Cationic Lipids and Their Use

Abstract

The present invention relates to novel cationic lipids of formula I, and more specifically formula IV, which are used, for example, in liposomes for delivering nucleic acids to cells.

Description

Technical Field

[0001] Priority This application claims priority to U.S. Provisional Application No. 62 / 870,245, filed Jul. 3, 2019; U.S. Provisional Application No. 62 / 880,435, filed Jul. 30, 2019; and U.S. Provisional Application No. 63 / 023,654, filed May 12, 2020, the contents of which are hereby incorporated by reference in their entirety.

[0002] The present invention relates in part to various novel lipids, including methods, compositions, and products for delivering nucleic acids to cells.

[0003] Description of Electronically Submitted Text Files This application includes a Sequence Listing that was electronically submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy created on Jul. 3, 2020 is named FAB-012PC_ST25.txt and is 1,441,597 bytes in size.

Background Art

[0004] Lipid-based materials such as liposomes are used as biological carriers for pharmaceutical and other biological applications, for example, to introduce drugs into cultured cell lines. Lipids are commonly used to deliver nucleic acids to cells in vitro under low serum or serum-free conditions, for example, in transfection. However, serum components inhibit the activity of many lipids both in vitro and in vivo, thus limiting their use in the presence of serum.

[0005] For example, in both in vitro and in vivo settings, an improved lipid delivery system for achieving higher levels of transfection is desirable. In particular, a lipid delivery system that is active in the presence of serum is needed. Improvement in the level of transfection enables treatment of medical conditions that currently require expression levels higher than those achievable with lipid delivery systems for therapeutic effect. Alternatively, higher transfection levels enable use of smaller amounts of material to achieve comparable expression levels, thereby reducing potential toxicity and cost.

[0006] In the art, there is a need for novel lipids, lipid-like materials, and lipid-based delivery systems. SUMMARY OF THE INVENTION

[0007] Accordingly, the present invention relates, inter alia, to novel lipids useful in improving the delivery of biological payloads, such as nucleic acids, to cells.

[0008] In an aspect, the present invention relates to a compound of formula (I),

[0009]

Chemical formula

[0010] In one aspect, the present invention relates to a compound of formula (II).

[0011]

Chemical formula

[0012] In one aspect, the present invention relates to a compound of formula (III),

[0013]

Chemical formula

[0014] In an embodiment, the present invention relates to a compound of formula (IV),

[0015]

Chemical formula

[0016] In an embodiment, the present invention relates to a compound of formula (V)

[0017]

Chemical formula

[0018] In an embodiment, the present invention relates to a compound of formula (VI)

[0019]

Chemical formula

[0020] In an embodiment, the present invention relates to a compound of formula (VII)

[0021] [Chemical formula]

[0022] In an embodiment, the present invention relates to a compound of formula (VIII)

[0023] [Chemical formula]

[0024] In an embodiment, the present invention relates to a compound of formula (IX)

[0025] [Chemical formula]

[0026] In an embodiment, the present invention relates to a compound of formula (X)

[0027] [Chemical formula]

[0028] In an embodiment, the present invention relates to a compound of formula (XI)

[0029] [Chemical formula]

[0030] In an embodiment, the present invention relates to a compound of formula (XII)

[0031] [Chemical formula]

[0032] In an embodiment, the present invention relates to a compound of formula (XIII)

[0033] [Chemistry]

[0034] In an embodiment, the present invention relates to a compound of formula (XIV).

[0035] [Chemistry]

[0036] In an embodiment, the present invention relates to a compound of formula (XV).

[0037] [Chemistry] wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0038] In an embodiment, the present invention relates to a compound of formula (XVI).

[0039] [Chemistry]

[0040] In an embodiment, the compounds of the present application (for example, those of formulas I to XVI) are components of pharmaceutical compositions and / or lipid aggregates and / or lipid carriers and / or lipid nucleic acid complexes and / or liposomes and / or lipid nanoparticles.

[0041] In embodiments, the compounds of the present application (e.g., those of Formulas I - XVI) are components of pharmaceutical compositions and / or lipid aggregates and / or lipid carriers and / or lipid nucleic acid complexes and / or liposomes and / or lipid nanoparticles that do not require additional lipids or helper lipids. In embodiments, the compounds of the present application (e.g., those of Formulas I - XVI) are components of pharmaceutical compositions and / or lipid aggregates and / or lipid carriers and / or lipid nucleic acid complexes and / or liposomes and / or lipid nanoparticles that further comprise neutral lipids (e.g., dioleoylphosphatidylethanolamine (DOPE), 1,2 - dioleoyl - sn - glycero - 3 - phosphocholine (DOPC), or cholesterol) and / or additional cationic lipids (e.g., N - [1 - (2,3 - dioleoyloxy)propyl] - N,N,N - trimethylammonium chloride (DOTMA), 1,2 - bis(oleoyloxy) - 3 - 3 - (trimethylammonium)propane (DOTAP), or 1,2 - dioleoyl - 3 - dimethylammonium - propane (DODAP)).

[0042] In embodiments, the compounds of the present application (e.g., those of Formulas I - XVI) are components of pharmaceutical compositions and / or lipid aggregates and / or lipid carriers and / or lipid nucleic acid complexes and / or liposomes and / or lipid nanoparticles having a defined particle size. In some embodiments, particles comprising the compounds of the present application (e.g., those of Formulas I - XVI) are characterized as determining an average particle size and polydispersity. In some embodiments, the particles comprising the compounds of the present application have an average hydrodynamic radius of less than 50 nm, 50 - 100 nm, 100 - 150 nm, 150 - 200 nm, 200 - 250 nm, 250 - 500 nm, or greater than 500 nm. In some embodiments, the average particle size depends on the conditions of the particle formation process (e.g., without limitation, temperature, incubation time, solution pH, or solution ionic strength). In some embodiments, the compounds of the present application (e.g., those of Formulas I - XVI) are components of pharmaceutical compositions and / or lipid aggregates and / or lipid carriers and / or lipid nucleic acid complexes and / or liposomes and / or lipid nanoparticles whose average particle size depends on the ionic strength of the solution or the pH of the solution.

[0043] In an embodiment, the compound of the present application (for example, those of Formulas I to XVI) is a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier and / or a lipid nucleic acid complex and / or a liposome and / or a lipid nanoparticle, which is a constituent of a composition containing a nucleic acid, for example, DNA (for example, without limitation, plasmid, cosmid, phage, recombinant virus or other vector) or RNA (for example, without limitation, siRNA, microRNA (miRNA), long non-coding RNA (lncRNA), in vitro transcribed RNA, synthetic RNA, and / or mRNA, but in any case, containing one or more non-standard nucleotides that confer stability, avoid degradation by one or more nucleases, and / or avoid substantial cytotoxicity, or not containing non-standard nucleotides). In an embodiment, the RNA encodes a protein of interest, for example, without limitation, one or more reprogramming factors or gene editing proteins. In an embodiment, the RNA encodes an antigen that elicits an immune response.

[0044] In embodiments, the present invention relates to the delivery of synthetic RNA molecules capable of inducing immune tolerance to the encoded protein (e.g., without limitation, synthetic RNA molecules containing microRNA binding sites (e.g., miR142 microRNA binding sites, etc.), such microRNA binding sites optionally being present (optionally in one or more copies) in the 3'-UTR of the synthetic RNA molecule). In embodiments, the present invention relates to the delivery of synthetic RNA molecules capable of inducing immune tolerance to the encoded protein by co-delivery of a tolerance-inducing factor (e.g., without limitation, IL2, IL10, and / or tgf-β). In certain embodiments, the co-delivered factor is expressed by the synthetic RNA molecule. In embodiments, the synthetic RNA molecule encodes one or more gene editing proteins described herein. In one embodiment, the synthetic RNA molecule induces immune tolerance to one or more gene editing proteins. In another embodiment, the synthetic RNA molecule encodes a protein that is not normally present in the subject. In some embodiments, the subject has dystrophic epidermolysis bullosa. In other embodiments, the synthetic RNA molecule encodes one or more gene editing proteins that create a double-strand break into which a genetic payload is inserted. In some embodiments, the genetic payload includes an expression cassette. In other embodiments, the expression cassette induces the cell to express a therapeutic protein. In yet other embodiments, the therapeutic protein is immunotolerant. In further embodiments, the one or more gene editing proteins induce the cell to express a functional or semi-functional form of the protein. In one embodiment, the protein is collagen 7. Certain embodiments are directed to a composition for treating a disease, disorder, or condition, such composition comprising a synthetic RNA molecule of the present invention. In one embodiment, the disease, disorder, or condition is dystrophic epidermolysis bullosa.

[0045] In an embodiment, the pharmaceutical composition and / or lipid aggregate and / or lipid carrier and / or lipid nucleic acid complex and / or liposome and / or lipid nanoparticle are suitable for administration by injection, for example, without limitation, subcutaneous injection, intradermal injection, subdermal injection, intramuscular injection, and / or intravenous, intrathecal, intratumoral, intravitreal, subretinal, intraventricular, and / or topical administration and / or infusion. In an embodiment, the pharmaceutical composition and / or lipid aggregate and / or lipid carrier are suitable for administration as one or more injections containing less than about 10 ng of RNA or from about 10 ng to about 2000 ng of RNA.

[0046] In an aspect, the present invention relates to a method for transfecting a cell with a nucleic acid, comprising contacting the cell with a complex of the nucleic acid and a compound described herein (e.g., those of formulas I - XVI), wherein the complex of the nucleic acid and a compound described herein (e.g., those of formulas I - XVI) is optionally formed prior to contact with the cell.

[0047] In one aspect, the present invention relates to a method for reprogramming differentiated cells into a more undifferentiated state, comprising: (a) providing differentiated cells; (b) culturing the differentiated cells; and (c) transfecting the differentiated cells with a complex of one or more synthetic RNA molecules and a compound described herein (e.g., those of formulas I-XVI), wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors, and transfection results in cells expressing one or more reprogramming factors, resulting in the cells being reprogrammed into a more undifferentiated state. In embodiments, step (c) is performed in the presence of a medium containing components that support the reprogramming of the differentiated cells into a more undifferentiated state. In embodiments, the method further comprises repeating step (c) at least twice during a consecutive five-day period. In embodiments, the amount of one or more synthetic RNA molecules transfected in one or more subsequent transfections is greater than the amount transfected in one or more previous transfections. In embodiments, steps (a)-(c) are performed without using feeder cells and in the presence of feeder cell-conditioned medium. In embodiments, step (c) is performed without using irradiated human neonatal fibroblast feeder cells and in the presence of feeder cell-conditioned medium. In embodiments, the synthetic RNA molecule encodes one or more reprogramming factors (s) selected from Oct4, Sox2, Klf4, c-Myc, l-Myc, Tert, Nanog, Lin28, Utf1, Aicda, miR200 microRNA, miR302 microRNA, miR367 microRNA, miR369 microRNA, and biologically active fragments, analogs, variants, and family members thereof.

[0048] In one aspect, the present invention relates to a method for reprogramming non-pluripotent cells, comprising: (a) providing non-pluripotent cells; (b) culturing the cells; and (c) transfecting the cells with a complex of one or more synthetic RNA molecules and a compound described herein (e.g., those of formulas I-XVI), wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors, and transfection results in cells expressing one or more reprogramming factors, such that the cells are reprogrammed. In embodiments, step (c) is performed in the presence of a medium containing components that support the reprogramming of non-pluripotent cells. In embodiments, the method further comprises repeating step (c) at least twice over a period of five consecutive days. In embodiments, the amount of one or more synthetic RNA molecules transfected in one or more subsequent transfections is greater than the amount transfected in one or more previous transfections. In embodiments, steps (a)-(c) are performed without using feeder cells and in the presence of feeder cell-conditioned medium. In embodiments, step (c) is performed without using irradiated human neonatal fibroblast feeder cells and in the presence of feeder cell-conditioned medium. In embodiments, the synthetic RNA molecule encodes one or more reprogramming factor(s) selected from Oct4, Sox2, Klf4, c-Myc, l-Myc, Tert, Nanog, Lin28, Utf1, Aicda, miR200 microRNA, miR302 microRNA, miR367 microRNA, miR369 microRNA, and biologically active fragments, analogs, variants, and family members thereof.

[0049] In one aspect, the present invention relates to a method for performing gene editing on a cell, comprising transfecting the cell with a complex of one or more synthetic RNA molecules and a compound described herein (e.g., those of formulas I - XVI), wherein the one or more synthetic RNA molecules comprise at least one RNA molecule encoding one or more gene editing proteins selected from nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases, meganucleases, nickases, gene editing proteins disclosed in WO2014 / 071219A1 (incorporated herein by reference in its entirety), clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins, CRISPR / Cas9, Cas9, xCas9, Cas12a (Cpf1), Cas13a, Cas14, CasX, CasY, class 1 Cas proteins, class 2 Cas proteins, and MAD7, or natural or engineered variants, family members, orthologs, fragments or fusion constructs thereof.

[0050] In one aspect, the present invention relates to a method for performing reprogramming and / or gene editing on a cell, comprising contacting the cell with a complex of a nucleic acid and a compound described herein (e.g., those of formulas I - XVI) and / or a compound disclosed in US2009 / 0143583A1, which is incorporated herein by reference in its entirety.

[0051] In one aspect, the present invention relates to a pharmaceutical formulation comprising a nucleic acid of the present invention and a compound described herein (e.g., those of formulas I - XVI) and / or a compound disclosed in US2009 / 0143583A1, which is incorporated herein by reference in its entirety.

[0052] In embodiments, the new lipid is useful for vaccine delivery. In aspects, the present invention relates to a method of vaccinating against an infectious disease (without limitation, coronavirus infection, such as COVID-19), comprising contacting cells with a complex of a nucleic acid encoding an antigen of an infectious agent and a compound described herein (e.g., those of Formulas I-XVI).

[0053] In aspects, the present invention relates to a method for extracting an organic compound from a reactant containing lithium aluminum hydride and a solvent, comprising (a) stopping the reaction with water, (b) removing the solvent, (c) removing excess water, and (d) extracting the organic compound with an alcohol (optionally isopropyl alcohol) so as to obtain the extracted organic compound.

[0054] In aspects, the present invention relates to a method for extracting an organic compound from a reactant containing a water-reactive compound and a first solvent, comprising (a) stopping the reaction with water, (b) removing the first solvent, (c) removing excess water, and (d) extracting the organic compound with a second solvent so as to obtain the extracted organic compound.

[0055] In aspects, the present invention relates to a method for purifying an organic compound from a mixture of an organic compound and a phthalimide or phthalimide derivative (optionally phthalhydrazine), comprising (a) dissolving the mixture in acetone to form a precipitate, (b) removing the precipitate by centrifugation, and (c) removing acetone so as to obtain the purified organic compound.

[0056] Details of the present invention are set forth in the following accompanying description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described herein. Other features, objects, and advantages of the present invention will become apparent from the description and claims. In this specification and the appended claims, the singular forms also include the plural forms unless the context clearly dictates otherwise. Unless defined otherwise, 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 invention belongs.

[0057] Any aspect or embodiment disclosed herein can be combined with any other aspect or embodiment disclosed herein.

Brief Description of the Drawings

[0058]

Figure 1

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Figure 5B

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Figure 11A

Figure 11B

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Figure 20A

Figure 20B

Mode for Carrying Out the Invention

[0059] The present invention is based in part on the discovery of novel lipids that exhibit an excellent ability to support the delivery of nucleic acids to cells, especially during transfection. The present invention provides such compositions, methods of making such compositions, and methods of using such compositions to introduce nucleic acids into cells, for example, for the treatment of diseases.

[0060] Compound and Synthesis Method In an aspect, the present invention relates to a compound of formula (I),

[0061]

Chemical formula

[0062] In an embodiment, Q1, Q2, Q3, and Q4 are each independently N, B, P, or Fe.

[0063] In an embodiment, Q1, Q2, Q3, and Q4 are N.

[0064] In an embodiment, Q1, Q2, Q3, and Q4 are each independently a primary amine, a secondary amine, or a tertiary amine.

[0065] In an embodiment, Q1 and Q4 are primary amines, and Q2 and Q3 are tertiary amines.

[0066] In an embodiment, L1, L2, and L3 are independently (C1-C6)alkanediyl or (hydroxy)(C1-C6)alkanediyl.

[0067] In an embodiment, L1 and L3 are independently (hydroxy)(C1-C6)alkanediyl, and L2 is (C1-C6)alkanediyl.

[0068] In an embodiment, L1 and L3 are 2-hydroxypropanediyl, and L2 is (C1-C6)alkanediyl.

[0069] In an embodiment, L1 and L3 are 2-hydroxypropanediyl, and L2 is butanediyl.

[0070] In an embodiment, one or more of R1, R2, R3, R4, R5, R6, R7, and R8 are independently selected from H, linoleyl, alpha-linolenyl, gamma-linolenyl, linoelaidyl, arachidonyl, eicosapentaenyl, and docosahexaenyl.

[0071] In an embodiment, two or more of R1, R2, R3, R4, R5, R6, R7, and R8 are independently selected from H, linoleoyl, alpha-linolenoyl, gamma-linolenoyl, linoleidoyl, arachidonoyl, eicosapentaenoyl, and docosahexaenoyl.

[0072] In an embodiment, one or more of R1, R2, R3, R4, R5, R6, R7, and R8 are independently selected from H, myristoleyl, palmitoleyl, sapienyl, oleyl, elaidyl, vaccenyl, erucyl, caprylyl, capryl, lauryl, myristyl, palmityl, stearyl, arachidyl, behenyl, lignoceryl, and cerotyl.

[0073] In an embodiment, two or more of R1, R2, R3, R4, R5, R6, R7, and R8 are independently selected from H, myristoleoyl, palmitoleoyl, sapienoyl, oleoyl, elaidoyl, vaccenoyl, erucoyl, capryloyl, caproyl, lauroyl, myristoyl, palmitoyl, stearoyl, arachidoyl, behenoyl, lignoceroyl, and cerotoyl.

[0074] In an embodiment, R1, R2, R3, R5, R7, and R8 are H, and R4 and R6 are independently selected from linoleoyl, alpha-linolenyl, gamma-linolenyl, linoelaidyl, arachidonyl, eicosapentaenyl, and docosahexaenyl.

[0075] In an embodiment, R1, R2, R3, R5, R7, and R8 are H, and R4 and R6 are alpha-linolenyl.

[0076] In an embodiment, R1, R2, R3, R5, R7, and R8 are H, and R4 and R6 are linoleoyl.

[0077] In an embodiment, x and z are independently 0 or 1, and y is 1.

[0078] In an embodiment, Q1 and Q4 are primary amines, Q2 and Q3 are tertiary amines, L1 and L3 are 2-hydroxypropanediyl, L2 is butanediyl, R1, R2, R3, R5, R7, and R8 are H, R4 and R6 are linoleoyl, and x, y, and z are 1.

[0079] In an embodiment, one or more of L1, L2, and L3 contain at least one ester moiety.

[0080] In an embodiment, one or more of R2 and R4 contain at least one ester moiety.

[0081] In the embodiment, A1 and A2 are H.

[0082] In an aspect, the present invention relates to a compound of formula (II),

[0083]

Chemical formula

[0084] In the embodiment, R 15 , R 16 , R 17 , R 18 R 19 , and R 20 are H, R9, R 10 , R 11 , R 12 , R 13 and R 14 are, independently, H or (C1-C6)alkyl, m is 8, i is 8, s is 1, j is 1, k is 4, and t is 4.

[0085] In the embodiment, R15 , R 16 , R 17 , R 18 R 19 , and R 20 is H, R9, R 10 , R 11 , R 12 , R 13 and R 14 are, independently, H or (C1-C6) alkyl, R 27 and R 28 are methyl, m is 8, i is 8, s is 1, j is 1, k is 4, and t is 4.

[0086] In an embodiment, R 15 , R 16 , R 17 , R 18 R 19 , and R 20 is H, R9, R 10 , R 11 , R 12 , R 13 and R 14 are, independently, H, R 27 and R 28 are methyl, m is 8, i is 8, s is 1, j is 1, k is 4, and t is 4.

[0087] In an embodiment, R 15 , R 16 , R 17 , R 18 R 19 , and R 20 is H, R9, R 10 , R 11 , R 12 , R 13 and R 14 are, independently, H, R 27 and R 28 are methyl, R 21 , R 22 , R 23 , R 24 , R 25 , and R 26 is H, m is 8, i is 8, s is 1, j is 1, k is 4, and t is 4.

[0088] In the embodiment, R 15 , R 16 , R 17 , R 18 R 19 , and R 20 are H, R9, R 10 , R 11 , R 12 , R 13 and R 14 are, independently, H, R 27 and R 28 are methyl, R 21 , R 22 , R 23 , R 24 , R 25 , and R 26 are H, m is 9, i is 9, s is 1, j is 1, k is 4, and t is 4.

[0089] In the embodiment, R 15 , R 16 , R 17 , R 18 R 19 , and R 20 are H, R9, R 10 , R 11 , R 12 , R 13 and R 14 are, independently, H, R 27 and R 28 are methyl, R 21 , R 22 , R 23 , R 24 , R 25 , and R 26 are H, m is 9, i is 9, s is 1, j is 1, k is 3, and t is 3.

[0090] In an aspect, the present invention relates to a compound of formula (III),

[0091] [Chemical formula] wherein, L4, L5, L6, and L7 are independently a bond, (C1-C 20 )alkanediyl, (halo)(C1-C 20 )alkanediyl, (hydroxy)(C1-C 20 )alkanediyl, (alkoxy)(C1-C 20 )alkanediyl, arylene, heteroarylene, cycloalkanediyl, heterocyclo-diyl, -(CH2) v1 -C(O)-, or -((CH2) v1 -O) v2- , or -((CH2) v1 -C(O)-O) v2 -, and R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R 35 are independently H, (C1-C 60 )alkyl, (halo)(C1-C 60 )alkyl, (hydroxy)(C1-C 60 )alkyl, (alkoxy)(C1-C 60 )alkyl, (C2-C 60 )alkenyl, (halo)(C2-C 60 )alkenyl, (hydroxy)(C2-C 60 )alkenyl, (alkoxy)(C2-C 60 )alkenyl, (C2-C 60 )alkynyl, (halo)(C2-C 60 )alkynyl, (hydroxy)(C2-C 60 )alkynyl, (alkoxy)(C2-C 60 )alkynyl, wherein at least one of R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R 35 contains at least two unsaturated bonds, v, v1, and v2 are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0092] In an embodiment, L4, L5, L6, and L7 are -(CH2)3-, and R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R 35 are independently H or (C2-C 60 ) alkenyl, v is 1, where at least two of R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R 35 contain at least two unsaturated bonds.

[0093] In an embodiment, L4 and L5 are -(CH2)3-, L6 and L7 are -(CH2)4-, and R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R 35 are independently H or (C2-C 60 ) alkenyl, v is 1, where at least two of R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R 35 contain at least two unsaturated bonds.

[0094] In an embodiment, L4 and L6 are -(CH2)3-, L5 and L7 are -(CH2)4-, and R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R 35 are independently H or (C2-C 60 ) alkenyl, v is 1, where at least two of R 29 , R30 , R 31 , R 32 , R 33 , R 34 , and R 35 At least two of them contain at least two unsaturated bonds.

[0095] In an embodiment, L4 and L7 are -(CH2)3-, L5 and L6 are -(CH2)4-, R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R 35 are independently H or (C2-C 60 ) alkenyl, v is 1, where R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R 35 At least two of them contain at least two unsaturated bonds.

[0096] In an embodiment, L4 and L6 are -(CH2)3-, L5 and L7 are -(CH2)5-, R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R 35 are independently H or (C2-C 60 ) alkenyl, v is 1, where R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R 35 At least two of them contain at least two unsaturated bonds.

[0097] In an embodiment, L4 and L7 are (hydroxy)(C1-C 20 )alkanediyl, L5 and L6 are -(CH2)3-, R 29 , R30 , R 31 , R 32 , R 33 , R 34 , and R 35 are, independently, H or (C2-C 60 ) alkenyl, v is 1, where at least two of R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R 35 contain at least two unsaturated bonds.

[0098] In an embodiment, L4 and L7 are (hydroxy)(C1-C 20 ) alkanediyl, L5 and L6 are -(CH2)3-, and R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R 35 are, independently, H or (C2-C 60 ) alkenyl, v is 1, where at least three of R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R 35 contain at least two unsaturated bonds.

[0099] In an embodiment, L4 and L7 are (hydroxy)(C1-C 20 ) alkanediyl, L5 and L6 are -(CH2)4-, and R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R 35 are, independently, H or (C2-C 60 ) alkenyl, v is 1, where R 29 , R 30 , R 31 , R 32, R 33 , R 34 , and R 35 at least three of which contain at least two unsaturated bonds.

[0100] In an embodiment, L4 and L7 are (hydroxy)(C1-C 20 )alkanediyl, L5 and L6 are -(CH2)5-, R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R 35 are independently H or (C2-C 60 )alkenyl, v is 1, where at least three of R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R 35 contain at least two unsaturated bonds.

[0101] In an embodiment, L4 and L7 are (hydroxy)(C1-C 20 )alkanediyl, L5 and L6 are -(CH2)6-, R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R 35 are independently H or (C2-C 60 )alkenyl, v is 1, where at least three of R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R 35 contain at least two unsaturated bonds.

[0102] In an embodiment, R 29 , R 30 , R 31 , R 32 , R 33, R 34 , and R 35 Two or more of which are independently selected from H, myristoleoyl, palmitoleoyl, sapienoyl, oleoyl, elaidoyl, vaccenoyl, erucoyl, capryloyl, caproyl, lauroyl, myristoyl, palmitoyl, stearoyl, arachidoyl, behenoyl, lignoceroyl, and cerotoyl.

[0103] In an embodiment, R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , and R 35 Two or more of which are independently selected from H, linoleyl, alpha-linolenyl, gamma-linolenyl, linoelaidyl, arachidonyl, eicosapentaenyl, and docosahexaenyl.

[0104] In an embodiment, L4 and L7 are 2-hydroxypropanediyl, L2 is butanediyl, R 29 , R 30 , R 31 , and R 32 are H, R 33 , R 34 , and R 35 are linoleyl, L5 and L6 are -(CH2)3-, and v is 1.

[0105] In an embodiment, L4 and L7 are 2-hydroxypropanediyl, L2 is butanediyl, R 29 , R 30 , R 31 , and R 32 are H, R 33 , R 34 , and R 35 are linoleyl, L5 and L6 are -(CH2)4-, and v is 1.

[0106] In an embodiment, L4 and L7 are 2-hydroxypropanediyl, L2 is butanediyl, R 29 , R30 , R 31 , and R 32 is H, R 33 , R 34 , and R 35 is linoleyl, L5 and L6 are -(CH2)5-, and v is 1.

[0107] In an embodiment, L4 and L7 are 2-hydroxypropanediyl, L2 is butanediyl, R 29 , R 30 , R 31 , and R 32 is H, R 33 , R 34 , and R 35 is linoleyl, L5 and L6 are -(CH2)6-, and v is 1.

[0108] In an embodiment, the present invention relates to a compound of formula (IV),

[0109] [Chemical formula] wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0110] In an embodiment, the present invention relates to a compound of formula (V)

[0111] [Chemical formula]

[0112] In an embodiment, the present invention relates to a compound of formula (VI)

[0113] [Chemical formula]

[0114] In an embodiment, the present invention relates to a compound of formula (VII)

[0115] [Chemical formula]

[0116] In an embodiment, the present invention relates to a compound of formula (VIII)

[0117] [Chemical formula]

[0118] In an embodiment, the present invention relates to a compound of formula (IX)

[0119] [Chemical formula]

[0120] In an embodiment, the present invention relates to a compound of formula (X)

[0121] [Chemical formula]

[0122] In an embodiment, the present invention relates to a compound of formula (XI)

[0123] [Chemical formula]

[0124] In an embodiment, the present invention relates to a compound of formula (XII)

[0125] [Chemical formula]

[0126] In an embodiment, the present invention relates to a compound of formula (XIII)

[0127] [Chemical formula]

[0128] In an embodiment, the present invention relates to a compound of formula (XIV)

[0129]

Chemical formula

[0130] In an embodiment, the present invention relates to a compound of formula (XV),

[0131]

Chemical formula

[0132] In an embodiment, the present invention relates to a compound of formula (XVI)

[0133]

Chemical formula

[0134] The present invention also relates to compounds (such as those of formulas I to XVI) and intermediates and synthetic methods for preparing the compositions of the present invention.

[0135] In an embodiment, a compound (such as those of formulas I to XVI), and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier and / or a lipid nucleic acid complex and / or a liposome and / or a lipid nanoparticle containing the compound (such as those of formulas I to XVI) is soluble in an alcohol (such as ethyl alcohol) at room temperature (such as about 20 - 25 °C) and / or at low temperature (such as about 0 °C, or about -10 °C, or about -20 °C, or about -30 °C, or about -40 °C, or about -50 °C, or about -60 °C, or about -70 °C, or about -80 °C).

[0136] Specific synthetic methods have been discovered that are useful, for example, in the preparation of the compounds described herein (e.g., those of Formulas I-XVI). Certain embodiments relate to methods for extracting organic compounds from reactants containing lithium and / or aluminum compounds. Other embodiments relate to methods for extracting organic compounds from reactants containing reducing agents. In one embodiment, the reducing agent is a metal hydride. In another embodiment, the reducing agent is lithium aluminum hydride. In some embodiments, the reaction is quenched with water to obtain a quenched reaction product. In other embodiments, one or more solvents are removed from the quenched reaction product, for example, by evaporation. In one embodiment, the quenched reaction product is dried, for example, under heating and / or reduced pressure. In another embodiment, the dried quenched reaction product is extracted with a solvent. In some embodiments, the solvent is an alcohol. In one embodiment, the alcohol is isopropyl alcohol. In another embodiment, the isopropyl alcohol is heated to about 80 °C. Further embodiments include decanting and / or filtering the solvent containing the organic compound. In yet another embodiment, the solvent is removed, for example, by evaporation, to obtain the organic compound.

[0137] Methods for purifying the compounds of the present invention (e.g., those of Formulas I - XVI) have also been discovered. Accordingly, certain embodiments are directed to methods for purifying a compound (e.g., those of Formulas I - XVI). In one embodiment, a sample containing a compound (e.g., those of Formulas I - XVI) and one or more impurities is suspended in a solvent. In one embodiment, the solvent is acetone. In another embodiment, the solvent is heated. In some embodiments, the compound is soluble in the solvent. In other embodiments, at least one of the one or more impurities is insoluble in the solvent. In one embodiment, the one or more impurities include phthalimide derivatives. In one embodiment, the one or more impurities include phthalhydrazine. In a further embodiment, the sample is suspended in acetone, the compound dissolves, and at least one of the one or more impurities forms a precipitate. In yet another embodiment, the precipitate is removed by decantation, filtration, and / or centrifugation. In yet another embodiment, the solvent is removed, e.g., by evaporation, to obtain the purified compound.

[0138] In an aspect, the present invention relates to a method for extracting an organic compound from a reactant containing lithium aluminum hydride and a solvent, comprising (a) quenching the reaction with water, (b) removing the solvent, (c) removing excess water, and (d) extracting the organic compound with an alcohol (optionally isopropyl alcohol) so as to obtain the extracted organic compound.

[0139] In an aspect, the present invention relates to a method for extracting an organic compound from a reactant containing a water - reactive compound and a first solvent, comprising (a) quenching the reaction with water, (b) removing the first solvent, (c) removing excess water, and (d) extracting the organic compound with a second solvent so as to obtain the extracted organic compound.

[0140] In one aspect, the present invention relates to a method for purifying an organic compound from a mixture of an organic compound and a phthalimide or phthalimide derivative (optionally phthalhydrazine), comprising (a) dissolving the mixture in acetone to form a precipitate, (b) removing the precipitate by centrifugation, and (c) removing the acetone, such that a purified organic compound is obtained.

[0141] Pharmaceutical composition, lipid aggregate, lipid carrier In embodiments, the present invention relates to a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier and / or a lipid nucleic acid complex and / or a liposome and / or a lipid nanoparticle comprising a compound described herein (e.g., those of formulas I - XVI).

[0142] In embodiments, the pharmaceutical composition and / or the lipid aggregate and / or the lipid carrier and / or the lipid nucleic acid complex and / or the liposome and / or the lipid nanoparticle can be in any physical form, including, for example, lipid nanoparticles, liposomes, micelles, interleaved bilayers, etc.

[0143] In embodiments, the pharmaceutical composition and / or the lipid aggregate and / or the lipid carrier is a liposome. In embodiments, the liposome is a large unilamellar vesicle (LUV), a multilamellar vesicle (MLV), or a small unilamellar vesicle (SUV). In embodiments, the liposome has a diameter of up to about 50 - 80 nm. In embodiments, the liposome has a diameter of about 80 - over 1000 nm, or more. In embodiments, the liposome has a diameter of about 50 - 1000 nm, e.g., about 200 nm or less. Size refers to the size (diameter) of the formed particles. Alternatively, size may refer to the hydrodynamic radius of the formed particles. The particle size distribution can be determined using quasi - elastic light scattering (QELS) with a sub - micron particle sizer Nicomp Model 370.

[0144] In certain embodiments, the present invention relates to methods and compositions for generating lipid-encapsulated nucleic acid particles in which the nucleic acid is encapsulated within a lipid layer. Such nucleic acid-lipid particles, including but not limited to those incorporating RNA, are characterized using various biophysical parameters, including drug-to-lipid ratio, encapsulation efficiency, particle size, and polydispersity index (PDI). High drug-to-lipid ratios, high encapsulation efficiencies, good nuclease resistance and serum stability, and a controllable particle size, generally less than 200 nm in diameter (but not limited thereto), are desirable.

[0145] In certain embodiments, the particle size that can affect transfection efficiency depends on the ionic strength of the complex formation medium. In other embodiments, the particle size can depend on the ionic strength of the body fluid. In embodiments, the particle size depends on the pH of the complex formation medium, or the pH of the body fluid. In various embodiments, the particle size depends on the time or temperature of complex formation, or the mixing ratio of the components of the pharmaceutical composition and / or lipid aggregate and / or lipid carrier and / or lipid nucleic acid complex and / or liposome and / or lipid nanoparticle. In some embodiments, the particle size can be measured as the Z-average particle size reporting the average size of the particle distribution. In some embodiments, the desired particle size is from about 100 nm to about 200 nm, or from about 150 nm to about 200 nm, or from about 150 nm to about 175 nm, or from about 155 to about 165 nm.

[0146] Another physical property of the lipid nanoparticles according to the present invention is the polydispersity index (PDI), which is an indicator of their particle size distribution. The term "polydispersity" (or "dispersion") is used to describe the degree of heterogeneity of the particle size distribution. The PDI is dimensionless and is scaled such that values less than 0.15 are considered monodisperse. Danaei et al., Pharmaceutics. 2018 May 18;10(2):57. PDI values greater than 0.7 indicate that the particle size distribution of the sample is very broad and may be suitable for analysis by dynamic light scattering (DLS) techniques. Ibid. PDI values less than 0.3 are considered acceptable for drug delivery applications using lipid-based carriers, and values less than 0.2 are generally considered acceptable for implementations with various polymer-based nanoparticles. Ibid.; see also Badran et al., Digest J. Nanomater. Biostruct. 2014, 9, 83-91, Chen et al., Int. J. Pharm. 2011, 408, 223-234, Putri et al., J. Pharm. Sci. Commun. 2017, 14, 79-85.

[0147] In some embodiments, the metrics of particle size (e.g., measured as the Z-average particle size (nm)) and PDI represent the suitability of the lipid nanoparticles for transfection. In embodiments, the lipid carriers according to the present invention are less than 200 nm in diameter and have a PDI of less than 0.2.

[0148] In some embodiments, the lipid carrier according to the present invention has a zeta potential that is pH-dependent. The zeta potential, measured in volts (V) or millivolts (mV), is determined for the characterization of nanoparticles and estimates the surface charge, which can be used to understand the physical stability of the nano-suspension. The zeta potential is an important indicator of the stability of a colloidal dispersion. The zeta potential is the potential at the boundary of the double layer (DL) or the shear plane of the particles and typically has values in the range of +100 to -100 mV. For electrostatically stabilized suspensions, zeta potential values of less than -20 mV or greater than +20 mV are typically desirable. The zeta potential is affected by the pH of the medium. Other factors include ionic strength, the concentration of additive(s), and temperature.

[0149] In some embodiments, the zeta potential of a pharmaceutical composition containing lipid particles is less than -20 mV at a pH greater than 7.0, or less than -20 mV at a pH greater than 7.1, or less than -20 mV at a pH greater than 7.2, or less than -20 mV at a pH greater than 7.3, or less than -20 mV at a pH greater than 7.4, or less than -20 mV at a pH greater than 7.5.

[0150] In embodiments, the compounds, pharmaceutical compositions, or lipid aggregates according to the present disclosure have a Z-average particle size of about 50 nm to about 2000 nm, or about 700 nm to about 1500 nm. In some embodiments, the Z-average particle size is about 750 nm. In some embodiments, the Z-average particle size is less than about 200 nm. In some embodiments, a titrable particle size is desirable to determine the ability of the compound, or pharmaceutical composition, or lipid aggregate to permeate in vivo from the site of administration, although not limited thereto.

[0151] In some embodiments, the Z-average particle size is controlled using the properties of the medium in which the compounds, pharmaceutical compositions, or lipid aggregates according to the present disclosure are formed.

[0152] In an embodiment, the Z-average particle size is controlled using the ionic strength of the medium in which the compound, pharmaceutical composition, or lipid aggregate according to the present disclosure is formed.

[0153] In an embodiment, the particle size is determined by controlling the concentration of any one or more solutes (e.g., without limitation, sodium chloride, calcium chloride, potassium chloride, or sodium phosphate) in the medium for forming the lipid aggregate. In an embodiment, the lipid aggregate is formed in deionized water without adding any solute, which can optionally be used to control the particle size.

[0154] In an embodiment, the ionic strength of the medium in which the compound, pharmaceutical composition, or lipid aggregate according to the present disclosure is formed is used to maintain the particle size at 200 nm or less.

[0155] In an embodiment, the pH of the medium in which the compound, pharmaceutical composition, or lipid aggregate according to the present disclosure is formed is used to control the Z-average particle size.

[0156] In an embodiment, the compound, pharmaceutical composition, or lipid aggregate according to the present disclosure comprises a stable particle dispersion. In some embodiments, the stable dispersion has a pH of about 7.0 to about 8.0 or a pH of about 7.4.

[0157] In some embodiments, nuclease resistance is conferred by the formation of a complex between a nucleic acid (e.g., small interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), long non-coding RNA (lncRNA), plasmid DNA, etc.) and lipid nanoparticles. In some embodiments, the nuclease is RNase, optionally RNase A. In some embodiments, the RNase is naturally present in vivo.

[0158] The nucleic acid to lipid ratio is the amount of nucleic acid in a preparation of defined volume divided by the amount of lipid in the same volume. This can be based on molar / molar, or weight / weight, or weight / molar, or molar / weight. For the final, immediately administrable formulation, the nucleic acid:lipid ratio can optionally be calculated after dialysis, chromatography and / or enzymatic (e.g., nuclease) digestion has been used to remove as much of the exogenous nucleic acid as possible.

[0159] The encapsulation efficiency refers to the ratio of the drug (including nucleic acid) to lipid ratio of the starting mixture divided by the drug (including nucleic acid) to lipid ratio of the final, administration-appropriate formulation. This is a measure of relative efficiency. For an absolute measure of efficiency, the total amount of nucleic acid added to the starting mixture until the final, administration-appropriate formulation is reached can also be calculated. The amount of lipid lost during the formulation process can also be calculated. Efficiency is a measure of formulation loss and cost.

[0160] Transfection In embodiments, the compounds of the present application (e.g., those of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers are useful in lipid aggregates for the delivery of macromolecules and other compounds to cells. In embodiments, the compounds of the present application (e.g., those of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers are useful for the delivery of nucleic acids to cells.

[0161] In embodiments, a method for transfecting a cell with a nucleic acid is provided, comprising contacting the cell with a complex of the nucleic acid and a compound of the present application (e.g., those of Formulas I-XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier. In embodiments, the complex of the nucleic acid and the compound of the present application (e.g., those of Formulas I-XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier is formed prior to contact with the cell.

[0162] In embodiments, the compounds of the present application (e.g., those of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers encapsulate nucleic acids with high efficiency and / or have a high drug:lipid ratio and / or protect the encapsulated nucleic acids from degradation and / or clearance in serum and / or are suitable for systemic delivery and / or result in intracellular delivery of the encapsulated nucleic acids. Further, the compounds of the present application (e.g., those of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers are highly tolerable and provide an appropriate therapeutic index, such that treatment of patients with an effective dose of nucleic acid is not associated with significant toxicity and / or risk to the patient.

[0163] In embodiments, the compounds of the present application (e.g., those of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers are polycationic. In embodiments, the compounds of the present application (e.g., those of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers form stable complexes with various anionic macromolecules, such as polyanions, such as nucleic acids, such as RNA or DNA. These compounds (e.g., those of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers have the property of forming strong lipid aggregates with polyanions via their cationic moieties when dispersed in water in various embodiments. By using an excess of cationic charge relative to the anionic compound, the polyanion-lipid complex can be adsorbed to the cell membrane, thereby facilitating uptake of the desired compound by the cell.

[0164] In embodiments, the compounds of the present application (e.g., those of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers and / or lipid nucleic acid complexes and / or liposomes and / or lipid nanoparticles mediate one or more of the following: (i) reducing the nucleic acid payload to be delivered, without wishing to be bound by theory, protecting it from nuclease degradation, and enhancing receptor-mediated uptake; (ii) improving the association with negatively charged cell membranes, without wishing to be bound by theory, by imparting a positive charge to the complex; (iii) promoting fusion with endosomal membranes, without wishing to be bound by theory, and promoting the release of the complex from the endosomal compartment; and (iv) enhancing transport from the cytoplasm to the nucleus.

[0165] In embodiments, the present invention relates to the compounds of the present application (e.g., those of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers for transfection, or methods of transfection, with high transfection efficiency. In embodiments, the transfection efficiency is measured by evaluating the percentage of transfected cells compared to the entire population during the transfection protocol. In various embodiments, the transfection efficiency of the compositions and methods of the present application is greater than about 30%, or greater than about 40%, or greater than about 50%, or greater than about 60%, or greater than about 70%, or greater than about 80%, or greater than about 90%, or greater than about 95%. In various embodiments, the transfection efficiency of the compositions and methods of the present application is higher than that of commercially available products (e.g., LIPOFECTIN, LIPOFECTAMINE, LIPOFECTAMINE 2000, LIPOFECTAMINE 3000 (Life Technologies)). In various embodiments, the transfection efficiency of the compositions and methods of the present application is about 5-fold, or 10-fold, or 15-fold, or 20-fold, or 30-fold higher than that of commercially available products (e.g., LIPOFECTIN, LIPOFECTAMINE, LIPOFECTAMINE 2000, LIPOFECTAMINE 3000 (Life Technologies)).

[0166] In embodiments, the present invention relates to the compounds of the present application (e.g., those of formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers for transfection, or methods of transfection, which enable high-level endosomal escape. In various embodiments, the endosomal escape of the compositions and methods of the present application is greater than that of commercially available products (e.g., LIPOFECTIN, LIPOFECTAMINE, LIPOFECTAMINE 2000, LIPOFECTAMINE 3000 (Life Technologies)). In various embodiments, the endosomal escape of the compositions and methods of the present application is about 5-fold, or 10-fold, or 15-fold, or 20-fold, or 30-fold greater than that of commercially available products (e.g., LIPOFECTIN, LIPOFECTAMINE, LIPOFECTAMINE 2000, LIPOFECTAMINE 3000 (Life Technologies)).

[0167] In embodiments, the compounds of the present application (e.g., those of formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers are serum-tolerant. In embodiments, the compounds of the present application (e.g., those of formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers are substantially stable in serum. In embodiments, the compounds of the present application (e.g., those of formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers are serum-tolerant. In embodiments, the transfection method of the present application can function in the presence of serum and / or does not require serum inactivation and / or medium exchange. In embodiments, the stability in serum and / or serum tolerance can be measured by in vitro assays. The transfection efficiency with various amounts of serum can be used, optionally, to evaluate the ability to transfect macromolecules (e.g., without limitation, DNA or RNA) by comparison with commercially available products (e.g., LIPOFECTIN, LIPOFECTAMINE, LIPOFECTAMINE 2000, LIPOFECTAMINE 3000 (Life Technologies)).

[0168] In embodiments, the present invention relates to the compounds of the present application (e.g., those of formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers for transfection, or methods of transfection, which have low or reduced toxic effects. In embodiments, the present invention relates to the compounds of the present application (e.g., those of formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers for transfection, or methods of transfection, which have reduced toxic effects as compared to commercially available products (e.g., LIPOFECTIN, LIPOFECTAMINE, LIPOFECTAMINE 2000, LIPOFECTAMINE 3000 (Life Technologies)). In various embodiments, with the compositions and methods of the present application, the cells can have a post-transfection survival rate of more than about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%. In various embodiments, with the compositions and methods of the present application, the cells can have a post-transfection survival rate that is 5-fold, or 10-fold, or 15-fold, or 20-fold, or 30-fold higher as compared to commercially available products (e.g., LIPOFECTIN, LIPOFECTAMINE, LIPOFECTAMINE 2000, LIPOFECTAMINE 3000 (Life Technologies)). In embodiments, toxic effects include disruption of cell morphology and / or viability or dysregulation of one or more genes.

[0169] In embodiments, the present invention relates to the compounds of the present application (e.g., those of formulas I - XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers for transfection, or methods of transfection that enable high - level protein expression from transfected nucleic acids (e.g., DNA or RNA). In various embodiments, the protein expression of the compositions and methods of the present application is more than about 30%, or more than about 40%, or more than about 50%, or more than about 60%, or more than about 70%, or more than about 80%, or more than about 90%, or more than about 95% higher than when not transfected. In various embodiments, the protein expression obtained with the compositions and methods of the present application is higher than that obtained with commercially available products (e.g., LIPOFECTIN, LIPOFECTAMINE, LIPOFECTAMINE 2000, LIPOFECTAMINE 3000 (Life Technologies)). In various embodiments, the protein expression obtained with the compositions and methods of the present application is about 5 - fold, or 10 - fold, or 15 - fold, or 20 - fold, or 30 - fold higher than that obtained with commercially available products (e.g., LIPOFECTIN, LIPOFECTAMINE, LIPOFECTAMINE 2000, LIPOFECTAMINE 3000 (Life Technologies)).

[0170] In embodiments, the present invention relates to the compounds of the present application (e.g., those of formulas I - XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers for transfection, or methods of transfection that enable transfection, including efficient transfection as described herein, in various cell types. In embodiments, the present invention relates to the compounds of the present application (e.g., those of formulas I - XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers for transfection, or methods of transfection that enable transfection, including efficient transfection as described herein, in established cell lines, difficult - to - transfect cells, primary cells, stem cells, and blood cells. In embodiments, the cell type is a keratinocyte, fibroblast, or PBMC.

[0171] In an embodiment, the compounds of the present application (for example, those of formulas I to XVI), pharmaceutical compositions and / or lipid aggregates and / or lipid carriers are suitable for transfection or delivery of compounds to target cells in vitro or in vivo.

[0172] In an embodiment, the present invention relates to the compounds of the present application (for example, those of formulas I to XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers for transfection, or methods of transfection, which do not require additional transfection reagents, such as LipofectAMINE PLUS Reagent (Life Technologies).

[0173] In an embodiment, the compounds of the present application are components of pharmaceutical compositions and / or lipid aggregates and / or lipid carriers that do not require additional lipids or helper lipids, for example, in the case of efficient transfection. For example, in an embodiment, pharmaceutical compositions and / or lipid aggregates and / or lipid carriers that do not require one or more of DOPE, DOPC, cholesterol, and polyethylene glycol (PEG)-modified lipids (including without limitation or PEGylation of DOPE, DOPC, and / or cholesterol) for efficient transfection.

[0174] In an embodiment, the compounds of the present application are components of pharmaceutical compositions and / or lipid aggregates and / or lipid carriers that further contain additional lipids or helper lipids.

[0175] In an embodiment, the additional lipids or helper lipids are selected from one or more of the categories of cationic lipids, anionic lipids, neutral lipids, polyvalently charged lipids, and zwitterionic lipids. In some cases, cationic lipids may be used to promote charge-charge interactions with nucleic acids.

[0176] In embodiments, the additional lipid or helper lipid is a neutral lipid. In embodiments, the neutral lipid is dioleoylphosphatidylethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), or cholesterol. In embodiments, the cholesterol is derived from a plant source. In other embodiments, the cholesterol is derived from an animal, fungal, bacterial, or archaeal source.

[0177] In embodiments, the additional lipid or helper lipid is a further cationic lipid. In embodiments, the cationic lipid is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-bis(oleoyloxy)-3-3-(trimethylammonia)propane (DOTAP), or 1,2-dioleoyl-3-dimethylammonium-propane (DODAP).

[0178] In embodiments, phospholipids, 18:0 PC, 18:1 PC, 18:2 PC, 18:2 PE, DSPE, DOPE, 18:2 PE, DMPE, or combinations thereof are used as helper lipids. In embodiments, the additional lipid or helper lipid is DOTMA and DOPE, optionally in a ratio of about 1:1. In embodiments, the additional lipid or helper lipid is DHDOS and DOPE, optionally in a ratio of about 1:1.

[0179] In embodiments, the additional lipid or helper lipid is a commercially available product (e.g., LIPOFECTIN, LIPOFECTAMINE, LIPOFECTAMINE 2000, LIPOFECTAMINE 3000 (Life Technologies)).

[0180] In embodiments, the additional lipid or helper lipid is a compound having formula (A).

[0181] [Chemical formula]

[0182] In that case, R1 and R4 are linear alkenyls having 17 carbon atoms, R2 and R5 are -(CH2)p-NH2, where p is from 1 to 4, l is from 1 to 10, and Xa is a physiologically acceptable anion.

[0183] In embodiments, the additional lipid or helper lipid is a PEGylated lipid. In embodiments, the PEGylated lipid has a PEG molecule covalently attached thereto, where the PEG has an average molecular weight of from about 10 kDa to about 400 kDa. In embodiments, the polyethylene glycol suitable for use in the present invention has an average molecular weight of at least 10,000 daltons to 40,000 daltons. In embodiments, the PEG has an average molecular weight in the range of 20,000 to 700,000 daltons, such as an average molecular weight of 20,000 daltons, for example, in the range of 35,000 to 500,000 daltons, such as in the range of 20,000 to 600,000 daltons, for example, in the range of 35,000 to 350,000 daltons, such as in the range of 35,000 to 400,000 daltons, for example, in the range of 100,000 to 300,000 daltons, such as in the range of 50,000 to 350,000 daltons, for example, in the range of 200,000 to 300,000 daltons, such as in the range of 150,000 to 350,000 daltons. In certain embodiments, the polyethylene glycol suitable for use in the compositions and methods described herein has an average molecular weight selected from about 10,000 daltons, about 15,000 daltons, about 20,000 daltons, about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 50,000 daltons, about 75,000 daltons, about 100,000 daltons, about 150,000 daltons, about 200,000 daltons, about 250,000 daltons, about 300,000 daltons, about 400,000 daltons, 150,000 daltons, 200,000 daltons, 250,000 daltons, 300,000 daltons, 400,000 daltons. In this context, "about" when referring to the average molecular weight of polyethylene glycol means + / - 30%. In embodiments with respect to the compositions and methods described herein, the covalently attached PEG has an average molecular weight of 10 kDa, 20 kDa, or 40 kDa. In embodiments, the PEG is branched PEG, star PEG, or comb PEG.

[0184] In one embodiment, the compounds of the present application (e.g., those of Formulas I to XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers contain one or more polyethylene glycol (PEG) chains, optionally selected from PEG200, PEG300, PEG400, PEG600, PEG800, PEG1000, PEG1500, PEG2000, PEG3000, and PEG4000. In an embodiment, the PEG is PEG2000. In an embodiment, the compounds of the present application (e.g., those of Formulas I to XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers contain 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) or a derivative thereof. In one embodiment, the compounds of the present application (e.g., those of Formulas I to XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers contain the PEGylated lipid 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG). In another embodiment, the compounds of the present application (e.g., those of Formulas I to XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers contain 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG). In yet another embodiment, the compounds of the present application (e.g., those of Formulas I to XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers contain 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG). In a further embodiment, the compounds of the present application (e.g., those of Formulas I to XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers contain a mixture of PEGylated lipids or free PEG chains.

[0185] In embodiments, the compounds of the present application (e.g., those of Formulas I - XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers comprise one or more of N-(carbonyl-ethoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG2000-DSPE), fully hydrogenated phosphatidylcholine, cholesterol, LIPOFECTAMINE 3000, cationic lipids, polycationic lipids, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-5000] (FA-MPEG5000-DSPE).

[0186] In some embodiments, one or more PEGylated helper lipids are incorporated into a pharmaceutical composition and / or lipid aggregate and / or lipid carrier and / or liposome and / or lipid nanoparticle comprising a compound of the present application (e.g., those of Formulas I - XVI). In some embodiments, the concentration of the PEGylated helper lipid, or the ratio of the PEGylated helper lipid to any one or more of the compounds of the present application (e.g., those of Formulas I - XVI), or the ratio of the PEGylated helper lipid to nucleic acid is used to affect the particle size. In some embodiments, the particle size is determined by other factors (e.g., without limitation, the ionic strength of the solution containing the particles, addition of salts to the solution containing the particles, addition of other small molecules to the solution containing the particles, adjustment of the pH of the solution containing the particles). In some embodiments, the control of particle size by other factors is used in combination with one or more PEGylated helper lipids or instead of one or more PEGylated helper lipids.

[0187] In one embodiment, the compound of the present application (for example, those of Formulas I to XVI) and / or the pharmaceutical composition and / or the lipid aggregate and / or the lipid carrier contain about 3.2 mg / mL of N-(carbonyl-ethoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG2000-DSPE), about 9.6 mg / mL of fully hydrogenated phosphatidylcholine, about 3.2 mg / mL of cholesterol, about 2 mg / mL of ammonium sulfate, and histidine as a buffer, and about 0.27 mg / mL of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate (polyethylene glycol)-5000] (FA-MPEG5000-DSPE) is added to the lipid mixture. In another embodiment, the nucleic acid is complexed by combining about 1 μL of LIPOFECTAMINE 3000 per about 1 μg of nucleic acid and incubating at room temperature for at least about 5 minutes. In one embodiment, LIPOFECTAMINE 3000 is a solution containing lipids at a concentration of about 1 mg / mL. In an embodiment, the nucleic acid is encapsulated by combining about 10 μg of the compound of the present application (for example, those of Formulas I to XVI) and / or the pharmaceutical composition and / or the lipid aggregate and / or the lipid carrier per about 1 μg of nucleic acid and incubating at room temperature for about 5 minutes.

[0188] In embodiments, the compounds of the present application (e.g., those of Formulas I - XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers comprise one or more nanoparticles. In one embodiment, the nanoparticles are polymer nanoparticles. In various embodiments, the compounds of the present application (e.g., those of Formulas I - XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers comprise one or more of diblock copolymers, triblock copolymers, tetrablock copolymers, and multiblock copolymers. In various embodiments, the compounds of the present application (e.g., those of Formulas I - XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers comprise one or more of polymer nanoparticles comprising polyethylene glycol (PEG)-modified polylactic acid (PLA) diblock copolymer (PLA - PEG), PEG - polypropylene glycol - PEG - modified PLA - tetrablock copolymer (PLA - PEG - PPG - PEG), and poly(lactic - co - glycolic acid) copolymer. In another embodiment, the compounds of the present application (e.g., those of Formulas I - XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers comprise a statistical copolymer, or an alternating copolymer, or a periodic copolymer, or any other arbitrary type of polymer.

[0189] In embodiments, the compounds of the present application (e.g., those of Formulas I - XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers comprise one or more lipids described in WO / 2000 / 027795, the entire content of which is incorporated herein by reference.

[0190] In embodiments, the compounds of the present application (e.g., those of Formulas I - XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers comprise Polybrene™ (hexadimethrine bromide) as described in U.S. Patent No. 5,627,159, the entire content of which is incorporated herein by reference.

[0191] In various embodiments, the compounds of the present application (e.g., those of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers include one or more polymers. Examples of polymers include hexadimethrine bromide (Polybrene™), DEAE-dextran, protamine, protamine sulfate, poly-L-lysine, or poly-D-lysine. These polymers may be used in combination with cationic lipids to provide synergistic effects on cellular uptake, stability of the compounds of the present application (e.g., those of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers, including serum stability (e.g., in vivo stability), endosomal escape, cell viability, and protein expression.

[0192] In an embodiment, the compound of the present application is a component of a pharmaceutical composition and / or lipid aggregate and / or lipid carrier that further comprises one or more additional lipids or polymers selected from Table 1. In other embodiments, the nucleic acid of the present invention is a component of a pharmaceutical composition and / or lipid aggregate and / or lipid carrier that further comprises one or more lipids or polymers selected from Table 1.

[0193] In various embodiments, one or more, or two or more, or three or more, or four or more, or five or more of the lipids in Table 1 are formulated with the compound of the present application, and they are components of a pharmaceutical composition and / or lipid aggregate and / or lipid carrier.

[0194]

Table 1-1

[0195]

Table 1-2

[0196]

Table 1-3

[0197] Additional components that may be present in the pharmaceutical composition and / or lipid aggregates and / or lipid carriers and / or lipid nucleic acid complexes and / or liposomes and / or lipid nanoparticles of the present invention include polyamide oligomers (see, for example, U.S. Patent No. 6,320,017), peptides, proteins, detergents, lipid-derivatives, such as PEG conjugated to phosphatidylethanolamine and PEG conjugated to ceramide (see U.S. Patent No. 5,885,613), and other components that stabilize the bilayer.

[0198] In embodiments, the lipids of the present application include additional cationic lipids, neutral lipids, sterols, and lipids selected to reduce aggregation of the lipid particles being formed, which can result from steric stabilization of the particles preventing charge-induced aggregation during formation. Examples of lipids that reduce aggregation of the particles being formed include polyethylene glycol (PEG)-modified lipids, monosialoganglioside Gm1, and polyamide oligomers (such as those described in U.S. Patent No. 6,320,017). Other compounds having an uncharged hydrophilic steric hindrance moiety that prevents aggregation during formulation, such as PEG, Gm1 or ATTA, can also be conjugated to the lipids for use as in the methods and compositions of the present invention. ATTA-lipids are described, for example, in U.S. Patent No. 6,320,017, and PEG-lipid complexes are described in, for example, U.S. Patents Nos. 5,820,873, 5,534,499, and 5,885,613. Typically, the concentration of the lipid component selected to reduce aggregation is about 0.1 to 15% (in mole percent of lipid). If the particles are stable after formulation, PEG or ATTA can be removed by dialysis prior to administration to the subject.

[0199] In embodiments, it is desirable to direct the compounds of the present application (e.g., those of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers using cell type- or tissue-specific targeting moieties. Targeted lipid particles using various targeting moieties such as ligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin), and monoclonal antibodies have been reported previously (see, e.g., U.S. Patent Nos. 4,957,773 and 4,603,044). The targeting moiety can include the whole protein or a fragment thereof. In the targeting mechanism, it is generally necessary for the targeting substance to be located on the surface of the lipid particle so that the target moiety is available for interaction with a target, e.g., a cell surface receptor. Various different targeting substances and methods are known and available in the art, such as those described in Sapra and Allen, Prog. Lipid Res. 42(5):439-62(2003), and Abra et al., J. Liposome Res. 12:1-3,(2002). Standard methods for attaching the targeting substance can be used. For example, phosphatidylethanolamine that can be activated for attachment of the targeting substance, or derivatized lipophilic compounds such as lipid-derivatized bleomycin can be used. Targeted liposomes with antibodies can be constructed, for example, using liposomes incorporating Protein A (see Renneisen, et al., J. Bio.Chem., 265:16337-16342(1990) and Leonetti, et al., Proc. Natl. Acad. Sci. (USA), 87:2448-2451(1990)). Other examples of antibody binding are disclosed in U.S. Patent No. 6,027,726, the teachings of which are incorporated herein by reference. Examples of targeting moieties can also include other proteins specific for cell components, such as antigens associated with a neoplasm or tumor.The proteins used as the target-directing moieties can be covalently attached to the liposomes (see Heath, Covalent Attachment of Proteins to Liposomes, 149 Methods in Enzymology 111-119 (Academic Press, Inc. 1987)). Other target-directing methods include the biotin-avidin system.

[0200] In embodiments, a complex formation medium is used in the compositions and methods of the present application. In one embodiment, the complex formation medium has a pH greater than about 7, or greater than about 7.2, or greater than about 7.4, or greater than about 7.6, or greater than about 7.8, or greater than about 8.0, or greater than about 8.2, or greater than about 8.4, or greater than about 8.6, or greater than about 8.8, or greater than about 9.0. In one embodiment, the complex formation medium contains transferrin. In a further embodiment, the complex formation medium contains DMEM. In yet another embodiment, the complex formation medium contains DMEM / F12.

[0201] Nucleic acid In embodiments, the compounds of the present application (e.g., those of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers are suitable for association with nucleic acids, including, for example, any oligonucleotide or polynucleotide.

[0202] In embodiments, the nucleic acid is RNA, small interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), long non-coding RNA (lncRNA), antisense oligonucleotide, ribozyme, plasmid, immunostimulatory nucleic acid, antisense, antagomir, antimir, microRNA mimic, supermir, U1 adapter, or aptamer.

[0203] In embodiments, the nucleic acid is completely encapsulated within the compounds of the present application (e.g., those of formulas I - XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers. In other embodiments, the nucleic acid is partially encapsulated within the compounds of the present application (e.g., those of formulas I - XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers. In still other embodiments, the nucleic acid as well as the compounds of the present application (e.g., those of formulas I - XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers all exist without the nucleic acid being encapsulated within the compounds of the present application (e.g., those of formulas I - XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers.

[0204] Being completely encapsulated indicates that the nucleic acid in the compounds of the present application (e.g., those of formulas I - XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers is not significantly degraded after exposure to serum or nuclease assays that would significantly degrade free nucleic acid. In embodiments, in a completely encapsulated system, less than about 25% of the particulate nucleic acid is degraded in a process where typically about 100% of the free nucleic acid is degraded. In embodiments, less than about 10% or less than about 5% of the particulate nucleic acid is degraded.

[0205] The degree of encapsulation can be determined by the Oligreen assay. Oligreen is a hypersensitive fluorescent nucleic acid stain for quantifying oligonucleotides and single-stranded DNA in solution (available from Invitrogen Corporation, Carlsbad, CA).

[0206] Being completely encapsulated also suggests that the compounds of the present application (e.g., those of formulas I - XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers are serum stable, i.e., they are not rapidly degraded into their respective components upon in vivo administration.

[0207] In an embodiment, the compound of the present application (for example, those of formulas I to XVI) and / or the pharmaceutical composition and / or the lipid aggregate and / or the lipid carrier are complexed with a nucleic acid (for example, DNA or RNA), and in this case, depending on the target cell type, different ratios, generally a ratio of lipid (ng):RNA (ng) in the range of about 1:16 to about 25:1, are used. Exemplary lipid:RNA ratios are about 1:1 to about 10:1, for example, without limitation, about 2.5:1, or about 5:1.

[0208] Additional parameters such as nucleic acid concentration, type and concentration of buffer, etc. affect the transfection efficiency, but they can be modified by those skilled in the art through routine experiments.

[0209] In an embodiment, the nucleic acid is selected from RNA or DNA.

[0210] In an embodiment, the DNA is a plasmid, cosmid, phage, recombinant virus or other vector. In an embodiment, a vector (or plasmid) refers to, for example, individual elements used for introducing them into cells for the expression or replication of heterologous nucleic acids, etc. A vector can remain episomal or can be designed to result in the integration of a gene or a portion thereof into the genomic chromosome. Vectors that are artificial chromosomes such as yeast artificial chromosomes and mammalian artificial chromosomes are also contemplated. The selection and use of such media are well known to those skilled in the art. Vectors that are capable of expressing DNA functionally linked to a control sequence such as a promoter region capable of effecting the expression of a functionally linked DNA fragment are included (for example, expression vectors). Therefore, a vector refers to a recombinant DNA or RNA construct such as a plasmid, phage, recombinant virus or other vector that results in the expression of its DNA upon introduction into an appropriate host cell. Appropriate vectors are well known to those skilled in the art and include those that are replicable in eukaryotic cells and / or prokaryotic cells and those that remain episomal or are integrated into the host cell genome.

[0211] In an embodiment, the RNA is synthetic RNA. In an embodiment, the RNA is chemically synthesized RNA. In an embodiment, the RNA is in vitro transcribed RNA.

[0212] In an embodiment, the RNA is selected from siRNA, lncRNA, antisense oligonucleotide, microRNA, antagomir, aptamer, ribozyme, and mRNA.

[0213] In an embodiment, the synthetic RNA (including, without limitation, mRNA) does not contain non-standard nucleotides.

[0214] In an embodiment, the synthetic RNA (including, without limitation, mRNA) contains one or more non-standard nucleotides.

[0215] In an embodiment, the one or more non-standard nucleotides avoid substantial cytotoxicity. In an embodiment, the one or more non-standard nucleotides substantially avoid in vivo cytotoxicity. In an embodiment, the one or more non-standard nucleotides substantially avoid an immune response in a human subject. For example, the immune response can be an immune response mediated by the innate immune system. Immune responses can be monitored using markers known in the art (e.g., cytokines, interferons, TLRs). In an embodiment, at an effective dose, the need for treatment of a human subject with an immunosuppressant (e.g., B18R) to mitigate residual toxicity is eliminated.

[0216] In embodiments, the immune response is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 99.9%, or about 99.9% lower compared to the immune response induced by the corresponding unmodified nucleic acid. In embodiments, the upregulation of one or more immune response markers is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 99.9%, or about 99.9% lower compared to the upregulation of one or more immune response markers induced by the corresponding unmodified nucleic acid. In embodiments, the immune response marker includes the mRNA or protein product of an interferon gene, which includes the interferon alpha gene, IFNB1, TLR3, RARRES3, EIF2AK2, STAT1, STAT2, IFIT1, IFIT2, IFIT3, IFIT5, OAS1, OAS2, OAS3, OASL, ISG20 or a fragment, variant, analog, or family member thereof. In embodiments, the immune response marker includes the mRNA or protein product of a TNF gene, which includes the TNF alpha gene, TNFRSF1A; TNFRSF1B; LTBR; TNFRSF4; CD40; FAS; TNFRSF6B; CD27; TNFRSF8; TNFRSF9; TNFRSF10A; TNFRSF10B; TNFRSF10C; TNFRSF10D; TNFRSF11A; TNFRSF11B; TNFRSF12A; TNFRSF13B; TNFRSF13C; TNFRSF14; NGFR; TNFRSF17; TNFRSF18; TNFRSF19; TNFRSF21; TNFRSF25; and EDA2R or a fragment, variant, analog, or family member thereof.In an embodiment, the immune response marker includes the mRNA or protein product of an interleukin gene, which includes the IL-6 gene, IL-1; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8 or CXCL8; IL-9; IL-10; IL-11; IL-12; IL-13; IL-14; IL-15; IL-16; IL-17; IL-18; IL-19; IL-20; IL-21; IL-22; IL-23; IL-24; IL-25; IL-26; IL-27; IL-28; IL-29; IL-30; IL-31; IL-32; IL-33; IL-35; IL-36 or a fragment, variant, analog, or family member thereof.

[0217] In an embodiment, cell death is about 10%, about 25%, about 50%, about 75%, about 85%, about 90%, about 95%, or more than about 95% less than the cell death observed with the corresponding unmodified nucleic acid. Further, cell death may invade cells that are less than about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 1%, about 0.1%, about 0.01%, or less than about 0.01% of the cells that have contacted the modified nucleic acid.

[0218] While not wishing to be bound by theory, certain non-standard nucleotides, when incorporated into an RNA molecule, can reduce the toxicity of the RNA molecule, in part, by interfering with the binding of proteins that detect foreign nucleic acids, such as protein kinase R, Rig-1, and proteins of the oligoadenylate synthetase family. Non-standard nucleotides that have been reported to reduce the toxicity of an RNA molecule when incorporated into the RNA molecule include pseudouridine, 5-methyluridine, 2-thiouridine, 5-methylcytidine, N6-methyladenosine, and certain combinations thereof. However, the chemical features of non-standard nucleotides that can reduce the in vivo toxicity of RNA molecules have remained unclear until now. Further, most non-standard nucleotides, such as 5-methyluridine, 2-thiouridine, 5-methylcytidine, and N6-methyladenosine, reduce the efficiency with which an RNA molecule can be translated into protein upon their incorporation in large amounts, and in applications where protein expression is required, the usefulness of RNA molecules containing these nucleotides can be limited. Further, while pseudouridine can completely replace uridine in an RNA molecule without reducing the efficiency with which the synthetic RNA molecule can be translated into protein, in certain situations, such as when performing repeated transfections at high frequency, synthetic RNA molecules containing only adenosine, guanosine, cytidine, and pseudouridine can exhibit transient toxicity.

[0219] In an embodiment, the non-standard nucleotide has one or more substitutions at a position selected from the 2C, 4C, and 5C positions for pyrimidines, or at a position selected from the 6C, 7N, and 8C positions for purines.

[0220] In an embodiment, the non-standard nucleotide comprises, optionally, one or more of 5-hydroxylcytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine in an amount of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100% of the non-standard nucleotide.

[0221] In an embodiment, at least about 50% of the cytidine residues are non-standard nucleotides, and they are selected from 5-hydroxylcytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, and 5-methoxycytidine.

[0222] In an embodiment, at least about 75% or at least about 90% of the cytidine residues are non-standard nucleotides, and the non-standard nucleotides are selected from 5-hydroxylcytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, and 5-methoxycytidine.

[0223] In an embodiment, at least about 20% of uridine, or at least about 40% of uridine residues, or at least about 50%, or at least about 75%, or at least about 90% are non-standard nucleotides, and such non-standard forms are selected from pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.

[0224] In an embodiment, at least about 40% of uridine residues, or at least about 50%, or at least about 75%, or at least about 90% are non-standard nucleotides, and the non-standard nucleotides are selected from pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.

[0225] In an embodiment, at least about 10% of guanine residues are non-standard nucleotides, and the non-standard nucleotides are optionally 7-deazaguanosine.

[0226] In an embodiment, the synthetic RNA contains about 50% or less of 7-deazaguanosine in place of guanosine residues.

[0227] In an embodiment, the synthetic RNA does not contain non-standard nucleotides in place of adenosine residues.

[0228] In an embodiment, the present invention relates to an RNA molecule containing one or more non-standard nucleotides having one or more substitutions at the 2′C position and / or 4′C position and / or 5′C position in the case of pyrimidines, or at the 6′C position and / or 7′N position and / or 8′C position in the case of purines. In part, the substitutions at these positions can prevent the recognition of the synthetic RNA molecule by a protein that detects foreign nucleic acids, so it may be less toxic than a synthetic RNA molecule containing only standard nucleotides. Further, in part, the substitutions at these positions do not prevent base pairing and base stacking interactions, so the effect on the efficiency with which the synthetic RNA molecule can be translated into protein can be minimal.

[0229]

Chemical formula

[0230] Examples of non-standard nucleotides containing one or more substitutions at the 2C position and / or 4C position and / or 5C position in the case of pyrimidines, or at the 6C position and / or 7N position and / or 8C position in the case of purines, include 2-thiouridine, 5-azauridine, pseudouridine, 4-thiouridine, 5-methyluridine, 5-methylpseudouridine, 5-aminouridine, 5-aminopseudouridine, 5-hydroxyuridine, 5-hydroxypseudouridine, 5-methoxyuridine, 5-methoxypseudouridine, 5-hydroxymethyluridine, 5-hydroxymethylpseudouridine, 5-carboxyuridine, 5-carboxypseudouridine, 5-formyluridine, 5-formylpseudouridine, 5-methyl-5-azauridine, 5-amino-5-azauridine, 5-hydroxy-5-azauridine, 5-methylpseudouridine, 5-aminopseudouridine, 5-hydroxypseudouridine, 4-thio-5-azauridine, 4-thiopseudouridine, 4-thio-5-methyluridine, 4-thio-5-aminouridine, 4-thio-5-hydroxyuridine, 4-thio-5-methyl-5-azauridine, 4-thio-5-amino-5-azauridine, 4-thio-5-hydroxy-5-azauridine, 4-thio-5-methylpseudouridine, 4-thio-5-aminopseudouridine, 4-thio-5-hydroxypseudouridine, 2-thiocytidine, 5-azacytidine, pseudoisocytidine, N4-methylcytidine, N4-aminocytidine, N4-hydroxycytidine, 5-methylcytidine, 5-aminocytidine, 5-hydroxycytidine, 5-methoxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methyl-5-azacytidine, 5-amino-5-azacytidine, 5-hydroxy-5-azacytidine, 5-methylpseudoisocytidine, 5-aminopseudoisocytidine, 5-hydroxypseudoisocytidine, N4-methyl-5-azacytidine, N4-methylpseudoisocytidine, 2-thio-5-azacytidine, 2-thiopseudoisocytidine, 2-thio-N4-methylcytidine, 2-thio-N4-aminocytidine, 2-thio-N4-hydroxycytidine, 2-thio-5-methylcytidine, 2-thio-5-aminocytidine,2-thio-5-hydroxythymidine, 2-thio-5-methyl-5-azacytidine, 2-thio-5-amino-5-azacytidine, 2-thio-5-hydroxy-5-azacytidine, 2-thio-5-methylpseudoisocytidine, 2-thio-5-aminopseudoisocytidine, 2-thio-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-azacytidine, 2-thio-N4-methylpseudoisocytidine, N4-methyl-5-methylcytidine, N4-methyl-5-aminocytidine, N4-methyl-5-hydroxythymidine, N4-methyl-5-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine, N4-methyl-5-hydroxy-5-azacytidine, N4-methyl-5-methylpseudoisocytidine, N4-methyl-5-aminopseudoisocytidine, N4-methyl-5-hydroxypseudoisocytidine, N4-amino-5-azacytidine, N4-aminopseudoisocytidine, N4-amino-5-methylcytidine, N4-amino-5-aminocytidine, N4-amino-5-hydroxythymidine, N4-amino-5-methyl-5-azacytidine, N4-amino-5-amino-5-azacytidine, N4-amino-5-hydroxy-5-azacytidine, N4-amino-5-methylpseudoisocytidine, N4-amino-5-aminopseudoisocytidine, N4-amino-5-hydroxypseudoisocytidine, N4-hydroxy-5-azacytidine, N4-hydroxypseudoisocytidine, N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, N4-hydroxy-5-hydroxythymidine, N4-hydroxy-5-methyl-5-azacytidine, N4-hydroxy-5-amino-5-azacytidine, N4-hydroxy-5-hydroxy-5-azacytidine, N4-hydroxy-5-methylpseudoisocytidine, N4-hydroxy-5-aminopseudoisocytidine, N4-hydroxy-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-methylcytidine, 2-thio-N4-methyl-5-aminocytidine, 2-thio-N4-methyl-5-hydroxythymidine, 2-thio-N4-methyl-5-methyl-5-azacytidine, 2-thio-N4-methyl-5-amino-5-azacytidine,2-Thio-N4-methyl-5-hydroxy-5-azacytidine, 2-thio-N4-methyl-5-methylpseudoisocytidine, 2-thio-N4-methyl-5-aminopseudoisocytidine, 2-thio-N4-methyl-5-hydroxypseudoisocytidine, 2-thio-N4-amino-5-azacytidine, 2-thio-N4-aminopseudoisocytidine, 2-thio-N4-amino-5-methylcytidine, 2-thio-N4-amino-5-aminocytidine, 2-thio-N4-amino-5-hydroxycytidine, 2-thio-N4-amino-5-methyl-5-azacytidine, 2-thio-N4-amino-5-amino-5-azacytidine, 2-thio-N4-amino-5-hydroxy-5-azacytidine, 2-thio-N4-amino-5-methylpseudoisocytidine, 2-thio-N4-amino-5-aminopseudoisocytidine, 2-thio-N4-amino-5-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, 2-thio-N4-hydroxy-5-hydroxycytidine, 2-thio-N4-hydroxy-5-methyl-5-azacytidine, 2-thio-N4-hydroxy-5-amino-5-azacytidine, 2-thio-N4-hydroxy-5-hydroxy-5-azacytidine, 2-thio-N4-hydroxy-5-methylpseudoisocytidine, 2-thio-N4-hydroxy-5-aminopseudoisocytidine, 2-thio-N4-hydroxy-5-hydroxypseudoisocytidine, N6-methyladenosine, N6-aminoadenosine, N6-hydroxyadenosine, 7-deazaadenosine, 8-azaadenosine, N6-methyl-7-deazaadenosine, N6-methyl-8-azaadenosine, 7-deaza-8-azaadenosine, N6-methyl-7-deaza-8-azaadenosine, N6-amino-7-deazaadenosine, N6-amino-8-azaadenosine, N6-amino-7-deaza-8-azaadenosine, N6-hydroxyadenosine, N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine, N6-hydroxy-7-deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine,6-Thio-7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine, 6-thio-7-deaza-8-azaguanosine, and 5-methoxyuridine are included, but not limited thereto.

[0231] In embodiments, the present invention relates to one or more non-standard nucleotides selected from 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-hydroxyuridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, pseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine. In embodiments, at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% of the non-standard nucleotides are one or more of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, pseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.

[0232] In embodiments, at least about 50%, or at least about 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% of the cytidine residues are non-standard nucleotides selected from 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, and 5-methoxycytidine.

[0233] In embodiments, at least about 20%, or about 30%, or about 40%, or about 50%, or at least about 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% of the uridine residues are non-standard nucleotides selected from 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, pseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.

[0234] In embodiments, at least about 10% (e.g., 10%, or about 20%, or about 30%, or about 40%, or about 50%) of the guanosine residues are non-standard nucleotides, which are optionally 7-deazaguanosine. In embodiments, the RNA contains about 50% or less 7-deazaguanosine in place of guanosine residues.

[0235] In embodiments, the RNA does not contain non-standard nucleotides in place of adenosine residues.

[0236] Note that alternative naming systems exist for certain non-standard nucleotides. For example, in certain situations, 5-methylpseudouridine may be referred to as "3-methylpseudouridine" or "N3-methylpseudouridine" or "1-methylpseudouridine" or "N1-methylpseudouridine".

[0237] A nucleotide containing the prefix "amino" may refer to any nucleotide containing a nitrogen atom bonded to the atom at the described position of the nucleotide. For example, 5-aminocytidine may refer to 5-aminocytidine, 5-methylaminocytidine, and 5-nitrosocytidine. Similarly, a nucleotide containing the prefix "methyl" may refer to any nucleotide containing a carbon atom bonded to the atom at the described position of the nucleotide. For example, 5-methylcytidine may refer to 5-methylcytidine, 5-ethylcytidine, and 5-hydroxymethylcytidine. A nucleotide containing the prefix "thio" may refer to any nucleotide containing a sulfur atom bonded to the atom at a given position of the nucleotide. A nucleotide containing the prefix "hydroxy" may refer to any nucleotide containing an oxygen atom bonded to the atom at a given position of the nucleotide. For example, 5-hydroxyuridine may refer to 5-hydroxyuridine and uridine having a methyl group bonded to the oxygen atom, where the oxygen atom is bonded to the atom at the 5C position of uridine.

[0238] Accordingly, certain embodiments are directed to an RNA comprising one or more non-standard nucleotides, in which case the RNA molecule contains one or more nucleotides having one or more substitutions at the 2C position and / or 4C position and / or 5C position in the case of pyrimidines, or at the 6C position and / or 7N position and / or 8C position in the case of purines.

[0239] In an embodiment, the non-standard nucleotide includes at least one of pseudouridine, 2-thiouridine, 4-thiouridine, 5-azauridine, 5-hydroxyuridine, 5-methyluridine, 5-aminouridine, 2-thiopseudouridine, 4-thiopseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-aminopseudouridine, pseudoisocytidine, N4-methylcytidine, 2-thiocytidine, 5-azacytidine, 5-hydroxycytidine, 5-aminocytidine, 5-methylcytidine, N4-methylpseudoisocytidine, 2-thiopseudoisocytidine, 5-hydroxypseudoisocytidine, 5-aminopseudoisocytidine, 5-methylpseudoisocytidine, 7-deazaadenosine, 7-deazaguanosine, 6-thioguanosine, and 6-thio-7-deazaguanosine. In another embodiment, one or more nucleotides include at least one of pseudouridine, 2-thiouridine, 4-thiouridine, 5-azauridine, 5-hydroxyuridine, 5-methyluridine, 5-aminouridine, 2-thiopseudouridine, 4-thiopseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, and 5-aminopseudouridine, and at least one of pseudoisocytidine, N4-methylcytidine, 2-thiocytidine, 5-azacytidine, 5-hydroxycytidine, 5-aminocytidine, 5-methylcytidine, N4-methylpseudoisocytidine, 2-thiopseudoisocytidine, 5-hydroxypseudoisocytidine, 5-aminopseudoisocytidine, and 5-methylpseudoisocytidine.In yet another embodiment, one or more nucleotides include at least one of pseudouridine, 2-thiouridine, 4-thiouridine, 5-azauridine, 5-hydroxyuridine, 5-methyluridine, 5-aminouridine, 2-thiopseudouridine, 4-thiopseudouridine, 5-hydroxypseudouridine, and 5-methylpseudouridine, 5-aminopseudouridine, and at least one of pseudoisocytidine, N4-methylcytidine, 2-thiocytidine, 5-azacytidine, 5-hydroxycytidine, 5-aminocytidine, 5-methylcytidine, N4-methylpseudoisocytidine, 2-thiopseudoisocytidine, 5-hydroxypseudoisocytidine, 5-aminopseudoisocytidine, and 5-methylpseudoisocytidine, and at least one of 7-deazaguanosine, 6-thioguanosine, 6-thio-7-deazaguanosine, and 5-methoxyuridine. In yet another embodiment, one or more nucleotides include 5-methylcytidine and 7-deazaguanosine. In another embodiment, one or more nucleotides also include pseudouridine or 4-thiouridine or 5-methyluridine or 5-aminouridine or 4-thiopseudouridine or 5-methylpseudouridine or 5-aminopseudouridine. In yet another embodiment, one or more nucleotides also include 7-deazaadenosine. In another embodiment, one or more nucleotides include pseudoisocytidine and 7-deazaguanosine and 4-thiouridine. In yet another embodiment, one or more nucleotides include pseudoisocytidine or 7-deazaguanosine and pseudouridine. In yet another embodiment, one or more nucleotides include 5-methyluridine and 5-methylcytidine and 7-deazaguanosine. In a further embodiment, one or more nucleotides include pseudouridine or 5-methylpseudouridine and 5-methylcytidine and 7-deazaguanosine. In another embodiment, one or more nucleotides include pseudoisocytidine and 7-deazaguanosine and pseudouridine. In one embodiment, an RNA containing one or more non-standard nucleotides exists in vivo.

[0240] Certain non-standard nucleotides can be incorporated into RNA molecules more efficiently by RNA polymerases commonly used in in vitro transcription than other non-standard nucleotides, which is due in part to the fact that these certain non-standard nucleotides participate in standard base-pairing interactions and base stacking interactions and tend to interact with RNA polymerase in a manner similar to how the corresponding standard nucleotides interact with RNA polymerase. As a result, certain nucleotide mixtures containing one or more non-standard nucleotides may be beneficial in part because a large amount of RNA can be produced by in vitro transcription reactions containing these nucleotide mixtures. Accordingly, certain embodiments are directed to nucleotide mixtures containing one or more nucleotides having one or more substitutions at the 2C position and / or 4C position and / or 5C position in the case of pyrimidines, or at the 6C position and / or 7N position and / or 8C position in the case of purines. Examples of nucleotide mixtures (the numbers preceding each nucleotide indicate the exemplary proportion of non-standard nucleotide triphosphates in the in vitro transcription reaction, e.g., 0.2 pseudoisocytidine refers to a reaction containing adenosine-5'-triphosphate, guanosine-5'-triphosphate, uridine-5'-triphosphate, cytidine-5'-triphosphate, and pseudoisocytidine-5'-triphosphate, where pseudoisocytidine-5'-triphosphate is present in the reaction in an amount approximately equal to 0.2 times the total amount of pseudoisocytidine-5'-triphosphate and cytidine-5'-triphosphate present during the reaction when measured by amount based on either moles or mass, and two or more numbers preceding a nucleoside indicate a range of exemplary proportions), 1.0 pseudouridine, 0.1 - 0.8 2-thiouridine, 0.1 - 0.8 5-methyluridine, 0.2 - 1.0 5-hydroxyuridine, 0.2 - 1.0 5-methoxyuridine, 0.1 - 1.0 5-aminouridine, 0.1 - 1.0 4-thiouridine, 0.1 - 1.0 2-thiopseudouridine, 0.1 - 1.0 4-thiopseudouridine, 0.1 - 1.0 5-hydroxypseudouridine, 0.2 - 1 5-methylpseudouridine, 0.2 - 1.0 5-methoxypseudouridine, 0.1 - 1.0 5-aminopseudouridine, 0.2 to 1.0 2-thiocytidine, 0.1 to 0.8 pseudoisocytidine, 0.2 to 1.0 5-methylcytidine, 0.2 to 1.0 5-hydroxycytidine, 0.2 to 1.0 5-hydroxymethylcytidine, 0.2 to 1.0 5-methoxycytidine, 0.1 to 1.0 5-aminocytidine, 0.2 to 1.0 N4-methylcytidine, 0.2 to 1.0 5-methylpseudoisocytidine, 0.2 to 1.0 5-hydroxypseudoisocytidine, 0.2 to 1.0 5-aminopseudoisocytidine, 0.2 to 1.0 N4-methylpseudoisocytidine, 0.2 to 1.0 2-thiopseudoisocytidine, 0.2 to 1.0 7-deazaguanosine, 0.2 to 1.0 6-thioguanosine, 0.2 to 1.0 6-thio-7-deazaguanosine, 0.2 to 1.0 8-azaguanosine, 0.2 to 1.0 7-deaza-8-azaguanosine, 0.2 to 1.0 6-thio-8-azaguanosine, 0.1 to 0.5 7-deazaadenosine, and 0.1 to 0.5 N6-methyladenosine are exemplified, but not limited thereto.

[0241] In embodiments, an RNA comprising one or more non-standard nucleotide compositions or synthetic polynucleotide compositions (which can be prepared, for example, by in vitro transcription) substantially or entirely contains standard nucleotides at positions having adenine or "A" in the genetic code. The term "substantially" in this context refers to at least 90%. In these embodiments, the RNA composition or synthetic polynucleotide composition may further contain (e.g., may consist of) 7-deazaguanosine and the corresponding standard nucleotide "G" at positions where there is "G" in the genetic code, and the standard and non-standard nucleotides at positions where there is "G" can be in the range of 5:1 to 1:5, or in some embodiments, in the range of 2:1 to 1:2. In these embodiments, the RNA composition or synthetic polynucleotide composition may further contain (e.g., may consist of) one or more (e.g., two, three, or four) of 5-hydroxylcytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, and the standard nucleotide "C" at positions where there is "C" in the genetic code, and the standard and non-standard nucleotides at positions where there is "C" can be in the range of 5:1 to 1:5, or in some embodiments, in the range of 2:1 to 1:2. In embodiments, the level of non-standard nucleotides at the position of "C" is as described in the preceding paragraph. In these embodiments, the RNA composition or synthetic polynucleotide composition may further contain (e.g., may consist of) one or more (e.g., two, three, or four) of 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, pseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine and the standard nucleotide "U" at positions where there is "U" in the genetic code, and the standard and non-standard nucleotides at positions where there is "U" can be in the range of 5:1 to 1:5, or in some embodiments, in the range of 2:1 to 1:2.In an embodiment, the level of the non-standard nucleotide at the position of "U" is as described in the preceding paragraph.

[0242] Combining certain non-standard nucleotides may be beneficial in part because the contribution of the non-standard nucleotides to reducing the toxicity of the RNA molecule can be additive. Thus, an embodiment is directed to a nucleotide mixture, where the nucleotide mixture contains two or more of the above-described non-standard nucleotides. For example, the nucleotide mixture contains both pseudoisocytidine and 7-deazaguanosine, or the nucleotide mixture contains both N4-methylcytidine and 7-deazaguanosine, etc. In one embodiment, the nucleotide mixture contains two or more of the above-described non-standard nucleotides, and each of such non-standard nucleotides is present in the mixture at the above-described ratios. For example, the nucleotide mixture contains 0.1 to 0.8 pseudoisocytidine and 0.2 to 1.0 7-deazaguanosine, or the nucleotide mixture contains 0.2 to 1.0 N4-methylcytidine and 0.2 to 1.0 7-deazaguanosine, etc.

[0243] In certain situations, for example, when it is not necessary or desirable to maximize the yield of an in vitro transcription reaction, ratios of nucleotides other than those above may be used. The above-exemplified ratios and ranges of ratios relate to nucleotide triphosphate solutions of typical purity (purity greater than 90%). Larger ratios of these and other nucleotides can be used by using nucleotide triphosphate solutions of higher purity, for example, purity greater than about 95% or purity greater than about 98% or purity greater than about 99% or purity greater than about 99.5%, which can be achieved, for example, by purifying the nucleotide triphosphate solution using existing chemical purification techniques such as high performance liquid chromatography (HPLC), or by other means. In one embodiment, a nucleotide having multiple isomers is purified to concentrate the desired isomer.

[0244] Another embodiment is a method for inducing cells in vivo to express a target protein, the method comprising contacting the cells with a compound of the present application (e.g., those of formulas I - XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier, and an RNA molecule containing one or more non-standard nucleotides having one or more substitutions at the 2C-position and / or 4C-position and / or 5C-position in the case of pyrimidines, or at the 6C-position and / or 7N-position and / or 8C-position in the case of purines. Still another embodiment is a method for performing transfection, reprogramming, and / or gene editing of cells in vivo, the method comprising contacting the cells with a compound of the present application (e.g., those of formulas I - XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier, and an RNA molecule containing one or more non-standard nucleotides having one or more substitutions at the 2C-position and / or 4C-position and / or 5C-position in the case of pyrimidines, or at the 6C-position and / or 7N-position and / or 8C-position in the case of purines. In one embodiment, the RNA molecule is generated by in vitro transcription. In one embodiment, the RNA molecule encodes one or more reprogramming factors. In another embodiment, the one or more reprogramming factors include the Oct4 protein. In another embodiment, the cells are also contacted with an RNA molecule encoding the Sox2 protein. In yet another embodiment, the cells are also contacted with an RNA molecule encoding the Klf4 protein. In yet another embodiment, the cells are also contacted with an RNA molecule encoding the c-Myc protein. In yet another embodiment, the cells are also contacted with an RNA molecule encoding the Lin28 protein.

[0245] Enzymes such as T7 RNA polymerase can selectively incorporate standard nucleotides in in vitro transcription reactions containing both standard and non-standard nucleotides. As a result, in in vitro transcription reactions containing a specific proportion of non-standard nucleotides, RNAs containing a proportion of non-standard nucleotides different from, and sometimes lower than, the proportion of non-standard nucleotides present during the reaction may be produced. Thus, in certain embodiments, a reference to the proportion of nucleotide incorporation (e.g., "synthetic RNA molecules containing 50% pseudoisocytidine" or "0.1 - 0.8 pseudoisocytidine") refers not only to RNA molecules containing the described proportion of nucleotides, but also to RNA molecules synthesized in such reactions that may contain a proportion of nucleotides different from the proportion of non-standard nucleotides present during the reaction, even if RNAs containing a proportion of nucleotides different from the described proportion of nucleotides (or nucleotide derivatives, e.g., nucleotide triphosphates) are produced.

[0246] By using alternative codons, different nucleotide sequences can encode the same protein. Thus, in certain embodiments, a reference to the proportion of nucleotide incorporation can refer to both RNA molecules containing the described proportion of nucleotides and RNA molecules encoding the same protein as the protein encoded by different RNA molecules containing the described proportion of nucleotides.

[0247] When non-standard nucleotide members of the 5-methylcytidine demethylation pathway are incorporated into synthetic RNA, they can enhance the efficiency with which the synthetic RNA can be translated into protein in vivo and can reduce the toxicity of the synthetic RNA in vivo. These non-standard nucleotides include, for example, 5-methylcytidine, 5-hydroxymethylcytidine, 5-formylcytidine, and 5-carboxycytidine (also known as "cytidine-5-carboxylic acid"). Accordingly, certain embodiments are directed to nucleic acids. In an embodiment, the nucleic acid is present in vivo. In one embodiment, the nucleic acid is a synthetic RNA molecule. In another embodiment, the nucleic acid comprises one or more non-standard nucleotides. In one embodiment, the nucleic acid comprises one or more non-standard nucleotide members of the 5-methylcytidine demethylation pathway. In another embodiment, the nucleic acid comprises at least one of 5-methylcytidine, 5-hydroxymethylcytidine, 5-formylcytidine, and 5-carboxycytidine or derivatives thereof. In a further embodiment, the nucleic acid comprises at least one of pseudouridine, 5-methylpseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-methylcytidine, 5-hydroxymethylcytidine, N4-methylcytidine, N4-acetylcytidine, and 7-deazaguanosine or derivatives thereof.

[0248] 5-methylcytidine demethylation pathway

[0249] [Chemical formula] Certain combinations of non-standard nucleotides can be particularly effective in enhancing the efficiency with which synthetic RNA can be translated into protein in vivo and in reducing the toxicity of synthetic RNA in vivo. For example, there are combinations of 5-methyluridine and 5-methylcytidine, 5-hydroxyuridine and 5-methylcytidine, 5-hydroxyuridine and 5-hydroxymethylcytidine, 5-methyluridine and 7-deazaguanosine, 5-methylcytidine and 7-deazaguanosine, 5-methyluridine, 5-methylcytidine, and 7-deazaguanosine, and 5-methyluridine, 5-hydroxymethylcytidine, and 7-deazaguanosine. Accordingly, certain embodiments are directed to nucleic acids comprising at least two of 5-methyluridine, 5-methylcytidine, 5-hydroxymethylcytidine, and 7-deazaguanosine, or one or more derivatives thereof. Other embodiments are directed to nucleic acids comprising at least three of 5-methyluridine, 5-methylcytidine, 5-hydroxymethylcytidine, and 7-deazaguanosine, or one or more derivatives thereof. Other embodiments are directed to nucleic acids comprising all of 5-methyluridine, 5-methylcytidine, 5-hydroxymethylcytidine, and 7-deazaguanosine, or one or more derivatives thereof. In one embodiment, the nucleic acid comprises one or more 5-methyluridine residues, one or more 5-methylcytidine residues, and one or more 7-deazaguanosine residues or one or more 5-methyluridine residues, one or more 5-hydroxymethylcytidine residues, and one or more 7-deazaguanosine residues.

[0250] Synthetic RNA molecules containing specific proportions of specific non-standard nucleotides and combinations thereof can exhibit particularly high translation efficiency and low toxicity in vivo. Accordingly, certain embodiments are directed to nucleic acids that include at least one of one or more uridine residues, one or more cytidine residues, and one or more guanosine residues, and that include one or more non-standard nucleotides. In one embodiment, about 20% to about 80% of the uridine residues are 5-methyluridine residues. In another embodiment, about 30% to about 50% of the uridine residues are 5-methyluridine residues. In a further embodiment, about 40% of the uridine residues are 5-methyluridine residues. In one embodiment, about 60% to about 80% of the cytidine residues are 5-methylcytidine residues. In another embodiment, about 80% to about 100% of the cytidine residues are 5-methylcytidine residues. In a further embodiment, about 100% of the cytidine residues are 5-methylcytidine residues. In yet another embodiment, about 20% to about 100% of the cytidine residues are 5-hydroxymethylcytidine residues. In one embodiment, about 20% to about 80% of the guanosine residues are 7-deazaguanosine residues. In another embodiment, about 40% to about 60% of the guanosine residues are 7-deazaguanosine residues. In a further embodiment, about 50% of the guanosine residues are 7-deazaguanosine residues. In one embodiment, about 20% to about 80% or about 30% to about 60% or about 40% of the cytidine residues are N4-methylcytidine and / or N4-acetylcytidine residues. In another embodiment, each cytidine residue is a 5-methylcytidine residue. In a further embodiment, about 100% of the cytidine residues are 5-methylcytidine residues and / or 5-hydroxymethylcytidine residues and / or N4-methylcytidine residues and / or N4-acetylcytidine residues and / or one or more derivatives thereof. In yet another embodiment, about 40% of the uridine residues are 5-methyluridine residues, about 20% to about 100% of the cytidine residues are N4-methylcytidine and / or N4-acetylcytidine residues, and about 50% of the guanosine residues are 7-deazaguanosine residues. In one embodiment, about 40% of the uridine residues are 5-methyluridine residues and about 100% of the cytidine residues are 5-methylcytidine residues.In another embodiment, about 40% of the uridine residues are 5-methyluridine residues, and about 50% of the guanosine residues are 7-deazaguanosine residues. In a further embodiment, about 100% of the cytidine residues are 5-methylcytidine residues, and about 50% of the guanosine residues are 7-deazaguanosine residues. In a further embodiment, about 100% of the uridine residues are 5-hydroxyuridine residues. In one embodiment, about 40% of the uridine residues are 5-methyluridine residues, about 100% of the cytidine residues are 5-methylcytidine residues, and about 50% of the guanosine residues are 7-deazaguanosine residues. In another embodiment, about 40% of the uridine residues are 5-methyluridine residues, about 20% to about 100% of the cytidine residues are 5-hydroxymethylcytidine residues, and about 50% of the guanosine residues are 7-deazaguanosine residues. In an embodiment, less than 100% of the cytidine residues are 5-methylcytidine residues. In other embodiments, less than 100% of the cytidine residues are 5-hydroxymethylcytidine residues. In one embodiment, each uridine residue of the synthetic RNA molecule is a pseudouridine residue or a 5-methylpseudouridine residue. In another embodiment, about 100% of the uridine residues are pseudouridine residues and / or 5-methylpseudouridine residues. In a further embodiment, about 100% of the uridine residues are pseudouridine residues and / or 5-methylpseudouridine residues, about 100% of the cytidine residues are 5-methylcytidine residues, and about 50% of the guanosine residues are 7-deazaguanosine residues.

[0251] Other non-standard nucleotides that can be used instead of or in combination with 5-methyluridine include, but are not limited to, pseudouridine, 5-hydroxyuridine, 5-hydroxypseudouridine, 5-methoxyuridine, 5-methoxypseudouridine, 5-carboxyuridine, 5-carboxypseudouridine, 5-formyluridine, 5-formylpseudouridine, 5-hydroxymethyluridine, 5-hydroxymethylpseudouridine, and 5-methylpseudouridine (“1-methylpseudouridine”, “N1-methylpseudouridine”) or one or more derivatives thereof. Other non-standard nucleotides that can be used instead of or in combination with 5-methylcytidine and / or 5-hydroxymethylcytidine include, but are not limited to, pseudoisocytidine, 5-methylpseudoisocytidine, 5-hydroxymethylcytidine, 5-formylcytidine, 5-carboxycytidine, 5-methoxycytidine, N4-methylcytidine, N4-acetylcytidine or one or more derivatives thereof. In certain embodiments, for example, when transfection, injection or delivery is performed only once, or when the cells, tissues, organs or patients being transfected, injected or delivered are not particularly sensitive to transfection-related toxicity or innate immune signaling, the proportion of non-standard nucleotides can be reduced. Reducing the proportion of non-standard nucleotides may be beneficial in part because it can reduce the cost of nucleic acids by reducing the proportion of non-standard nucleotides. In certain situations, for example, when minimal immunogenicity of the nucleic acid is desired, the proportion of non-standard nucleotides can be increased.

[0252] Enzymes such as T7 RNA polymerase can selectively incorporate standard nucleotides in in vitro transcription reactions containing both standard and non-standard nucleotides. As a result, in in vitro transcription reactions containing a specific proportion of non-standard nucleotides, RNAs may be produced that contain a different, and sometimes lower, proportion of non-standard nucleotides than the proportion of non-standard nucleotides that were present during the reaction. Thus, in certain embodiments, a reference to the proportion of nucleotide incorporation (e.g., "50% 5-methyluridine") refers not only to a nucleic acid containing the stated proportion of nucleotides, but also to a nucleic acid synthesized in such a reaction that may contain a different proportion of nucleotides than the proportion of nucleotides (or nucleotide derivatives, e.g., nucleotide triphosphates) that were present during the reaction, even if the reaction produces a nucleic acid that contains a different proportion of non-standard nucleotides than the proportion that was present during the reaction. Further, by using alternative codons, different nucleotide sequences can encode the same protein. Thus, in certain embodiments, a reference to the proportion of nucleotide incorporation can refer to both a nucleic acid containing the stated proportion of nucleotides and a nucleic acid that encodes the same protein as the protein encoded by a different nucleic acid containing the stated proportion of nucleotides.

[0253] Certain embodiments are directed to a nucleic acid or derivative thereof comprising a 5'-cap structure selected from cap 0, cap 1, cap 2, and cap 3. In one embodiment, the nucleic acid comprises one or more UTRs. In another embodiment, the one or more UTRs enhance the stability of the nucleic acid. In a further embodiment, the one or more UTRs comprise the 5'-UTR of alpha globin or beta globin. In yet another embodiment, the one or more UTRs comprise the 3'-UTR of alpha globin or beta globin. In yet another embodiment, the synthetic RNA molecule comprises the 5'-UTR of alpha globin or beta globin and the 3'-UTR of alpha globin or beta globin. Certain embodiments are directed to a nucleic acid comprising a post-transcriptional regulatory element. In one embodiment, the post-transcriptional regulatory element is selected from the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE), the hepatitis B virus post-transcriptional regulatory element (HPRE), the matrix attachment region of chicken lysozyme (cMAR), and the 5'-DNase I hypersensitive site 4 (cHS4). In another embodiment, the one or more UTRs comprise WPRE. In a further embodiment, the synthetic RNA molecule comprises the 5'-UTR of alpha globin or beta globin and a 3'-UTR comprising WPRE. In yet another embodiment, the synthetic RNA molecule comprises the 5'-UTR of alpha globin or beta globin and the 3'-UTR of alpha globin or beta globin, in addition to one or more copies of WPRE. Exemplary WPRE elements of the invention are SEQ ID NO: 813, SEQ ID NO: 814, SEQ ID NO: 815, SEQ ID NO: 816, SEQ ID NO: 817, and SEQ ID NO: 818.

[0254] In one embodiment, the 5'-UTR contains a Kozak sequence that is substantially similar to the Kozak consensus sequence. In another embodiment, the nucleic acid contains a 3'-poly(A) tail. In a further embodiment, the 3'-poly(A) tail is about 20 nt to about 250 nt or about 120 nt to about 150 nt in length. In a further embodiment, the 3'-poly(A) tail is about 20 nt, or about 30 nt, or about 40 nt, or about 50 nt, or about 60 nt, or about 70 nt, or about 80 nt, or about 90 nt, or about 100 nt, or about 110 nt, or about 120 nt, or about 130 nt, or about 140 nt, or about 150 nt, or about 160 nt, or about 170 nt, or about 180 nt, or about 190 nt, or about 200 nt, or about 210 nt, or about 220 nt, or about 230 nt, or about 240 nt, or about 250 nt in length.

[0255] The poly(A) tail generated by poly(A) polymerase may vary in length depending on reaction conditions including reaction time and enzyme activity, and in an enzymatic tailing reaction, a mixture of RNA molecules with different lengths of poly(A) tails may be generated. Certain embodiments are directed to synthetic RNA molecules containing tails that are about 10, about 20, about 30, about 40, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, or about 400, or more than about 400 nucleotides. In one embodiment, the tail is a poly(A) tail. Other embodiments are directed to tails containing fewer than about 10 nucleotides.

[0256] By synthesizing RNA using a template that codes for a tail, increased control over the length of the tail and reduced variability within or between reactions can be achieved. Accordingly, certain embodiments are directed to a template that codes for a tail. In certain embodiments, the tail contains about 10, about 20, about 30, about 40, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, or about 400 nucleotides. Other embodiments are directed to synthetic RNA molecules synthesized using a template that codes for a tail.

[0257] Including nucleotides other than adenosine within the tail can enhance the stability and / or translation efficiency of synthetic RNA molecules and improve the fidelity of template DNA generation in bacteria. Accordingly, some embodiments are directed to synthetic RNA molecules comprising a tail, where the tail comprises adenosine nucleotides and one or more other nucleotides. Other embodiments are directed to templates encoding a tail, where the tail comprises deoxyadenosine nucleotides and one or more other nucleotides. In one embodiment, the tail comprises guanosine nucleotides. In another embodiment, the tail comprises cytosine nucleotides. In further embodiments, the tail comprises uridine nucleotides. In yet another embodiment, the tail comprises one or more chemically modified nucleotides and / or non-standard nucleotides. In various embodiments, the other nucleotides are incorporated at regular intervals, or at random intervals, or in pairs or groups of adjacent nucleotides separated by one or more adenosine nucleotides. In one embodiment, the tail comprises deoxyguanosine nucleotides. In another embodiment, the tail comprises deoxycytosine nucleotides. In further embodiments, the tail comprises deoxythymidine nucleotides. In various embodiments, the other nucleotides are incorporated at regular intervals, or at random intervals, or in pairs or groups of adjacent nucleotides separated by one or more deoxyadenosine nucleotides. In embodiments, the tail region is composed of about 2% to 10% non-uridine nucleotides, about 10% to 20% non-uridine nucleotides, about 20% to 35% non-uridine nucleotides.

[0258] Including a stem-loop structure before or after the tail can enhance the stability and / or translation efficiency of synthetic RNA molecules. Accordingly, some embodiments are directed to synthetic RNA molecules that include a tail and a stem-loop structure. In various embodiments, the stem-loop structure appears before the tail, after the tail, or both before and after the tail. In certain embodiments, the stem-loop structure is a histone 3’UTR stem-loop. In certain embodiments, the sequence of the stem-loop structure is A(G(Y(Y(Y(UUYUNA)R)R)R)C)A or M(G(G(C(Y(C(UUUUMA)G)R)G)C)C)A or A(G(G(Y(Y(Y(UHHUHA)R)R)R)C)C)A.

[0259] Aspects of the invention include a composition comprising a DNA template that includes (a) a sequence encoding a protein, (b) a tail region comprising deoxyadenosine nucleotides and one or more other nucleotides, and (c) a restriction enzyme binding site.

[0260] In embodiments, the length of the tail region is from about 80 base pairs to about 120 base pairs, from about 120 base pairs to about 160 base pairs, from about 160 base pairs to about 200 base pairs, from about 200 base pairs to about 240 base pairs, from about 240 base pairs to about 280 base pairs, or from about 280 base pairs to about 320 base pairs.

[0261] In embodiments, the length of the tail region is greater than 320 base pairs.

[0262] In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% guanosine residues.

[0263] In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% cytosine residues.

[0264] In embodiments, the tail region contains about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% uridine residues.

[0265] In any of the preceding embodiments and aspects, the tail region contains about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% adenosine residues.

[0266] In embodiments, the synthetic RNA contains from about 200 nucleotides to about 5000 nucleotides.

[0267] In embodiments, the synthetic RNA contains from about 500 to about 2000 nucleotides, or from about 500 to about 1500 nucleotides, or from about 500 to about 1000 nucleotides.

[0268] The protein of interest In embodiments, the compounds, pharmaceutical compositions, or lipid aggregates described herein form a complex or associate with a nucleic acid (e.g., DNA or RNA, e.g., mRNA), and such nucleic acid encodes a recombinant protein of interest.

[0269] In embodiments, the recombinant protein of interest is a soluble protein.

[0270] In embodiments, the protein of interest is selected from Table 2B.

[0271] In embodiments, the soluble protein is one or more reprogramming factors. In embodiments, the one or more reprogramming factors are selected from Oct4, Sox2, Klf4, c-Myc, l-Myc, Tert, Nanog, Lin28, Utf1, Aicda, miR200 microRNA, miR302 microRNA, miR367 microRNA, miR369 microRNA, and biologically active fragments, analogs, variants, and family members thereof.

[0272] In an embodiment, the target recombinant protein is a gene editing protein. In an embodiment, the gene editing protein is selected from a nuclease, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease, a meganuclease, a nickase, a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein, CRISPR / Cas9, Cas9, xCas9, Cas12a (Cpf1), Cas13a, Cas14, CasX, CasY, a class 1 Cas protein, a class 2 Cas protein, and MAD7, or a natural or engineered variant, family member, ortholog, fragment or fusion construct thereof.

[0273] In an embodiment, the gene editing protein comprises a DNA binding domain comprising a plurality of repeat sequences, wherein at least one of the repeat sequences comprises the amino acid sequence LTPvQVVAIAwxyzα (SEQ ID NO: 819), where each of "v", "w", "x", and "y" is independently selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y and null, "z" is selected from GGRPALE (SEQ ID NO: 820), GGKQALE (SEQ ID NO: 821), GGKQALETVQRLLPVLCQD (SEQ ID NO: 630), GGKQALETVQRLLPVLCQA (SEQ ID NO: 631), GKQALETVQRLLPVLCQD (SEQ ID NO: 824), and GKQALETVQRLLPVLCQA (SEQ ID NO: 825), and "α" is any 4 consecutive amino acids or null. In an embodiment, "v" is Q, D or E, "w" is S or N, "x" is I, H, N, or I, "y" is D, A, I, N, H, K, S, G or null, and in an embodiment, the repeat sequence is 36-39 amino acids in length (e.g., 36, or 37, or 38, or 39 amino acids in length).

[0274] In embodiments, α contains at least one glycine (G) residue. In embodiments, α contains at least one histidine (H) residue. In embodiments, α contains at least one histidine (H) residue at any one of positions 33, 34, or 35. In embodiments, α contains at least one aspartic acid (D) residue. In embodiments, α contains at least one, or two, or three of glycine (G) residue, histidine (H) residue, and aspartic acid (D) residue.

[0275] In embodiments, α optionally contains one or more hydrophilic residues selected from the following: optionally, a positively charged polar hydrophilic amino acid selected from arginine (R) and lysine (K); optionally, an electrically neutral polar hydrophilic amino acid selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C); optionally, a negatively charged polar hydrophilic amino acid selected from aspartic acid (D) and glutamate (E), and optionally, a positively charged polar hydrophilic aromatic amino acid selected from histidine (H).

[0276] In some embodiments, α contains one or more positively charged polar hydrophilic amino acids selected from arginine (R) and lysine (K). In some embodiments, α contains one or more electrically neutral polar hydrophilic amino acids selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C). In some embodiments, α contains one or more negatively charged polar hydrophilic amino acids selected from aspartic acid (D) and glutamate (E). In some embodiments, α contains one or more positively charged polar hydrophilic aromatic amino acids selected from histidine (H).

[0277] In embodiments, α comprises one or more hydrophobic residues and is optionally selected from the following: optionally a hydrophobic aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and optionally a hydrophobic aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y). In some embodiments, α comprises one or more hydrophobic aliphatic amino acids selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V). In some embodiments, α comprises one or more aromatic amino acids selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).

[0278] In embodiments, α is defined by GabG, where "a" and "b" are independently selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, and null.

[0279] In embodiments, α is selected from GHGG (SEQ ID NO: 828), HGSG (SEQ ID NO: 829), HGGG (SEQ ID NO: 830), GGHD (SEQ ID NO: 831), GAHD (SEQ ID NO: 832), AHDG (SEQ ID NO: 833), PHDG (SEQ ID NO: 834), GPHD (SEQ ID NO: 835), GHGP (SEQ ID NO: 836), PHGG (SEQ ID NO: 837), PHGP (SEQ ID NO: 838), AHGA (SEQ ID NO: 839), LHGA (SEQ ID NO: 840), VHGA (SEQ ID NO: 841), IVHG (SEQ ID NO: 842), IHGM (SEQ ID NO: 843), RDHG (SEQ ID NO: 845), RHGE (SEQ ID NO: 846), time GE (SEQ ID NO: 847), RHGD (SEQ ID NO: 848), time GD (SEQ ID NO: 849), GPYE (SEQ ID NO: 850), NHGG (SEQ ID NO: 851), THGG (SEQ ID NO: 852), GTHG (SEQ ID NO: 853), GSGS (SEQ ID NO: 854), GSGG (SEQ ID NO: 855), GGGG (SEQ ID NO: 856), GRGG (SEQ ID NO: 857), and GKGG (SEQ ID NO: 858).

[0280] In an embodiment, the gene editing protein includes a DNA binding domain containing a plurality of repeat sequences, at least one of the repeat sequences includes the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 629), and is 36 to 39 amino acids in length, where "v" is Q, D or E, "w" is S or N, "x" is H, N, or I, "y" is D, A, I, N, G, H, K, S, or null, and "z" is GGKQALETVQRLLPVLCQD (SEQ ID NO: 630) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 631).

[0281] In an embodiment, the gene editing protein further includes a nuclease domain containing a catalytic domain of a nuclease.

[0282] In an embodiment, the gene editing protein is capable of creating a single-stranded or double-stranded break in a gene.

[0283] In an embodiment, the single-stranded or double-stranded break causes a persistent splicing change in the gene.

[0284] It has now been discovered that by incorporating microRNA binding sites into the sequence of a synthetic RNA molecule, the encoded protein can be made immunotolerant. Accordingly, certain embodiments are directed to synthetic RNA molecules that include a microRNA binding site. In one embodiment, the microRNA binding site is a miR223 microRNA binding site. In another embodiment, the microRNA binding site is a miR142 microRNA binding site (mIR-142 has the sequence of SEQ ID NO: 810). In some embodiments, the microRNA binding site is present within the 3'-UTR of the synthetic RNA molecule. In other embodiments, the 3'-UTR of the synthetic RNA molecule includes multiple microRNA binding sites. In yet other embodiments, the 3'-UTR of the synthetic RNA molecule includes multiple miR142 microRNA binding sites. In certain embodiments, the 3'-UTR of the synthetic RNA molecule includes multiple miR223 microRNA binding sites. In one embodiment, the miR142 microRNA binding site is TCCATAAAGTAGGAAACACTACA (SEQ ID NO: 811). In another embodiment, the 3'-UTR of the synthetic RNA molecule includes 4 copies of the miR142 microRNA binding site (SEQ ID NO: 812). In further embodiments, the microRNA binding sites are separated by two or more nucleotides. In one embodiment, the encoded protein is a gene editing protein. In another embodiment, the encoded protein is selectively expressed in non-hematopoietic cells. In certain embodiments, the encoded protein is made immunotolerant. In other embodiments, the encoded protein is absent or present at lower levels than normal in a subject. In yet other embodiments, the encoded protein is a gene editing protein and a vector for repairing or inserting a gene is delivered to a cell. In some embodiments, the gene product is made immunotolerant.

[0285] Some embodiments are directed to the induction and / or stimulation and / or propagation of an immune response. In some embodiments, the recombinant protein of interest is an antigen. In some embodiments, the recombinant protein of interest provides acquired immunity against one or more diseases, optionally infectious diseases. In some embodiments, the immune system initiates a response to the recombinant protein of interest. In some embodiments, the recombinant protein of interest is derived from a pathogen or toxin, and in some other embodiments, the recombinant protein of interest is a synthetic mimetic of a pathogen or toxin. In some embodiments, the recombinant protein of interest is a fusion protein and optionally independently includes a sequence derived from or mimicking a pathogen or toxin and / or a sequence known to stimulate an immune response. In some embodiments, the subject becomes immune or resistant to a particular pathogen or toxin after administration of synthetic RNA encoding the antigen of interest.

[0286] In various embodiments, the pathogen is a coronavirus. Coronaviruses (CoV) are members of the family Coronaviridae, which includes beta-coronaviruses and alpha-coronaviruses - respiratory pathogens.

[0287] In embodiments, the coronavirus protein is a beta-coronavirus protein or an alpha-coronavirus protein. In some embodiments, the beta-coronavirus protein is selected from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, and HCoV-OC43 proteins, or antigenic fragments thereof. In some embodiments, the alpha-coronavirus protein is selected from HCoV-NL63 and HCoV-229E proteins, or antigenic fragments thereof.

[0288] In some embodiments, the coronavirus is a beta coronavirus or an alpha coronavirus. In some embodiments, the beta coronavirus is selected from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, and HCoV-OC43. In an embodiment, the alpha coronavirus is selected from HCoV-NL63 and HCoV-229E. In an embodiment, the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0289] In some embodiments, the recombinant protein of interest is a coronavirus protein. In various embodiments, the coronavirus protein is a protein from SARS-CoV-2, such as, for example, the SARS-CoV-2 spike protein. In an embodiment, the SARS-CoV-2 protein is selected from the spike surface glycoprotein, membrane glycoprotein M, envelope protein E, and nucleocapsid phosphorylated protein N, or antigenic fragments thereof. In an embodiment, the SARS-CoV-2 protein selected from the spike surface glycoprotein comprises S1, S2, and S2'.

[0290] In some embodiments, the SARS-CoV-2 spike protein comprises the following amino acid sequence:

[0291] In some embodiments, the envelope protein comprises the following amino acid sequence: MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPSFYVYSRVKNLNSSRVPDLLV (SEQ ID NO: 101).

[0292] In some embodiments, the membrane protein comprises the following amino acid sequence: MADSNGTITVEELKKLLEQWNLVIGFLFLTWICLLQFAYANRNRFLYIIKLIFLWLLWPVTLACFVLAAVYRINWITGGIAIAMACLVGLMWLSYFIASFRLFARTRSMWSFNPETNILLNVPLHGTILTRPLLESELVIGAVILRGHLRIAGHHLGRCDIKDLPKEITVATSRTLSYYKLGASQRVAGDSGFAAYSRYRIGNYKLNTDHSSSSDNIALLVQ (SEQ ID NO: 102).

[0293] In some embodiments, the nucleocapsid phosphoprotein N comprises the following amino acid sequence: MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA (SEQ ID NO: 103).

[0294] In an embodiment, the spike surface glycoprotein comprises the amino acid sequence of SEQ ID NO: 100, the membrane glycoprotein precursor M comprises the amino acid sequence of SEQ ID NO: 101, the envelope protein E comprises the amino acid sequence of SEQ ID NO: 102, and the nucleocapsid phosphoprotein N comprises the amino acid sequence of SEQ ID NO: 103, or an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to any of the foregoing, or an antigenic fragment of any of the foregoing.

[0295] In various embodiments, the SARS-CoV-2 protein has an amino acid sequence that has at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with any known wild-type amino acid sequence of the SARS-CoV-2 protein, or the SARS-CoV-2 amino acid sequences disclosed herein (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity), e.g., SEQ ID NO: 100,It may be related to any one of 101, 102, 103, or may include antigenic fragments thereof.

[0296] In embodiments, a nucleic acid encoding a coronavirus protein, or an antigenic fragment thereof, is provided. The coronavirus protein can be any coronavirus protein, including but not limited to a beta coronavirus protein or an alpha coronavirus protein. The nucleic acid includes, but is not limited to, RNA such as mRNA. In some embodiments, the nucleic acid includes DNA. The DNA can be associated with an AAV encoding a coronavirus protein, or an antigenic fragment thereof.

[0297] In some embodiments, the nucleic acid includes a vector that can include any type of nucleotide, including but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthetic, or partially obtained from natural substances, and in an exemplary embodiment, contains natural, non-natural, or modified nucleotides.

[0298] In embodiments, the vaccine is provided against one or more diseases, pathogens, or toxins. In some embodiments, the vaccine according to the embodiments of the present disclosure elicits a protective antibody titer and / or a T cell response against the encoded antigen. The encoded antigen can be, for example, an infectious agent antigen, such as a SARS-CoV-2 antigen. In some embodiments, the vaccine according to the embodiments of the present disclosure elicits an antigen-specific antibody titer (e.g., IgG, IgM, and / or IgA) specific for the encoded antigen, which can be an infectious agent antigen, such as a SARS-CoV-2 antigen.

[0299] It has now been discovered that the encoded protein can be made immunotolerant by expressing specific factors in combination with the protein. In embodiments, the present invention relates to the delivery of synthetic RNA molecules capable of inducing immunotolerance to an encoded protein by co-delivery of a factor that induces tolerance. In certain embodiments, the co-delivered factor is expressed by the synthetic RNA molecule. In some embodiments, the co-delivered factor is IL2 (SEQ ID NO: 548). In some embodiments, the co-delivered factor is IL10 (SEQ ID NO: 272 or SEQ ID NO: 273) (e.g., without limitation, IL2, IL10, and / or tgf-β). In some embodiments, the co-delivered factor is TGFβ-1 (SEQ ID NO: 190). In some embodiments, the co-delivered factor is TGFβ-2 (SEQ ID NO: 191).

[0300] Gene editing In an aspect, the present invention relates to a complex of one or more synthetic RNA molecules and a compound described herein (e.g., those of Formulas I - XVI), wherein the one or more synthetic RNA molecules comprise at least one RNA molecule encoding one or more gene editing proteins selected from nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases, meganucleases, nickases, clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins, CRISPR / Cas9, Cas9, xCas9, Cas12a (Cpf1), Cas13a, Cas14, CasX, CasY, Class 1 Cas proteins, Class 2 Cas proteins, and MAD7, or natural or engineered variants, family members, orthologs, fragments or fusion constructs thereof.

[0301] In one aspect, the present invention relates to a method for performing gene editing on a cell, comprising transfecting the cell with a complex of one or more synthetic RNA molecules and a compound described herein (e.g., those of formulas I-XVI), wherein the one or more synthetic RNA molecules comprise at least one RNA molecule encoding one or more gene editing proteins selected from nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases, meganucleases, nickases, clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins, CRISPR / Cas9, Cas9, xCas9, Cas12a (Cpf1), Cas13a, Cas14, CasX, CasY, class 1 Cas proteins, class 2 Cas proteins, and MAD7, or natural or engineered variants, family members, orthologs, fragments or fusion constructs thereof.

[0302] Some naturally occurring proteins contain DNA-binding domains that can recognize specific DNA sequences, such as zinc fingers (ZF) and transcription activator-like effectors (TALE). Using a fusion protein containing one or more of these DNA-binding domains and the cleavage domain of the FokI endonuclease, double-strand breaks can be created in the desired region of a cell's DNA (see, for example, US Patent Application Publication No. US2012 / 0064620, US Patent Application Publication No. US2011 / 0239315, US Patent No. 8,470,973, US Patent Application Publication No. US2013 / 0217119, US Patent No. 8,420,782, US Patent Application Publication No. US2011 / 0301073, US Patent Application Publication No. US2011 / 0145940, US Patent No. 8,450,471, US Patent No. 8,440,431, US Patent No. 8,440,432, and US Patent Application Publication No. 2013 / 0122581, all of which are incorporated herein by reference in their entirety). Other gene editing proteins include clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins. However, current methods for gene editing in cells are inefficient and carry a risk of uncontrolled mutagenesis, making them undesirable for research, therapeutic, or cosmetic use.

[0303] Some embodiments are directed to methods of gene editing and / or gene modification using the compounds of the present application (e.g., those of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers. For example, in embodiments, the compounds of the present application (e.g., those of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers associate with synthetic RNA encoding a gene editing protein, and the resulting composition is used to effect gene editing and / or gene modification in cells, e.g., ex vivo or in vivo.

[0304] Some embodiments include, for example, gene editing and / or gene modification using synthetic RNA that encodes one or more of: RNA containing non-standard nucleotides, such as nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases, meganucleases, nickases, clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins, DNA repair proteins, DNA-modifying proteins, base-modifying proteins, DNA methyltransferases, proteins that cause DNA demethylation, DNA-substrate enzymes, or natural or engineered variants, family members, orthologs, fragments or fusion constructs thereof. In embodiments, the efficiency of gene editing and / or gene modification is high, for example, higher than gene editing and / or gene modification using DNA. In embodiments, the methods herein for gene editing and / or gene modification are sufficiently efficient for in vivo applications. In embodiments, the methods herein for gene editing and / or gene modification are sufficiently efficient such that there is no need for cell selection (e.g., selection of edited cells).

[0305] In embodiments, the efficiency of gene editing of the methods herein is about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 100%. In various embodiments, the efficiency of gene modification of the methods herein is about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 100%.

[0306] Some embodiments are directed to gene editing proteins that include a highly efficient, engineered nuclease cleavage domain or DNA modification domain. Other embodiments are directed to gene editing proteins that include a highly faithful, engineered nuclease cleavage domain or DNA modification domain. Various embodiments are directed to gene editing proteins that include a highly efficient, engineered DNA binding domain. Other embodiments are directed to gene editing proteins that include a highly faithful, engineered DNA binding domain. Still other embodiments are directed to gene editing proteins that include engineered repeat sequences. Some embodiments are directed to gene editing proteins that include one or more CRISPR-related family members. Some embodiments are directed to methods for changing the DNA sequence of a cell by transfecting the cell with a gene editing protein or inducing the cell to express it. Other embodiments are directed to methods for changing the DNA sequence of cells present in an in vitro culture. In yet another further embodiment, methods for changing the DNA sequence of cells present in vivo are targeted.

[0307] A gene editing protein comprising the StsI endonuclease cleavage domain (SEQ ID NO: 1) can exhibit substantially lower off-target activity in vivo than previously disclosed gene editing proteins while maintaining high levels of on-target activity in vivo. Other novel engineered proteins (StsI-HA (SEQ ID NO: 2), StsI-HA2 (SEQ ID NO: 3), StsI-UHA (SEQ ID NO: 4), StsI-UHA2 (SEQ ID NO: 5), StsI-HF (SEQ ID NO: 6), and StsI-UHF (SEQ ID NO: 7)) have also been found to exhibit high on-target activity in vivo, low off-target activity in vivo, small size, solubility, and other desirable properties when used as the nuclease domain of a gene editing protein. StsI-HA, StsI-HA2 (high activity), StsI-UHA, and StsI-UHA2 (ultra-high activity) can exhibit higher on-target activity in vivo than either wild-type StsI and wild-type FokI, in part due to specific amino acid substitutions at positions 34 and 61 within the N-terminal region, and StsI-HF (high fidelity) and StsI-UHF (ultra-high fidelity) can exhibit lower off-target activity in vivo than either wild-type StsI and wild-type FokI, in part due to specific amino acid substitutions at positions 141 and 152 within the C-terminal region.

[0308] Accordingly, certain embodiments are directed to a protein. In an embodiment, the protein is present in vivo. In other embodiments, the protein comprises a nuclease domain. In one embodiment, the nuclease domain comprises the cleavage domain of the FokI endonuclease (SEQ ID NO: 53), the cleavage domain of the StsI endonuclease (SEQ ID NO: 1), StsI-HA (SEQ ID NO: 2), StsI-HA2 (SEQ ID NO: 3), StsI-UHA (SEQ ID NO: 4), StsI-UHA2 (SEQ ID NO: 5), StsI-HF (SEQ ID NO: 6), and StsI-UHF (SEQ ID NO: 7) or one or more of their biologically active fragments or variants.

[0309] Engineered gene editing proteins that include a DNA binding domain containing a specific novel repeat array can exhibit lower off-target activity in vivo than previously disclosed gene editing proteins while maintaining high levels of on-target activity in vivo. Certain ones of these engineered gene editing proteins can provide several advantages over previously disclosed gene editing proteins, such as an increase in the flexibility of the linker region connecting the repeat arrays, which can result in increased binding efficiency. Accordingly, certain embodiments are directed to proteins that include multiple repeat arrays. In one embodiment, at least one of the repeat arrays contains the amino acid sequence: GabG, where "a" and "b" each represent any amino acid. In one embodiment, the protein is a gene editing protein. In another embodiment, one or more of the repeat arrays are present within the DNA binding domain. In a further embodiment, "a" and "b" are each independently selected from the group consisting of H and G. In yet another embodiment, "a" and "b" are H and G, respectively. In one embodiment, the amino acid sequence is present within about 5 amino acids of the C-terminus of the repeat array. In another embodiment, the amino acid sequence is present at the C-terminus of the repeat array. In an embodiment, one or more of the Gs of the amino acid sequence GabG are replaced with one or more amino acids other than G, such as A, H, or GG. In one embodiment, the repeat array is about 32 to about 40 amino acids in length or about 33 to about 39 amino acids or about 34 to 38 amino acids or about 35 to about 37 amino acids or about 36 amino acids or greater than about 32 amino acids or greater than about 33 amino acids or greater than about 34 amino acids or greater than about 35 amino acids. Other embodiments are directed to proteins that include one or more transcription activator-like effector domains. In one embodiment, at least one of the transcription activator-like effector domains includes a repeat array. Other embodiments are directed to proteins that include multiple repeat arrays generated by inserting one or more amino acids between at least two of the repeat arrays of a transcription activator-like effector domain.In one embodiment, one or more amino acids are inserted from the C-terminus of at least one repeat sequence by about 1 amino acid or about 2 amino acids or about 3 amino acids or about 4 amino acids or about 5 amino acids. Still other embodiments are directed to proteins comprising multiple repeat sequences, where approximately every other repeat sequence is of a different length than the repeat sequence immediately before or after it. In one embodiment, every other repeat sequence is about 36 amino acids in length. In another embodiment, every other repeat sequence is 36 amino acids in length. Still other embodiments are directed to proteins comprising multiple repeat sequences, where the multiple repeat sequences include at least two repeat sequences each of at least 36 amino acids in length, and at least two of the repeat sequences of at least 36 amino acids in length are separated by at least one repeat sequence of less than 36 amino acids in length. Some embodiments are directed to proteins comprising one or more sequences selected, for example, from SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60.

[0310] Other embodiments are directed to proteins comprising a DNA binding domain. In an embodiment, the DNA binding domain comprises a plurality of repeat sequences. In one embodiment, the plurality of repeat sequences enables highly specific recognition of a binding site within a target DNA molecule. In another embodiment, at least two of the repeat sequences have at least about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 98%, or about 99% homology to each other. In a further embodiment, at least one of the repeat sequences comprises one or more regions capable of binding to a binding site within the target DNA molecule. In yet another embodiment, the binding site comprises a defined sequence that is about 1 to about 5 bases in length. In one embodiment, the DNA binding domain comprises a zinc finger. In another embodiment, the DNA binding domain comprises a transcription activator-like effector (TALE). In a further embodiment, the plurality of repeat sequences includes at least one repeat sequence having at least about 50% or about 60% or about 70% or about 80% or about 90% or about 95% or about 98%, or about 99% homology to a TALE. In yet another embodiment, the gene editing protein comprises a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein. In one embodiment, the gene editing protein comprises a nuclear localization sequence. In another embodiment, the nuclear localization sequence comprises the amino acid sequence: PKKKRKV (SEQ ID NO: 471). In one embodiment, the gene editing protein comprises a mitochondrial localization sequence. In another embodiment, the mitochondrial localization sequence comprises the amino acid sequence: LGRVIPRKIASRASLM (SEQ ID NO: 472). In one embodiment, the gene editing protein comprises a linker. In another embodiment, the linker connects the DNA binding domain and the nuclease domain. In a further embodiment, the linker is about 1 to about 10 amino acids in length. In an embodiment, the linker is about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10 amino acids in length. In one embodiment, the gene editing protein is capable of generating a nick or a double-strand break in the target DNA molecule.

[0311] In an embodiment, the gene editing protein comprises: (a) a DNA binding domain comprising a plurality of repeat sequences, wherein at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 629), where "v" is Q, D or E, "w" is S or N, "x" is H, N, or I, "y" is D, A, I, N, G, H, K, S, or null, and "z" is GGKQALETVQRLLPVLCQD (SEQ ID NO: 630) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 631); and optionally, (b) a nuclease domain comprising a catalytic domain of a nuclease. In an embodiment, the nuclease domain is capable of forming a dimer with another nuclease domain. In an embodiment, the nuclease domain comprises the catalytic domain of a protein comprising the amino acid sequence of SEQ ID NO: 632. In an embodiment, at least one of the repeat sequences comprising the amino acid sequence LTPvQVVAIAwxyzGHGG (SEQ ID NO: 629) is 36 to 39 amino acids in length.

[0312] Certain embodiments are directed to a method for modifying the genome of a cell in vivo, the method comprising introducing into a cell in vivo a nucleic acid molecule encoding a non-naturally occurring fusion protein comprising an artificial transcription activator-like (TAL) effector repeat domain comprising one or more repeating units 36 amino acids in length and a nuclease domain, wherein the repeat domain is engineered for recognition of a predetermined nucleotide sequence and the fusion protein recognizes such predetermined nucleotide sequence. In one embodiment, the cell is a eukaryotic cell. In another embodiment, the cell is an animal cell. In a further embodiment, the cell is a mammalian cell. In yet another embodiment, the cell is a human cell. In one embodiment, the cell is a plant cell. In another embodiment, the cell is a prokaryotic cell. In an embodiment, the fusion protein introduces nuclease-mediated cleavage into the nucleic acid of the cell, thereby modifying the genome of the cell.

[0313] Certain embodiments are directed to compositions for altering the DNA sequence of cells in vivo that include a nucleic acid, where the nucleic acid encodes a gene editing protein. Other embodiments are directed to compositions for altering the DNA sequence of cells in vivo that include a nucleic acid mixture, where the nucleic acid mixture includes a first nucleic acid that encodes a first gene editing protein and a second nucleic acid that encodes a second gene editing protein. In one embodiment, the binding site of the first gene editing protein and the binding site of the second gene editing protein are present within the same target DNA molecule. In another embodiment, the binding site of the first gene editing protein and the binding site of the second gene editing protein are separated by fewer than about 50 bases, or fewer than about 40 bases, or fewer than about 30 bases or fewer than about 20 bases, or fewer than about 10 bases, or from about 10 bases to about 25 bases or about 15 bases. In one embodiment, the nuclease domain of the first gene editing protein and the nuclease domain of the second gene editing protein are capable of dimer formation. In another embodiment, the dimer is capable of generating a nick or double-strand break in the target DNA molecule.

[0314] Certain embodiments are directed to therapeutic compositions. Other embodiments are directed to cosmetic compositions. In an embodiment, the composition includes a repair template. In a further embodiment, the repair template is a single-stranded DNA molecule or a double-stranded DNA molecule.

[0315] Other embodiments are directed to articles of manufacture for synthesizing a protein or a nucleic acid encoding a protein. In one embodiment, the article is a nucleic acid. In another embodiment, the protein comprises a DNA binding domain. In a further embodiment, the nucleic acid comprises a nucleotide sequence encoding a DNA binding domain. In one embodiment, the protein comprises a nuclease domain. In another embodiment, the nucleic acid comprises a nucleotide sequence encoding a nuclease domain. In one embodiment, the protein comprises a plurality of repeat sequences. In another embodiment, the nucleic acid encodes a plurality of repeat sequences. In a further embodiment, the nuclease domain is selected from FokI, StsI, StsI-HA, StsI-HA2, StsI-UHA, StsI-UHA2, StsI-HF, and StsI-UHF or a native or engineered variant or biologically active fragment thereof. In one embodiment, the nucleic acid comprises an RNA polymerase promoter. In another embodiment, the RNA polymerase promoter is a T7 promoter or an SP6 promoter. In a further embodiment, the nucleic acid comprises a viral promoter. In one embodiment, the nucleic acid comprises an untranslated region. In another embodiment, the nucleic acid is a template for in vitro transcription.

[0316] Certain embodiments are directed to methods for inducing cells to express proteins in vivo. Other embodiments are directed to methods for altering the DNA sequence of cells in vivo, including transfecting cells in vivo with gene editing proteins or inducing cells in vivo to express gene editing proteins. Still other embodiments are directed to methods for reducing the expression of a target protein in cells in vivo. In one embodiment, the cells are induced to express a gene editing protein, where the gene editing protein is capable of generating a nick or double-strand break in a target DNA molecule. In another embodiment, the nick or double-strand break results in gene inactivation. Still other embodiments are directed to methods for generating inactive, hypoactive, or dominant-negative forms of proteins in vivo. In one embodiment, the protein is survivin. Still other embodiments are directed to methods for repairing one or more mutations in cells in vivo. In one embodiment, the cells are contacted with a repair template. In another embodiment, the repair template is a DNA molecule. In a further embodiment, the repair template does not contain a binding site for the gene editing protein. In yet another embodiment, the repair template encodes an amino acid sequence encoded by a DNA sequence that includes a binding site for the gene editing protein.

[0317] In various embodiments, the repair template is about 20 nucleotides, or about 30 nucleotides, or about 40 nucleotides, or about 50 nucleotides, or about 60 nucleotides, or about 70 nucleotides, or about 80 nucleotides, or about 90 nucleotides, or about 100 nucleotides, or about 150 nucleotides, or about 200 nucleotides, or about 300 nucleotides, or about 400 nucleotides, or about 500 nucleotides, or about 750 nucleotides, or about 1000 nucleotides. In various embodiments, the repair template is about 20 - 1000 nucleotides, or about 20 - 500 nucleotides, or about 20 - 400 nucleotides, or about 20 - 200 nucleotides, or about 20 - 100 nucleotides, or about 80 - 100 nucleotides, or about 50 - 100 nucleotides.

[0318] In various embodiments, the mass ratio of RNA (e.g., synthetic RNA encoding a gene editing protein) to the repair template is about 1:10, or about 1:9, or about 1:8, or about 1:7, or about 1:6, or about 1:5, or about 1:4, or about 1:3, or about 1:2, or about 1:1, or about 2:1, or about 3:1, or about 4:1, or about 5:1, or about 6:1, or about 7:1, or about 8:1, or about 9:1, or about 10:1.

[0319] In various embodiments, the molar ratio of RNA (e.g., synthetic RNA encoding a gene editing protein) to the repair template is about 1:10, or about 1:9, or about 1:8, or about 1:7, or about 1:6, or about 1:5, or about 1:4, or about 1:3, or about 1:2, or about 1:1, or about 2:1, or about 3:1, or about 4:1, or about 5:1, or about 6:1, or about 7:1, or about 8:1, or about 9:1, or about 10:1.

[0320] In various embodiments, the repair template has a dual function, causing repair to occur at the target sequence where the gene has been edited and preventing further binding of the gene editing protein, thereby suppressing or eliminating further gene editing (e.g., via a repair template that causes a repair that makes what was once a binding site for the gene editing protein no longer suitable for binding of the gene editing protein). Thus, in some embodiments, the gene editing method of the present application is adjustable to ensure a single gene editing for each target site.

[0321] Reprogramming In one aspect, the present invention relates to a method for reprogramming differentiated cells into a more undifferentiated state, comprising: (a) providing a differentiated cell or a non-pluripotent cell; (b) culturing the differentiated cell or the non-pluripotent cell; and (c) transfecting the differentiated cell or the non-pluripotent cell with a complex of one or more synthetic RNA molecules and a compound described herein (e.g., those of Formulae I-XVI), wherein the one or more synthetic RNA molecules comprise at least one RNA molecule encoding one or more reprogramming factors, and transfection results in cells expressing the one or more reprogramming factors, resulting in the cells being reprogrammed into a more undifferentiated state. In an embodiment, step (c) is performed in the presence of a medium containing components that support the reprogramming of the differentiated cells into a more undifferentiated state. In an embodiment, the method further comprises repeating step (c) at least twice during a consecutive five-day period. In an embodiment, the amount of the one or more synthetic RNA molecules transfected in one or more subsequent transfections is greater than the amount transfected in one or more previous transfections. In an embodiment, steps (a)-(c) are performed without using feeder cells and in the presence of feeder cell-conditioned medium. In an embodiment, step (c) is performed without using feeder cells of irradiated human neonatal fibroblasts and in the presence of feeder cell-conditioned medium. In an embodiment, the synthetic RNA molecule encodes one or more reprogramming factors selected from Oct4, Sox2, Klf4, c-Myc, l-Myc, Tert, Nanog, Lin28, Utf1, Aicda, miR200 microRNA, miR302 microRNA, miR367 microRNA, miR369 microRNA, and biologically active fragments, analogs, variants, and family members thereof.

[0322] In embodiments, the differentiated or non-pluripotent cells are derived from a biopsy. In embodiments, the differentiated or non-pluripotent cells are from a human subject. In embodiments, the differentiated or non-pluripotent cells are derived from a punch biopsy specimen of the skin. In embodiments, the differentiated or non-pluripotent cells are keratinocytes, fibroblasts, or PBMCs.

[0323] In embodiments, the method for reprogramming further comprises contacting the cells with at least one member of the group consisting of poly-L-lysine, poly-L-ornithine, RGD peptide, fibronectin, vitronectin, collagen, and laminin.

[0324] In embodiments, the method for reprogramming uses a medium that is substantially free of immunosuppressive agents.

[0325] Cells can be reprogrammed by exposing them to specific extracellular cues and / or by ectopic expression of specific proteins, microRNAs, etc. Although several reprogramming methods have been reported, most of those that rely on ectopic expression require the introduction of exogenous DNA, which can carry a risk of mutations. DNA-free reprogramming methods based on the direct delivery of reprogramming proteins have been reported. However, these methods are too inefficient and unreliable for commercial use. Furthermore, RNA-based reprogramming methods have been reported (see, for example, Angel. MIT Thesis. 2008. 1-56, Angel et al. PLoS ONE. 2010. 5, 107, Warren et al. Cell Stem Cell. 2010. 7, 618-630; Angel. MIT Thesis. 2011. 1-89, and Lee et al., Cell. 2012. 151, 547-558, all of which are incorporated herein by reference). However, existing RNA-based reprogramming methods are time-consuming, unreliable, and inefficient when performed on mature cells, require multiple transfections (resulting in significant expense and opportunities for error), can reprogram only a limited number of cell types, require the use of immunosuppressive agents, and require the use of multiple human-derived components such as blood-derived HSA and human fibroblast feeders. Many of the drawbacks of the RNA-based reprogramming methods disclosed to date make them undesirable for research, therapeutic, or cosmetic use.

[0326] Reprogramming can be carried out by transfecting a cell with one or more nucleic acids encoding one or more reprogramming factors. Examples of reprogramming factors include, but are not limited to, Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Utf1 protein, Aicda protein, miR200 microRNA, miR302 microRNA, miR367 microRNA, miR369 microRNA, and biologically active fragments, analogs, variants, and family members thereof. Accordingly, certain embodiments are directed to methods for reprogramming cells in vivo. In one embodiment, a cell in vivo is reprogrammed by transfecting the cell with one or more nucleic acids encoding one or more reprogramming factors. In one embodiment, the one or more nucleic acids include an RNA molecule encoding Oct4 protein. In another embodiment, the one or more nucleic acids also include one or more RNA molecules encoding Sox2 protein, Klf4 protein, and c-Myc protein. In yet another embodiment, the one or more nucleic acids also include an RNA molecule encoding Lin28 protein. In one embodiment, the cell is a human skin cell, and such human skin cell is reprogrammed into a pluripotent stem cell. In another embodiment, the cell is a human skin cell, and such human skin cell is reprogrammed into a glucose-responsive insulin-producing cell. Examples of other cells that can be reprogrammed and other cells into which cells can be reprogrammed include, but are not limited to, skin cells, pluripotent stem cells, mesenchymal stem cells, β-cells, retinal pigment epithelial cells, hematopoietic cells, heart cells, airway epithelial cells, neural stem cells, neurons, glial cells, bone cells, blood cells, and dental pulp stem cells. In one embodiment, the cell is contacted with a medium that supports the reprogrammed cell. In one embodiment, the medium also supports the cell.

[0327] Importantly, it has been reported that infecting skin cells with viruses encoding Oct4, Sox2, Klf4, and c-Myc and culturing the cells in a medium that supports cardiomyocyte growth results in reprogramming of the skin cells into cardiomyocytes without first reprogramming the skin cells into pluripotent stem cells (see Efs et al., Nat Cell Biol. 2011;13:215-22, which is incorporated herein by reference). In certain situations, direct reprogramming (reprogramming a somatic cell into another somatic cell without first reprogramming the somatic cell into a pluripotent stem cell, also known as "transdifferentiation") may be desirable, in part because culturing pluripotent stem cells can be time-consuming and expensive, the additional handling associated with the establishment and characterization of stable pluripotent stem cell lines can increase the risk of contamination, and the additional time in culture associated with first generating pluripotent stem cells can increase the risk of genomic instability and the acquisition of mutations, including point mutations, copy number polymorphisms, and karyotypic abnormalities. Accordingly, certain embodiments are directed to methods for reprogramming somatic cells in vivo, where the cells are reprogrammed into somatic cells and no characterized pluripotent stem cell lines are generated.

[0328] In certain situations, the total number of transfection times required to reprogram cells according to the method of the present invention may be less than that according to other methods. Thus, certain embodiments are directed to methods for reprogramming cells in vivo, where from about 1 to about 12 transfections are performed over about 20 consecutive days, or from about 4 to about 10 transfections are performed over about 15 consecutive days, or from about 4 to about 8 transfections are performed over about 10 consecutive days. It is recognized that when a cell is contacted with a medium containing a nucleic acid molecule, the cell can be considered to contact and / or take up two or more nucleic acid molecules simultaneously or at different times. Thus, even if a cell is contacted with a nucleic acid-containing medium only once, the cell can be contacted with the nucleic acid two or more times, for example, repeatedly.

[0329] Notably, nucleic acids can contain one or more non-standard or “modified” residues as described herein. For example, any of the non-standard nucleotides described herein can be used in the reprogramming methods of the present application. In one embodiment, uridine-5'-triphosphate can be replaced with pseudouridine-5'-triphosphate in an in vitro transcription reaction to generate synthetic RNA, where up to 100% of the uridine residues of the synthetic RNA can be replaced with pseudouridine residues. In in vitro transcription, residual immunogenic RNA can be generated even when uridine and cytidine are completely replaced with pseudouridine and 5-methylcytidine, respectively (see, for example, Angel. Reprogramming Human Somatic Cells to Pluripotency Using RNA [Doctoral Thesis]. Cambridge, MA: MIT; 2011, the contents of which are incorporated herein by reference). Therefore, when transfecting cells with RNA, it is common to add an immunosuppressant to the transfection medium. In certain situations, it may not be desirable to add an immunosuppressant to the transfection medium, in part because the B18R of recombinant immunosuppressants most commonly used for such purposes can be expensive and difficult to manufacture. Cells can be transfected and / or reprogrammed in vivo according to the methods of the present invention without using B18R or any other immunosuppressant. Reprogramming cells in vivo according to the methods of the present invention without using an immunosuppressant can be efficient and reliable in a short time. Accordingly, certain embodiments are directed to methods for transfecting cells in vivo, where the transfection medium does not contain an immunosuppressant. Other embodiments are directed to methods for reprogramming cells in vivo, where the transfection medium does not contain an immunosuppressant. In certain situations, for example, when using a high cell density, it may be beneficial to add an immunosuppressant to the transfection medium.Accordingly, certain embodiments are directed to methods for transfecting cells in vivo, where the transfection medium contains an immunosuppressive agent. Other embodiments are directed to methods for reprogramming cells in vivo, where the transfection medium contains an immunosuppressive agent. In one embodiment, the immunosuppressive agent is B18R or a biologically active fragment, analog, variant or family member thereof or dexamethasone or a derivative thereof. In one embodiment, the transfection medium does not contain an immunosuppressive agent and the nucleic acid dosage is selected to prevent transient toxicity. In another embodiment, the nucleic acid dosage is about less than 1 mg per 1 cm of tissue. 2 is less than about 1 mg per 100,000 cells or less than about 1 mg per kg or less than about 10 mg per kg.

[0330] Reprogrammed cells generated according to certain embodiments of the present invention are suitable for therapeutic and / or cosmetic uses because they do not contain unwanted exogenous DNA sequences and because they are not exposed to animal- or human-derived products that may be undefined and may contain toxic and / or pathogenic contaminants. Furthermore, the speed, high efficiency, and high reliability of certain embodiments of the present invention can reduce the risk of acquisition and accumulation of mutations and other chromosomal abnormalities. Thus, certain embodiments of the present invention can be used to generate cells having an appropriate safety profile for use in therapeutic and / or cosmetic applications. For example, cells that have not been exposed to allogeneic materials can be obtained by reprogramming cells using RNA and a medium of the present invention that does not contain components derived from animals or humans. Thus, certain embodiments are directed to reprogrammed cells having a desirable safety profile. In one embodiment, the reprogrammed cells have a normal karyotype. In another embodiment, the reprogrammed cells have a copy number polymorphism (CNV) of less than about 5, e.g., less than about 3, or no copy number polymorphism compared to the genome of the patient. In yet another embodiment, the reprogrammed cells have a normal karyotype and have less than about 100 single nucleotide variants in the coding region, or less than about 50 single nucleotide variants in the coding region, or less than about 10 single nucleotide variants in the coding region compared to the genome of the patient.

[0331] Endotoxins and nucleases can co-purify and / or be in a bound state with other proteins such as serum albumin. In particular, recombinant proteins may, in part, due to cell lysis that can occur during their production, often have high levels of bound endotoxins and nucleases. Endotoxins and nucleases can be reduced, removed, replaced, or otherwise inactivated by many of the methods of the present invention, such as by acetylation, addition of a stabilizer such as sodium octanoate followed by heat treatment, addition of a nuclease inhibitor to an albumin solution and / or medium, by crystallization, by contact with one or more ion exchange resins, by contact with charcoal, by preparative electrophoresis, or by affinity chromatography. By partially or completely reducing, removing, replacing, or otherwise inactivating endotoxins and / or nucleases from the medium and / or one or more components of the medium, the efficiency by which cells can be transfected and reprogrammed can be increased. Accordingly, certain embodiments are directed to methods for transfecting one or more nucleic acids into cells in vivo, where the transfection medium is treated such that one or more endotoxins and / or nucleases are partially or completely reduced, removed, replaced, or otherwise inactivated. Other embodiments are directed to media that minimally cause degradation of nucleic acids. In one embodiment, the medium contains less than about 1 EU / mL, or less than about 0.1 EU / mL, or less than about 0.01 EU / mL.

[0332] In certain situations, protein-based lipid carriers such as serum albumin can be replaced with non-protein-based lipid carriers such as methyl-β-cyclodextrin. The medium of the present invention can also be used, for example, in methods where the presence of a lipid carrier may not be necessary or may not be beneficial, such as methods using one or more lipid-based transfection reagents, polymer-based transfection reagents, or peptide-based transfection reagents, or methods using electroporation, without using a lipid carrier when transfection is performed. Many protein-binding molecules such as metals can be highly toxic to cells in vivo. This toxicity can cause a decrease in survival rate and the acquisition of mutations. Thus, in certain embodiments, there is an additional benefit of generating cells that do not contain toxic molecules.

[0333] The binding molecule component of the protein can be measured by suspending the protein in a solution and measuring the conductivity of the solution. Thus, certain embodiments are directed to a medium containing a protein, where an approximately 10% aqueous solution of the protein has a conductivity of less than approximately 500 μmho / cm. In one embodiment, the solution has a conductivity of less than approximately 50 μmho / cm. In another embodiment, less than approximately 0.65% of the dry weight of the protein is composed of lipids and / or less than approximately 0.35% of the dry weight of the protein is composed of free fatty acids.

[0334] To enhance the desired effect of a nucleic acid, the amount of nucleic acid delivered to cells in vivo can be increased. However, increasing the amount of nucleic acid delivered to cells in vivo beyond a certain point can, in part, due to the toxicity of the transfection reagent, cause a decrease in cell viability. When a nucleic acid is delivered to a cell population in vivo (e.g., cells within a region of tissue) of a certain volume, the amount of nucleic acid delivered to each cell can vary depending on the total amount of nucleic acid delivered to the cell population and also in accordance with the cell density, with the higher the cell density, the less nucleic acid is delivered to each cell. In certain embodiments, cells in vivo are transfected with one or more nucleic acids two or more times. Under certain circumstances, e.g., when growing cells, the cell density can change with each transfection. Accordingly, certain embodiments are directed to methods for transfecting cells in vivo, where the cells are transfected two or more times and the amount of nucleic acid delivered to the cells is different for two of the transfections. In one embodiment, the cells grow between two of the transfections and the amount of nucleic acid delivered to the cells is greater for the second of the two transfections than for the first of the two transfections. In another embodiment, the cells are transfected three or more times and the amount of nucleic acid delivered to the cells is greater for the second of the three transfections than for the first of the three transfections and the amount of nucleic acid delivered to the cells is greater for the third of the three transfections than for the second of the three transfections. In yet another embodiment, the cells are transfected two or more times and the maximum amount of nucleic acid delivered to the cells during each transfection is a sufficiently low amount such that at least about 80% viability is obtained in at least two consecutive transfections.

[0335] By regulating the amount of nucleic acid delivered to a population of cells in vivo that is growing in a series of transfections, both increased nucleic acid effect and increased cell viability can be achieved. In certain situations, when cells in vivo are contacted with one or more nucleic acids encoding one or more reprogramming factors in a series of transfections, for at least a portion of that series of transfections, if the amount of nucleic acid delivered in a later transfection is greater than the amount of nucleic acid delivered in a previous transfection, the efficiency of reprogramming can be increased. Accordingly, certain embodiments are directed to methods for reprogramming cells in vivo, where one or more nucleic acids are repeatedly delivered to the cells in a series of transfections, and the amount of nucleic acid delivered to the cells is greater in at least one later transfection than in at least one previous transfection. In one embodiment, the cells are transfected about 2 to about 10 times, or about 3 to about 8 times, or about 4 to about 6 times. In another embodiment, the one or more nucleic acids include at least one RNA molecule, the cells are transfected about 2 to about 10 times, and the amount of nucleic acid delivered to the cells in each transfection is the same as or greater than the amount of nucleic acid most recently delivered to the cells in a previous transfection. In yet another embodiment, the amount of nucleic acid delivered to the cells in the first transfection is about 20 ng / cm 2 ~ about 250 ng / cm 2 or 100 ng / cm 2 ~ 600 ng / cm 2 . In yet another embodiment, the cells are transfected about 5 times at intervals of about 12 to about 48 hours, and the amount of nucleic acid delivered to the cells is about 25 ng / cm 2 in the first transfection, about 50 ng / cm 2 in the second transfection, about 100 ng / cm 2 in the third transfection, about 200 ng / cm 2 in the fourth transfection, and about 400 ng / cm 2It is. In yet another embodiment, the cells are transfected at least one more time after the fifth transfection, and the amount of nucleic acid delivered to the cells is about 400 ng / cm 2 It is.

[0336] Certain embodiments are directed to methods for transfecting cells with nucleic acids in vivo, where the amount of nucleic acid is determined by measuring the cell density and selecting the amount of nucleic acid to be transfected based on the measured cell density. In one embodiment, the cell density is measured by optical means. In another embodiment, the cells are transfected repeatedly, the cell density increases between two transfections, and the amount of nucleic acid transfected is greater in the second of the two transfections than in the first of the two transfections.

[0337] The amount of blood protein produced in a patient can be increased by administering nucleic acid to the patient at a plurality of administration sites. In certain embodiments, the amount of blood protein is increased compared to the amount of blood protein produced in a patient by administering nucleic acid to the patient at a single injection site. In one embodiment, the administration is by injection. In another embodiment, the injection is an intradermal injection. In yet another embodiment, the injection is a subcutaneous injection or an intramuscular injection. In embodiments, the plurality of administration sites includes administration sites on the skin. In other embodiments, the plurality of administration sites is at least about 1 or at least about 2 or at least about 5 or at least about 10 or at least about 20 or at least about 50 or at least about 100 administration sites. In one embodiment, the administration is performed within at least about 5 minutes or at least about 10 minutes or at least about 30 minutes or at least about 1 hour or at least about 2 hours or at least about 5 hours or at least about 12 hours or at least about 1 day. In certain embodiments, the amount of blood protein is increased by at least about 10 percent or at least about 20 percent or at least about 50 percent or at least about 100 percent or at least about 3-fold or at least about 5-fold or at least about 10-fold or at least about 20-fold or at least about 50-fold or at least about 100-fold or at least about 500-fold or at least about 1000-fold or more than 1000-fold.

[0338] In certain circumstances, the in vivo transfection efficiency and viability of cells contacted with the medium of the present invention can be improved by conditioning the medium. Accordingly, certain embodiments are directed to methods for conditioning the medium. Other embodiments are directed to the conditioned medium. In one embodiment, the feeder is a fibroblast and the medium is conditioned for about 24 hours. Other embodiments are directed to methods for transfecting cells in vivo, where the transfection medium is conditioned. Other embodiments are directed to methods for reprogramming and / or gene editing cells in vivo, where the medium is conditioned. In one embodiment, the feeder is mitotically inactivated by exposure to a chemical such as mitomycin C or by exposure to gamma rays. In certain embodiments, although not wishing to be bound by theory in part, it may be beneficial to use only autologous materials, for example, to avoid the risk of disease transmission from the feeder to the cells or the patient. Accordingly, certain embodiments are directed to methods for transfecting cells in vivo, where the transfection medium is conditioned and the feeder is derived from the same individual as the individual in which the cells are transfected. Other embodiments are directed to methods for reprogramming and / or gene editing cells in vivo, where the medium is conditioned and the feeder is derived from the same individual as the individual in which the cells are reprogrammed and / or gene edited.

[0339] Some molecules can be added to the medium conditionally. Thus, certain embodiments are directed to a medium to which one or more molecules present in conditioned medium are added. In one embodiment, Wnt1, Wnt2, Wnt3, Wnt3a or a biologically active fragment, analog, variant, agonist, or family member thereof is added to the medium. In another embodiment, TGF-β or a biologically active fragment, analog, variant, agonist, or family member thereof is added to the medium. In yet another embodiment, cells in vivo are reprogrammed according to the method of the invention, wherein TGF-β is not added to the medium for about 1 to about 5 days and then TGF-β is added for at least about 2 days. In yet another embodiment, IL-6, IL-6R or a biologically active fragment, analog, variant, agonist, or family member thereof is added to the medium. In yet another embodiment, sphingolipids or fatty acids are added to the medium. In yet another embodiment, the sphingolipid is lysophosphatidic acid, lysosphingomyelin, sphingosine-1-phosphate or a biologically active analog, variant or derivative thereof.

[0340] In addition to inactivating cell mitosis, irradiation can, under certain conditions, alter the gene expression of cells, causing cells to produce less of certain proteins, such as proteins of the Wnt family members, etc., than non-irradiated cells, and more of other specific proteins. Furthermore, proteins of certain Wnt family members can promote cell proliferation and transformation. In certain situations, the efficiency of reprogramming can be greatly enhanced by contacting cells in vivo with a conditioned medium using irradiated feeders instead of mitomycin c-treated feeders. The increase in reprogramming efficiency observed when using irradiated feeders is, in part, caused by Wnt proteins secreted by the feeders. Accordingly, certain embodiments are directed to a method for reprogramming cells in vivo, wherein the cells are contacted with an agonist such as Wnt1, Wnt2, Wnt3, Wnt3a or a biologically active fragment, analog, variant, family member thereof or an agonist of a downstream target of the Wnt protein, and / or an agent that mimics one or more of the biological actions of the Wnt protein, such as 2-amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyl)pyrimidine.

[0341] Many reprogramming methods using DNA are inefficient, so these methods may be difficult or impossible to use with cells derived from patient samples that may contain only a small number of cells. In contrast, the high efficiency of certain embodiments of the present invention enables reliable reprogramming of a small number of cells, including single cells. Certain embodiments are directed to methods for reprogramming a small number of cells. Other embodiments are directed to methods for reprogramming single cells. In one embodiment, the cells are contacted with one or more enzymes. In another embodiment, the enzyme is collagenase. In yet another embodiment, the collagenase is free of animal-derived components. In one embodiment, the collagenase is present at a concentration of about 0.1 mg / mL to about 10 mg / mL, or about 0.5 mg / mL to about 5 mg / mL. In another embodiment, the cells are blood cells. In yet another embodiment, the cells are contacted with a medium containing one or more proteins derived from the patient's blood. In yet another embodiment, the cells are contacted with a medium containing DMEM / F12 + 2 mM L-alanyl-L-glutamine + about 5% to about 25% patient-derived serum, or about 10% to about 20% patient-derived serum, or about 20% patient-derived serum.

[0342] In certain situations, by using the medium of the present invention to transfect a mixture of RNAs encoding Oct4, Sox2, Klf4, and c-Myc into cells in vivo, the cell growth rate can be increased. If the amount of RNA delivered to the cells is too small to ensure transfection of all cells, only a small amount of cells may show an increase in growth rate. In certain situations, such as when preparing personalized therapeutic agents, it may be desirable to increase the cell growth rate, partly because this can shorten the time required for preparing the therapeutic agent and thus reduce the cost of the therapeutic agent. Accordingly, certain embodiments are directed to methods for transfecting cells in vivo with a mixture of RNAs encoding Oct4, Sox2, Klf4, and c-Myc. In one embodiment, the cells show an increased growth rate. In another embodiment, the cells are reprogrammed.

[0343] Also provided are methods for performing somatic gene editing and reprogramming simultaneously or sequentially as described herein.

[0344] Therapeutic methods In embodiments, the present invention relates to a method for treating a disease or disorder by delivering a therapeutic agent having the compounds of the present application (e.g., those of formulas I - XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier.

[0345] In embodiments, the present invention relates to a method for treating a disease or disorder by delivering a nucleic acid therapeutic agent having the compounds of the present application (e.g., those of formulas I - XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier.

[0346] In embodiments, the present invention relates to a method for treating a disease or disorder by delivering a therapeutic agent having an RNA, e.g., a synthetic RNA, the compounds of the present application (e.g., those of formulas I - XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier.

[0347] In an embodiment, the present invention relates to a method for treating a disease or disorder by delivering a therapeutic agent, such as a nucleic acid, such as RNA, such as synthetic RNA, the compounds of the present application (such as those of formulas I to XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier, by a method for expressing a target protein having a therapeutic effect.

[0348] In an embodiment, the present invention relates to a method for treating a disease or disorder by delivering a therapeutic agent, such as a nucleic acid, such as RNA, such as synthetic RNA, the compounds of the present application (such as those of formulas I to XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier, by a method for expressing a gene editing protein having a therapeutic effect (such as gene editing or gene modification in vivo and / or ex vivo, etc.).

[0349] In an embodiment, the present invention relates to a method for treating a disease or disorder by delivering a therapeutic agent, such as a nucleic acid, such as RNA, such as synthetic RNA, the compounds of the present application (such as those of formulas I to XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier, by a method for expressing a reprogramming factor having a therapeutic effect (such as ex vivo).

[0350] In an embodiment, the present invention relates to a method for treating a disease or disorder by delivering a therapeutic agent, such as a nucleic acid, such as RNA, such as synthetic RNA, the compounds of the present application (such as those of formulas I to XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier, by a method for expressing a gene editing protein having a therapeutic effect (such as gene editing or gene modification in vivo and / or ex vivo, etc.) and expressing a reprogramming factor having a therapeutic effect (such as ex vivo).

[0351] Certain embodiments are directed to methods for treating a patient, comprising a. inducing a cell to express a protein of interest by transfecting the cell in vivo with a nucleic acid encoding the protein of interest, and / or b. reprogramming the cell in vivo. In one embodiment, the cell is reprogrammed to a more undifferentiated state. In another embodiment, the cell is reprogrammed by transfecting the cell with one or more synthetic RNA molecules encoding one or more reprogramming proteins. In a further embodiment, the cell is differentiated. In yet another embodiment, the cell is differentiated into one of a skin cell, a glucose-responsive insulin-producing cell, a hematopoietic cell, a heart cell, a retinal cell, a kidney cell, a nerve cell, a stromal cell, an adipocyte, an osteocyte, a muscle cell, an oocyte, and a sperm cell. Other embodiments are directed to methods for treating a patient, comprising a. inducing a cell to express a gene editing protein by transfecting the cell in vivo with a nucleic acid encoding the gene editing protein, and / or b. reprogramming the cell in vivo.

[0352] In an embodiment, the treatment results in the improvement of one or more of the patient's symptoms.

[0353] Certain embodiments are directed to methods and compositions for the treatment of rare diseases. In an embodiment, the rare disease is one or more of a rare metabolic disease, a rare cardiovascular disease, a rare skin disease, a rare neurological disease, a rare developmental disorder, a rare genetic disease, a rare lung disease, a rare liver disease, a rare kidney disease, a rare psychiatric disease, a rare reproductive disorder, a rare musculoskeletal disease, a rare orthopedic disease, an inborn error of metabolism, a lysosomal storage disorder, and a rare eye disease.

[0354] Examples of diseases that can be treated by the present invention include, but are not limited to, Alzheimer's disease, spinal cord injury, amyotrophic lateral sclerosis, cystic fibrosis, heart diseases including ischemic and dilated cardiomyopathy, macular degeneration, Parkinson's disease, Huntington's disease, diabetes, sickle cell anemia, thalassemia, Fanconi anemia, xeroderma pigmentosum, muscular dystrophy, severe combined immunodeficiency, hereditary sensory neuropathy, cancer, and HIV / AIDS.

[0355] Further examples of diseases that can be treated by the present invention include, but are not limited to, type 1 diabetes, heart diseases including ischemic and dilated cardiomyopathy, macular degeneration, Parkinson's disease, cystic fibrosis, sickle cell anemia, thalassemia, Fanconi anemia, severe combined immunodeficiency, hereditary sensory neuropathy, xeroderma pigmentosum, Huntington's disease, muscular dystrophy, amyotrophic lateral sclerosis, Alzheimer's disease, cancer, and infectious diseases such as hepatitis and HIV / AIDS.

[0356] In embodiments, examples of diseases that can be treated by the present invention include infectious diseases. In some embodiments, the infectious disease is an infectious disease caused by a pathogen, and optionally is selected from bacteria, viruses, fungi, or parasites. In some embodiments, the virus is (a) an influenza virus, and optionally is selected from influenza A, B, C, and D viruses, or (b) a member of the family Coronaviridae, and optionally is selected from beta coronaviruses, and optionally is selected from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), HCoV-HKU1, and HCoV-OC43 or alpha coronaviruses, and optionally is selected from HCoV-NL63 and HCoV-229E. In some embodiments, the virus is SARS-CoV-2. In some embodiments, the protein of interest is an antigen such as a 2019-nCoV protein, an antigenic fragment thereof, or a nucleic acid encoding the same, as described elsewhere herein, and optionally is selected from a spike surface glycoprotein, membrane glycoprotein M, envelope protein E, and nucleocapsid phosphorylated protein N. In some embodiments, the protein of interest is an antigen such as the S1 or S2 subunit of the spike surface glycoprotein, or an antigenic fragment thereof, as described elsewhere herein.

[0357] In various embodiments, the subject has coronavirus disease 2019 (COVID-19). In additional embodiments, the subject is elderly and / or has one or more comorbidities including, but not limited to, hypertension and / or diabetes. A subject with coronavirus infection can experience symptoms including, but not limited to, fever, fatigue, dry cough, chills and pain, shortness of breath and other breathing difficulties, diarrhea, upper respiratory symptoms (e.g., sneezing, runny nose, nasal congestion, cough, sore throat), pneumonia, pneumonia respiratory failure, liver failure and kidney failure, acute respiratory distress syndrome (ARDS), and disruption of cytokine balance.

[0358] In some embodiments, the virus is an influenza virus. In some embodiments, the protein of interest is an antigen such as an influenza virus antigen as described elsewhere herein, and optionally is selected from hemagglutinin (HA) protein, matrix 2 (M2) protein, and neuraminidase, or antigenic fragments thereof, or nucleic acids encoding them.

[0359] In embodiments, the disease or disorder is selected from diphtheria, tetanus, pertussis, influenza, pneumonia, hepatitis A, hepatitis B, polio, yellow fever, human papillomavirus (HPV) infection, anthrax, rabies, Japanese encephalitis, meningitis, measles, mumps, rubella, gastroenteritis, smallpox, typhoid fever, varicella (chickenpox), rotavirus, and herpes zoster. In some embodiments, the present invention relates to the treatment of hepatitis. Exemplary hepatitis that can be treated includes, but is not limited to, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, autoimmune hepatitis, alcoholic hepatitis, acute hepatitis, and chronic hepatitis.

[0360] In embodiments, the disease or disorder is a metabolic disorder. In embodiments, the metabolic disorder is selected from disorders of carbohydrate metabolism, amino acid metabolism, urea cycle, fatty acid metabolism, porphyrin metabolism, lysosomal storage disorders, peroxisomal biosynthesis disorders, and purine or pyrimidine metabolism disorders.

[0361] In an embodiment, the metabolic disorder is a disorder of carbohydrate metabolism, where the disease is galactosemia and the defective gene is optionally GALT, GALK1, or GALE, or the disease is essential fructosuria and the defective gene is optionally KHK, or the disease is hereditary fructose intolerance and the defective gene is optionally ALDOB, or the disease is glycogenosis type I and the defective gene is optionally G6PC, SLC37A4, or SLC17A3, or the disease is glycogenosis type II and the defective gene is optionally GAA, or the disease is glycogenosis type III and the defective gene is optionally AGL, or the disease is glycogenosis type IV and the defective gene is optionally GBE1, or the disease is glycogenosis type V and the defective gene is optionally PYGM, or the disease is glycogenosis type VI and the defective gene is optionally PYGL, or the disease is glycogenosis type VII and the defective gene is optionally PYGM, or the disease is glycogenosis type IX and the defective gene is optionally PHKA1, PHKA2, PHKB, PHKG1, or PHKG2, or the disease is glycogenosis type XI and the defective gene is optionally SLC2A2, or the disease is glycogenosis type XII and the defective gene is optionally ALDOA, or the disease is glycogenosis type XIII and the defective gene is optionally ENO1, ENO2, or ENO3, or the disease is glycogenosis type 0 and the defective gene is optionally GYS1 or GYS2, or the disease is pyruvate carboxylase deficiency and the defective gene is optionally PC, or the disease is pyruvate kinase deficiency and the defective gene is optionally PKLR, or the disease is transaldolase deficiency and the defective gene is optionally TALDO1, or the disease is triosephosphate isomerase deficiency and the defective gene is optionally TPI1, or the disease is fructose bisphosphatase deficiency and the defective gene is optionally FBP1, or the disease is hyperoxaluria and the defective gene is optionally AGXT or GRHPR, or the disease is hexokinase deficiency and the defective gene is optionally HK1, or the disease is glucose-galactose malabsorption and the defective gene is optionally SLC5A1,Or the disease is glucose-6-phosphate dehydrogenase deficiency, and the defective gene is optionally G6PD.,

[0362] In an embodiment, the metabolic disorder is a disorder of amino acid metabolism, wherein the disease is alkaptonuria and the defective gene is optionally HGD, the disease is aspartylglucosaminuria and the defective gene is optionally AGA, the disease is methylmalonic acidemia and the defective gene is optionally MUT, MCEE, MMAA, MMAB, MMACHC, MMADHC, or LMBRD1, the disease is maple syrup urine disease and the defective gene is optionally BCKDHA, BCKDHB, DBT, or DLD, the disease is homocystine the disease is tyrosinemia, the defective gene is optionally CBS, the disease is tyrosinemia, the defective gene is optionally FAH, TAT, or HPD, the disease is trimethylaminuria, the defective gene is optionally FMO3, the disease is Hartnup disease, the defective gene is optionally SLC6A19, the disease is biotinidase deficiency, the defective gene is optionally BTD, the disease is ornithine carbamoyltransferase deficiency, the defective gene is optionally OTC, the disease is carbamoyl phosphate synthase I deficiency, wherein the defective gene is optionally CPS1, wherein the disease is citrullinemia, wherein the defective gene is optionally ASS or SLC25A13, wherein the disease is hyperargininemia, wherein the defective gene is optionally ARG1, wherein the disease is hyperhomocysteinemia, wherein the defective gene is optionally MTHFR, wherein the disease is hypermethioninemia, wherein the defective gene is optionally MAT1A, GNMT, or AHCY, wherein the disease is hyperlysinemia, wherein the defective gene is optionally AASS, wherein the disease is nonketotic hyperglycinemia. and the defective gene is optionally GLDC, AMT, or GCSH, the disease is propionic acidemia, the defective gene is optionally PCCA or PCCB, the disease is hyperprolinemia, the defective gene is optionally ALDH4A1 or PRODH, the disease is cystinuria, the defective gene is optionally SLC3A1 or SLC7A9, the disease is dicarboxylic aminoaciduria, the defective gene is optionally SLC1A1, the disease is glutaric acidemia type 2, and the defective gene is optionally ETFA,It is ETFB, or ETFDH, or the disease is isovaleric academia, and the defective gene is optionally IVD, or the disease is 2-hydroxyglutaric aciduria, and the defective gene is optionally L2HGDH or D2HGDH.,

[0363] In an embodiment, the metabolic disorder is a disorder of the urea cycle, where the disease is N-acetylglutamate synthase deficiency, and the defective gene is optionally NAGS, or the disease is argininosuccinic aciduria, and the defective gene is optionally ASL, or the disease is arginemia, and the defective gene is optionally ARG1.

[0364] In the embodiment, the metabolic disorder is a disorder of fatty acid metabolism, where the disease is very long-chain acyl-CoA dehydrogenase deficiency, and the defective gene is optionally ACADVL, or the disease is long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency, and the defective gene is optionally HADHA, or the disease is medium-chain acyl-CoA dehydrogenase deficiency, and the defective gene is optionally ACADM, or the disease is short-chain acyl-CoA dehydrogenase deficiency, and the defective gene is optionally ACADS, or the disease is 3-hydroxyacyl-CoA dehydrogenase deficiency, and the defective gene is optionally HADH, or the disease is 2,4-dienoyl-CoA reductase deficiency, and the defective gene is optionally NADK2, or the disease is 3-hydroxy-3-methylglutaryl-CoA lyase deficiency, and the defective gene is optionally HMGCL, or the disease is malonyl-CoA decarboxylase deficiency, and the defective gene is optionally MLYCD, or the disease is systemic primary carnitine deficiency, and the defective gene is optionally SLC22A5, or the disease is carnitine-acylcarnitine translocase deficiency, and the defective gene is optionally SLC25A20, or the disease is carnitine palmitoyltransferase I deficiency, and the defective gene is optionally CPT1A, or the disease is carnitine palmitoyltransferase II deficiency, and the defective gene is optionally CPT2, or the disease is lysosomal acid lipase deficiency, and the defective gene is optionally LIPA, or the disease is Gaucher disease, and the defective gene is optionally GBA.

[0365] In an embodiment, the metabolic disorder is a disorder of porphyrin metabolism, where the disease is acute intermittent porphyria and the defective gene is optionally HMBS, or the disease is Gunther's disease and the defective gene is optionally UROS, or the disease is porphyria cutanea tarda and the defective gene is optionally UROD, or the disease is hepatoerythropoietic porphyria and the defective gene is optionally UROD, or the disease is hereditary coproporphyria and the defective gene is optionally CPOX, or the disease is variegate porphyria and the defective gene is optionally PPOX, or the disease is erythropoietic protoporphyria and the defective gene is optionally FECH, or the disease is aminolevulinic acid dehydratase-deficient porphyria and the defective gene is optionally ALAD.

[0366] In an embodiment, the metabolic disorder is a lysosomal storage disorder, where the disease is Fabry disease and the defective gene is optionally ASAH1, or the disease is Krabbe disease and the defective gene is optionally GALC, or the disease is galactosialidosis and the defective gene is optionally CTSA, or the disease is Fabry disease and the defective gene is optionally GLA, or the disease is Schindler disease and the defective gene is optionally NAGA, or the disease is GM1 gangliosidosis and the defective gene is optionally GLB1, or the disease is Tay-Sachs disease and the defective gene is optionally HEXA, or the disease is Sandhoff disease and the defective gene is optionally HEXB, or the disease is GM2-gangliosidosis, AB variant and the defective gene is optionally GM2A, or the disease is Niemann-Pick disease and the defective gene is optionally SMPD1, NPC1, or NPC2, or the disease is metachromatic leukodystrophy and the defective gene is optionally ARSA or PSAP, or the disease is multiple sulfatase deficiency and the defective gene is optionally SUMF1, or the disease is Hurler syndrome and the defective gene is optionally IDUA, or the disease is Hunter syndrome and the defective gene is optionally IDS, or the disease is Sanfilippo syndrome and the defective gene is optionally SGSH, NAGLU, HGSNAT, or GNS, or the disease is Morquio syndrome and the defective gene is optionally GALNS or GLB1, or the disease is Maroteaux-Lamy syndrome and the defective gene is optionally ARSB, or the disease is Sly syndrome and the defective gene is optionally GUSB, or the disease is sialidosis and the defective gene is optionally NEU1, NEU2, NEU3, or NEU4, or the disease is I-cell disease and the defective gene is optionally GNPTAB or GNPTG, or the disease is mucolipidosis type IV and the defective gene is optionally MCOLN1, or the disease is infantile neuronal ceroid lipofuscinosis and the defective gene is optionally PPT1 or PPT2, or the disease is Jansky-Bielschowsky disease and the defective gene is optionally TPP1, or the disease is Batten disease,The defective gene is optionally CLN1, CLN2, CLN3, CLN5, CLN6, MFSD8, CLN8, or CTSD, the disease is Kufs disease type A, the defective gene is optionally CLN6 or PPT1, the disease is Kufs disease type B, the defective gene is optionally DNAJC5 or CTSF, the disease is alpha-mannosidosis, the defective gene is optionally MAN2B1, MAN2B2, or MAN2C1, the disease is beta-mannosidosis, the defective gene is optionally MANBA, the disease is fucosidosis, the defective gene is optionally FUCA1, the disease is cystinosis, the defective gene is optionally CTNS, the disease is pycnodysostosis, the defective gene is optionally CTSK, the disease is sialidosis, the defective gene is optionally SLC17A5, the disease is infantile free sialic acid storage disease, the defective gene is optionally SLC17A5, or the disease is Danon disease, the defective gene is optionally LAMP2.

[0367] In an embodiment, the metabolic disorder is a peroxisome biogenesis disorder, where the disease is Zellweger syndrome and the defective gene is optionally PEX1, PEX2, PEX3, PEX5, PEX6, PEX12, PEX14, or PEX26; or the disease is infantile Refsum disease and the defective gene is optionally PEX1, PEX2, or PEX26; or the disease is neonatal adrenoleukodystrophy and the defective gene is optionally PEX5, PEX1, PEX10, PEX13, or PEX26; or the disease is RCDP1 type and the defective gene is optionally PEX7; or the disease is pipecolic acidemia and the defective gene is optionally PAHX; or the disease is acatalasemia and the defective gene is optionally CAT; or the disease is primary hyperoxaluria type 1 and the defective gene is optionally AGXT; or the disease is acyl-CoA oxidase deficiency and the defective gene is optionally ACOX1; or the disease is D-bifunctional protein deficiency and the defective gene is optionally HSD17B4; or the disease is dihydroxyacetone phosphate acyltransferase deficiency and the defective gene is optionally GNPAT; or the disease is X-linked adrenoleukodystrophy and the defective gene is optionally ABCD1; or the disease is α-methylacyl-CoA racemase deficiency and the defective gene is optionally AMACR; or the disease is RCDP2 type and the defective gene is optionally DHAPAT; or the disease is RCDP3 type and the defective gene is optionally AGPS; or the disease is adult Refsum disease-1 and the defective gene is optionally PHYH; or the disease is Marinesco-Sjögren syndrome and the defective gene is optionally TRIM37.

[0368] In an embodiment, the metabolic disorder is a disorder of purine or pyrimidine metabolism, where the disease is Lesch-Nyhan syndrome and the defective gene is optionally HPRT, or the disease is adenine phosphoribosyltransferase deficiency and the defective gene is optionally APRT, or the disease is adenosine deaminase deficiency and the defective gene is optionally ADA, or the disease is adenylate deaminase deficiency type 1 and the defective gene is optionally AMPD1, or the disease is adenylosuccinate lyase deficiency and the defective gene is optionally ADSL, or the disease is dihydropyrimidine dehydrogenase deficiency and the defective gene is optionally DPYD, or the disease is Miller syndrome and the defective gene is optionally DHODH, or the disease is orotic aciduria and the defective gene is optionally UMPS, or the disease is purine nucleoside phosphorylase deficiency and the defective gene is optionally PNP, or the disease is xanthinuria and the defective gene is optionally XDH, MOCS1, or MOCS2, GEPH.

[0369] In an embodiment, the present invention relates to a method for modulating transthyretin (TTR). The method includes administering to a subject an effective amount of synthetic RNA encoding TTR, wherein the synthetic RNA includes one or more non-standard nucleotides that avoid substantial cytotoxicity.

[0370] In an embodiment, modulating results in an increase in the amount of TTR in the subject.

[0371] In an embodiment, modulating results in a decrease in the amount of TTR in the subject.

[0372] In an embodiment, modulating results in treatment of one or more of amyloidosis, senile systemic amyloidosis (SSA), familial amyloid polyneuropathy (FAP), and familial amyloid cardiomyopathy (FAC).

[0373] In an embodiment, the non-standard nucleotide has one or more substitutions at a position selected from the 2C position, 4C position, and 5C position for pyrimidine, or at a position selected from the 6C position, 7N position, and 8C position for purine.

[0374] In an embodiment, the present invention treats or reduces pain, such as postoperative pain and / or chronic pain, in a subject in need thereof by administering an effective amount of a synthetic RNA encoding a gene editing protein capable of creating a double-strand break in a voltage-gated sodium channel type 1 (NaV1) gene, such as NaV1.3, NaV1.7, NaV1.8, and / or NaV1.9.

[0375] In an embodiment, the administration targets the central nervous system (CNS) or the peripheral nervous system (PNS), such as neurons and glial cells of the CNS or PNS.

[0376] In an embodiment, the disease or disorder is a pulmonary disease or disorder. In an embodiment, the disease or disorder is an inflammation associated with a pulmonary disease or disorder. In an embodiment, the pulmonary disease or disorder is selected from asbestosis, asthma, bronchiectasis, bronchitis, chronic cough, chronic obstructive pulmonary disease (COPD), cold, croup, cystic fibrosis, hantavirus, idiopathic pulmonary fibrosis, influenza, lung cancer, novel influenza, pertussis, pleurisy, pneumonia, pulmonary embolism, pulmonary hypertension, respiratory syncytial virus (RSV), sarcoidosis, sleep apnea, spirometry, sudden infant death syndrome (SIDS), and tuberculosis.

[0377] Furthermore, in some embodiments, the compositions of the present application are for acne vulgaris, summer acne, acne agminata, cosmetic acne, electric acne, acne keloidalis nuchae, acne mechanica, drug-induced acne, acne miliaris necrotica, acne necrotica, rosacea, actinic keratosis, acne vulgaris, summer acne, acne agminata, cosmetic acne, electric acne, acne keloidalis nuchae, acne mechanica, drug-induced acne, acne miliaris necrotica, acne necrotica, rosacea, acute urticaria, allergic contact dermatitis, alopecia areata, angioedema, tinea pedis, atopic dermatitis, autoeczematization, baby acne, hair loss, blastomycosis, blackheads, birthmarks and other skin pigmentation problems, boils, contusions, insect bites and stings, burns, cellulitis, scabies, chloracne, cholinergic or stress urticaria, chronic urticaria, cold urticaria, confluent and reticulated papillomatosis, verruca, cysts, dandruff, dermatitis herpetiformis, dermatographism, dyshidrotic eczema, diaper dermatitis, dry skin, hyperhidrosis, ectodermal dysplasia, for example, anhidrotic ectodermal dysplasia and X-linked anhidrotic ectodermal dysplasia, eczema, verruciform epidermal dysplasia, erythema nodosum, excoriated acne, exercise-induced anaphylactic folliculitis, seborrhea, folliculitis, freckles, frostbite, onychomycosis, hair density, hair growth rate, halogen acne, hair loss, miliaria rubra, hematoma, herpes simplex infection (e.g., other than genital), hidradenitis suppurativa, urticaria, hyperhidrosis, hyperpigmentation, anhidrotic ectodermal dysplasia, hypopigmentation, impetigo, ingrown hair, heat urticaria, pitted nails, neonatal acne or baby acne, pruritus, irritant contact dermatitis, lice, keloids, porokeratosis, lichen planus, lichen sclerosus, lupus miliaris disseminatus faciei, melasma, moles, molluscum contagiosum, nail growth rate, nail health, neurodermatitis, nummular eczema, occupational acne, oil acne, onychomycosis, physical urticaria, hair follicle cysts, pityriasis versicolor, vitiligo, poison ivy, pomade acne, pseudofolliculitis barbae or acne keloidalisnuchae), psoriasis, psoriatic arthritis, pressure urticaria or delayed pressure urticaria, puncture wounds such as cuts and abrasions, rashes, rare or aqueous urticaria, rhinoplasty, tinea, rosacea, Rothmund-Thomson syndrome, skin laxity, scabies, scars, seborrhea, seborrheic dermatitis, herpes zoster, skin cancer, skin tags, solar urticaria, spider bites, skin streaks, sunburn, tar acne, tropical acne, thinning of the skin, thrush, vitiligo, transient acantholytic dermatosis, tycoon’s cap or acne necrotica, uneven skin tone, varicose veins, venous eczema, vibratory angioedema, leukoderma, warts, Weber-Christian disease, wrinkles, X-linked anhidrotic ectodermal dysplasia, xerotic eczema, yeast infections, and age-related systemic signs, among others, but not limited to, diseases, disorders, and / or conditions that can be used for the treatment, management, or prevention of, and / or can modify, adorn, or change the appearance of members of the integumentary system of a subject suffering from the disease, disorder, and / or condition.

[0378] Exemplary cancers and / or tumors of the present invention include basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intraepithelial neoplasia; kidney cancer or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell lung cancer); melanoma; multiple myeloma; neuroblastoma; oral cancer (lips, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine cancer or endometrial cancer; urinary system cancer; vulvar cancer; lymphomas including Hodgkin lymphoma and non-Hodgkin lymphoma, and B cell lymphomas (low grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate / follicular NHL; intermediate diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; large lesion NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myelogenous leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), and also include phakomatosis, edema (such as those associated with brain tumors), and angiogenic abnormalities associated with Meigs syndrome, but are not limited thereto.

[0379] In embodiments, one or more rare diseases are treated, managed or prevented by the compositions of the present application, and these include, by way of example, erythropoietic protoporphyria, Hailey-Hailey disease, epidermolysis bullosa (EB), xeroderma pigmentosum, Ehlers-Danlos syndrome, cutis laxa, protein C & protein S deficiency, Alport syndrome, linear palmoplantar keratoderma, lethal acantholytic EB, pseudoxanthoma elasticum (PXE), ichthyosis vulgaris, pemphigus vulgaris, and basal cell nevus syndrome.

[0380] In an embodiment, the composition of the present application is used for treating, managing or preventing one or more inflammatory diseases or conditions such as inflammation, acute inflammation, chronic inflammation, respiratory diseases, atherosclerosis, restenosis, asthma, allergic rhinitis, atopic dermatitis, septic shock, rheumatoid arthritis, inflammatory bowel disease, inflammatory pelvic disease, pain, ocular inflammatory diseases, celiac disease, Reye's syndrome, glycerol kinase deficiency, familial eosinophilia (FE), autosomal recessive spastic ataxia, laryngeal inflammatory diseases; tuberculosis, chronic cholecystitis, bronchiectasis, silicosis and other pneumoconioses, etc.

[0381] In an embodiment, the composition of the present application is used for treating, managing or preventing one or more autoimmune diseases or conditions such as multiple sclerosis, type 1 diabetes, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain - Barré syndrome, scleroderma, Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen encephalitis, primary sclerosing cholangitis, sclerosing cholangitis, autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, fibromyalgia, Ménière's syndrome; transplant rejection (e.g., prevention of allograft rejection), pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, Graves' disease, and other autoimmune diseases, etc.

[0382] In the embodiments, the composition of the present application is applicable to ADHD, AIDS-neurological complications, agenesis of the septum pellucidum, acquired epileptic aphasia, acute disseminated encephalomyelitis, adrenoleukodystrophy, agenesis of the corpus callosum, agnosia, Aicardi syndrome, Alexander disease, Alpers disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis, anencephaly, aneurysm, Angelman syndrome, angiomatosis, anoxia, aphasia, apraxia, arachnoid cyst, arachnoiditis, Arnold-Chiari malformation, arteriovenous malformation, aspartame, Asperger syndrome, ataxia telangiectasia, ataxia, attention deficit hyperactivity disorder, autism, autonomic dysfunction, back pain, Barth syndrome, Batten disease, Behcet's disease, Bell palsy, benign essential blepharospasm, benign focal muscular atrophy, benign intracranial hypertension, Bernhardt-Roth syndrome, Binswanger disease, blepharospasm, Bloch-Sulzberger syndrome, brachial plexus birth injury, brachial plexus injury, Rudberg-Eggleston syndrome, cerebral aneurysm, brain injury, tumors of the brain and spinal cord, Brown-Séquard syndrome, bulbospinal muscular atrophy, Canavan disease, carpal tunnel syndrome, causalgia, cavernoma, cavernous hemangioma, cavernous malformation, central cervical spinal cord syndrome, spinal cord central syndrome, central pain syndrome, head injury, cerebellar degeneration, cerebellar hypoplasia, cerebral aneurysm, cerebral arteriosclerosis, cerebral aneurysm, cerebral beriberi, cerebral gigantism, cerebral hypoxia, cerebral palsy, cerebro-ocular-facial-skeletal syndrome, Charcot-Marie-Tooth disease, Chiari malformation, chorea, acanthocytic chorea, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic orthostatic intolerance, chronic pain, cocaine syndrome type II, Coffin-Lowry syndrome, coma including persistent vegetative state, complex regional pain syndrome, congenital bilateral facial nerve palsy, congenital myasthenia, congenital myopathy, congenital cavernous vascular malformation, corticobasal degeneration, cranial arteritis, craniosynostosis, Creutzfeldt-Jakob disease, progressive posttraumatic syndrome, Cushing syndrome, cytomegalovirus inclusion disease (CIBD), cytomegalovirus infection, dancing eyes-dancing feet syndrome, Dandy-Walker syndrome, Dawson Disease, Dormusia (DeMorsier's syndrome, Dejerine-Klumpke paralysis, multi-infarct dementia, subcortical dementia, Lewy body dementia, dermatomyositis, developmental coordination disorder, Devic's disease, diabetic neuropathy, multiple sclerosis, Dravet syndrome, autonomic neuropathy, dysgraphia, dyslexia, dysphagia, dyspraxia, dystonia, early infantile epileptic encephalopathy, empty sella syndrome, encephalitis lethargica, encephalitis and meningitis, brain tumor, brain disorder, encephalotrigeminal angiomatosis, epilepsy, Erb's palsy, Erb-Duchenne paralysis and Dejerine-Klumpke paralysis, Fabry disease, Fahr syndrome, absence, familial autonomic neuropathy, familial angioedema, familial idiopathic basal ganglia calcification, familial spastic paralysis, febrile seizures (e.g., GEFS and GEFS plus), Fisher syndrome, hypotonic child syndrome, Friedreich's ataxia, Gaucher disease, Gerstmann syndrome, Gerstmann-Straussler-Scheinker disease, giant cell arteritis, giant cell inclusion disease, globoid cell leukodystrophy, glossopharyngeal neuralgia, Guillain-Barré syndrome, HTLV-1 associated myelopathy, Hallervorden-Spatz disease, head trauma, headache, persistent unilateral headache, hemifacial spasm, alternating hemiplegia, hereditary neuropathy, hereditary spastic paraplegia, hereditary polyneuropathic disorder, herpes zoster oticus, herpes zoster, Hirayama syndrome, holoprosencephaly, Huntington's disease, anencephaly, normal pressure hydrocephalus, hydrocephalus, hydromyelia, hyperadrenocorticism, hypersomnia, hypertonia, hypotonia, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, infantile hypotonia, infantile phytanic acid storage disease, infantile Refsum disease, infantile spasms, inflammatory myopathy, intestinal lipodystrophy, intracranial cyst, intracranial hypertension, Isaac's syndrome, Joubert syndrome, Kearns-Sayre syndrome, Kennedy disease, Kinsbourne syndrome, Kleine-Levin syndrome, Klippel-Feil syndrome, Klippel-Trenaunay-Weber syndrome (KTS), Klüver-Bucy syndrome, Korsakoff amnestic syndrome, Krabbe disease, Kugelberg-Welander disease, kuru, Lambert-Eaton myasthenic syndrome, Landau-Kleffner syndrome, lateral femoral cutaneous nerve entrapment, lateral medullary syndrome, learning disorder, Leigh disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, leukodystrophy, Levine-CritchleySyndrome), Dementia with Lewy Bodies, Alyssencephaly, Locked-in syndrome, Lou Gehrig's disease, Lupus - neurological sequelae, Lyme disease - neurological complications, Machado-Joseph disease, Megaencephaly, Melkersson-Rosenthal syndrome, Meningitis, Menkes disease, Dysensory femoral neuralgia, Metachromatic leukodystrophy, Microcephaly, Migraine, Miller Fisher syndrome, Mild stroke, Mitochondrial myopathy, Moebius syndrome, Unilateral crural muscular atrophy, Motor neuron disease, Moyamoya disease, Mucolipidosis, Mucopolysaccharidoses, Multi-infarct dementia, Multifocal motor neuropathy, Multiple sclerosis, Multiple system atrophy with orthostatic hypotension, Multiple system atrophy myasthenia, muscular dystrophy, congenital myasthenia, myasthenia gravis, demyelinating diffuse sclerosis, infantile myoclonic encephalopathy, myoclonus, myopathy-congenital, myopathy-thyrotoxicity, myopathy, myotonia congenita, myotonia, narcolepsy, neuroacanthocytosis, neurodegeneration with cerebral iron deposition, neurofibromatosis, neuroleptic malignant syndrome, neurological complications of AIDS, neurological manifestations of Pompe disease, neuromyelitis optica, neuromyotonia, neuronal ceroid lipofuscinosis, neuronal migration disorder, hereditary neuropathies, neurosarcoidosis, neurotoxicity, cavernous nevus, Niemann-Pick disease, O'Sullivan-McLeod syndrome Syndrome, Occipital Neuralgia, Occult Spinal Dysraphism Sequence, Ohtahara Syndrome, Olivopontocerebellar Atrophy, Opsoclonus-Myoclonus, Orthostatic Hypotension, Overuse Syndrome, Chronic Pain, Paraneoplastic Syndrome, Paresthesia, Parkinson's Disease, Congenital ParnyotoniaCongenita), paroxysmal choreoathetosis, paroxysmal hemicrania, Parry-Romberg, Pelizaeus-Merzbacher disease, Pena-Shokker II syndrome, perineural cysts, periodic paralysis, peripheral neuropathy, periventricular leukomalacia, persistent vegetative state, pervasive developmental disorder, phytanic acid storage disease, Pick's disease, piriformis syndrome, pituitary tumors, polymyositis, Pompe disease, syringomyelia, post-polio syndrome, postherpetic neuralgia, postinfectious encephalomyelitis, orthostatic hypotension, postural orthostatic tachycardia syndrome, postural tachycardia syndrome, primary lateral sclerosis, prion disease, progressive facial hemiatrophy, progressive gait ataxia, progressive multifocal leukoencephalopathy, progressive sclerosing leukodystrophy, progressive supranuclear palsy, pseudotumor cerebri, pyridoxine-dependent and pyridoxine-responsive seizure disorders, Ramsay Hunt syndrome type I, Ramsay Hunt syndrome type II, Rasmussen encephalitis and other autoimmune epilepsy, reflex sympathetic dystrophy syndrome, infantile Refsum disease, Refsum disease, repetitive movement disorder, repetitive stress disorder, restless legs syndrome, retrovirus-associated myelopathy, Rett syndrome, Reye syndrome, Riley-Day syndrome, SUNCT headache, sacral nerve root cysts, chorea (Saint Vitus Dance), salivary gland diseases, Sandhoff disease, Schilder disease, split-brain syndrome, seizure disorder, septo-optic dysplasia, severe myoclonic epilepsy in infancy (SMEI), shaken baby syndrome, herpes zoster, Shy-Drager syndrome, Sjogren syndrome, sleep apnea, sleeping sickness, Sotos syndrome, spasticity, diastematomyelia, spinal cord infarction, spinal cord injury, spinal cord tumors, spinal muscular atrophy, spinocerebellar atrophy, Steele-Richardson-Olszewski syndrome, stiff-person syndrome, striatonigral degeneration, stroke, Sturge-Weber syndrome, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, dysphagia, Sydenham chorea, absence seizures, tabes dorsalis, syringohydromyelia, syringomyelia, systemic lupus erythematosus, tabes dorsalis, tardive dyskinesia, tarlo cysts, Tay-Sachs disease, temporal arteritis, tethered cord syndrome, Tommasen disease, thoracic outlet syndrome, thyrotoxic myopathy, painful tic, Todd paralysis, Tourette syndrome, transient ischemic attack, transmissible spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremor, trigeminal neuralgia, tropical spastic paraparesis, tuberous sclerosis, vascular erectileIt is used for treating, managing or preventing one or more neurological diseases including Tumor, vasculitis including temporal arteritis, von Economo disease, von Hippel-Lindau disease (VHL), von Recklinghausen disease, Wallenberg syndrome, Werdnig-Hoffmann disease, Wernicke-Korsakoff syndrome, West syndrome, Whipple disease, Williams syndrome, Wilson disease, X-linked spinal muscular atrophy, and Zellweger syndrome.

[0383] In embodiments, the composition of the present application is used for treating and reducing pain, such as postoperative pain or chronic pain.

[0384] In embodiments, the composition of the present application is used for treating Huntington's disease.

[0385] In embodiments, the composition of the present application is used for treating sickle cell anemia or thalassemia.

[0386] In embodiments, the composition of the present application changes the RNA splicing of exons related to a disease or disorder. In embodiments, the disease or disorder is selected from Alport syndrome, Alzheimer's disease, Bethlem myopathy and Ullrich scleroatonic muscular dystrophy, Duchenne muscular dystrophy, dystrophic epidermolysis bullosa, Friedreich's ataxia, Huntington's disease, junctional epidermolysis bullosa, Leber congenital amaurosis (LCA), and various myopathies and dystrophies.

[0387] In an embodiment, the composition of the present application is used for treating one or more respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis, allergic rhinitis, sinusitis, pulmonary vasoconstriction, inflammation, allergy, impeded respiration, respiratory distress syndrome, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, hantavirus pulmonary syndrome (HPS), Reye's syndrome, Goodpasture's syndrome, pleurisy, pneumonia, pulmonary edema, pulmonary fibrosis, sarcoidosis, complications associated with respiratory syncytial virus infection, and other respiratory diseases, etc.

[0388] In an embodiment, the composition of the present application is used for treating, managing or preventing cardiovascular diseases such as diseases or conditions affecting the heart and vasculature including, but not limited to, coronary heart disease (CHD), cerebrovascular disease (CVD), aortic valve stenosis, peripheral vascular disease, atherosclerosis, arteriosclerosis, myocardial infarction (heart attack), cerebrovascular disease (stroke), transient ischemic attack (TIA), angina (stable and unstable), atrial fibrillation, arrhythmia, valvular disease, and / or congestive heart failure.

[0389] In embodiments, the compositions of the present application are used for treating, managing, or preventing one or more metabolic-related disorders. In embodiments, the present invention is useful for the treatment, management, or prevention of diabetes, including type 1 diabetes, type 2 diabetes, and diabetes associated with obesity. The compositions and methods of the present invention are useful for the treatment or prevention of diabetic nephropathy, hyperglycemia, glucose intolerance, insulin resistance, obesity, dyslipidemia, lipid abnormalities, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, low HDL, high LDL, atherosclerosis and its sequelae, vascular restenosis, irritable bowel syndrome, inflammatory bowel diseases including Crohn's disease and ulcerative colitis, other inflammatory conditions, pancreatitis, abdominal obesity, neurodegenerative diseases, retinopathy, neoplastic diseases, adipocytic tumors, adipocytic cancers such as liposarcoma, prostate cancer, and other cancers (including gastric cancer, breast cancer, bladder cancer, and colon cancer), angiogenesis, Alzheimer's disease, psoriasis, hypertension, metabolic syndrome (e.g., a person having three or more of the following disorders: abdominal obesity, hypertriglyceridemia, low HDL cholesterol, hypertension, and high fasting plasma glucose concentration), ovarian hyperandrogenism (polycystic ovary syndrome), and other disorders in which insulin resistance is a factor, such as sleep apnea, but are not limited thereto. The compositions and methods of the present invention are useful for the treatment, management, or prevention of obesity and obesity-related disorders, including genetic or environmentally induced obesity. Obesity-related disorders here are related to, caused by, or resulting from obesity. Examples of obesity-related disorders include obesity, diabetes, overeating, binge eating, and bulimia, hypertension, elevated plasma insulin concentration and insulin resistance, lipid abnormalities, hyperlipidemia, endometrial cancer, breast cancer, prostate cancer, kidney cancer, and colon cancer, osteoarthritis, obstructive sleep apnea, gallstones, heart disease, cardiac arrhythmias and dysrhythmias, myocardial infarction, congestive heart failure, coronary heart disease, sudden death, stroke, polycystic ovary disease, craniopharyngioma, Prader-Willi syndrome, Fröhlich syndrome, GH deficiency subjects, normal variant short stature, Turner syndrome, and other pathological conditions showing a decrease in metabolic activity or resting energy expenditure expressed as a percentage of total fat-free body mass, such as children with acute lymphoblastic leukemia.Further examples of obesity-related disorders include metabolic syndrome, insulin resistance syndrome, reproductive hormone abnormalities, sexual and reproductive dysfunction (such as reproductive disorders, infertility, male hypogonadism, and hirsutism in women), fetal defects related to maternal obesity, gastrointestinal motility disorders such as obesity-related gastroesophageal reflux, respiratory disorders such as obesity-hypoventilation syndrome (Pickwickian syndrome), shortness of breath, cardiovascular diseases, inflammation, for example, systemic inflammation of the vascular system, atherosclerosis, hypercholesterolemia, lower back pain, gallbladder diseases, hyperuricemia, gout, and kidney cancer, as well as an increased risk of anesthesia. The compositions and methods of the present invention are also useful for treating Alzheimer's disease.

[0390] Nucleic acids, including liposomal formulations containing nucleic acids, can accumulate in the liver and / or spleen when delivered in vivo. It has now been discovered that nucleic acids encoding proteins can regulate protein expression in the liver and spleen, and that nucleic acids so used can be potential therapeutic agents for the treatment of liver and spleen diseases. Accordingly, certain embodiments are directed to methods for treating liver and / or spleen diseases by delivering to a patient a nucleic acid encoding a protein of interest. Other embodiments are directed to therapeutic compositions containing a nucleic acid encoding a protein of interest for the treatment of liver and / or spleen diseases. Liver and / or spleen diseases and conditions that can be treated include, but are not limited to, hepatitis, alcohol-induced liver disease, drug-induced liver disease, Epstein-Barr virus infection, adenovirus infection, cytomegalovirus infection, toxoplasmosis, Rocky Mountain spotted fever, non-alcoholic fatty liver disease, hemochromatosis, Wilson's disease, Gilbert's disease, and cancers of the liver and / or spleen.

[0391] In some embodiments, the compositions and methods of the present application relate to the treatment of type 1 diabetes, heart diseases including ischemic and dilated cardiomyopathy, macular degeneration, Parkinson's disease, cystic fibrosis, sickle cell anemia, thalassemia, Fanconi anemia, severe combined immunodeficiency, hereditary sensory neuropathy, xeroderma pigmentosum, Huntington's disease, muscular dystrophy, amyotrophic lateral sclerosis, Alzheimer's disease, cancer, and infectious diseases such as hepatitis and HIV / AIDS.

[0392] In embodiments, the methods and compositions of the present application are used to treat or prevent one or more metabolic diseases or disorders. In embodiments, the methods and compositions of the present application are used to treat or prevent one or more of diseases or disorders of carbohydrate metabolism, diseases or disorders of amino acid metabolism, diseases or disorders of the urea cycle, diseases or disorders of fatty acid metabolism, diseases or disorders of porphyrin metabolism, lysosomal storage diseases, peroxisomal biosynthetic disorders, and diseases or disorders of purine or pyrimidine metabolism.

[0393] In embodiments, the methods and compositions of the present application are used to treat or prevent one or more eye diseases or disorders, including but not limited to diabetic retinopathy, dry eye, cataract, retinal vein occlusion, macular edema, macular degeneration (exudative and atrophic), refractive and accommodative disorders, keratoconus, amblyopia, glaucoma, Stargardt disease, endophthalmitis, conjunctivitis, uveitis, retinal detachment, corneal ulcer, dacryocystitis, Duane retraction syndrome, and optic neuritis.

[0394] In embodiments, the eye disease or disorder is central serous chorioretinopathy (CSR), adult vitelliform disease, uveitis, primary and secondary systemic diseases (e.g., as non-limiting examples, sarcoid, rheumatoid-like diseases, arthritis, etc.), white dot syndromes (including MEWDS (multiple evanescent white dot syndrome)), serpiginous choroidopathy, acute posterior multifocal placoid epitheliopathy (AMPPE), presumed ocular histoplasmosis (POHS), or serpiginous retinochoroidopathy. In some embodiments, the disease can be a macular disease or cone dystrophy, such as, as non-limiting examples, Stargardt disease, etc. In some embodiments, the disease can be a hereditary degenerative disease, such as retinitis pigmentosa (RP). In some embodiments, the eye disease can be an eye melanoma, an eye tumor, or an infiltrative tumor.

[0395] In embodiments, the eye disease or disorder is Fuchs corneal dystrophy. In embodiments, the eye disease or disorder is Leber congenital cataract.

[0396] In embodiments, the methods and compositions of the present application target any of the proteins in Table 2A, Table 2B, and / or Table 2C or are used to treat any disease or disorder.

[0397] In various embodiments, the methods and compositions of the present application include using nucleic acid drugs, including synthetic RNA, in the diagnosis, treatment, prevention, or amelioration of the diseases, disorders, and / or conditions described herein. In various embodiments, the methods and compositions of the present application include using nucleic acid drugs, including synthetic RNA, in the modification, alteration, and / or change of tissues (e.g., cosmetically).

[0398] Generally speaking, in various embodiments, the synthetic RNA described herein is administered to humans at the specific dosages described herein and the synthetic RNA contains a sequence called a target sequence that encodes a protein of interest that can be a therapeutic protein.

[0399] Synthetic RNAs containing only standard nucleotides can bind to pattern recognition receptors, can be recognized as pathogen-associated molecular patterns, and can induce a strong immune response in cells, thereby resulting in translation arrest, secretion of inflammatory cytokines, and cell death. Synthetic RNAs containing specific non-standard nucleotides can evade detection by the innate immune system and can be efficiently translated into proteins in humans and the like. For example, at least one of the non-standard nucleotides described herein, such as members of the group consisting of 5-methylcytidine, 5-hydroxylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-methoxyuridine, 5-formyluridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-methoxypseudouridine, and 5-formylpseudouridine, can evade detection by the innate immune system and can be efficiently translated into proteins in humans and the like. Accordingly, certain embodiments are directed to methods for inducing cells to express a protein of interest, including contacting the cells with a synthetic RNA. Other embodiments are directed to methods for transfecting cells with a synthetic RNA, including contacting the cells with a solution containing one or more synthetic RNA molecules. Still other embodiments are directed to methods for treating a patient, including administering a synthetic RNA to the patient.In one embodiment, the synthetic RNA comprises at least one of the non-standard nucleotides described herein, such as members of the group of 5-methylcytidine, 5-hydroxylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-methoxyuridine, 5-formyluridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-methoxypseudouridine, and 5-formylpseudouridine. In another embodiment, the synthetic RNA encodes a protein of interest. Exemplary RNAs can contain non-standard nucleotides and combinations of non-standard nucleotides at levels as described elsewhere herein. In an embodiment, the method results in the expression of a protein of interest. In an embodiment, the method results in the expression of a protein of interest in a patient's skin.

[0400] Other embodiments are directed to methods for delivering nucleic acids to cells in vivo. Still other embodiments are directed to methods for inducing cells in vivo to express a protein of interest. Still other embodiments are directed to methods for treating a patient. In one embodiment, the method includes disrupting the stratum corneum. In another embodiment, the method includes contacting cells with a nucleic acid. In yet another embodiment, the method results in cells that take up the nucleic acid. In a further embodiment, the method results in cells that express a protein of interest. In yet another embodiment, the method results in the expression of a protein of interest in a patient. In yet another embodiment, the method results in the improvement of one or more of the patient's symptoms. In yet another embodiment, the patient is in need of a protein of interest. In yet another embodiment, the patient is deficient in a protein of interest.

[0401] Still other embodiments are directed to methods for treating a patient, including delivering a composition to the patient. In another embodiment, the composition comprises one or more nucleic acid molecules. In still another embodiment, at least one of the one or more nucleic acid molecules encodes a protein of interest. In an embodiment, the nucleic acid is synthetic RNA. In other embodiments, the method results in improvement of one or more of the patient's symptoms. Other embodiments are directed to methods for treating an indication by delivering to a cell or patient a nucleic acid encoding a protein or peptide. Still other embodiments are directed to compositions comprising a nucleic acid encoding a protein or peptide. Indications that can be treated using the methods and compositions of the invention, as well as proteins and peptides that can be encoded by the compositions of the invention, are listed in Table 2A, Table 2B, and / or Table 2C, but they are presented by way of example and are not limiting. In one embodiment, the indication is selected from Table 2A, Table 2B, and / or Table 2C. In another embodiment, the protein or peptide is selected from Table 2A, Table 2B, and / or Table 2C. In still another embodiment, the indication and the protein or peptide are selected from the same row of Table 2A, Table 2B, and / or Table 2C. In another embodiment, the protein is a gene editing protein. In still another embodiment, the gene editing protein targets a gene that is at least partially involved in the phenotype of the disease. In still another embodiment, the gene editing protein targets a gene encoding a protein selected from Table 2A, Table 2B, and / or Table 2C. In still another embodiment, the gene editing protein, alone or in combination with one or more other molecules or gene editing proteins, corrects or removes a mutation that is at least partially involved in the phenotype of the disease.

[0402] In various embodiments, the present invention contemplates any precursor form and / or mature form and / or isoform and / or variant of the proteins disclosed in Table 2A, Table 2B, and / or Table 2C and targeting such proteins. In embodiments, any of the precursor form and / or mature form and / or isoform and / or variant has enhanced secretion compared to the corresponding wild-type protein. In embodiments, any of the precursor form and / or mature form and / or isoform and / or variant has an altered half-life (e.g., in serum, plasma, intracellular), e.g., a longer or shorter half-life. In embodiments, this is compared to the wild type.

[0403]

Table 2-1

[0404]

Table 2-2

[0405]

Table 2-3

[0406]

Table 2-4

[0407] Table 2B are all exemplary identifiers (e.g., gene sequence numbers and references are incorporated herein by reference in their entirety).

[0408]

Table 3-1

[0409]

Table 3-2

[0410]

Table 3-3

[0411]

Table 3-4

[0412]

Table 3-5

[0413]

Table 3-6

[0414]

Table 3-7

[0415]

Table 3-8

[0416]

Table 3-9

[0417]

Table 3-10

[0418]

Table 3-11

[0419]

Table 3-12

[0420]

Table 3-13

[0421]

Table 3-14

[0422]

Table 3-15

[0423]

Table 3-16

[0424]

Table 3-17

[0425]

Table 3-18

[0426]

Table 3-19

[0427]

Table 3-20

[0428]

Table 3-21

[0429]

Table 3-22

[0430]

Table 3-23

[0431]

Table 3-24

[0432]

Table 3-25

[0433]

Table 3-26

[0434]

Table 3-27

[0435]

Table 3-28

[0436] In various embodiments, the methods and compositions of the present application are used to treat or prevent one or more of the diseases or disorders in the following table. In various embodiments, the methods and compositions of the present application are used to treat or prevent one or more of the diseases or disorders in the following table, for example, by regulating genes associated with the diseases in the following table. In an embodiment, the methods and compositions of the present application are used to gene-edit the genes described in Table 2C below using the compositions of the present application.

[0437]

Table 4-1

[0438]

Table 4-2

[0439]

Table 4-3

[0440]

Table 4-4

[0441]

Table 4-5

[0442]

Table 4-6

[0443]

Table 4-7

[0444] The Entrez entries described in the above table are hereby incorporated by reference in their entirety into this specification.

[0445] Additional exemplary targets of the present invention include the cosmetic targets described in Table 6 of International Patent Publication No. WO2013 / 151671, the contents of which are hereby incorporated by reference in their entirety into this specification.

[0446] Furthermore, in some embodiments, the methods and compositions of the present application target any of the proteins in Table 2A, Table 2B, and / or Table 2C, or are used to treat any disease or disorder. In various embodiments, the present invention contemplates targeting the full-length and / or truncated forms of any of the proteins disclosed in Table 2B. In various embodiments, the present invention contemplates targeting the precursor form and / or mature form and / or isoform of any of the proteins disclosed in Table 2A, Table 2B, and / or Table 2C.

[0447] In various embodiments, the invention contemplates targeting proteins that have a sequence identity of about 60% (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) with any of the protein sequences disclosed herein (e.g., within Table 2A, Table 2B, and / or Table 2C).

[0448] In various embodiments, the invention contemplates targeting proteins that include an amino acid sequence having one or more amino acid mutations relative to any of the protein sequences described herein (e.g., within Table 2A, Table 2B, and / or Table 2C). For example, the invention contemplates targeting proteins that include an amino acid sequence having one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or eleven, or twelve amino acid mutations relative to any of the protein sequences described herein (e.g., within Table 2A, Table 2B, and / or Table 2C). In embodiments, the one or more amino acid mutations can be independently selected from substitutions, insertions, deletions, and cleavages.

[0449] In embodiments, the amino acid mutation is an amino acid substitution and can include conservative and / or non-conservative substitutions.

[0450] "Conservative substitution" can be carried out, for example, based on the similarity of the amino acid residues involved in terms of polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity. The 20 naturally occurring amino acids can be classified into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0451] As used herein, "conservative substitution" is defined as exchanging one amino acid with another amino acid described within the same group of the above six standard amino acid groups. For example, in the exchange of Asp with Glu, one negative charge is retained in the polypeptide so modified. Further, glycine and proline can be substituted for each other based on their ability to disrupt the α-helix.

[0452] As used herein, "non-conservative substitution" is defined as exchanging one amino acid with another amino acid described in a different group of the above six standard amino acid groups (1)-(6).

[0453] In various embodiments, substitutions can also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, β-methylamino acids, Cα-methylamino acids, Nα-methylamino acids, and designer amino acids such as common amino acid analogs).

[0454] Manufacturing method Methods for the chemical synthesis of the compounds of the present application (e.g., those of formulas I to XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers are described elsewhere in this specification.

[0455] In the methods and compositions of the present invention, specific cationic lipids are used, and their synthesis, preparation and characterization are described elsewhere in this specification and in the accompanying examples. Furthermore, the present invention provides methods for preparing lipid aggregates and / or lipid carriers, including those associated with therapeutic agents, e.g., nucleic acids.

[0456] In embodiments, the lipid aggregates and / or lipid carriers of the present application, such as liposomes, are produced using microfluidics. In embodiments, the lipid aggregates and / or lipid carriers of the present application are manufactured using a Nanoassemblr device (Precision Nanosystems). In embodiments, a syringe pump is used to mix an organic solution and an aqueous solution at a specific flow rate. Optionally, the ratio of the flow rate of the aqueous solution to the flow rate of the organic solution can be selected from about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 8:1, or about 10:1. In embodiments, the organic solution contains one or more of ethanol, acetone, acetonitrile, dimethyl sulfoxide, toluene, and chloroform, or a mixture thereof. In other embodiments, other solvents are used.

[0457] In embodiments, the lipid aggregates and / or lipid carriers of the present application are produced by dropwise mixing one solution into another solution. In embodiments, the lipid aggregates and / or lipid carriers of the present application are produced using a spraying mechanism, or by solvent evaporation, or by sonication, or by extrusion through one or more membranes, or by a self-assembly process, or by a combination of methods.

[0458] In the methods described herein, a mixture of lipids is combined with a buffered aqueous solution of nucleic acid to produce an intermediate mixture containing nucleic acid encapsulated within lipid particles, where the encapsulated nucleic acid is present at a nucleic acid / lipid ratio of about 3 wt% to about 25 wt%, for example, 5 - 15 wt%. The intermediate mixture can optionally be sized to obtain nucleic acid-in-lipid particles where the lipid moiety is a unilamellar vesicle having a diameter of, for example, 30 - 150 nm, for example, about 40 - 90 nm. Subsequently, by raising the pH to neutralize at least a portion of the surface charge on the lipid-nucleic acid particles, a composition of nucleic acid-in-lipid with at least partially neutralized surface is provided.

[0459] Some cationic lipids are amino lipids that are charged at a pH below the pKa of the amino group and substantially neutral at a pH above the pKa. These cationic lipids are referred to as titratable cationic lipids and can be used in the compounds of the present application (e.g., those of formulas I - XVI), and / or pharmaceutical compositions, and / or lipid aggregates, and / or lipid carriers using a two-step process. First, titratable cationic lipids and other vesicle components can be used in the presence of nucleic acid to form lipid vesicles at a lower pH. By doing so, the nucleic acid is encapsulated and trapped in the vesicles. Second, the surface charge of the newly formed vesicles can be neutralized by increasing the pH of the medium to a level above the pKa of the titratable cationic lipid present, i.e., to a level above physiological pH. Particularly advantageous aspects of this process include both the ease of removing any adsorbed nucleic acid from the surface and the resulting nucleic acid delivery vehicle having a neutral surface. The compounds of the present application (e.g., those of formulas I - XVI), and / or pharmaceutical compositions, and / or lipid aggregates, and / or lipid carriers, and / or liposomes, and / or lipid particles having a neutral surface are expected to avoid rapid clearance from the circulation and to avoid certain toxicities associated with cationic liposome preparations. Additional details regarding the use of such titratable cationic lipids in this manner in the formulation of nucleic acid-lipid particles are described in U.S. Patent No. 6,287,591 and U.S. Patent No. 6,858,225, which are incorporated herein by reference.

[0460] Vesicles formed in such a manner provide a formulation with a high nucleic acid content and a uniform vesicle size. Further, the vesicles are in the size range of about 30 to about 150 nm, e.g., about 30 to about 90 nm.

[0461] Without being bound by any particular theory, highly efficient nucleic acid encapsulation is thought to be the result of electrostatic interactions at low pH. At acidic pH (e.g., pH 4.0), the vesicle surface is charged and binds to a portion of the nucleic acid via electrostatic interactions. Exchanging the external acidic buffer with a more neutral buffer (e.g., pH 7.5) neutralizes the surface of the lipid particles or liposomes and allows any external nucleic acid to be removed. Further detailed information regarding the formulation process is described in various publications (e.g., U.S. Patent No. 6,287,591 and U.S. Patent No. 6,858,225).

[0462] In embodiments, the present invention provides a method for preparing a lipid / nucleic acid formulation. In the method described herein, a mixture of lipids is combined with an aqueous buffered solution of nucleic acid to produce an intermediate mixture containing nucleic acid encapsulated within lipid particles, where, for example, the encapsulated nucleic acid is present at a nucleic acid / lipid ratio of from about 10 wt% to about 50 wt%. The intermediate mixture can optionally be sized to obtain nucleic acid-in-lipid particles in which the lipid moiety is a unilamellar vesicle having a diameter of, for example, 30 - 150 nm, or about 40 - 90 nm. Subsequently, by raising the pH to neutralize at least a portion of the surface charge on the lipid-nucleic acid particles, a composition of nucleic acid-in-lipid with at least partially neutralized surface is provided.

[0463] In embodiments, the mixture of lipids includes at least two lipid components, namely, a first lipid component of the present invention selected from lipids having a pKa such that the lipid is cationic at a pH below the pKa and neutral at a pH above the pKa, and a second lipid component selected from lipids that prevent particle aggregation during lipid-nucleic acid particle formation. In embodiments, the amino lipid is the novel cationic lipid of the present invention.

[0464] In the preparation of the nucleic acid-lipid particles of the present invention, the lipid mixture is typically a lipid solution in an organic solvent. Subsequently, this lipid mixture can be dried to form a thin film or lyophilized to form a powder, and then hydrated with an aqueous buffer to form liposomes. Alternatively, in one method, the lipid mixture can be solubilized in a water-miscible alcohol such as ethanol, and this ethanol solution can be added to an aqueous buffer to cause spontaneous liposome formation. In embodiments, the alcohol is used in the form in which it is commercially available. For example, ethanol can be used as absolute ethanol (100%) or as 95% ethanol with the balance being water. This method is detailed in U.S. Patent No. 5,976,567, which is hereby incorporated by reference in its entirety.

[0465] In an embodiment, the lipid mixture is a mixture of a cationic lipid, a neutral lipid (other than the cationic lipid), a sterol (e.g., cholesterol), and a PEG-modified lipid (e.g., PEG-DMG or PEG-DMA) in an alcohol solvent. In an embodiment, the lipid mixture consists essentially of a cationic lipid, a neutral lipid, cholesterol, and a PEG-modified lipid in an alcohol, e.g., ethanol. In an embodiment, the first solution consists of the above lipid mixture, and the molar ratio is about 20-70% cationic lipid: 5-45% neutral lipid: 20-55% cholesterol: 1: 0.5-15% PEG-modified lipid. In an embodiment, the first solution consists essentially of a lipid selected from Table 1, DSPC, Chol, and PEG-DMG or PEG-DMA, e.g., the molar ratio is about 20-60% cationic lipid: 5-25% DSPC: 25-55% Chol: 0.5-15% PEG-DMG or PEG-DMA. In an embodiment, the lipid molar ratio is about 40 / 10 / 40 / 10 (mol% of cationic lipid / DSPC / Chol / PEG-DMG or PEG-DMA), 35 / 15 / 40 / 10 (mol% of cationic lipid / DSPC / Chol / PEG-DMG or PEG-DMA), or 52 / 13 / 30 / 5 (mol% of cationic lipid / DSPC / Chol / PEG-DMG or PEG-DMA). In an embodiment, the neutral lipid in these compositions is replaced with POPC, DPPC, DOPE, or SM.

[0466] In an embodiment, a lipid mixture is combined with a buffered aqueous solution that may contain nucleic acid. The buffered aqueous solution is typically a solution in which the buffer has a pH lower than the pKa of the protonatable lipid in the lipid mixture. Examples of suitable buffers include citrate, phosphate, acetate, and MES. The buffer is an anion in the range of 1 to 1000 mM depending on the chemistry of the nucleic acid to be encapsulated, and optimization of the buffer concentration may be important to achieve a high loading level (see, for example, U.S. Patent No. 6,287,591 and U.S. Patent No. 6,858,225). Alternatively, pure water acidified to pH 5-6 with chloride, sulfate, etc. may be useful. In this case, it may be suitable to add 5% glucose, or dialyze the particles to remove ethanol and raise the pH, or add another non-ionic solute to balance the osmotic potential through the particle membrane when mixing with a pharmaceutically acceptable carrier such as normal saline. The amount of nucleic acid in the buffer can vary, but is typically from about 0.01 mg / mL to about 200 mg / mL, for example, from about 0.5 mg / mL to about 50 mg / mL.

[0467] A mixture of lipids is combined with a buffered aqueous solution of a therapeutic nucleic acid to obtain an intermediate mixture. The intermediate mixture is typically a mixture of lipid particles having encapsulated nucleic acid. Additionally, the intermediate mixture may also contain some nucleic acid bound to the surface of the lipid particles (liposomes or lipid vesicles) by ionic attraction between the negatively charged nucleic acid and the positively charged lipid on the surface of the lipid particles (amino lipids or other lipids that make up the protonatable first lipid component have a positive charge in a buffer having a pH lower than the pKa of the protonatable group on the lipid). In an embodiment, the lipid mixture is an alcoholic solution of lipids, and the volume of each solution is adjusted such that when combined, the resulting alcohol content is about 20% to about 45% by volume. The method of combining the mixtures can include any of a wide variety of processes, which often vary depending on the scale of the formulation being produced. For example, when the total volume is about 10 to 20 mL or less, the solutions can be combined in a test tube and stirred together using a vortex mixer. Large-scale processes can be carried out in glassware of a suitable production scale.

[0468] Optionally, the lipid-encapsulated therapeutic agent (e.g., nucleic acid) complex generated by combining a lipid mixture with a buffered aqueous solution of a therapeutic agent (e.g., nucleic acid) can be size-adjusted to achieve a desired size range and a relatively narrow distribution of lipid particle sizes. In embodiments, the compositions provided herein are size-adjusted to an average diameter of about 70 to about 200 nm, such as about 90 to about 130 nm. Several techniques are available for size-adjusting liposomes to a desired size. One size-adjusting method is described in U.S. Patent No. 4,737,323, which is hereby incorporated by reference in its entirety. By subjecting the liposome suspension to sonication by bath-type or probe-type sonication, the size is gradually reduced, and small unilamellar vesicles (SUVs) with a size of less than about 0.05 microns are generated. Homogenization is another method that relies on shear energy to fragment large liposomes into smaller liposomes. In a typical homogenization technique, multilamellar vesicles are passed through a standard emulsion homogenizer and recycled until a selected liposome size, typically about 0.1 to 0.5 microns, is observed. In either method, the particle size distribution can be monitored by conventional laser beam particle size measurement. In the case of the specific method herein, extrusion is used to obtain a uniform vesicle size.

[0469] Extrusion of the liposome composition through a small pore polycarbonate membrane or an asymmetric ceramic membrane results in a relatively well-defined particle size distribution. Typically, the suspension is circulated through the membrane one or more times until the desired particle size distribution of the liposome complex is achieved. The liposomes can be continuously extruded through smaller pore membranes to achieve a stepwise reduction in liposome size. In some cases, the formed lipid-nucleic acid composition can be used without size adjustment.

[0470] In an embodiment, the method of the present invention further includes a step of neutralizing at least a part of the surface charge of the lipid moiety of the lipid-nucleic acid composition. By at least partially neutralizing the surface charge, unencapsulated nucleic acids are removed from the lipid particle surface and can be removed from the composition using prior art techniques. In an embodiment, nucleic acids adsorbed on the surface without encapsulation are removed from the resulting composition by buffer exchange. For example, replacement of a citrate buffer (pH about 4.0, used for formation of the composition) with a HEPES buffered saline (HBS pH about 7.5) solution results in neutralization of the liposome surface and release of nucleic acids from the surface. The free nucleic acids are then removed by chromatography using standard methods and the buffer can then be switched to a buffer at a pH above the pKa of the lipid used.

[0471] Optionally, lipid vesicles (i.e., lipid particles) can be formed by hydration in an aqueous buffer, sized using any of the above methods, and then nucleic acids added. As described above, the aqueous buffer should be at a pH below the pKa of the amino lipid. Thereafter, a solution of the nucleic acid can be added to these sized preformed vesicles. In order to enable encapsulation of the nucleic acid into such preformed vesicles, the mixture should contain an alcohol such as ethanol. In the case of ethanol, it should be present at a concentration of about 20% (w / w) to about 45% (w / w). Further, depending on the composition of the lipid vesicles and the nature of the nucleic acid, it may be necessary to warm the mixture of the preformed vesicles and the nucleic acid-containing aqueous buffer-ethanol mixture to a temperature of about 25°C to about 50°C. It will be apparent to those skilled in the art that optimization of the encapsulation process to achieve the desired level of nucleic acid in the lipid vesicles requires manipulation of variable factors such as the concentration of ethanol and temperature. Once the nucleic acid is encapsulated within the vesicles performed, the external pH can be raised to at least partially neutralize the surface charge. Thereafter, nucleic acids adsorbed on the surface without encapsulation can be removed.

[0472] Vaccines and adjuvants In some embodiments, the compounds and compositions of the invention are associated with antigenic compositions of proteins or nucleic acids. For example, the lipids of the present application (e.g., any one of Formulas I-XVI) can be associated with a protein or nucleic acid vaccine antigen (e.g., any of the proteins of interest described herein). For example, the invention can relate to a method of vaccinating against a disease comprising any of the infectious diseases described herein by administering a lipid of the present application (e.g., any one of Formulas I-XVI) in combination with an antigenic composition of a protein or nucleic acid (e.g., any of the proteins of interest described herein).

[0473] In embodiments, the antigen of the protein or nucleic acid is a beta-coronavirus protein or an alpha-coronavirus protein, or an antigenic fragment thereof, or a nucleic acid encoding the same (e.g., RNA (e.g., mRNA) or DNA). In some embodiments, the beta-coronavirus protein is selected from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, and HCoV-OC43 proteins, or antigenic fragments thereof, or a nucleic acid encoding the same (e.g., RNA (e.g., mRNA) or DNA). In some embodiments, the alpha-coronavirus protein is selected from HCoV-NL63 and HCoV-229E proteins, or antigenic fragments thereof, or a nucleic acid encoding the same (e.g., RNA (e.g., mRNA) or DNA).

[0474] In some embodiments, the antigen of the protein or nucleic acid is a coronavirus protein, or an antigenic fragment thereof, or a nucleic acid encoding the same (e.g., RNA (e.g., mRNA) or DNA). In various embodiments, the coronavirus protein is a protein from SARS-CoV-2, such as the SARS-CoV-2 spike protein. In an embodiment, the SARS-CoV-2 protein is selected from the spike surface glycoprotein, membrane glycoprotein M, envelope protein E, and nucleocapsid phosphorylated protein N, or an antigenic fragment thereof, or a nucleic acid encoding the same (e.g., RNA (e.g., mRNA) or DNA). In an embodiment, the SARS-CoV-2 protein selected from the spike surface glycoprotein comprises S1, S2 and S2’.

[0475] In some embodiments, the coronavirus is a betacoronavirus or an alphacoronavirus. In some embodiments, the betacoronavirus is selected from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, and HCoV-OC43. In an embodiment, the alphacoronavirus is selected from HCoV-NL63 and HCoV-229E. In an embodiment, the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0476] In an embodiment, the antigen of the protein or nucleic acid is an influenza virus antigen, and optionally, is selected from hemagglutinin (HA) protein, matrix 2 (M2) protein, and neuraminidase, or an antigenic fragment thereof, or a nucleic acid encoding the same (e.g., RNA (e.g., mRNA) or DNA).

[0477] In embodiments, the disease or disorder for which vaccination is performed is selected from diphtheria, tetanus, pertussis, influenza, pneumonia, hepatitis A, hepatitis B, polio, yellow fever, human papillomavirus (HPV) infection, anthrax, rabies, Japanese encephalitis, meningitis, measles, mumps, rubella, gastroenteritis, smallpox, typhoid fever, varicella (chickenpox), rotavirus, and herpes zoster. In some embodiments, the present invention relates to the treatment of hepatitis. Exemplary hepatitis that can be treated includes, but is not limited to, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, autoimmune hepatitis, alcoholic hepatitis, acute hepatitis, and chronic hepatitis.

[0478] In some embodiments, the compound, composition, or vaccine of the present invention further comprises an adjuvant, optionally selected from aluminum gels or salts. In embodiments, the aluminum gel or salt is selected from aluminum hydroxide, aluminum phosphate, and aluminum sulfate. In some embodiments, the adjuvant is a nucleic acid encoding a chimeric protein or chimeric protein complex as described herein.

[0479] In some embodiments, the additional adjuvant is selected from water-in-oil emulsion formulations, saponin adjuvants, ovalbumin, Freund's adjuvant, cytokines, and chitosan. Exemplary additional adjuvants include, but are not limited to: (1) Ovalbumin (e.g., ENDOFIT), which is often used in biochemical research; (2) Water-in-oil emulsion formulations (with or without other specific immunostimulants such as muramyl peptides or bacterial cell wall components), for example, (a) MF59 (PCT Publication No. WO90 / 14837), which contains 5% squalane, 0.5% Tween80, and 0.5% Span85 (optionally containing various amounts of MTP-PE) and is formulated into submicron particles using a microfluidizer such as a Model HOy microfluidizer (Microfluidics, Newton, Mass.); (b) SAF, which contains 10% squalane, 0.4% Tween80, 5% pluronic block polymer L121, and thr-MDP and is microfluidized into a submicron emulsion or vortexed to produce an emulsion with a larger particle size; (c) RIBI adjuvant system (RAS) (RIBI IMMUNOCHEM, Hamilton, MO.), which contains 2% squalane, 0.2% Tween80, and one or more bacterial cell wall components from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), optionally including MPL + CWS (DETOX (trademark)); and (d) Water-in-oil emulsion formulations such as ADDAVAX (Invitrogen); (3) STIMULON (Cambridge Bioscience, Worcester, Mass.)Can adjuvants such as saponin be used, or particles such as ISCOM (immunostimulatory complex) generated therefrom; (4) complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (5) interleukins (non-limiting examples include IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., gamma interferon), macrophage colony-stimulating factor (M-CSF), cytokines such as tumor necrosis factor (TNF), etc.; (6) chitosan and other derivatives of chitin or poly-N-acetyl-D-glucosamine in which a high proportion of N-acetyl groups have been removed by hydrolysis (see, for example, European Patent Application No. EP0460020, which is hereby incorporated by reference in its entirety for disclosing pharmaceutical formulations containing chitosan as a mucosal absorption enhancer); and (7) other substances that act as immunostimulants to enhance the effectiveness of the composition, such as monophosphoryl lipid A. In other embodiments, the additional adjuvant is one or more of an aluminum salt or gel, a pattern recognition receptor (PRR) agonist, a CpG ODN, and an imidazoquinoline. In some embodiments, the additional adjuvant is cyclic [G(3’,5’)pA(3’,5’)] (e.g., 3’3’-cGAMP VACCIGRADE); cyclic [G(2’,5’)pA(3’,5’)]2’3’ (e.g., 2’3’ cGAMP VACCIGRADE); cyclic [G(2’,5’)pA(2’,5’)] (e.g., 2’2’-cGAMP VACCIGRADE), cyclic diadenylic acid monophosphate (e.g., c-di-AMP VACCIGRADE); cyclic diguanylic acid monophosphate (e.g., c-di-GMP VACCIGRADE); TLR7 agonist - imidazoquinoline compounds (e.g., TLR7 agonists such as gardiquimod VACCIGRADE, imiquimod VACCIGRADE, R848 VACCIGRADE, etc.); lipopolysaccharide (e.g., TLR4 agonist), e.g., E.From the E. coli 0111:B4 strain (e.g., LPS-EB VACCIGRADE); monophosphoryl lipid A (e.g., MPLA-SM VACCIGRADE and MPLA synthetic VACCIGRADE); N-glycolyl muramyl dipeptide (e.g., N-glycolyl-MDP VACCIGRADE); CpG ODN, class A and / or CpG ODN, class B and / or CpG ODN, class C (e.g., ODN1585 VACCIGRADE, ODN1826 VACCIGRADE, ODN2006 VACCIGRADE, ODN2395 VACCIGRADE), triacylated lipoprotein (e.g., Pam3CSK4 VACCIGRADE); polyinosinic acid-polycytidylic acid (e.g., poly(I:C)(HMW)VACCIGRADE); and cord factor (i.e., the mycobacterial cell wall component trehalose 6,6’ dimycolate (TDM,)) or an analog thereof (e.g., TDB VACCIGRADE, TDB-HS15 VACCIGRADE), and is one or more of these. In some embodiments, the additional adjuvant is a TLR agonist (e.g., TLR1, and / or TLR2, and / or TLR3, and / or TLR4, and / or TLR5, and / or TLR6, and / or TLR7, and / or TLR8, and / or TLR9, and / or TLR10, and / or TLR11, and / or TLR12, and / or TLR13), a nucleotide-binding oligomerization domain (NOD) agonist, an interferon gene stimulator (STING) ligand, or a related substance.

[0480] In some embodiments, the additional adjuvant is one or more of a mineral adjuvant, a gel-based adjuvant, a tensoactive agent, a bacterial product, an oil emulsion, a particulate adjuvant, a fusion protein, and a lipopeptide. Other inorganic salt adjuvants other than the aluminum adjuvant described elsewhere include salts of calcium (e.g., calcium phosphate), salts of iron, and salts of zirconium. Other gel-based adjuvants other than the aluminum gel-based adjuvant described elsewhere include acemannan. Tensoactive agents include Quil A, a saponin derived from an aqueous extract of Quillaja saponaria bark; a tensoactive glycoside containing a hydrophobic nucleus with a triterpenoid structure having a carbohydrate chain linked to the nucleus; and QS-21. Bacterial products include cell wall peptidoglycans or lipopolysaccharides of Gram-negative bacteria (e.g., those from Mycobacterium spp., Corynebacterium parvum, C. granulosum, Bordetella pertussis, and Neisseria meningitidis), N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), different compounds derived from MDP (e.g., threonyl-MDP), lipopolysaccharide (LPS) (e.g., from the cell wall of Gram-negative bacteria), trehalose dimycolate (TDM), and CpG motif-containing DNA. Oil emulsions include FIA, Montanide, Adjuvant 65, Lipovant, the montanide family of oil adjuvants, and various liposomes. Among particulate and polymeric systems, poly(DL-lactide-co-glycolide) microparticles have been widely studied and are utilized herein.

[0481] In some embodiments, additional adjuvants are described in Jennings et al. Adjuvants and Delivery Systems for Viral Vaccines - Mechanisms and Potential. In: Brown F, Haaheim LR, (eds). Modulation of the Immune Response to Vaccine Antigens. Dev. Biol. Stand, Vol. 92. Basel: Karger 1998; 19 - 28 and / or Sayers et al. J Biomed Biotechnol. 2012; 2012:831486, and / or Petrovsky and Aguilar, Immunology and Cell Biology (2004) 82, 488 - 496, the contents of which are hereby incorporated by reference in their entirety.

[0482] Administration / Dosage / Additives Administration of these compositions according to the invention may be by any common route, provided that the target tissue is accessible via that route. This includes oral, nasal, or buccal. Alternatively, administration may be by injection into the dermis, subcutis, muscle, peritoneal cavity, portal vein or vein, or by direct injection into the affected tissue, for example, cancer tissue. The agents disclosed herein may also be administered by catheter systems. Such compositions are usually administered as pharmaceutically acceptable compositions as described herein.

[0483] In embodiments, the compositions of the present application are administered parenterally, for example, intra - articular, intravenous, intra - peritoneal, subcutaneous, or intramuscular. In embodiments, the pharmaceutical composition is administered by bolus injection into the vein or peritoneal cavity.

[0484] In some embodiments, the lipid-nucleic acid composition can be administered to the lungs by aerosol inhalation (see Brigham, et al., Am. J. Sci. 298(4):278-281(1989)), and optionally, can be performed by using a vibrating mesh nebulizer (e.g., without limitation, AEROGEN SOLO (Galway, Ireland)). In some embodiments, the lipid-nucleic acid composition can be administered by direct injection at the affected site (Culver, Human Gene Therapy, MaryAnn Liebert, Inc., Publishers, New York. pp. 70-71(1994)).

[0485] Administration of the compositions described herein can be, for example, by injection, topical administration, ophthalmic administration, and intranasal administration. In some embodiments, the injection can be coupled to an electric force (e.g., including using a device used in electrochemotherapy, electroporation (e.g., CLINIPORATOR, IGEA Srl, Carpi [MO], Italy)). Topical administration can be, but is not limited to, creams, lotions, ointments, gels, sprays, solutions, etc. Topical administration further includes penetration enhancers, such as, without limitation, surfactants, fatty acids, bile salts, chelating agents, non-chelating non-surfactants, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, combinations of fatty acids and / or salts and bile acids and / or salts, combinations of sodium salts with lauric acid, capric acid, and UDCA, etc. Topical administration can also include fragrances, colorants, sunscreens, antibacterial agents, and / or moisturizing agents. The compositions described herein can be administered to at least one site, such as, without limitation, the forehead, scalp, hair follicles, hair, upper eyelid, lower eyelid, eyebrows, eyelashes, infraorbital region, periorbital region, temporal, nose, nasal bridge, cheek, tongue, nasolabial fold, lips, periorbital region, jaw line, ear, neck, breast, forearm, upper arm, palm, hand, fingers, nails, back, abdomen, sides, buttocks, thighs, calves, feet, toes, etc.

[0486] Routes of administration include, for example, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, intravaginal, transdermal, intraportal, intrarectal, inhalation, or topical, particularly administration to the ear, nose, eye, or skin. In embodiments, administration is effected by oral or parenteral injection.

[0487] In formulating, the solution can be administered in a manner compatible with the dosage form and in an amount therapeutically effective, as described herein. The formulation can be readily administered in various dosage forms such as injection solutions, drug-release capsules, etc. In the ...

Claims

1. A compound of formula (IV) 【Chemical 1】 [wherein, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15].

2. The compound according to claim 1, wherein n is 2 to 15.

3. The compound according to claim 1, wherein n is 2 to 12.

4. The compound according to claim 1, wherein the compound is 【Chemical 2】 .

5. The compound according to claim 1, wherein the compound is 【Chemical Formula 3】 .

6. The compound according to claim 1, wherein the compound is 【Chemical Formula 4】 .

7. The compound according to claim 1, wherein the compound is 【Chemical Formula 5】 .

8. The compound according to claim 1, wherein the compound is 【Chemical Formula 6】 .

9. The compound according to claim 1, wherein the compound is 【Chemical Formula 7】 .

10. The compound according to claim 1, wherein the compound is 【Chemical 8】 .

11. The compound according to claim 1, wherein the compound is 【Chemical Formula 9】 .

12. A pharmaceutical composition comprising the compound according to claim 1 and a pharmaceutically acceptable carrier or additive.

13. A lipid aggregate comprising the compound according to claim 1.

14. The lipid aggregate according to claim 13, wherein the lipid aggregate does not contain one or more additional lipids or polymers.

15. The lipid aggregate according to claim 13, further comprising a nucleic acid selected from DNA or RNA molecules.

16. An in vitro method for transfecting a cell with a nucleic acid, the method comprising contacting the cell with a complex of the nucleic acid and a compound of formula (IV) 【Chemical 10】 [wherein, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15].[

17. A pharmaceutical composition comprising a complex of a nucleic acid and a compound of formula (IV) for treating a disease by transfecting a cell, wherein the compound of formula (IV) is 【Chemical Formula 11】 [wherein, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15] having a structure, a pharmaceutical composition.

18. The compound according to claim 17, wherein the compound is 【Chemical Formula 12-1】 【Chemical Formula 12-2】 [Chemical 12-3] selected from.

19. The compound according to claim 16, wherein the compound is 【Chemical Formula 13-1】 【Chemical 13-2】 【Chemical Formula 13-3】 selected from.

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