2.46 (m, 3H), 2.27 (dtd, J= 12.8, 7.5, 2.7 Hz, 4H), 2.19 - 1.99 (m, 2H), 1.62 - 1.51 (m, 6H), 1.26 (d, J= 6.3 Hz, 42H), 0.88 (t, J= 6.8 Hz, 9H). MS: m / z 800.4 (M+Na).

[0440] Example 23 — Preparation of COMPOUND NO. 23

[0441] COMPOUND NO. 23 was prepared in accordance with the General Scheme (A).1H NMR (500 MHz, CDCl3) 85.24 - 5.08 (m, 1H), 5.06 (t, J= 9.6 Hz, 1H), 4.91 (dd, J= 9.1, 8.0 Hz, 1H), 4.73 (dd, J= 12.2, 4.0 Hz, 1H), 4.44 (d, J= 7.9 Hz, 1H), 4.17 (dd, J= 12.2, 2.6 Hz, 1H), 3.84 (dt, J= 9.6, 6.4 Hz, 1H), 3.65 (t, J= 9.5 Hz, 1H), 3.52 (ddd, J= 9.7, 4.0, 2.5 Hz, 1H), 3.44 (dt, J= 9.6, 6.8 Hz, 1H), 2.95 (ddd, J= 12.0, 8.0, 6.4 Hz, 1H), 2.89 - 2.79 (m, 3H), 2.76 (dt, J= 12.2, 6.3 Hz, 1H), 2.66 - 2.49 (m, 3H), 2.27 (dtd, J= 12.2, 7.5, 2.9 Hz, 4H), 2.19 - 1.98 (m, 2H), 1.62 - 1.52 (m, 6H), 1.32 - 1.22 (m, 42H), 0.88 (td, J = 7.0, 1.3 Hz, 9H). MS: m / z 649.3 (M+Na). MS: m / z 800.3 (M+Na).

[0442] Example 24 — Preparation of COMPOUND NO. 24

[0443] COMPOUND NO. 24 was prepared in accordance with the General Scheme (A).1H NMR (500 MHz, CDCl3) 85.08 (t, J= 9.6 Hz, 1H), 4.92 (dd, J= 9.8, 7.9 Hz, 1H), 4.77 (dd, J = 12.1, 4.1 Hz, 1H), 4.45 (d, J = 8.0 Hz, 1H), 4.28 (d, J = 7.6 Hz, 1H), 4.13 (dd, J = 12.0, 3.6 Hz, 1H), 3.84 (dt, J= 9.6, 6.5 Hz, 1H), 3.74 (t, J= 9.5 Hz, 1H), 3.57 (dt, J= 9.6, 3.8 Hz, 1H), 3.44 (dt, J= 9.6, 6.9 Hz, 1H), 3.09 - 2.95 (m, 2H), 2.85 (d, J= 10.4 Hz, 1H), 2.66 - 2.50 (m, 3H), 2.41 (dd,.7 = 10.4, 5.5 Hz, 1H), 2.37 -2.19 (m, 4H), 2.11 (q,.7= 8.6 Hz, 1H), 1.70 (dtd,,7= 13.6, 8.0, 2.6 Hz, 2H), 1.66 - 1.47 (m, 6H), 1.34 - 1.17 (m, 42H), 0.88 (td, J= 7.0, 1.2 Hz, 9H). MS: m / z 798.3 (M+Na).

[0444] Example 25 — Preparation of COMPOUND NO. 25

[0445] COMPOUND NO. 25 was prepared in accordance with the General Scheme (A). 'll NMR (500 MHz, CDCl3): 85.06-5.03 (t, J=10 Hz, 1H), 4.94-4.90 (t, J=10 Hz, 1H), 4.47-4.43 (m, 2H), 4.40-4.38 (m, 1H), 4.14-4.11 (m, 1H), 3.85-3.83 (m, 1H), 3.62-3.48 (m, 4H), 3.47-3.42 (m,2H), 3.42 (m, 9H), 2.80-2.77 (t, 1H, J=10 Hz), 2.68-2.88 (t, 1H, J=10 Hz), 2.54-2.51 (t, 1H, J=10 Hz), 2.34-2.23 (m, 4H), 1.77-1.47(m, 13H), 1.45-1.27 (m, 56H), 0.89-0.86 (m, 9H). MS found 956.2 [M] - calcd for [C55HIO6N09S+=956],

[0446] Example 26 — Preparation of COMPOUND NO. 26

[0447] COMPOUND NO. 26 was prepared in accordance with the General Scheme (A). ' H NMR (500 MHz, CDCl3): 55.10-5.06 (t, J=10 Hz, 1H), 4.92-4.88 (t, J=10 Hz, 1H), 4.62-4.59 (dd, 1H, J=5Hz, 10Hz), 4.46 (d, 1H, J=10 Hz), 4.29-4.27 (m, 1H), 4.17-4.15 (m, 1H), 4.04 (m, 1H), 3.85-3.80 (m, 2H), 3.57-3.54 (m, 1H,), 3.43-3.34 (m,2H), 3.31-3.20 (m, 2H), 3.0-2.95(m, 1H), 2.53-2.41 (br, 1H), 2.36-2.15 (m, 6H), 1.77-1.44 (m, 10H), 1.34-1.15 (m, 44H), 0.89-0.86 (m, 9H). MS found 808.2 [M+H]+> calcd for [C47H85NO9=807].

[0448] Example 27 — Preparation of COMPOUND NO. 27

[0449] COMPOUND NO. 27 was prepared in accordance with the General Scheme (A).1H NMR (500 MHz, CDCl3): 85.09-5.07 (t, J=10 Hz, 1H), 4.97 (br, 1H), 4.89-4.86 (t, J=10 Hz, 1H), 4.74-4.70 (dd, 1H, J=5Hz, 10Hz), 4.45 (d, 1H, J=10Hz), 4.20-4.17 (m, 1H), 4.17-4.15 (m, 1H), 3.86-3.82 (m, 1H), 3.68 (t, 1H, J=10HZ), 3.51-3.41 (m,2H), 3.31-3.20 (m, 2H), 3.35 (s, 2H), 2.81-2.55 (m, 6H), 2.31-2.25 (m, 2H), 2.2-1.86 (m, 4H), 1.62-1.45 (m, 6H), 1.34-1.15 (m, 44H), 0.89-0.86 (m, 9H). MS found 867.3 [M+H] - calcd for [C48H86N2On=866].

[0450] Example 28 — Preparation of COMPOUND NO. 28

[0451] COMPOUND NO. 28 was prepared in accordance with the General Scheme (A).1H NMR (500 MHz, CDCl3): 85.12-5.08 (t, J=10 Hz, 1H), 5.08-5.05 (t, 1H, J=10Hz), 4.94-4.89 (m, 2H), 4.68-4.65 (dd, 1H, J=5Hz, 10Hz), 1.47- 4.44 (m, 2H), 4.45 (d, 1H, J=10 Hz), 4.35-4.32 (m, 1H), 4.20-4.18 (m, 1H), 3.88-3.82 (m, 2H), 3.68 (t, 1H, J=10HZ), 3.64 (t, 1H, J=10 HZ), 3.56-3.53 (m,2H), 3.46-3.41 (m, 2H), 2.83-2.15 (m, 8H), 1.81-1.55 (m, 7H), 1.34-1.15 (m, 44H), 0.89-0.86 (m, 9H). MS found 851.3 [M+H] - calcd for [C4SH86N2OIO=850].

[0452] Example 29 — Preparation of COMPOUND NO. 29

[0453] COMPOUND NO. 29 was prepared in accordance with the General Scheme (A).1H NMR (500 MHz, CDCl3): 8 11.19 (br, 1H), 5.10-5.05 (m, 1H), 4.94-4.88 (m, 1H), 4.74-4.5 (br, 1H), 4.45 (d, 1H, J=10 Hz), 4.32-4.15 (br, 1H), 3.86-3.82 (m, 1H), 3.7-3.65 (m, 2H), 3.54-3.52 (m, 1H), 3.44-3.43 (m, 2H), 2.36-3.15 (m, 3H), 2.24-1.78 (m, 6H), 1.56-1.45 (m, 12H), 1.43-1.42 (m, 4H), 1.34-1.15 (m, 44H), 0.89-0.86 (m, 9H). MS found 880.3 [M+H]+- calcd for [C5OH9ON2OIO=879].

[0454] Example 30 — Preparation of COMPOUND NO, 30

[0455] COMPOUND NO. 30 was prepared in accordance with the General Scheme (A).JH NMR (500 MHz, CDCl3): 85.08-5.05 (t, 1H, J=10Hz), 5.04-5.02 (t, 1H, J=10Hz), 4.95-4.86 (m, 2H), 4.73-4.70 (m, 1H), 4.51-4.47 (dd, 1H, J=10Hz, 5Hz), 4.45 (d, 1H, J=10 Hz), 4.35-4.33 (m,1H), 4.19-4.17 (m,lH), 3.86-3.82 (m, 1H), 3.51-3.41 (m, 3H), 3.54-3.52 (m, 1H), 2.61-2.21(m, 4H), 2.16-1.75 (m, 5H), 1.56-1.45 (m, 9H), 1.34-1.15 (m, 44H), 1.14-1.12 (t, J=5.0Hz, 4H), 0.89-0.86 (m, 9H). MS found 853.1 [M+H] - calcd for [C49H89NOio=852].

[0456] Example 31 — Preparation of COMPOUND NO. 31

[0457] COMPOUND NO. 31 was prepared in accordance with the General Scheme (A).JH NMR (500 MHz, CDCl3) 55.08 (t, J= 9.6 Hz, 1H), 4.92 (dd, J= 9.8, 8.0 Hz, 1H), 4.83 (dd, J = 12.3, 4.1 Hz, 1H), 4.44 (d, J= 8.0 Hz, 1H), 4.10 (dd, J= 12.2, 2.4 Hz, 1H), 3.84 (dt, J= 9.6, 6.4 Hz, 1H), 3.68 (t, J= 9.6 Hz, 1H), 3.54 (ddd, J= 9.6, 4.1, 2.4 Hz, 1H), 3.44 (dt, J= 9.6, 6.8 Hz, 1H), 3.01 (dq, J= 11.8, 6.7 Hz, 1H), 2.78 (br, 1H), 2.70 - 2.48 (m, 5H), 2.34 (d, J= 7.2 Hz, 1H), 2.31 - 2.20 (m, 4H), 1.68 (dd, J = 13.9, 2.5 Hz, 4H), 1.64 - 1.49 (m, 12H), 1.26 (d, J= 8.8 Hz, 42H), 0.87 (td, J= 7.0, 1.6 Hz, 9H). MS: m / z 836.4 (M+Na).

[0458] Example 32 — Preparation of COMPOUND NO. 32

[0459] COMPOUND NO. 32 was prepared in accordance with the General Scheme (A). ’H NMR (500 MHz, CDCl3) 85.08 (t, J= 9.6 Hz, 1H), 4.92 (dd, J= 9.7, 8.0 Hz, 1H), 4.85 - 4.74 (m, 1H), 4.44 (d, J = 8.0 Hz, 1H), 4.11 (d, J = 12.2 Hz, 1H), 3.84 (dt, J = 9.6, 6.5 Hz, 1H), 3.73 -3.64 (m, 1H), 3.53 (ddd, J= 9.7, 4.1, 2.4 Hz, 1H), 3.44 (dt, J= 9.7, 6.9 Hz, 1H), 3.02 (br, 1H), 2.75 -2.48 (m, 6H), 2.32 -2.20 (m, 5H), 1.56 (dt, J= 13.2, 6.7 Hz, 12H), 1.40(m, 6H), 1.26 (d, J = 6.2 Hz, 42H), 0.88 (td, J = 7.0, 1.6 Hz, 9H). MS: m / z 850.5 (M+Na).

[0460] Example 33 — Preparation of COMPOUND NO. 33

[0461] COMPOUND NO. 33 was prepared in accordance with the General Scheme (A). 'H NMR (500 MHz, CDCl3) 85.06 (q, J = 9.0 Hz, 1H), 4.92 (dd, J = 9.7, 8.0 Hz, 1H), 4.71 (dd, J = 12.4, 3.9 Hz, 1H), 4.45 (dd, J = 7.9, 4.5 Hz, 1H), 4.25 - 4.13 (m, 1H), 3.84 (dt, J = 9.7, 6.4 Hz, 1H), 3.70 (t, J = 9.5 Hz, 1H), 3.59 - 3.49 (m, 1H), 3.44 (dtd, J= 9.8, 6.9, 3.2 Hz, 1H), 3.09 (br, 2H), 2.83 -2.59 (m, 3H), 2.37 - 2.20 (m, 5H), 1.73(br, 2H), 1.62 - 1.44 (m, 6H), 1.26 (d, J= 7.2 Hz, 42H), 1.13 (d, J = 3.8 Hz, 6H), 0.87 (td, J = 7.0, 1.6 Hz, 9H). MS: m / z 810.4 (M+Na).

[0462] Example 34 — Preparation of COMPOUND NO. 34

[0463] COMPOUND NO. 34 was prepared in accordance with the General Scheme (A).1H NMR (499 MHz, CDCl3) 85.07 (d, J = 9.6 Hz, 1H), 4.97 - 4.88 (m, 1H), 4.80 (dd, J = 12.2, 4.3 Hz, 1H), 4.45 (dd, J= 7.9, 4.2 Hz, 1H), 4.10 (dd, J = 12.2, 2.7 Hz, 1H), 3.84 (dt, J = 9.6, 6.4 Hz, 1H), 3.70 (t, J = 9.5 Hz, 1H), 3.55 (ddd, J = 9.6, 4.3, 2.7 Hz, 1H), 3.43 (dt, J = 9.9, 6.8 Hz, 1H),2.86 (d, J= 8.4 Hz, 2H), 2.70- 2.41 (m, 5H), 2.37 - 2.20 (m, 5H), 1.66(br, 6H), 1.61 - 1.46 (m, 8H), 1.25 (d, J= 11.5 Hz, 42H), 0.87 (td, J= 7.0, 1.4 Hz, 9H). MS: m / z 822.3 (M+Na).

[0464] Example 35 — Preparation of COMPOUND NO. 35

[0465] COMPOUND NO. 35 was prepared in accordance with the General Scheme (A). 'H NMR (500 MHz, CDCl3) 55.08 (t, J= 9.6 Hz, 1H), 4.92 (dd, J= 9.7, 7.9 Hz, 1H), 4.80 (dd, J = 12.3, 4.2 Hz, 1H), 4.44 (d, <7= 7.9 Hz, 1H), 4.10 (dd, J= 12.2, 2.5 Hz, 1H), 3.84 (dt,<7= 9.5, 6.5 Hz, 1H), 3.67 (t, J= 9.6 Hz, 1H), 3.53 (ddd, J= 9.6, 4.2, 2.6 Hz, 1H), 3.43 (dt, J= 9.6, 6.9 Hz, 1H), 2.86 (dd, J= 8.0, 5.1 Hz, 1H), 2.63 - 2.53 (m, 3H), 2.47 (s, 2H), 2.37 (s, 2H), 2.32 - 2.21 (m, 4H), 1.54 (dt, J = 8.7, 6.0 Hz, 10H), 1.39 (d, J= 3.1 Hz, 8H), 1.34 - 1.18 (m, 44H), 0.88 (td, J= 7.0, 1.5 Hz, 9H). MS: m / z 864.4 (M+Na).

[0466] Example 36 — Preparation of COMPOUND NO. 36

[0467] COMPOUND NO. 36 was prepared in accordance with the General Scheme (A). ’H NMR (500 MHz, CDCl3) 85.42 - 5.29 (m, 4H), 5.08 (t, J= 9.6 Hz, 1H), 4.96 - 4.87 (m, 1H), 4.83 (dd, J = 12.3, 4.1 Hz, 1H), 4.44 (d, <7= 8.1 Hz, 1H), 4.08 (dd, J= 12.2, 2.4 Hz, 1H), 3.84 (dt, J= 9.6, 6.4 Hz, 1H), 3.66 (t, J= 9.6 Hz, 1H), 3.57 - 3.48 (m, 1H), 3.44 (dt, J= 9.6, 6.8 Hz, 1H), 3.03 - 2.90 (m, 1H), 2.77 (d, J= 9.3 Hz, 1H), 2.68 (dt, J= 9.2, 6.9 Hz, 1H), 2.59 (ddt, J = 10.8, 7.9, 5.6 Hz, 2H), 2.55 - 2.41 (m, 3H), 2.30 - 2.23 (m, 4H), 2.00 (q, J= 6.6 Hz, 8H), 1.97 - 1.88 (m, 7H), 1.85 (dp, <7= 8.4, 2.8 Hz, 3H), 1.85-1.83(M, 2H), 1.83 - 1.75 (m, 2H), 1.56 (dt,<7 = 14.2, 7.2 Hz, 6H), 1.33 - 1.24 (m, 44H), 0.88 (td, J= 7.0, 2.4 Hz, 9H). MS: m / z 986.5 (M+Na).

[0468] Example 37 — Preparation of COMPOUND NO. 37

[0469] COMPOUND NO. 37 was prepared in accordance with the General Scheme (A).JH NMR (500 MHz, CDCl3) 85.45 - 5.25 (m, 8H), 5.08 (t, J= 9.6 Hz, 1H), 4.92 (dt, J= 9.7, 7.2 Hz, 1H), 4.83 (dd, <7= 12.3, 4.1 Hz, 1H), 4.44 (d, <7= 8.0 Hz, 1H), 4.08 (dd, J= 2.2, 2A.z, 1H), 3.84 (dt, <7= 9.6, 6.4 Hz, 1H), 3.66 (t, <7= 9.5 Hz, 1H), 3.53 (ddd, <7= 9.6, 4.2, 2.5 Hz, 1H), 3.44 (dt, <7 = 9.7, 6.8 Hz, 1H), 3.02 - 2.92 (m, 1H), 2.80 - 2.74 (m, 4H), 2.71 - 2.64 (m, 1H), 2.59 (dtd, <7= 10.3, 8.8, 5.1 Hz, 2H), 2.55 - 2.42 (m, 2H), 2.30 - 2.23 (m, 2H), 2.08 - 2.01 (m, 8H), 2.01 - 1.89 (m, 4H), 1.89 - 1.75 (m, 6H), 1.56 (dd, J= 17.8, 10.6 Hz, 8H), 1.39 - 1.24 (m, 38H), 0.88 (q, J = 6.8 Hz, 9H). MS: m / z 982.5 (M+Na).

[0470] Example 38 — Preparation of COMPOUND NO. 38

[0471] COMPOUND NO. 38 was prepared in accordance with the General Scheme (A). 'H NMR (500 MHz, CDCl3) 85.06 (t, <7= 9.5 Hz, 1H), 4.90 (dd, J= 9.8, 7.9 Hz, 1H), 4.56 - 4.42(m, 2H), 4.37 (dd, J= 12.1, 2.4 Hz, 1H), 3.84 (dt, J= 9.7, 6.4 Hz, 1H), 3.73 - 3.60 (m, 1H), 3.59 - 3.51 (m, 1H), 3.43 (dt, J = 9.6, 6.8 Hz, 1H), 3.08 (q, J= 7.4 Hz, 1H), 2.91 - 2.81 (m, 2H), 2.75 - 2.60 (m, 9H), 2.34 (tt, J= 15.7, 7.6 Hz, 2H), 2.25 (tt, J= 7.4, 3.2 Hz, 2H), 2.08 (p, J= 7.4 Hz, 2H),1.63 - 1.52 (m, 6H), 1.27 (dd, J= 12.4, 6.5 Hz, 32H), 0.87 (t, J= 6.8 Hz, 9H). MS: m / z 746.3 (M+Na).

[0472] Example 39 — Preparation of COMPOUND NO. 39

[0473] COMPOUND NO. 39 was prepared in accordance with the General Scheme (A). 'H NMR (499 MHz, CDCl3) 55.07 (t, J= 9.6 Hz, 1H), 4.91 (dd, J= 9.9, 8.0 Hz, 1H), 4.56 (dd, J= 12.1, 3.1 Hz, 1H), 4.45 (d, J= 8.0 Hz, 1H), 4.23 (dd, J= 12.1, 2.5 Hz, 1H), 3.84 (dt, J= 9.6, 6.4 Hz, 1H), 3.71 (t, J= 9.5 Hz, 1H), 3.53 - 3.36 (m, 2H), 2.78 (ddd, J= 12.4, 7.9, 4.9 Hz, 1H), 2.68 - 2.58 (m, 1H), 2.56 (td, J= 7.3, 4.9 Hz, 1H), 2.49 (ddd, J= 14.7, 7.8, 5.1 Hz, 1H), 2.43 - 2.32 (m, 3H), 2.32 - 2.16 (m, 14H), 1.57 (td, J= 15.3, 7.5 Hz, 8H), 1.31 - 1.22 (m, 30H), 0.91 - 0.84 (m, 9H). MS: m / z 743.4 (M+Na).

[0474] Example 40 — Preparation of COMPOUND NO. 40

[0475] COMPOUND NO. 40 was prepared in accordance with the General Scheme (A).1H NMR (500 MHz, CDCl3) 85.08 (t, J= 9.6 Hz, 1H), 4.92 (dd, J= 9.8, 8.0 Hz, 1H), 4.79 (dd, J = 12.3, 4.0 Hz, 1H), 4.44 (d, J = 8.0 Hz, 1H), 4.13 (dd, J= 12.3, 2.3 Hz, 1H), 3.84 (dt, J= 9.6, 6.4 Hz, 1H), 3.71 (t, J= 9.6 Hz, 1H), 3.56 - 3.49 (m, 1H), 3.44 (dt, J= 9.7, 6.8 Hz, 1H), 2.84 (t, J = 6.7 Hz, 2H), 2.64 (dt, J= 12.8, 6.3 Hz, 1H), 2.55 (dt, J= 14.8, 6.8 Hz, 1H), 2.36 (s, 3H), 2.34 -2.30 (m, 2H), 2.26 (qd, J= 7.5, 2.0 Hz, 4H), 1.65 - 1.45 (m, 6H), 1.38 - 1.13 (m, 31H), 0.87 (t, J = 6.8 Hz, 12H), 0.61 - 0.47 (m, 2H), 0.12 (p, J= 4.9 Hz, 2H). MS: m / z 712.3 (M+Na).

[0476] Example 41 — Preparation of COMPOUND NO. 41

[0477] COMPOUND NO. 41 was prepared in accordance with the General Scheme (A). 'H NMR (499 MHz, CDCl3) 85.06 (t, J= 9.5 Hz, 1H), 4.91 (dd, J= 9.9, 7.9 Hz, 1H), 4.46 (d, J = 7.9 Hz, 1H), 4.43 - 4.26 (m, 2H), 3.84 (dt, J= 9.7, 6.4 Hz, 1H), 3.75 (t, J= 9.4 Hz, 1H), 3.53 -3.36 (m, 2H), 2.81 (ddd, J= 12.8, 9.8, 5.8 Hz, 1H), 2.68 (ddd, J= 12.8, 7.0, 4.4 Hz, 1H), 2.51 (td, J= 7.5, 4.3 Hz, 1H), 2.49 - 2.30 (m, 7H), 2.30 - 2.24 (m, 4H), 2.20 (s, 12H), 1.62 - 1.51 (m, 10H), 1.32 - 1.21 (m, 32H), 0.87 (t, J = 6.8 Hz, 9H). MS: m / z 814.3 (M+Na).

[0478] Example 42 — Preparation of COMPOUND NO. 42

[0479] COMPOUND NO. 42 was prepared in accordance with the General Scheme (A).1H NMR (500 MHz, CDCl3) 85.06 (td, J = 9.5, 3.2 Hz, 1H), 4.91 (ddd, J= 9.7, 7.9, 1.6 Hz, 1H),4.72 - 4.51 (m, 1H), 4.45 (d, J= 8.0 Hz, 1H), 4.33 - 4.17 (m, 1H), 3.89 - 3.79 (m, 2H), 3.72 -3.57 (m, 3H), 3.52 (ddd, J= 9.7, 4.1, 2.6 Hz, 1H), 3.43 (dtd, J= 9.1, 6.8, 2.1 Hz, 1H), 2.89 (dt, J = 11.1, 2.1 Hz, 1H), 2.79 (dt, J= 12.0, 7.2 Hz, 1H), 2.69 (tdd, J= 14.6, 6.0, 1.9 Hz, 2H), 2.65 -2.50 (m, 7H), 2.36-2.23 (m, 4H), 2.23 - 2.13 (m, 1H), 1.89- 1.74 (m, 2H), 1.55 (dq, J= 13.7, 7.5 Hz, 6H), 1.34 - 1.19 (m, 30H), 1.02 (q, J= 6.9 Hz, 6H), 0.87 (t, J= 6.9 Hz, 9H). MS: m / z 799.3 (M+Na).

[0480] Example 43 — Preparation of COMPOUND NO. 43

[0481] COMPOUND NO. 43 was prepared in accordance with the General Scheme (A).1H NMR (500 MHz, CDCl3) 85.08 (t, J= 9.6 Hz, 1H), 4.91 (dd, J= 9.7, 8.0 Hz, 1H), 4.83 (dd, J = 12.2, 4.1 Hz, 1H), 4.44 (d, J= 8.0 Hz, 1H), 4.08 (dd, J= 12.3, 2.4 Hz, 1H), 3.84 (dt, J= 9.6, 6.5 Hz, 1H), 3.66 (t, J= 9.5 Hz, 1H), 3.53 (ddd, J= 9.6, 4.1, 2.4 Hz, 1H), 3.44 (dt, J= 9.5, 6.8 Hz, 1H), 3.02 - 2.92 (m, 1H), 2.77 (d, J= 9.4 Hz, 1H), 2.72 - 2.64 (m, 1H), 2.64 - 2.55 (m, 2H), 2.55 - 2.41 (m, 3H), 2.26 (tt, J= 10.7, 7.5 Hz, 4H), 2.03 - 1.88 (m, 4H), 1.88 - 1.74 (m, 4H), 1.55 (dq, J= 13.9, 7.1 Hz, 6H), 1.27 (t, J= 9.4 Hz, 30H), 0.87 (t, J= 6.8 Hz, 9H). MS: m / z 738.3 (M+Na).

[0482] Example 44 — Preparation of COMPOUND NO. 44

[0483] COMPOUND NO. 44 was prepared in accordance with the General Scheme (A).1H NMR (500 MHz, CDCl3) 85.08 (t, J= 9.6 Hz, 1H), 4.92 (dd, J= 9.8, 8.0 Hz, 1H), 4.70 (dd, J = 12.2, 3.4 Hz, 1H), 4.45 (d, J= 7.9 Hz, 1H), 4.15 (dd, J= 12.2, 2.3 Hz, 1H), 3.85 (dt, J= 9.6, 6.4 Hz, 1H), 3.77 - 3.65 (m, 5H), 3.53 - 3.38 (m, 2H), 2.82 (dd,,7 = 13.6, 7.2 Hz, 2H), 2.68 - 2.53 (m, 5H), 2.53 - 2.38 (m, 7H), 2.26 (qd, J = 7.6, 1.6 Hz, 4H), 1.65 - 1.45 (m, 6H), 1.34 - 1.20 (m, 30H), 1.03 (t, J= 7.1 Hz, 3H), 0.87 (t, J= 6.8 Hz, 9H). MS: m / z 785.3 (M+Na).

[0484] Example 45 — Preparation of COMPOUND NO. 45

[0485] COMPOUND NO. 45 was prepared in accordance with the General Scheme (A). 'll NMR (500 MHz, CDCl3) 85.08 (t, J= 9.6 Hz, 1H), 4.92 (dd, J= 9.7, 7.9 Hz, 1H), 4.84 (dd, J = 12.3, 4.0 Hz, 1H), 4.45 (d, J= 8.0 Hz, 1H), 4.11 (dd, J= 12.3, 2.1 Hz, 1H), 3.85 (dt, J= 9.6, 6.4 Hz, 1H), 3.69 (t,.7= 9.6 Hz, 1H), 3.52 (ddd, J= 9.7, 4.1, 2.2 Hz, 1H), 3.44 (dt, J= 9.6, 6.9 Hz, 1H), 2.82 -2.68 (m, 1H), 2.62 (tq, J= 11.5, 5.1 Hz, 2H), 2.57 - 2.44 (m, 1H), 2.32 - 2.22 (m, 10H), 1.56 (dt, J= 14.4, 7.2 Hz, 6H), 1.34- 1.16 (m, 30H), 0.91 - 0.81 (m, 9H). MS: m / z 672.4 (M+Na).

[0486] Example 46 — Preparation of COMPOUND NO. 46

[0487] COMPOUND NO. 46 was prepared in accordance with the General Scheme (A). ' H NMR (500 MHz, CDCl3) 85.04 (t, J= 9.4 Hz, 1H), 4.93 (dd, J= 9.8, 7.9 Hz, 1H), 4.52 (dd, J = 12.2, 4.1 Hz, 1H), 4.45 (d, J= 7.9 Hz, 1H), 4.33 (dd, J= 12.1, 2.3 Hz, 1H), 3.85 (dt, J= 9.6, 6.4 Hz, 1H), 3.71 (q, J= 4.3 Hz, 4H), 3.63 (t, J= 9.4 Hz, 1H), 3.57 - 3.37 (m, 2H), 2.84 (t, J= 7.1 Hz, 2H), 2.75 - 2.63 (m, 4H), 2.63 - 2.54 (m, 2H), 2.47 (t, J= 4.6 Hz, 4H), 2.40 - 2.20 (m, 4H), 1.66 - 1.49 (m, 6H), 1.35 - 1.18 (m, 30H), 0.88 (t, J= 6.9 Hz, 9H).

[0488] Example 47 — Preparation of COMPOUND NO. 47

[0489] COMPOUND NO. 47 was prepared in accordance with the General Scheme (A).1H NMR (500 MHz, CDCl3) 65.08 (t, J= 9.6 Hz, 1H), 4.95 - 4.89 (m, 1H), 4.80 (dd, J= 12.2, 4.2 Hz, 1H), 4.44 (d, J= 7.9 Hz, 1H), 4.10 (dd, J= 12.2, 2.5 Hz, 1H), 3.84 (dt, J=9.7, 6.5 Hz, 1H), 3.67 (t, J= 9.5 Hz, 1H), 3.53 (ddd, J= 9.6, 4.2, 2.5 Hz, 1H), 3.44 (dt, J= 9.8, 6.9 Hz, 1H), 2.92 -2.80 (m, 1H), 2.60 - 2.50 (m, 5H), 2.40-2.32(m, 2H), 2.31 - 2.21 (m, 4H), 1.57 (dt, J= 10.7, 5.1 Hz, 12H), 1.41 - 1.33 (m, 6H), 1.31 - 1.20 (m, 30H), 0.87 (t, J= 6.8 Hz, 9H). MS: m / z 766.3 (M+Na).

[0490] Example 48 — Preparation of COMPOUND NO. 48

[0491] COMPOUND NO. 48 was prepared in accordance with the General Scheme (C). MS found 822 [M+H calcd for [C48H88N2O7=820.6].

[0492] Example 49 — Preparation of COMPOUND NO. 49

[0493] COMPOUND NO. 49 was prepared in accordance with the General Scheme (C). MS found 8501 [M+H] - calcd for [C5oH92N208=848.7].

[0494] Example 50 — Preparation of COMPOUND NO. 50

[0495] COMPOUND NO. 50 was prepared in accordance with the General Scheme (C).1H NMR (500 MHz, CDCl3): 85.10-5.01 (t, J=10 Hz, 1H), 4.94-4.86 (t, J=10 Hz, 1H), 4.49-4.46 (m,l H), 4.45 (d, 1H, J=10Hz), 4.32-4.29 (m,lH), 3.87-3.82 (m, 1H), 3.62-3.58 (t, 1H, t=10Hz), 3.50-3.42 (m,3H), 2.41-2.24 (m, 8 H), 1.85-1.83 (m,2H), 1.59-1.47 (m, 16H), 1.4-1.37 (m, 4H), 1.25-1.21 (m, 44H), 0.89-0.86 (m, 9H). MS found 865.1 [M+H]+’ calcd for [C5IH93NO9=864].

[0496] Example 51 — Preparation of COMPOUND NO. 51

[0497] COMPOUND NO. 51 was prepared in accordance with the General Scheme (C).1H NMR (500 MHz, CDCl3): 85.06-5.02 (t, J=10 Hz, 1H), 4.94-4.90 (t, J=10 Hz, 1H), 4.47-4.44 (m,l H), 4.45 (d, 1H, J=10Hz), 4.36-4.33 (m,lH), 3.86-3.83 (m, 1H), 3.72-3.68 (t, 1H, t=10Hz),3.54-3.42 (m,2H), 2.55-2.24 (m, 6 H), 2.04-1.83 1.59-1.53 (m, 7H), 1.4-1.37 (tn, 3H), 1.25-1.21 (m, 44H), 0.89-0.86 (m, 9H). MS found 837.3 [M+H]+’ calcd for [C4sH89NO9=836],

[0498] Example 52 — Preparation of COMPOUND NO. 52

[0499] COMPOUND NO. 52 was prepared in accordance with the General Scheme (C).NMR (500 MHz, CDCl3): 85.06-5.02 (t, J=10 Hz, 1H), 4.93-4.91 (t, J=10 Hz, 1H), 4.48-4.43 (m,2 H), 4.34-4.31 (m, 1H), 3.85-3.82 (m, 1H), 3.64-3.60 (t, 1H, t=10Hz), 3.52-3.50 (m,lH), 3.44-3.40 (m, 1H), 2.56-2.42 (m, 8 H), 2.25-2.14 (m,3H), 1.95-1.80(m, 6H), 1.79-1.77 (m, 5H), 1.59-1.52 (m, 12H), 1.4-1.17 (m, 37H), 0.89-0.86 (m, 9H). MS found 861.2[M+H]+’ calcd for [C5lH89NO9=860],

[0500] Example 53 — Preparation of COMPOUND NO. 53

[0501] COMPOUND NO. 53 was prepared in accordance with the General Scheme (C).1H NMR (500 MHz, CDCl3): 85.06-5.02 (t, J=10 Hz, 1H), 4.94-4.90 (m, 1H), 4.49-4.43(m,2 H), 4.37-4.35 (m,lH), 3.86-3.82 (m, 2H), 3.72-3.61 (m, 2H), 3.47-3.42 (m,3H), 2.65-2.43 (m, 2 H), 2.32-2.15 (m,6H), 2.10-1.73 (m, 18H), 1.65-1.47 (m, 2H), 1.25-1.21 (m, 32H), 1.18-1.12 (m,8H), 0.89-0.86 (m, 9H). MS found 833.3 [M+H]+- calcd for [C49H89NO9=832].

[0502] Example 54 — Preparation of COMPOUND NO. 54

[0503] COMPOUND NO. 54 was prepared in accordance with the General Scheme (C).1H NMR (500 MHz, CDCl3): 85.15-5.07 (t, J=10 Hz, 1H), 4.92-4.88 (t, J=10 Hz, 1H), 4.47-4.46 (d, 1H, J=10Hz), 3.80-3.78 (m, 1H), 3.64 (t, 1H, t=10Hz), 3.44-3.42 (m,lH), 2.25-2.14 (m, 2 H), 1.93-1.76 (m,4H), 1.79-1.76 (m, 4H), 1.6-1.50 (m, 14H), 1.40-1.32 (m, 9H), 1.30-1.21 (m, 26H), 1.17-1.09 (m, 8H), 0.89-0.86 (m, 9H). MS found 775 [M+H]+’ calcd for [C47H83NO7=774].

[0504] Example 55 — Preparation of COMPOUND NO. 55

[0505] COMPOUND NO. 55 was prepared in accordance with the General Scheme (B). 'H NMR (500 MHz, CDCl3) 85.33 (t, J= 9.8 Hz, 2H), 5.22 (d, J= 3.9 Hz, 2H), 4.96 (dd, J= 10.2, 3.9 Hz, 2H), 4.62 (dd, J= 12.2, 4.5 Hz, 2H), 4.03 (dd, J= 12.2, 2.4 Hz, 2H), 3.91 (ddd, J= 10.0, 4.6, 2.4 Hz, 2H), 3.60 (t, J= 9.6 Hz, 2H), 2.87 (dt, J= 11.9, 7.5 Hz, 2H), 2.71 (d, J= 9.3 Hz, 2H), 2.69 - 2.44 (m, 10H), 2.39 - 2.22 (m, 6H), 2.03 - 1.89 (m, 8H), 1.86 (t, J= 7.0 Hz, 4H), 1.84 - 1.74 (m, 4H), 1.74 - 1.53 (m, 12H), 1.27 (dd, J= 15.1, 6.1 Hz, 64H), 0.88 (td, J= 7.0, 1.0 Hz, 12H). ). MS: m / z 1401.7 (M+H).

[0506] Example 56 — Preparation of COMPOUND NO. 56

[0507] COMPOUND NO. 56 was prepared in accordance with the General Scheme (B). ' H NMR (500 MHz, CDCl3) 5’H NMR (500 MHz, CDCl3) 85.38 - 5.28 (m, 2H), 5.27 - 5.21 (m, 2H), 5.02 - 4.92 (m, 2H), 4.26 - 4.17 (m, 2H), 4.17 - 4.09 (m, 2H), 3.90 (tdd, J= 10.2, 5.3, 2.6 Hz, 2H), 3.75 - 3.57 (m, 2H), 2.81 - 2.56 (m, 12H), 2.56 - 2.48 (m, 4H), 2.40 - 2.24 (m, 8H), 1.72 - 1.52 (m, 18H), 1.33 - 1.20 (m, 62H), 0.88 (td, J= 7.0, 1.2 Hz, 12H). MS: m / z 1459.6 (M+H).

[0508] Example 57 — Preparation of COMPOUND NO. 57

[0509] COMPOUND NO. 57 was prepared in accordance with the General Scheme (B). 'H NMR (500 MHz, CDCl3) 65.37 - 5.25 (m, 2H), 5.23 (dd, J= 3.8, 1.8 Hz, 2H), 4.95 (ddt, J= 14.2, 10.3, 3.6 Hz, 2H), 4.22 (dd, J= 12.0, 2.7 Hz, 2H), 4.09 (d, J= 12.1 Hz, 2H), 3.84 (dd, J = 10.0, 4.4 Hz, 2H), 3.71 - 3.53 (m, 4H), 3.45 (d, J= 16.1 Hz, 2H), 2.87 (t, J= 14.8 Hz, 2H), 2.80 - 2.47 (m, 8H), 2.41 -2.21 (m, 10H), 2.21 -2.00 (m, 2H), 1.72- 1.55 (m, 16H), 1.27 (dd, J = 14.9, 5.7 Hz, 68H), 1.11 (d, J= 6.2 Hz, 6H), 0.88 (t, J= 6.8 Hz, 12H). MS: m / z 1461.8 (M+H).

[0510] Example 58 — Preparation of COMPOUND NO. 58

[0511] COMPOUND NO. 58 was prepared in accordance with the General Scheme (B). 'H NMR (500 MHz, CDCl3) 85.37 (t, J= 9.8 Hz, 1H), 5.28 - 5.21 (m, 3H), 4.96 (ddd, J= 10.6, 7.2, 3.8 Hz, 2H), 4.66 (dd, J= 12.2, 4.4 Hz, 1H), 4.01 (dd, J= 12.4, 2.3 Hz, 1H), 3.92 (ddd, J = 9.9, 4.3, 2.4 Hz, 1H), 3.83 - 3.74 (m, 3H), 3.71 - 3.58 (m, 2H), 2.42 - 2.21 (m, 9H), 2.06 - 1.86 (m, 7H), 1.86 - 1.75 (m, 3H), 1.61 (ddt, J= 15.2, 12.3, 7.5 Hz, 10H), 1.27 (dd, J= 12.3, 7.1 Hz, 67H), 0.88 (t, J= 6.8 Hz, 12H). ). MS: m / z 1236.5 (M+H).

[0512] Example 59 — Preparation of COMPOUND NO. 59

[0513] COMPOUND NO. 59 was prepared in accordance with the General Scheme (D).1H NMR (500 MHz, CDCl3): 85.40-5.31 (m, 8H), 4.13-4.12 (m, J=5.0 Hz, 6H), 2.78-2.75 (t, 4H, J=10Hz), 2.40-2.36 (m, 6H), 3.30 (t, 4H, t=10Hz), 2.70-2.03 (dd,8H, J=5Hz, 10Hz), 1.60-1.56 (m, 16 H), 1.43-1.39 (m,5H), 1.37-1.26 (m, 28H), 0.89-0.85 (m, 6H). MS found 839.2 [M+H] calcd for [C53H91NO6=838].

[0514] Example 60 — Preparation of COMPOUND NO. 60

[0515] COMPOUND NO. 60 was prepared in accordance with the General Scheme (F). 'H NMR (500 MHz, CDCl3) 87.27 (s, 2H), 4.38-4.37 (m, 1H), 4.26-4.23 (m, 1H), 4.02-3.99 (m, 7H), 2.63-2.37(m, 6H), 1.83-1.70 (m, 6H), 1.60-1.58 (m, 4H), 1.51-1.34 (m, 14H), 1.34-1.26 (m, 54H), 0.88 (t, J=5Hz, 9 H). MS found 870.7 [M+H] - calcd for [C55H99NO6=869.7]

[0516] Example 61 — Preparation of COMPOUND NO. 61

[0517] COMPOUND NO. 61 was prepared in accordance with the General Scheme (F). MS found 1527.4 [M+H]+> calcd for [C98H175NO10=1526.3]

[0518] Example 62 — Preparation of COMPOUND NO. 62

[0519] COMPOUND NO. 62 was prepared in accordance with the General Scheme (E). MS found 950.8 [M+H] ‘ calcd for [C61H107NO6=949.8].

[0520] Example 63 — Preparation of COMPOUND NO. 63

[0521] COMPOUND NO. 63 was prepared in accordance with the General Scheme (E).rH NMR (500 MHz, CDCl3) 84.12 (d, J= 6.0 Hz, 6H), 2.48 - 2.24 (m, 10H), 1.67 - 1.51 (m, 12H), 1.51 - 1.34 (m, 10H), 1.25 (s, 6H), 0.88 (t, J= 6.9 Hz, 12H). MS found 958.9 [M+H]+’ calcd for [C6iHii5NO6=957.9],

[0522] Example 64 — Preparation of COMPOUND NO.64

[0523] COMPOUND NO. 64 was prepared in accordance with the General Scheme (E). MS found 1071 [M+H]1,calcd for [C69H131NO6=1070.0].

[0524] Example 65 — Preparation of COMPOUND NO. 65

[0525] COMPOUND NO. 65 was prepared in accordance with the General Scheme (E). MS found 846.68 [M+H]+> calcd for [C53H99NO6=845.6].

[0526] Example 66 — Preparation of COMPOUND NO. 66

[0527] COMPOUND NO. 66 was prepared in accordance with the General Scheme (E). MS found 790.5 [M+H] - calcd for [C49H9iNO6=789.7].

[0528] Example 67 — Preparation of COMPOUND NO. 67

[0529] COMPOUND NO. 67 was prepared in accordance with the General Scheme (E).1H NMR (500 MHz, CDCl3) 85.46 - 5.25 (m, 8H), 4.21 -4.04 (m, 4H), 3.84 (ddt, J= 9.7, 5.4, 4.2 Hz, 1H), 3.51 (d, J= 6.1 Hz, 2H), 3.42 (h, J= 5.6 Hz, 2H), 2.77 (t, J= 6.4 Hz, 4H), 2.56 (s, 2H), 2.41 - 2.25 (m, 8H), 2.09 - 2.01 (m, 8H), 1.66 - 1.54 (m, 8H), 1.47 (td, J= 7.3, 4.0 Hz, 5H), 1.43 - 1.22 (m, 32H), 0.94 - 0.83 (m, 6H). MS found 826.6 [M+H] - calcd for [C52H91NO6=825.7]

[0530] Example 68 — Preparation of COMPOUND NO, 68

[0531] COMPOUND NO. 68 was prepared in accordance with the General Scheme (F). 'H NMR (500 MHz, DMSO) 86.11 (d, J=5.0Hz, 1H), 5.72 (d, J=10 Hz, 1H), 5.21 (t, J=5.0Hz, 1H), 4.83 (dd, 1H, J=2.5, 5.0 Hz, 1H), 4.31(m, 1H), 4.10-4.08 (m, 1H), 3.89-3.85 (m, 1H), 3.58-3.53(m, 1H), 2.39-2.17 (m, 11H), 1.99-1.64 (m, 14H), 1.38-1.13 (m, 45H), 0.99-0.83 (m, 15H). MS found 927.8 [M+H]+’ calcd for [C55H93NO10=927.7]

[0532] Example 69 — Preparation of COMPOUND NO. 69

[0533] COMPOUND NO. 69 was prepared in accordance with the General Scheme (F).rH NMR(500MHz, CDCl3) 55.71 (d, J=10 Hz, 1H), 5.10-5.08 (m, 2H), 4.52-4.50 (m, 1H), 4.27-4.25 (m, 1H), 3.64 (m, 2H), 2.43-2.12(m, 11H), 1.95-1.79 (m, 14H), 1.59-1.11 (m, 45H), 0.94-0.83 (m, 15H). MS found 927.8 [M+H]’ calcd for [C55H93NO10=927.7]

[0534] Example 70 — Preparation of COMPOUND NO. 70

[0535] COMPOUND NO. 70 was prepared in accordance with the General Scheme (E). MS (ES): m / z calcd for C52H91O6NNa (M+Na), 848.67; found, 848.6.

[0536] Example 71 — Preparation of COMPOUND NO. 71

[0537] COMPOUND NO. 71 was prepared in accordance with the General Scheme (D). MS found 786.5 [M+H]+- calcd for [C45H83NO7=785.6].

[0538] Example 72 — Preparation of COMPOUND NO. 72

[0539] COMPOUND NO. 72 was prepared in accordance with the General Scheme (D). MS found 786.4 [M+H]+- calcd for [C45H83NO7=785.6].

[0540] Example 73 — Preparation of COMPOUND NO, 73

[0541] COMPOUND NO. 73 was prepared in accordance with the General Scheme (D). MS found 814.6 [M+H]+- calcd for [C50H87NO7=813.6],

[0542] Example 74 — Preparation of COMPOUND NO. 74

[0543] COMPOUND NO. 74 was prepared in accordance with the General Scheme (D). MS found 800.5 [M+H] calcd for [C49H85NO7=799.6].

[0544] Example 75 — Preparation of COMPOUND NO. 75

[0545] COMPOUND NO. 75 was prepared in accordance with the General Scheme (D). MS found 800.7 [M+H] - calcd for [C49H85NO7=799.6].

[0546] Example 76 — Preparation of COMPOUND NO. 76

[0547] COMPOUND NO. 76 was prepared in accordance with the General Scheme (D). MS found 828.6 [M+HJ - calcd for [C51H89NO7=827.6].

[0548] Example 77 — Preparation of COMPOUND NO. 77

[0549] COMPOUND NO. 77 was prepared in accordance with the General Scheme (G).1H NMR (499 MHz, CDCl3) 56.29 (d, J= 3.7 Hz, 1H), 5.49 (t, J= 9.9 Hz, 1H), 5.13 - 5.05 (m,2H), 4.28 (dd, J= 12.5, 4.4 Hz, 1H), 4.08 - 3.99 (m, 2H), 2.86 (s, 3H), 2.45 (t, J= 6.7 Hz, 2H), 2.34 (tt, J= 12.3, 3.6 Hz, 1H), 2.25 -2.07 (m, 5H), 2.03 - 1.95 (m, 3H), 1.87 - 1.76 (m, 15H), 1.65 - 1.61 (m, 4H), 1.49 - 1.39 (m, 6H), 1.26 (qd, J= 13.7, 8.2 Hz, 36H), 1.16 (d, J= 7.9 Hz, 11H), 0.88 (q, J= 7.2 Hz, 20H). MS found 1108.7 [M+H]+> calcd for [ C67H113NO11= 1107.8314],

[0550] Example 78 — Preparation of COMPOUND NO. 78

[0551] COMPOUND NO. 78 was prepared in accordance with the General Scheme (H).1H NMR (499 MHz, CDCl3) 55.30 (s, 1H), 4.10 (s, 6H), 3.83 - 3.78 (m, 1H), 3.47 (s, 2H), 3.44 -3.36 (m, 2H), 2.54 (s, 2H), 2.33 - 2.27 (m, 9H), 1.59 (dt, J= 7.5, 3.8 Hz, 9H), 1.50 - 1.42 (m, 4H), 1.41 - 1.37 (m, 4H), 1.31 - 1.22 (m, 50H), 0.87 (t, J= 7.0 Hz, 9H). MS found 879 [M+H] -calcd for [ C53H99NO8= 877.7371],

[0552] Example 79 — Preparation of COMPOUND NO. 79

[0553] COMPOUND NO. 79 was prepared in accordance with the General Scheme (H).JH NMR (499 MHz, CDCl3) 8: 4.10 (s, 6H), 3.87 - 3.81 (m, 1H), 3.48 - 3.37 (m, 4H), 2.37 (d, J = 6.4 Hz, 2H), 2.29 (t, J = 7.6 Hz, 6H), 1.59 (t, J = 7.2 Hz, 10H), 1.49 (q, J= 6.7Hz, 3H), 1.42-1.38 (m, 4H), 1.25 (s, 66H), 0.88 (t, J= 7.0 Hz, 9H). MS found 963 [M+H] calcd for [ C59H111NO8= 961.8310]

[0554] Example 80 — Preparation of COMPOUND NO. 80

[0555] COMPOUND NO. 80 was prepared in accordance with the General Scheme (H).1H NMR (499 MHz, CDCl3) 84.09 (s, 6H), 3.87 (q, J= 6.4 Hz, 1H), 3.46 (s, 2H), 3.44 - 3.35 (m, 2H), 2.63 (s, 2H), 2.42 (d, J= 6.8 Hz, 2H), 2.21 (ddd, J= 12.2, 8.7, 3.6 Hz, 3H), 1.93 (dd, J= 13.8, 3.6 Hz, 6H), 1.81 - 1.77 (m, 6H), 1.61 - 1.57 (m, 4H), 1.52 (q, J= 5.9 Hz, 3H), 1.42 - 1.35 (m, 10H), 1.31 - 1.13 (m, 30H), 0.88 (dd, J= 14.3, 4.7 Hz, 15H). MS found 873 [M+H]+’calcd for [ C53H93NO8 = 871.6901],

[0556] Example 81 — Preparation of COMPOUND NO, 81

[0557] COMPOUND NO. 81 was prepared in accordance with the General Scheme (I). 'H NMR (500 MHz, CDCl3)t7: 5.30-5.37 (m, 6H), 4.10 (s, 6H), 3.79-3.84 (m, 1H), 3.46 (s, 2H), 3.36-3.43 (m, 2H), 2.56 (br, 1H, OH), 2.33-2.37 (m, 3H), 2.29 (t, 6H, J = 7.9Hz), 2.00 (dd, J = 5.75, 6.85 Hz, 11H), 1.57-1.62 (m, 10H), 1.45-1.49 (m, 4H), 1.39 (t, 4H, J = 7.3Hz), 1.24-1.35 (m, 63H), 0.86-0.89 (m, 9H). MS found 1125 [M+H]+’ calcd for [ C71H129NO8= 1123.9718],

[0558] Example 82 — Preparation of COMPOUND NO. 82

[0559] COMPOUND NO. 82 was prepared in accordance with the General Scheme (I). 'H NMR (500 MHz, CDCl3> / : 5.29-5.40 (m, 12H), 4.10 (s, 6H), 3.85-3.90 (m, 1H), 3.46 (s, 2H), 3.35-3.45 (m, 2H), 2.76 (t, J = 6.4 Hz, 6H), 2.64 (br, 1H), 2.40-2.45 (m, 3H), 2.29 (t, J = 7.4 Hz, 6H), 2.04 (dd, J = 6.3, 7.15 Hz, 12H), 1.58-1.62 (m, 10H), 1.49-1.55 (m, 4H), 1.38-1.42 (m, 4H), 1.24-1.36 (m, 45H), 0.88 (t, J = 6.85 Hz, 9H). MS found 1119.2 [M+H]+’ calcd for [ C71H123NO8= 1117.9249],

[0560] Example 83 — Preparation of COMPOUND NO. 83

[0561] COMPOUND NO. 83 was prepared in accordance with the General Scheme (J).1H NMR (499 MHz, MeOD) 65.44 - 5.32 (m, 2H), 4.14 (d, J= 23.8 Hz, 6H), 3.90 (d, J= 6.6 Hz, 6H), 3.67 - 3.39 (m, 9H), 3.28 - 3.10 (m, 3H), 2.64 (s, 12H), 2.08 - 1.93 (m, 5H), 1.89 - 1.38 (m, 38H), 1.34- 1.18 (m, 11H), 1.01 (dd, J= 22.9, 11.4 Hz, 9H), 0.96 - 0.88 (m, 18H). MS found 1251.2 [M+H]+- calcd for [ C74H123NO14= 1249.8944],

[0562] Example 84 — Preparation of COMPOUND NO. 84

[0563] COMPOUND NO. 84 was prepared in accordance with the General Scheme (J).1H NMR (499 MHz, MeOD) 85.47 - 5.28 (m, 6H), 4.17 (s, 6H), 3.54 - 3.38 (m, 6H), 3.27 - 3.08 (m, 3H), 2.35 (t, J= 7.4 Hz, 6H), 2.11 -2.01 (m, 12H), 1.67 (q, J = 7.4Hz, 10H), 1.56 (dt, J = 46.0, 6.9 Hz, 5H), 1.32 (dd, J= 13.6, 8.1 Hz, 30H), 0.91 (t, J= 6.8 Hz, 9H). MS found 872.6 [M+H]+’ calcd for [C53H93NO8= 871.6901],

[0564] Example 85 — Preparation of COMPOUND NO. 85

[0565] COMPOUND NO. 85 was prepared in accordance with the General Scheme (K).1H NMR (499 MHz, CDCI3) 85.22 - 5.15 (m, 2H), 4.62 (dtd, J= 24.8, 12.3, 2.6 Hz, 2H), 4.21 (dt, J = 12.0, 5.9 Hz, 2H), 3.89 (s, 2H), 3.64 - 3.58 (m, 4H), 3.52 - 3.45 (m, 2H), 2.62 (s, 3H), 2.43 (s, 6H), 2.20 (ddt, J= 15.7, 12.2, 3.4 Hz, 4H), 1.93 (d, J= 13.6 Hz, 8H), 1.80 (d, J= 12.2 Hz, 8H), 1.62 - 1.57 (m, 8H), 1.51 (s, 7H), 1.43 - 1.32 (m, 17H), 1.31 - 1.21 (m, 27H), 1.20 - 1.13 (m, 12H), 0.88 (t, J= A Hz, 22H). MS found 1293.9 [M+H] calcd for [ C78H136N2O12= 1293.0093],

[0566] Example 86 — Preparation of COMPOUND NO. 86

[0567] COMPOUND NO. 86 was prepared in accordance with the General Scheme (K).1H NMR (499 MHz, CDCI3) 85.25 - 5.18 (m, 2H), 4.67 -4.52 (m, 2H), 4.21 (td, J= 12.3, 7.0 Hz, 2H), 3.90 (s, 2H), 3.66 - 3.53 (m, 6H), 2.52 (d, J= 94.6 Hz, 8H), 2.32 - 2.25 (m, 8H), 1.58 (d, J= 7.1 Hz, 16H), 1.50 (s, 6H), 1.41 (d, J= 7.3 Hz, 8H), 1.28 (d, J = 19.0 Hz, 72H), 0.87 (t, J= 7.0 Hz, 12H). MS found 1302 [M+H]+- calcd for [ C78H144N2O12= 1301.0719],

[0568] Example 87 — Preparation of COMPOUND NO. 87

[0569] COMPOUND NO. 87 was prepared in accordance with the General Scheme (K).1H NMR (499 MHz, CDCl3) 55.20 (d, J= 7.5 Hz, 2H), 4.52 (dd, J= 24.4, 10.7 Hz, 8H), 4.27 (s, 2H), 4.12 (ddd, J= 28.1, 12.3, 7.2 Hz, 2H), 3.73 - 3.53 (m, 9H), 3.16 (s, 2H), 2.90 (d, J= 22.7 Hz, 4H), 2.32 - 2.25 (m, 7H), 1.97 (q, J= 13.7 Hz, 4H), 1.70 - 1.55 (m, 19H), 1.50 (q, J= 7.3 Hz, 8H), 1.33 - 1.21 (m, 81H), 0.88 (t, J= 7.0 Hz, 12H). MS found 1414.4 [M+H]+’calcd for [ C86H160N2O12= 1413.1971],

[0570] Example 88 — Preparation of COMPOUND NO. 88

[0571] COMPOUND NO. 88 was prepared in accordance with the General Scheme (L). 'H NMR (499 MHz, CDCl3) 5 'HNMR (499 MHz, CDCl3) 85.38 - 5.20 (m, 10H), 4.62 (ddd, J= 24.4, 12.3, 2.7 Hz, 2H), 4.27 - 4.18 (m, 2H), 3.85 (s, 2H), 3.71 - 3.49 (m, 5H), 2.62 - 2.21 (m, 18H), 2.00 (q, J = 6.3 Hz, 16H), 1.58 (p, J= 3.6 Hz, 15H), 1.47 (s, 8H), 1.39 (t, J= 5.5 Hz, 7H), 1.35 - 1.23 (m, 87H), 0.89 - 0.86 (m, 12H). MS found 1630 [M+H] - calcd for [ C102H184N2O12= 1629.3849],

[0572] Example 89 — Preparation of COMPOUND NO, 89

[0573] COMPOUND NO. 89 was prepared in accordance with the General Scheme (L).1H NMR (499 MHz, CDCl3) 85.39 - 5.30 (m, 6H), 4.14 (s, 6H), 3.80 - 3.30 (m, 18H), 2.76 (t, J = 6.7 Hz, 3H), 2.53 (q, J= 6.2 Hz, 1H), 2.35 - 2.25 (m, 8H), 2.19 - 2.12 (m, 2H), 2.04 (q,,7= 7.0 Hz, 7H), 1.66 - 1.45 (m, 35H), 1.36 - 1.25 (m, 51H), 0.88 (d, J= 7.1 Hz, 9H). MS found 1360 [M+H] calcd for [ C84H146N2O10= 1359.0927],

[0574] Example 90 — Preparation of COMPOUND NO. 90

[0575] COMPOUND NO. 90 was prepared in accordance with the General Scheme (M). 'll NMR (499 MHz, CDCl3) 85.27 (ddd, J= 8.4, 5.8, 2.7 Hz, 1H), 5.17 (dd, J= 8.1, 1.5 Hz, 1H), 4.82 (dt, J= 10.0, 3.0 Hz, 1H), 4.43 - 4.27 (m, 3H), 4.11 (dd, J= 12.5, 5.9 Hz, 1H), 3.80 (d, J = 10.0 Hz, 1H), 2.40- 2.14 (m, 7H), 2.01 - 1.89 (m, 9H), 1.80 (d, J= 14.0 Hz, 8H), 1.68 - 1.54 (m, 11H), 1.48 - 1.11 (m, 56H), 0.88 (t, J = 7.0 Hz, 20H). MS found 1138.8 [M+H]+calcd for [ C69H119NO11 =1137.8783],

[0576] Example 91 — Preparation of COMPOUND NO. 91

[0577] COMPOUND NO. 91 was prepared in accordance with the General Scheme (M). 'H NMR (499 MHz, CDCl3) 85.28 (td, J= 5.5, 2.7 Hz, 1H), 5.17 (d, J= 8.1 Hz, 1H), 4.85 (dt, J= 10.0, 3.2 Hz, 1H), 4.43 (dd, J= 12.4, 2.5 Hz, 1H), 4.30 (ddd, J= 14.5, 12.3, 3.1 Hz, 2H), 4.15 (dd, J= 12.4, 5.5 Hz, 1H), 3.80 (d, J= 10.0 Hz, 1H), 2.31 - 2.15 (m, 5H), 1.99 - 1.90 (m, 11H), 1.80 (d, J= 13.4 Hz, 10H), 1.61 (p, J= 3.7 Hz, 5H), 1.43 (q, J= 6.0 Hz, 5H), 1.37 (dd, J= 12.5, 3.5 Hz, 6H), 1.30 - 1.22 (m, 32H), 1.15-1.18 (m, 12H), 0.88 (t, J= 7.4 Hz, 20H). MS found 1111.0 [M+H]- calcd for [ C67H115NO11= 1109.8470],

[0578] Example 92 — Preparation of COMPOUND NO. 92

[0579] COMPOUND NO. 92 was prepared in accordance with the General Scheme (M). 'H NMR (499 MHz, CDCl3) 85.34 - 5.18 (m, 2H), 4.87 (dt, J= 9.6, 3.8 Hz, 1H), 4.54 - 4.21 (m, 3H), 4.15 -4.05 (m, 1H), 3.89 - 3.79 (m, 1H), 2.37 - 2.25 (m, 11H), 1.62-1.58 (m, 19H), 1.41 (d, J= 5.5 Hz, 4H), 1.30-1.27 (m, 73H), 0.88 (t, J= 7.0 Hz, 12H). MS found 1147.1 [M+H] -calcd for [ C69H127NO11 =1145.9409],

[0580] Example 93 — Preparation of COMPOUND NO. 93

[0581] COMPOUND NO. 93 was prepared in accordance with the General Scheme (M).1H NMR (499 MHz, CDCl3) 85.37 (d, J= 9.0 Hz, 2H), 5.03 - 4.97 (m, 2H), 4.20 (dd, J= 12.5, 2.5 Hz, 2H), 3.98 (dd, J= 12.5, 4.9 Hz, 2H), 3.01 (d, J= 5.9 Hz, 5H), 2.38 (td, J= 7.3, 2.5 Hz, 4H), 2.20 (dtt, J= 15.7, 12.2, 3.6 Hz, 6H), 2.02 - 1.91 (m, 14H), 1.86 - 1.77 (m, 10H), 1.70 - 1.62 (m, 14H), 1.54- 1.48 (m, 9H), 1.42 - 1.15 (m, 54H), 0.93 -0.86 (m, 20H). MS found 1373.9 [M+H] ‘ calcd for [ C84H144N2O12 = 1373.0719],

[0582] Example 94- Preparation of LNPs of Present Disclosure Comprising DNA and In Vivo Screening

[0583] The following is a nonlimiting example that provides exemplary methods for formulating a plurality of multi-component LNP compositions comprising exemplary compounds of the present disclosure and DNA.

[0584] To formulate the LNPs, one of COMPOUND NOS. 1-76, the phospholipid DOPC, the structural lipid cholesterol (Choi) and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (DMG-PEG2000; Avanti Polar Lipids, Alabaster, Alabama, USA) were combined to prepare LNP compositions.

[0585] Individual 25 mg / ml stock solutions were prepared by solubilizing the lipids in 200-proof HPLC-grade ethanol and stock solutions were stored at -80°C until formulated. At the time offormulation, the lipid stock solutions were briefly allowed to equilibrate to room temp and then placed on a hot plate maintained at a temperature range of 50-55°C. Subsequently, the hot lipid stock solutions were combined to yield desired final mol percentages. The LNP compositions are shown in Table 4.

[0586] Table 4

[0587] A 1 mg / ml solution of nanoplasmid DNA encoding firefly luciferase under the control of a CMV promoter (Nature Technology Corporation) 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 ethanol phase was vigorously mixed with the nucleic acid in sodium acetate phase using the Precision Nanoassemblr instrument.

[0588] The resultant LNP compositions were then transferred to a Repligen Float-A-Lyzer dialysis device- having a molecular weight cut off (MWCO) of 8-10kDa (Spectrum Chemical Mfg. Corp, CA, USA) and processed by dialysis against phosphate buffered saline (PBS) (dialysate: dialysis buffer volume at least 1:200 v / v), pH 7.4 overnight at 4°C (or alternatively room temperature for at least 4 hours), to remove the 25% ethanol and achieve a complete buffer exchange. In some experiments the LNPs were further concentrated by in an Amicon® Ultra-4 centrifugal filter unit, MWCO-30kDa (Millipore Sigma, USA) spun at -4100 x g in an ultracentrifuge. The LNPs were then stored at 4°C until further use.

[0589] Adult BALB / C mice (n=3) were administered 0.5 mg / kg of DNA formulated in an LNP composition listed in Table 4. One group of mice was treated with vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control.

[0590] The location and extent of luciferase expression in treated and control mice were determined at 48 hr for DNA delivery by bioluminescent 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 supine on a heated stage. Mice were then administered D-luciferin (Perkin-Elmer #122799) IP, and BLI was performed. The results are shown in Table 5.

[0591] Table 5

[0592] Visual analysis of the BLI images revealed that the BLI signal was predominantly located in the liver. Accordingly, as shown in Table 5, LNP compositions of the present disclosure successfully delivered DNA to liver cells and the encoded transgene was expressed in the liver cells. In addition, administration of LNP compositions of Table 4 were well tolerated in the mice. Most of the mice lost some weight 24 hours after administration but then recovered.

[0593] Example 95- Preparation of LNPs of Present Disclosure Comprising RNA and In Vivo Screening

[0594] The following is a nonlimiting example that provides exemplary methods for formulating a plurality of multi-component LNP compositions comprising exemplary compounds of the present disclosure and mRNA.

[0595] LNP compositions of the present disclosure comprising COMPOUND NO. 52 and RNA encoding firefly luciferase (TriLink BioTechnologies) were prepared as described in Example 94. The LNP compositions are shown in Table 6.

[0596] Table 6

[0597] Adult female BALB / C mice (n=3 / group) were intravenously administered 0.5 mg / kg of 5’-CleanCap— fLuciferase mRNA (TriLink Biotech) formulated with the LNP compositions shown in Table 6. One group of mice was treated with vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control.

[0598] The location and extent of luciferase expression in treated and control mice were determined at 4 hr by bioluminescent 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 supine on a heated stage. Mice were then administered D-luciferin (Perkin-Elmer #122799) IP, and BLI was performed. The results are shown in Table 7.

[0599] Table 7

[0600] Visual analysis of the BLI images revealed that the BLI signal was predominantly located in the liver. Accordingly, as shown in Table 7, LNP compositions of the present disclosure successfully delivered mRNA in vivo, predominantly to cells in the liver, and the encoded transgene was subsequently expressed by the cells.

[0601] Example 96- Preparation of LNPs of Present Disclosure Comprising RNA and Tn Vivo Screening

[0602] The following is a nonlimiting example that provides exemplary methods for formulating a plurality of multi-component LNP compositions comprising exemplary compounds of the present disclosure and mRNA.

[0603] LNP compositions of the present disclosure comprising COMPOUND NOS. 54 or 59 and RNA encoding firefly luciferase (TriLink BioTechnologies) were prepared as described in Example 94. The LNP compositions are shown in Table 8.

[0604] Table 8

[0605] Adult female BALB / C mice (n=3 / group) were intravenously administered 0.5 mg / kg of 5’-CleanCap— fLuciferase mRNA (TriLink Biotech) formulated with the LNP compositions shown in Table 8. One group of mice was treated with vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control.

[0606] The location and extent of luciferase expression in treated and control mice were determined at 4 hr by bioluminescent 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 supine on a heated stage. Mice were then administered D-luciferin (Perkin-Elmer #122799) IP, and BLI was performed. The results are shown in Table 9.

[0607] Table 9

[0608] Visual analysis of the BLI images revealed that the BLI signal was predominantly located in the liver. Accordingly, as shown in Table 9, LNP compositions of the present disclosure successfully delivered mRNA in vivo, predominantly to cells in the liver, and the encoded transgene was subsequently expressed by the cells.

[0609] Example 97- Preparation of LNPs of Present Disclosure Comprising RNA and Tn Vivo Screening

[0610] The following is a nonlimiting example that provides exemplary methods for formulating a plurality of multi-component LNP compositions comprising exemplary compounds of the present disclosure and mRNA.

[0611] LNP compositions of the present disclosure comprising one of COMPOUND NOS. 1-76 and RNA encoding firefly luciferase (TriLink BioTechnologies) were prepared as described in Example 94. The LNP compositions are shown in Table 10.

[0612] Table 10

[0613] In two separate experiments, adult female BALB / C mice (n=3 / group) were intravenously administered 0.5 mg / kg of 5’-CleanCap— fLuciferase mRNA (TriLink Biotech) formulated with the LNP compositions shown in Table 10. In each experiment, one group of mice was treated with vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control.

[0614] The location and extent of luciferase expression in treated and control mice were determined at 4 hr by bioluminescent 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 supine on a heated stage. Mice were then administered D-luciferin (Perkin-Elmer #122799) IP, and BLI was performed. The results are shown in Tables 11 and 12.

[0615] Table 11

[0616] Table 12

[0617] Visual analysis of the BLI images revealed that the BLI signal was predominantly located in the liver. Accordingly, as shown in Tables 11 and 12, LNP compositions of the present disclosure successfully delivered mRNA in vivo, predominantly to cells in the liver, and the encoded transgene was subsequently expressed by the cells.

[0618] Example 98- Preparation of LNPs of Present Disclosure Comprising DNA and In Vivo Screening

[0619] The following is a nonlimiting example that provides exemplary methods for formulating a plurality of multi-component LNP compositions comprising exemplary compounds of the present disclosure and DNA.

[0620] LNP compositions of the present disclosure comprising COMPOUND NOS. 67 or 69 and DNA encoding firefly luciferase were prepared as described in Example 94. The LNP compositions are shown in Table 13.

[0621] Table 13

[0622] Adult female BALB / C mice (n=3 / group) were intravenously administered 0.5 mg / kg of DNA formulated with the LNP compositions shown in Table 13. One group of mice was treated with vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control.

[0623] The location and extent of luciferase expression in treated and control mice were determined at 48 hr for DNA delivery by bioluminescent 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 supine on a heated stage. Mice were then administered D-luciferin (Perkin-Elmer #122799) IP, and BLI was performed. The results are shown in Table 14.

[0624] Table 14

[0625] Visual analysis of the BLI images revealed that the BLI signal was predominantly located in the liver. Accordingly, as shown in Table 14, LNP compositions of the present disclosure successfully delivered DNA in vivo, predominantly to cells in the liver, and the encoded transgene was subsequently expressed by the cells.

[0626] Example 99 - LNP compositions of present disclosure deliver RNA with high specificity to the liver in vivo

[0627] This experiment shows the ability of LNP compositions of the present disclosure to deliver Cas-CLOVER mRNA to the liver, targeted by a pair of gRNAs to the human KLKB1gene, resulting in subsequent in vivo gene editing of the KLKB1 gene. The KLKB1 gene encodes for the protease kallikrein, which is elevated during attacks of hereditary angioedema, a rare genetic disorder characterized by recurrent episodes of the accumulation of fluids outside of the blood vessels, blocking the normal flow of blood or lymphatic fluid and causing rapid swelling of tissue in the hands, feet, limbs, face, intestinal tract or airways.

[0628] In this experiment, each group of adult female BALB / C mice (n=2 / group) was intravenously co-administered mRNA encoding 5’-CleanCapM6-Cas-CLOVER v.3 (SEQ ID NO: 5) and a pair of gRNAs (SEQ ID NOs: 6 and 7) targeted to exon 11 of the mouse KLKB1 gene. SEQ ID NO: 5 is entered as the DNA sequence in electronic sequence listing, but it represents an mRNA sequence. "U" was replaced by "T" to comply with sequence listing requirements. The mRNA and gRNA molecules were formulated within LNP compositions of the present disclosure as shown in Table 15. All uridine residues in the mRNA were Nl-methylpsuedouridine.

[0629] Table 15

[0630] LNP compositions of the present disclosure (0.5 mg / kg) were administered to the mice from each group. One group of mice was treated with vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control.

[0631] Target site gene editing by the Cas-CLOVER mRNA delivered to the mice was measured by Droplet Digital PCR (ddPCR). Briefly, 50 ng of genomic DNA, primers and labeled probes were fractionated into 20000 droplets and PCR amplified with the following cycling parameters: 95C for 10 min, 40 cycles of 94C (30 sec), 55C (1 min), and a final step of 98C for 10 min. Then, samples were read with a QX200 droplet reader (Biorad) following manufacturer instructions. Percentage of indels was calculated as (# ofNEHJ droplets x 100) / (# ofNHEJ + # of reference droplets). Results of the ddPCR are provided in Table 16 as indel percentages found at the KLKB 1 intron 11 insertion site.

[0632] Table 16

[0633] As shown in Table 16, LNP compositions of the present disclosure successfully delivered Cas-CLOVER mRNA to the liver as shown by subsequent gene editing of the KLKB1 gene, with a range of activities from about 13% to greater than 34% edited KLKB1 loci.

[0634] Example 100 - LNP compositions comprising a polyphenol additive enhance delivery of DNA to the liver in vivo

[0635] This experiment shows the ability of LNP compositions of the present disclosure that comprise at least one polyphenol additive to enhance delivery of DNA to liver cells in vivo.

[0636] A series of LNP compositions of the present disclosure were prepared comprising the following components at the following % moles: COMPOUND NO. 69 (50% moles): DOPC (10% moles): cholesterol (38.5% moles): PEG-DMG (1.5% moles) and a DNA nanoplasmid encoding the flue gene operably associated with the constitutive CMV promoter, with a lipid: DNA (w / w) of 80: 1. Additionally, each LNP composition comprised a polyphenol additive compound of the following structure:at varying additive:total lipid weight ratios (0.1, 0.15, 0.2). One LNP composition was prepared without adding the additive. The compositions of the LNP compositions are listed in Table 17.

[0637] Table 17

[0638] In this experiment, each group of adult female BALB / C mice (n=3 / group) was intravenously administered the DNA nanoplasmid via tail vein injection. The DNA molecules were formulated within LNP compositions of the present disclosure as described in Table 17. The LNP compositions of the present disclosure (0.5 mg / kg) were administered to the mice from each of the groups. One group of mice was treated with vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control.

[0639] Luciferase expression was measured by whole body luminescence imaging (BLI) of anesthetized mice at 48 hours post-administration 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 supine on a heated stage. Mice were then administered D-luciferin (Perkin-Elmer #122799) IP, and BLI was performed. The results of BLI measurements (total flux [p / s]) are shown in Table 18.

[0640] Table 18

[0641] As shown in Table 18, the addition of at least one polyphenol additive to the LNP compositions resulted in an increase in BLI of up to 10-fold compared to the LNP composition lacking the additive.

[0642] Example 101- Preparation of LNPs of Present Disclosure Comprising RNA and In Vivo Screening

[0643] The following is a nonlimiting example that provides exemplary methods for formulating a plurality of multi-component LNP compositions comprising exemplary compounds of the present disclosure and mRNA.

[0644] LNP compositions of the present disclosure comprising one of COMPOUND NOS. 77-93 and RNA encoding firefly luciferase (TriLink BioTechnologies) were prepared as described in Example 94. The LNP compositions are shown in Table 19.

[0645] Table 19

[0646] In two separate experiments, adult female BALB / C mice (n=3 / group) were intravenously administered 0.5 mg / kg of 5’-CleanCap— fLuciferase mRNA (TriLink Biotech) formulated with the LNP compositions shown in Table 18. In each experiment, one group of mice was treated with vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control.

[0647] The location and extent of luciferase expression in treated and control mice were determined at 4 hr by bioluminescent 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 supine on a heated stage. Mice were then administered D-luciferin (Perkin-Elmer #122799) IP, and BLI was performed. The results are shown in Tables 19 and 20.

[0648] Table 19

[0649] Table 20

[0650] Visual analysis of the BLI images revealed that the BLI signal was predominantly located in the liver. Accordingly, as shown in Tables 19 and 20, LNP compositions of the present disclosure successfully delivered mRNA in vivo, predominantly to cells in the liver, and the encoded transgene was subsequently expressed by the cells.

[0651] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

1. What is claimed is:

1. A compound of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII) or Formula (IX):

6. 8.Formula (V) Formula (VI)10.

12. 14.Formula (IX)15.or a salt thereof,16.wherein:

18.

19. each R2 is independently: (i) C5-C20 alkyl with optionally one or more alkenyl or (ii)22.

23. each R5 is independently: (i) C5-C20 alkyl with optionally one or more alkenyl, (ii)27. 29.each R7 is independently:

30.

31. each Rs is independently: (i) C5-C20 alkyl with optionally one or more alkenyl, (ii)33.

34. each R9 is independently: (i) C5-C20 alkyl with optionally one or more alkenyl or (ii)36. 38.o40.each Rio is independently: (i) -H or (ii)41.

42. ;43.each Rn is independently: (i) C5-C20 alkyl with optionally one or more alkenyl or (ii)47. 49.each R13 is independently: (i) C5-C20 alkyl with optionally one or more alkenyl, (ii)50.-(C5-C20 alkyl)52.

53. (iii)54.

57.

58. n is an integer ranging from 0 to 6;59.p is 0, 1 or 2;60.each Ryis independently: C5-C20 alkyl with optionally one or more cyclohexyl; and each Rzis independently:

63.

2. The compound of claim 1, wherein Ri is66.

3. The compound of claim 2, wherein Rz is69.

4. The compound of any one of claims 1-3, wherein each R2 is independently73.

5. The compound of any one of claims 1-4, wherein n is 0.

6. The compound of any one of claims 1-4, wherein n is 1.

7. The compound of any one of claims 1-6, wherein each R3 is independently C5-C20 alkyl.

8. The compound of any one of claims 1-6, wherein each R3 is independently80.

9. A compound selected from:

83.

85.

87.

89.

91.

94.

96.

98.

101.

104.

106.

108.

111.

114.

116.

118.

121.

10. A lipid nanoparticle comprising at least one compound of any one of claims 1-9.

11. A lipid nanoparticle comprising at least one compound of any one of claims 1-9, at least one phospholipid, at least one structural lipid, and at least one PEGylated lipid.

12. The lipid nanoparticle of claim 11, further comprising at least one nucleic acid molecule.

13. The lipid nanoparticle of claim 12, wherein the at least one nucleic acid molecule is a RNA molecule.

14. The lipid nanoparticle of claim 12, wherein the at least one nucleic acid molecule is a DNA molecule.

15. The lipid nanoparticle of claim 12, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is from about 40:1 to about 100:1 (w / w).

16. A composition comprising at least one lipid nanoparticle comprising at least one compound of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII) and / or Formula (IX) of any one of claims 1- 9.

17. The composition of claim 16, wherein the at least one lipid nanoparticle comprises about 50% of the at least one compound of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII) and / or Formula (IX) by moles and at least one nucleic acid molecule,130.wherein the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule,131.wherein the at least one lipid nanoparticle further comprises:132.about 38.5% of cholesterol by moles,133.about 10% of DOPC by moles, and134.about 1.5% of DMG-PEG2000 by moles; and135.wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 40: 1, about 50:1 or about 80:1 (w / w).

18. The composition of claim 16, wherein the at least one lipid nanoparticle comprises about 54% of the at least one compound of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII) and / or Formula (IX) by moles and at least one nucleic acid molecule,137.wherein the at least one nucleic acid molecule comprises at least one RNA molecule and / or at least one DNA molecule,138.wherein the at least one lipid nanoparticle further comprises:139.about 35% of cholesterol by moles,140.about 10% of DOPC by moles, and141.about 1% of DMG-PEG2000 by moles; and142.wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 40: 1 or about 100:1 (w / w).

19. The composition according to any one of claims 17 or 18, wherein the RNA molecule is an mRNA molecule, preferably wherein the mRNA molecule further comprises a 5’- CAP.

20. The composition according to any one of claims 17 or 18 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 Beautytransposase, a Hyperactive Sleeping Beauty (SB100X) transposase, a helitron transposase, a Tol2 transposase, a TcBuster transposase or a mutant TcBuster transposase.

21. The composition according to any one of claims 17 or 18, wherein the DNA molecule is a circular DNA molecule, DoggyBone DNA molecule, a DNA plasmid, a DNA nanoplasmid, or a linearized DNA molecule, preferably wherein the DNA molecule is a DoggyBone DNA molecule or a DNA nanoplasmid.

22. The composition according to any one of claims 17 or 18, wherein the at least one DNA molecule comprises a nucleic acid sequence encoding at least one transposon.

23. The composition according to any one of claims 17-22, wherein the at least one nucleic acid molecule comprises a nucleic acid sequence encoding at least one therapeutic protein.

24. The composition according to of any one of claims 17-22, wherein the at least one nucleic acid molecule comprises a nucleic acid sequence encoding at least one transposon, wherein the transposon comprises a nucleic acid sequence encoding at least one therapeutic protein.

25. A pharmaceutical composition, comprising a composition of any one of claims 17-24 and at least one pharmaceutically-acceptable excipient or diluent.

26. A method of delivering at least one nucleic acid to at least one target cell comprising contacting the at least one target cell in vivo with at least one composition of any of claims 17-24.

27. A method of delivering at least one nucleic acid to at least one target cell comprising contacting the at least one target cell ex vivo with at least one composition of any of claims 17-24.

28. The method of claims 26 or 27, wherein the target cell is selected from a group consisting of immune cells, stem cells, epithelial cells, and liver cells.

29. A method of genetically modifying at least one cell comprising contacting the at least one cell with at least one composition of any of claims 17-24.

30. The method of claims 28 or 29 wherein the at least one cell is a liver cell.

31. The method of claim 30, wherein the liver cell is a hepatocyte, a hepatic stellate cell, Kupffer cell or liver sinusoidal endothelial cell.

32. At least one cell modified according to the method of any one of claims 29-31.

33. A method of treating at least one disease or disorder in a subject in need thereof comprising administering to the subject at least one therapeutically effective amount of the composition of any one of claims 17-24, the pharmaceutical composition of claim 25, or the at least one cell of claim 32.

34. The method of claim 33, wherein the at least one disease or disorder is a liver disease or disorder.

35. The method of claim 33, wherein the at least one disease of disorder is a genetic disorder.

36. The method of claim 33, wherein the at least one disease of disorder is cancer.

37. The lipid nanoparticle composition of claim 16, wherein the lipid nanoparticle composition comprises at least one genomic editing composition, wherein the at least one genomic 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 an inactivated nuclease domain thereof, (ii) a Clo051 protein or a nuclease domain thereof; and b) at least one gRNA molecule.

38. The lipid nanoparticle of any one of claims 10-15, wherein the lipid nanoparticle retains at least 80% activity after storage at 4°C for six months.

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