Cationic lipids and uses thereof

Abstract

The present invention relates to novel cationic lipids of Formula I, and more particularly to Formula IV. These novel cationic lipids are useful for delivery of nucleic acids to cells, for example in the form of liposomes.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application date of July 3, 2020, application number 202080061516.0, and invention name “Cationic lipids and their uses”.

[0002] priority

[0003] This application claims priority to U.S. Provisional Application No. 62 / 870,245 filed on July 3, 2019, U.S. Provisional Application No. 62 / 880,435 filed on July 30, 2019, and U.S. Provisional Application No. 63 / 023,654 filed on May 12, 2020, the contents of which are incorporated herein by reference in their entirety. Field of the Invention

[0004] The present invention relates in part to various novel lipids, including methods, compositions, and products for delivering nucleic acids to cells.

[0005] Instructions for electronically submitting text documents

[0006] This application contains a sequence listing, which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy created on July 3, 2020 is named FAB-012PC_ST25.txt and is 1,441,597 bytes in size. Background Art

[0007] Lipid-based materials (such as liposomes) are used as bio-carriers for drugs and other biological applications, for example, to introduce agents into cultured cell lines. Lipids are commonly used, for example, in transfections, to deliver nucleic acids to cells in vitro under low serum or serum-free conditions. However, serum components can inhibit the activity of many lipids, which limits the use of the lipids in vitro and in vivo in the presence of serum.

[0008] Improved lipid delivery systems are needed, for example, to achieve higher levels of transfection in vitro and in vivo. Specifically, active lipid delivery systems are needed in the presence of serum. The increased transfection levels will allow treatment of disease states for which expression levels higher than those currently achievable with lipid delivery systems are required for therapeutic effect. Alternatively, higher transfection levels will allow comparable expression levels to be achieved using less material, thereby reducing potential toxicity and reducing costs.

[0009] There is a need in the art for new lipids, lipid-like materials, and lipid-based delivery systems. Summary of the invention

[0010] Thus, the present invention relates to novel lipids that are particularly useful for improving the delivery of biological payloads, such as nucleic acids, to cells.

[0011] In various aspects, the present invention relates to compounds of formula (I):

[0012]

[0013] Wherein: Q1, Q2, Q3 and Q4 are independently atoms or groups capable of carrying a positive charge;

[0014] A1 and A2 are independently empty, H or optionally substituted C1-C6 alkyl;

[0015] L1, L2 and L3 are independently empty, a bond, (C1-C 20 )alkanediyl, (halogen)(C1-C 20 )alkanediyl, (hydroxy) (C1-C 20 )alkanediyl, (alkoxy) (C1-C 20 ) alkanediyl, arylene, heteroarylene, cycloalkanediyl, heterocyclo-diyl, or any combination thereof optionally linked by one or more of ether, ester, anhydride, amide, carbamate, secondary amine, tertiary amine, quaternary ammonium, thioether, urea, carbonyl, or imine;

[0016] R1, R2, R3, R4, R5, R6, R7 and R8 are independently empty, H, (C1-C 60 )alkyl, (halogen) (C1-C 60 )alkyl, (hydroxy) (C1-C 60 )alkyl, (alkoxy) (C1-C 60 )alkyl, (C2-C 60 )alkenyl, (halogen)(C2-C 60 )alkenyl, (hydroxy) (C2-C 60 )alkenyl, (alkoxy) (C2-C 60 )alkenyl, (C2-C 60 )alkynyl, (halogen)(C2-C 60 )alkynyl, (hydroxy) (C2-C 60 )alkynyl, (alkoxy) (C2-C 60 ) alkynyl, wherein at least one of R1, R2, R3, R4, R5, R6, R7 and R8 contains at least two unsaturated bonds; and x, y and z are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0017] In various aspects, the present invention relates to compounds of formula (II):

[0018]

[0019] wherein: R9, R10, R11, R12, R13, R14, R15, R16, R17, R18, R19, R20, R21, R22, R23, R24, R25, R26, R27 and R28 are independently H, halo, OH, (C1-C6)alkyl, (halo)(C1-C6)alkyl, (hydroxy)(C1-C6)alkyl, (alkoxy)(C1-C6)alkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; and

[0020] i, j, k, m, s and t are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0021] In various aspects, the present invention relates to compounds of formula (III):

[0022]

[0023] Wherein L4, L5, L6 and L7 are independently a bond, (C1-C 20 )alkanediyl, (halogen)(C1-C 20 )alkanediyl, (hydroxy) (C1-C 20 )alkanediyl, (alkoxy) (C1-C 20 ) alkanediyl, arylene, heteroarylene, cycloalkanediyl, heterocyclo-diyl, -(CH2) v1 -C(O)-、-((CH2) v1 -O) v2 -or–((CH2) v1 -C(O)-O) v2 -;

[0024] R 29 , R 30 , R 31 , R 32 , R 33 , R 34 and R 35 are independently H, (C1-C 60 )alkyl, (halogen) (C1-C 60 )alkyl, (hydroxy) (C1-C 60 )alkyl, (alkoxy) (C1-C 60 )alkyl, (C2-C 60 )alkenyl, (halogen)(C2-C 60 )alkenyl, (hydroxy) (C2-C 60 )alkenyl, (alkoxy) (C2-C 60 )alkenyl, (C2-C 60 )alkynyl, (halogen)(C2-C60 )alkynyl, (hydroxy) (C2-C 60 )alkynyl, (alkoxy) (C2-C 60 ) alkynyl, wherein R 29 , R 30 , R 31 , R 32 , R 33 , R 34 and R 35 At least one of the comprises at least two unsaturated bonds;

[0025] v, v1 and v2 are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0026] In an embodiment, the invention relates to compounds of formula (IV):

[0027]

[0028] wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0029] In an embodiment, the invention relates to compounds of formula (V):

[0030]

[0031] In an embodiment, the invention relates to compounds of formula (VI):

[0032]

[0033] In an embodiment, the present invention relates to compounds of formula (VII):

[0034]

[0035] In an embodiment, the invention relates to compounds of formula (VIII):

[0036]

[0037] In an embodiment, the present invention relates to compounds of formula (IX):

[0038]

[0039] In an embodiment, the present invention relates to compounds of formula (X):

[0040]

[0041] In an embodiment, the present invention relates to compounds of formula (XI):

[0042]

[0043] In an embodiment, the present invention relates to compounds of formula (XII):

[0044]

[0045] In an embodiment, the present invention relates to compounds of formula (XIII):

[0046]

[0047] In an embodiment, the present invention relates to compounds of formula (XIV):

[0048]

[0049] In an embodiment, the invention relates to compounds of formula (XV):

[0050]

[0051] (XV) wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0052] In an embodiment, the present invention relates to compounds of formula (XVI):

[0053]

[0054] In an embodiment, a compound of the invention (e.g., a compound of Formula I-XVI) is a component of 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.

[0055] In an embodiment, the compound of the invention (e.g., a compound of Formula I-XVI) is a component of a pharmaceutical composition and / or lipid aggregate and / or lipid carrier and / or lipid nucleic acid complex and / or liposome and / or lipid nanoparticle that does not require additional lipids or helper lipids. In an embodiment, a compound of the invention (e.g., a compound of Formula I-XVI) is a component of a pharmaceutical composition and / or lipid aggregate and / or lipid carrier and / or lipid nucleic acid complex and / or liposome and / or lipid nanoparticle that also comprises a neutral lipid (e.g., dioleoylphosphatidylethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or cholesterol) and / or an additional cationic lipid (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)).

[0056] In an embodiment, the compound of the invention (e.g., a compound of Formula I-XVI) is a component of a pharmaceutical composition and / or lipid aggregate and / or lipid carrier and / or lipid nucleic acid complex and / or liposome and / or lipid nanoparticle having a specified particle size. In some embodiments, particles comprising a compound of the invention (e.g., a compound of Formula I-XVI) are characterized to determine the average particle size and polydispersity. In some embodiments, particles comprising a compound of the invention have an average hydrodynamic radius of <50 nm, 50-100 nm, 100-150 nm, 150-200 nm, 200-250 nm, 250-500 nm, or >500 nm. In some embodiments, the average particle size depends on the conditions of the particle formation process (e.g., but not limited to, temperature, incubation time, solution pH, or solution ionic strength). In some embodiments, the compound of the invention (e.g., a compound of Formula I-XVI) is a component of a pharmaceutical composition and / or lipid aggregate and / or lipid carrier and / or lipid nucleic acid complex and / or liposome and / or lipid nanoparticle whose average particle size depends on the ionic strength of the solution or the pH of the solution.

[0057] In an embodiment, a compound of the invention (e.g., a compound of Formula I-XVI) is a component of 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 comprises a nucleic acid such as DNA (e.g., but not limited to a plasmid, cosmid, phage, recombinant virus or other vector) or RNA (e.g., but not limited to siRNA, microRNA (miRNA), long non-coding RNA (lncRNA), in vitro transcribed RNA, synthetic RNA and / or mRNA, in each case comprising one or more atypical nucleotides that confer stability, avoid degradation by one or more nucleases, and / or avoid substantial cytotoxicity, or does not comprise an atypical nucleotide). In an embodiment, the RNA encodes a target protein, such as, but not limited to, one or more reprogramming factors or gene editing proteins. In an embodiment, the RNA encodes an antigen that provokes an immune response.

[0058] In embodiments, the present invention relates to the delivery of synthetic RNA molecules (e.g., but not limited to, synthetic RNA molecules comprising microRNA binding sites (such as, for example, miR142 microRNA binding sites)) capable of inducing immune tolerance to encoded proteins, such microRNA binding sites are optionally present in the 3'-UTR of the synthetic RNA molecules (optionally in one or more copies). In embodiments, the present invention relates to the delivery of synthetic RNA molecules capable of inducing immune tolerance to encoded proteins by co-delivering factors (e.g., but not limited to IL2, IL10 and / or tgf-β) that induce tolerance. In certain embodiments, the co-delivered factors are expressed by synthetic RNA molecules. In embodiments, synthetic RNA molecules encode one or more gene editing proteins described herein. In one embodiment, synthetic RNA molecules induce immune tolerance of one or more gene editing proteins. In another embodiment, synthetic RNA molecules encode proteins that are not normally present in a subject. In some embodiments, the subject suffers from dystrophic epidermolysis bullosa. In other embodiments, synthetic RNA molecules encode one or more gene editing proteins that produce double-strand breaks, in which a genetic payload is inserted. In some embodiments, the genetic payload comprises an expression cassette. In other embodiments, the expression cassette induces cells to express therapeutic proteins. In yet other embodiments, the therapeutic protein is immunotolerant. In other embodiments, one or more gene editing proteins induce cells to express a functional or semi-functional form of a protein. In one embodiment, the protein is collagen 7. Certain embodiments relate to a composition for treating a disease, disorder, or condition, wherein the composition comprises a synthetic RNA molecule of the present invention. In one embodiment, the disease, disorder, or condition is dystrophic epidermolysis bullosa.

[0059] In embodiments, pharmaceutical compositions and / or lipid aggregates and / or lipid carriers and / or lipid nucleic acid complexes and / or liposomes and / or lipid nanoparticles are suitable for administration by injection, such as, but not limited to, subcutaneous injection, intradermal injection, subdermal injection, intramuscular injection and / or intravenous, intrathecal, intratumoral, intravitreal, subretinal, intraventricular and / or local administration and / or infusion. In embodiments, pharmaceutical compositions and / or lipid aggregates and / or lipid carriers are suitable for administration as one or more injections containing less than about 10 ng of RNA or about 10 ng to about 2000 ng of RNA.

[0060] In various aspects, 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., a compound of Formula I-XVI), wherein the complex of the nucleic acid and the compound described herein (e.g., a compound of Formula I-XVI) is optionally formed prior to contacting the cell.

[0061] In many aspects, the present invention relates to a method for reprogramming differentiated cells to a less differentiated state, comprising (a) providing differentiated cells; (b) culturing differentiated cells; and (c) transfecting differentiated cells with a complex of one or more synthetic RNA molecules and a compound described herein (e.g., a compound of Formula I-XVI), wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors and wherein the transfection causes the cell to express one or more reprogramming factors, so that the cell is reprogrammed to a less differentiated state. In embodiments, step (c) is performed in the presence of a culture medium containing a component that supports the reprogramming of differentiated cells to a less differentiated state. In embodiments, step (c) is also included in repeating step (c) at least twice within 5 consecutive days. In embodiments, the amount of one or more synthetic RNA molecules transfected in one or more later transfections is greater than the amount transfected in one or more earlier transfections. In embodiments, steps (a)-(c) are performed without using feeder cells in the presence of feeder cell conditioned medium. In embodiments, step (c) is performed without using irradiated human neonatal fibroblast feeder cells in the presence of feeder cell conditioned medium. In an embodiment, the synthetic RNA molecule encodes one or more reprogramming factors selected from the group consisting of 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.

[0062] In many aspects, 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 compounds described herein (e.g., compounds 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 wherein the transfection causes the cells to express one or more reprogramming factors, so that the cells are reprogrammed. In embodiments, step (c) is carried out in the presence of a culture medium containing a component supporting reprogramming of non-pluripotent cells. In embodiments, it is also included to repeat step (c) at least twice within 5 consecutive days. In embodiments, the amount of one or more synthetic RNA molecules transfected in one or more later transfections is greater than the amount transfected in one or more earlier transfections. In embodiments, steps (a)-(c) are carried out without using feeder cells and in the presence of feeder cell conditioned medium. In embodiments, step (c) is carried out without using irradiated human neonatal fibroblast feeder cells and in the presence of feeder cell conditioned medium. In an embodiment, the synthetic RNA molecule encodes one or more reprogramming factors selected from the group consisting of 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.

[0063] In various aspects, the present invention relates to a method for gene editing cells, comprising transfecting cells with a complex of one or more synthetic RNA molecules and a compound described herein (e.g., a compound of Formulae I-XVI), wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more gene editing proteins selected from the following: nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases, large-range nucleases, nickases, gene editing proteins disclosed in WO2014 / 071219A1 (the entire contents of which are incorporated herein by reference), 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.

[0064] In various aspects, the present invention relates to methods for reprogramming and / or gene editing cells, comprising contacting the cells with a complex of a nucleic acid and a compound described herein (e.g., a compound of Formula I-XVI) and / or a compound disclosed in US2009 / 0143583A1, the entire contents of which are incorporated herein by reference.

[0065] In various aspects, the invention relates to pharmaceutical formulations comprising a nucleic acid of the invention and a compound described herein (e.g., a compound of Formula I-XVI) and / or a compound disclosed in US 2009 / 0143583 A1, the entire contents of which are incorporated herein by reference.

[0066] In embodiments, the novel lipids can be used for delivery of vaccines. In various aspects, the present invention relates to a method for vaccination against infectious diseases (but not limited to coronavirus infections, such as COVID-19), comprising contacting a cell with a complex of a nucleic acid encoding an infectious agent antigen and a compound described herein (e.g., a compound of Formula I-XVI).

[0067] In various aspects, the present invention relates to a method for extracting an organic compound from a reaction 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 isopropanol); to obtain an extracted organic compound.

[0068] In various aspects, the present invention relates to a method for extracting an organic compound from a reaction 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; to obtain an extracted organic compound.

[0069] In various aspects, the present invention relates to a method for purifying an organic compound from a mixture of the organic compound and phthalimide or a 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; to obtain a purified organic compound.

[0070] The details of the present invention are set forth in the following attached specific embodiments. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, illustrative methods and materials are now described. Other features, objects and advantages of the present invention will be apparent from the specific embodiments and claims. In the specification and the appended claims, unless the context clearly provides otherwise, the singular form also includes the plural form. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0071] Any aspect or embodiment disclosed herein may be combined with any other aspect or embodiment disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 Depicted are primary human epidermal keratinocytes cultured in 24-well plates and transfected with 400 ng of in vitro transcribed RNA encoding green fluorescent protein (GFP) per well, complexed with the indicated lipids and having the indicated lipid to RNA mass ratio. Complexation was performed in DMEM and transfection was performed in 100% fetal bovine serum (FBS). Images were taken eight hours after transfection.

[0073] Figure 2 Depicted Figure 1 For experiments, fluorescence was measured at the indicated time points after transfection with DHDLinS.

[0074] Figure 3 Describes Figure 1 Results from experiments performed in , but with the indicated RNA amounts (in nanograms) and the indicated lipid to RNA mass ratios (in micrograms lipid per microgram RNA). Images were taken 16 hours after transfection. As shown, all RNA amounts and lipid to RNA mass ratios tested produced fluorescent signals.

[0075] Figure 4 Describes as Figure 1 Results of experiments performed in , but using human peripheral blood mononuclear cells (hPBMC) instead of keratinocytes. Images were taken 16 hours after transfection. "LF3000" indicates cells transfected with LIPOFECTAMINE 3000 commercial transfection reagent. "Neg." indicates non-transfected cells compared to cells transfected with DHDLinS.

[0076] Figure 5A Describes as Figure 4 Results from experiments performed in but using confluent layers of primary human epidermal keratinocytes instead of hPBMCs. Images were taken 24 hours after transfection.

[0077] Figure 5B Depicted Figure 5A Experiments are shown at higher magnification.

[0078] Figure 6 Describes as Figure 4 Results from experiments performed in but using primary adult human dermal fibroblasts instead of hPBMCs. Images were taken 16 hours after transfection.

[0079] Figure 7 Depicted Figure 6 Experiments are shown at higher magnification.

[0080] Figure 8 Describes as Figure 3 The results of the experiments performed in , but using DHDLinS purified by extraction with acetone as described in Example 6.

[0081] Fig. 9 Depicted are the results of experiments in which primary human epidermal keratinocytes were transfected with in vitro transcribed RNA encoding a gene editing protein complexed with DHDLinS.

[0082] Fig.10 Depict the results of an experiment in which primary human epidermal keratinocytes were transfected with in vitro transcribed RNA encoding GFP and complexed with DLinDHS. The RNA-DLinDHS complex was added to 20,000 human epidermal keratinocytes in serum-free medium (0% fetal bovine serum (FBS)) or 50% FBS. Images were taken 16 hours after transfection. "MC3LNP" means cells transfected with GFP RNA prepared using a microfluidic device into lipid nanoparticles consisting of DLin-MC3-DMA, DOPE, cholesterol and DMPE-PEG in a molar ratio of 30:30:38.5:1.5.

[0083] Fig.11A Illustrate the results of an experiment in which primary human dermal fibroblasts were transfected with in vitro transcribed RNA encoding GFP and complexed with DLinDHS. Of the GFP encoding RNA, 20% was labeled with Cy5. The RNA-DLinDHS complex was added to 20,000 human dermal fibroblasts in 10% FBS. Images were taken at 50 minutes, 100 minutes, 150 minutes, 200 minutes, 250 minutes, and 300 minutes after transfection.

[0084] Fig. 11BThe results of an experiment are illustrated in which primary human dermal fibroblasts were transfected with in vitro transcribed RNA encoding GFP and complexed with DLinDHS. In the DLinDHS, 10% was labeled with the BODIPY fluorescent tag. The RNA-DLinDHS complex was added to 20,000 human dermal fibroblasts in 10% FBS. Images were taken in the BODIPY channel at the time of transfection ("0 min") and 20 minutes, 40 minutes, 60 minutes, 80 minutes, 100 minutes, 120 minutes, and 140 minutes after transfection.

[0085] Fig.12 Schematic diagram of the synthesis of DLinDHS is illustrated.

[0086] Fig.13 Illustrated are the results of experiments in which primary human epidermal keratinocytes were transfected with in vitro transcribed RNA encoding GFP and complexed with compounds of the invention comprising a dihydroxyspermine head group conjugated to different fatty acid derived tails including myristyl, oleyl, linoleyl and linoleyl. This figure illustrates the fluorescence (au) of the complexes studied and RNA complexed with LIPOFECTAMINE 2000 versus time after transfection (hours).

[0087] Fig.14 The results of an experiment are illustrated in which primary human epidermal keratinocytes were transfected with in vitro transcribed RNA encoding GFP and complexed with a compound of the invention comprising a dilinoleyl fatty acid derived tail structure. As shown, the structure of the test compound is bis-(2-hydroxy-3-aminopropyl)-N,N'-dilinoleyl-diamine, where the label represents the length of the carbon chain between the central amino groups of the head group (n on the structure). This figure illustrates the relationship between the fluorescence (au) of the studied complex and RNA complexed with LIPOFECTAMINE 2000 and the time (hours) after transfection.

[0088] Fig.15 Depicted are graphs illustrating the Z-average particle size (nm) and polydispersity index (PDI) of several DLinDHS / RNA complexes versus the ionic strength ([NaCl], mM) of the complexing medium. The complexes were formed by diluting lipids into the complexing medium, which is an aqueous RNA solution with a specified ionic strength.

[0089] Fig.16 Depicted are graphs illustrating the zeta potential (mV) of DLinDHS / RNA complexes diluted into 20 mM buffer titrated to the indicated pH.

[0090] Fig.17Images of DLinDHS / RNA complexes that were not treated with RNase A ("untreated"), DLinDHS / RNA complexes that were treated with RNase A after complexation ("RNase after complexation"), and DLinDHS complexed with RNA that was treated with RNase A before complexation ("RNase before complexation") are illustrated.

[0091] Fig.18 The results of transfection of in vitro transcribed GFP-encoding RNA complexed with DLinDHS in primary human dermal fibroblasts, activated human peripheral blood mononuclear cells (PBMCs), human lung adenocarcinoma cells, pluripotent stem cells (iPS cells), and rat embryonic cortical neurons are illustrated.

[0092] Fig.19 Describe the result of experiment, wherein the RNA of in vitro transcription is compounded with DLinDHS and is administered to rat by intradermal injection.The tissue at injection site is fixed and mounted on a slide, then stained with hematoxylin (blue) and rabbit anti-GFP antibody (brown).Two different magnifications of the same tissue are shown.

[0093] Fig. 20A Depicted are biopsied human skin imaged by fluorescence confocal microscopy 48 hours after administration of RNA encoding GFP complexed with DLinDHS by intradermal injection.

[0094] Fig. 20B Depicted are experimental results of in vivo delivery of mRNA to human subjects via intradermal injection. DETAILED DESCRIPTION

[0095] The present invention is based in part on the discovery of novel lipids that exhibit, among other things, an exceptional ability to support delivery of nucleic acids to cells, such as during transfection. The present invention provides such compositions, methods of preparing the compositions, and methods of using the compositions to introduce nucleic acids into cells, including for use in treating disease.

[0096] Compounds and Synthesis Methods

[0097] In various aspects, the present invention relates to compounds of formula (I):

[0098]

[0099] Wherein: Q1, Q2, Q3 and Q4 are independently atoms or groups capable of carrying a positive charge;

[0100] A1 and A2 are independently null, H or optionally substituted C1-C6 alkyl;

[0101] L1, L2 and L3 are independently empty, a bond, (C1-C 20 )alkanediyl, (halogen)(C1-C20 )alkanediyl, (hydroxy) (C1-C 20 )alkanediyl, (alkoxy) (C1-C 20 ) alkanediyl, arylene, heteroarylene, cycloalkanediyl, heterocyclo-diyl, or any combination thereof optionally linked by one or more of ether, ester, anhydride, amide, carbamate, secondary amine, tertiary amine, quaternary ammonium, thioether, urea, carbonyl, or imine;

[0102] R1, R2, R3, R4, R5, R6, R7 and R8 are independently empty, H, (C1-C 60 )alkyl, (halogen) (C1-C 60 )alkyl, (hydroxy) (C1-C 60 )alkyl, (alkoxy) (C1-C 60 )alkyl, (C2-C 60 )alkenyl, (halogen)(C2-C 60 )alkenyl, (hydroxy) (C2-C 60 )alkenyl, (alkoxy) (C2-C 60 )alkenyl, (C2-C 60 )alkynyl, (halogen)(C2-C 60 )alkynyl, (hydroxy) (C2-C 60 )alkynyl, (alkoxy) (C2-C 60 ) alkynyl, wherein at least one of R1, R2, R3, R4, R5, R6, R7 and R8 contains at least two unsaturated bonds; and

[0103] x, y and z are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0104] In an embodiment, Q1, Q2, Q3 and Q4 are independently N, B, P or Fe.

[0105] In an embodiment, Q1, Q2, Q3 and Q4 are N.

[0106] In an embodiment, Q1, Q2, Q3 and Q4 are independently primary, secondary or tertiary amines.

[0107] In an embodiment, Q1 and Q4 are primary amines, and Q2 and Q3 are tertiary amines.

[0108] In an embodiment, L1, L2 and L3 are independently (C1-C6)alkanediyl or (hydroxy)(C1-C6)alkanediyl.

[0109] In an embodiment, L1 and L3 are independently (hydroxy)(C1-C6)alkanediyl, and L2 is (C1-C6)alkanediyl.

[0110] In an embodiment, L1 and L3 are 2-hydroxypropanediyl, and L2 is (C1-C6)alkanediyl.

[0111] In an embodiment, L1 and L3 are 2-hydroxypropanediyl, and L2 is butanediyl.

[0112] In an embodiment, one or more of R1, R2, R3, R4, R5, R6, R7 and R8 are independently selected from H, linoleyl, α-linolenyl, γ-linolenyl, linoleyl, arachidonic acid, eicosapentaenyl and docosahexaenyl.

[0113] In an embodiment, two or more of R1, R2, R3, R4, R5, R6, R7 and R8 are independently selected from H, linoleoyl, α-linolenoyl, γ-linolenoyl, linolenoyl, arachidonic acid, eicosapentaenoyl and docosahexaenoyl.

[0114] In an embodiment, one or more of R1, R2, R3, R4, R5, R6, R7 and R8 are independently selected from H, myristyl, palmyl, sapienyl, oleyl, elaidyl, vaccenyl, erucyl, caprylyl, capryl, lauryl, myristyl, palmityl, stearyl, arachidyl, behenyl, lignoceryl and cerotyl.

[0115] In an embodiment, two or more of R1, R2, R3, R4, R5, R6, R7 and R8 are independently selected from H, myristoyl, palmitoyl, sapienoyl, oleoyl, elaidoyl, vaccenoyl, erucoyl, capryloyl, caproyl, lauroyl, myristoyl, palmitoyl, stearoyl, arachidoyl, behenoyl, lignoceroyl and cerotoyl.

[0116] In an embodiment, R1, R2, R3, R5, R7, R8 are H, and R4 and R6 are independently selected from linoleyl, alpha-linolenyl, gamma-linolenyl, linoleyl, arachidonic acid, eicosapentaenyl and docosahexenyl.

[0117] In an embodiment, R1, R2, R3, R5, R7 and R8 are H, and R4 and R6 are α-linolenyl.

[0118] In an embodiment, R1, R2, R3, R5, R7 and R8 are H, and R4 and R6 are linoleyl.

[0119] In an embodiment, x and z are independently 0 or 1, and y is 1.

[0120] In an embodiment, Q1 and Q4 are primary amines; Q2 and Q3 are tertiary amines; L1 and L3 are 2-hydroxypropylene; L2 is butanediyl; R1, R2, R3, R5, R7 and R8 are H, and R4 and R6 are linoleyl; and x, y and z are 1.

[0121] In an embodiment, one or more of L1, L2, and L3 comprises at least one ester moiety.

[0122] In an embodiment, one or more of R2 and R4 comprises at least one ester moiety.

[0123] In an embodiment, A1 and A2 are H.

[0124] In various aspects, the present invention relates to compounds of formula (II)

[0125]

[0126] Among them: R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 and R 28are independently H, halo, OH, (C1-C6)alkyl, (halo)(C1-C6)alkyl, (hydroxy)(C1-C6)alkyl, (alkoxy)(C1-C6)alkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl;

[0127] i, j, k, m, s and t are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0128] 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 or (C1-C6)alkyl; m is 8; i is 8; s is 1; j is 1; k is 4; and t is 4.

[0129] 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 or (C1-C6) alkyl; R 27 and R 28 is methyl; m is 8; i is 8; s is 1; j is 1; k is 4; and t is 4.

[0130] 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 Independently H; R 27 and R 28 is methyl; m is 8; i is 8; s is 1; j is 1; k is 4; and t is 4.

[0131] In an embodiment, R 15 , R16 , R 17 , R 18 R 19 and R 20 is H; R9, R 10 , R 11 , R 12 , R 13 and R 14 Independently H; R 27 and R 28 is 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.

[0132] 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 Independently H; R 27 and R 28 is methyl; R 21 , R 22 , R 23 , R 24 , R 25 and R 26 is H; m is 9; i is 9; s is 1; j is 1; k is 4; and t is 4.

[0133] 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 Independently H; R 27 and R 28 is methyl; R 21 , R 22 , R 23 , R 24 , R 25 and R 26is H; m is 9; i is 9; s is 1; j is 1; k is 3; and t is 3.

[0134] In various aspects, the present invention relates to compounds of formula (III):

[0135]

[0136] Wherein L4, L5, L6 and L7 are independently a bond, (C1-C 20 )alkanediyl, (halogen)(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 -;

[0137] R 29 , R 30 , R 31 , R 32 , R 33 , R 34 and R 35 are independently H, (C1-C 60 )alkyl, (halogen) (C1-C 60 )alkyl, (hydroxy) (C1-C 60 )alkyl, (alkoxy) (C1-C 60 )alkyl, (C2-C 60 )alkenyl, (halogen)(C2-C 60 )alkenyl, (hydroxy) (C2-C 60 )alkenyl, (alkoxy) (C2-C 60 )alkenyl, (C2-C 60 )alkynyl, (halogen)(C2-C 60 )alkynyl, (hydroxy) (C2-C 60 )alkynyl, (alkoxy) (C2-C 60 ) alkynyl, wherein R 29 , R 30 , R 31 , R 32 , R 33 , R 34 and R 35 At least one of the comprises at least two unsaturated bonds;

[0138] v, v1 and v2 are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0139] In an embodiment, L4, L5, L6 and L7 are -(CH2)3-; 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; wherein R 29 , R 30 , R 31 , R 32 , R 33 , R 34 and R 35 At least two of the two contain at least two unsaturated bonds.

[0140] In an embodiment, L4 and L5 are -(CH2)3-; L6 and L7 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; wherein R 29 , R 30 , R 31 , R 32 , R 33 , R 34 and R 35 At least two of the two contain at least two unsaturated bonds.

[0141] In an embodiment, L4 and L6 are -(CH2)3-; L5 and L7 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; wherein R 29 , R 30 , R 31 , R 32 , R 33 , R 34 and R 35At least two of the two contain at least two unsaturated bonds.

[0142] 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; wherein R 29 , R 30 , R 31 , R 32 , R 33 , R 34 and R 35 At least two of the two contain at least two unsaturated bonds.

[0143] 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; wherein R 29 , R 30 , R 31 , R 32 , R 33 , R 34 and R 35 At least two of the two contain at least two unsaturated bonds.

[0144] In an embodiment, L4 and L7 are (hydroxy) (C1-C 20 )alkanediyl; L5 and L6 are -(CH2)3-; 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; wherein R 29 , R 30 , R 31 , R 32 , R 33 , R 34 and R 35 At least two of the two contain at least two unsaturated bonds.

[0145] In an embodiment, L4 and L7 are (hydroxy) (C1-C 20 )alkanediyl; L5 and L6 are -(CH2)3-; 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; wherein R 29 , R 30 , R 31 , R 32 , R 33 , R 34 and R 35 At least three of them contain at least two unsaturated bonds.

[0146] In an embodiment, L4 and L7 are (hydroxy) (C1-C 20 )alkanediyl; 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; wherein R 29 , R 30 , R 31 , R 32 , R 33 , R 34 and R 35 At least three of them contain at least two unsaturated bonds.

[0147] 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; wherein R 29 , R 30 , R 31 , R 32 , R 33 , R 34 and R 35At least three of them contain at least two unsaturated bonds.

[0148] 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; wherein R 29 , R 30 , R 31 , R 32 , R 33 , R 34 and R 35 At least three of them contain at least two unsaturated bonds.

[0149] In an embodiment, R 29 , R 30 , R 31 , R 32 , R 33 , R 34 and R 35 wherein two or more of the at least one aryl radicals are independently selected from H, myristoyl, palmitoleoyl, sapienoyl, oleoyl, elaidoyl, vaccenoyl, erucoyl, capryloyl, caproyl, lauroyl, myristoyl, palmitoyl, stearoyl, arachidoyl, behenoyl, lignoceroyl and cerotoyl.

[0150] In an embodiment, R 29 , R 30 , R 31 , R 32 , R 33 , R 34 and R 35 Two or more of are independently selected from H, linoleyl, α-linolenyl, γ-linolenyl, linoleyl, arachidonic acid, eicosapentaenyl and docosahexaenyl.

[0151] In an embodiment, L4 and L7 are 2-hydroxypropanediyl; L2 is butanediyl; R29 , R 30 , R 31 and R 32 Yes H ； R 33 , R 34 and R 35 is linoleyl; L5 and L6 are –(CH2)3-; and v is 1.

[0152] In an embodiment, L4 and L7 are 2-hydroxypropanediyl; L2 is butanediyl; R 29 , R 30 , R 31 and R 32 Yes H ； R 33 , R 34 and R 35 is linoleyl; L5 and L6 are –(CH2)4-; and v is 1.

[0153] In an embodiment, L4 and L7 are 2-hydroxypropanediyl; L2 is butanediyl; R 29 , R 30 , R 31 and R 32 Yes H ； R 33 , R 34 and R 35 is linoleyl; L5 and L6 are –(CH2)5-; and v is 1.

[0154] In an embodiment, L4 and L7 are 2-hydroxypropanediyl; L2 is butanediyl; R 29 , R 30 , R 31 and R 32 Yes H ； R 33 , R 34 and R 35 is linoleyl; L5 and L6 are –(CH2)6-; and v is 1.

[0155] In an embodiment, the invention relates to compounds of formula (IV):

[0156]

[0157] wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0158] In an embodiment, the invention relates to compounds of formula (V):

[0159]

[0160] In an embodiment, the invention relates to compounds of formula (VI):

[0161]

[0162] In an embodiment, the present invention relates to compounds of formula (VII):

[0163] In an embodiment, the invention relates to compounds of formula (VIII):

[0164]

[0165] In an embodiment, the present invention relates to compounds of formula (IX):

[0166]

[0167] In an embodiment, the present invention relates to compounds of formula (X):

[0168]

[0169] In an embodiment, the present invention relates to compounds of formula (XI):

[0170]

[0171] In an embodiment, the present invention relates to compounds of formula (XII):

[0172]

[0173] In an embodiment, the present invention relates to compounds of formula (XIII):

[0174]

[0175] In an embodiment, the present invention relates to compounds of formula (XIV):

[0176]

[0177] In an embodiment, the invention relates to compounds of formula (XV):

[0178]

[0179] (XV) wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0180] In an embodiment, the present invention relates to compounds of formula (XVI):

[0181]

[0182] The present invention also relates to intermediates and synthetic methods for preparing the compounds (eg, compounds of Formulae I-XVI) and compositions of the present invention.

[0183] In an embodiment, the compound (e.g., a compound of Formula I-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 comprising a compound (e.g., a compound of Formula I-XVI) is soluble in alcohol (e.g., ethanol) at room temperature (e.g., about 20-25°C) and / or at low temperatures (e.g., 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).

[0184] Certain synthetic methods have been found, for example, synthetic methods that can be used to prepare compounds described herein (e.g., compounds of Formula I-XVI). Certain embodiments relate to methods for extracting organic compounds from reactions containing lithium and / or aluminum compounds. Other embodiments relate to methods for extracting organic compounds from reactions 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 produce a quenched reaction. In other embodiments, one or more solvents are removed from the quenched reaction, for example by evaporation. In one embodiment, the quenched reaction is dried, for example, under heating and / or under reduced pressure. In another embodiment, the dried, quenched reaction is extracted with a solvent. In some embodiments, the solvent is an alcohol. In one embodiment, the alcohol is isopropanol. In another embodiment, the isopropanol is heated to about 80° C. In another embodiment, the solvent containing the organic compound is decanted and / or filtered. In yet another embodiment, the solvent is removed, for example, by evaporation to produce the organic compound.

[0185] Also found is a method for purifying a compound of the present invention (e.g., a compound of formula I-XVI). Therefore, certain embodiments relate to a method for purifying a compound (e.g., a compound of formula I-XVI). In one embodiment, a sample containing a compound (e.g., a compound of formula 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 a solvent. In other embodiments, at least one of the one or more impurities is insoluble in a solvent. In one embodiment, one or more impurities include phthalimide derivatives. In one embodiment, one or more impurities include phthalic acid hydrazine. In another embodiment, the sample is suspended in acetone, the compound is dissolved, and at least one of the one or more impurities forms a precipitate. In another embodiment, the precipitate is removed by decantation, filtration, and / or centrifugation. In another embodiment, the solvent is removed, for example, by evaporation to produce a purified compound.

[0186] In various aspects, the present invention relates to a method for extracting an organic compound from a reaction 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 isopropanol); to obtain an extracted organic compound.

[0187] In various aspects, the present invention relates to a method for extracting an organic compound from a reaction 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; to obtain an extracted organic compound.

[0188] In various aspects, the present invention relates to a method for purifying an organic compound from a mixture of the organic compound and phthalimide or a 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; to obtain a purified organic compound.

[0189] Pharmaceutical composition, lipid aggregate, lipid carrier

[0190] In an embodiment, the invention relates to pharmaceutical compositions and / or lipid aggregates and / or lipid carriers and / or lipid nucleic acid complexes and / or liposomes and / or lipid nanoparticles comprising a compound as described herein (e.g., a compound of Formula I-XVI).

[0191] 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 is in any physical form, including, for example, lipid nanoparticles, liposomes, micelles, staggered bilayers, etc.

[0192] In embodiments, the pharmaceutical composition and / or lipid aggregates and / or lipid carriers are liposomes. In embodiments, the liposomes are large unilamellar vesicles (LUVs), multilamellar vesicles (MLVs) or small unilamellar vesicles (SUVs). In embodiments, the liposomes have a diameter of at most about 50 to 80 nm. In embodiments, the liposomes have a diameter greater than about 80 to 1000 nm or greater. In embodiments, the diameter of the liposomes is about 50 to 1000 nm, for example, about 200 nm or less. Size represents the size (diameter) of the particles formed. Size alternatively represents the hydrodynamic radius of the particles formed. Size distribution can be determined using quasi-elastic light scattering (QELS) on a Nicomp 370 submicron particle size analyzer.

[0193] In certain embodiments, the present invention relates to methods and compositions for producing lipid-encapsulated nucleic acid particles, wherein the nucleic acid is encapsulated in a lipid layer. Such nucleic acid-lipid particles (including but not limited to nucleic acid-lipid particles incorporating RNA) are characterized using a variety of biophysical parameters, including: drug to lipid ratio; Encapsulation efficiency; Particle size and polydispersity index (PDI). High drug to lipid ratio, high encapsulation efficiency, good nuclease resistance and serum stability and controllable particle size, usually less than 200nm in diameter is desirable (but not limited to).

[0194] In certain embodiments, the particle diameter that can affect transfection efficiency depends on the ionic strength of the complex culture medium. In other embodiments, the particle diameter may depend on the ionic strength of body fluids. In embodiments, the particle diameter depends on the pH of the complex culture medium, or depends on the pH of body fluids. In multiple embodiments, the particle diameter depends on the compounding time or temperature, or depends on the mixing rate of the components of the pharmaceutical composition and / or lipid aggregates and / or lipid carriers and / or lipid nucleic acid complexes and / or liposomes and / or lipid nanoparticles. In some embodiments, the particle diameter can be measured as the Z average particle diameter, which reports the average size of the particle distribution. In some embodiments, the desired size of the particle is about 100nm to about 200nm, or about 150nm to about 200nm, or about 150nm to about 175nm, or about 155nm to about 165nm.

[0195] Another physical property of the lipid nanoparticles according to the present invention is the polydispersity index (PDI), which is an indication of the size distribution of the lipid nanoparticles. The term "polydispersity" (or "dispersity") is used to describe the degree of unevenness of the particle size distribution. PDI is dimensionless and scaled so that values ​​less than 0.15 are considered monodisperse. Danaei et al., Pharmaceutics. May 18, 2018; 10(2): 57. PDI values ​​greater than 0.7 indicate that the sample has a very wide particle size distribution and may be suitable for analysis by dynamic light scattering (DLS) technology. Same as above. In drug delivery applications using lipid-based carriers, PDI values ​​below 0.3 are considered acceptable, while for various polymer-based nanoparticles, values ​​below 0.2 are generally considered acceptable in practice. 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.

[0196] In some embodiments, the measurement of particle size (e.g., measured as Z-average particle size (nm)) and the measurement of PDI represent the suitability of lipid nanoparticles for transfection. In embodiments, the lipid carrier according to the present invention has a diameter size of less than 200 nm and a PDI of less than 0.2.

[0197] In some embodiments, lipid carriers according to the present invention have a pH-dependent zeta potential. Zeta potentials in volts (V) or millivolts (mV) are determined for the characterization of nanoparticles to estimate surface charge, which can be used to understand the physical stability of nanosuspensions. Zeta potential is a key indicator of colloidal dispersion stability. Zeta potential is the potential at the boundary of the double layer (DL) or the shear plane of the particles, and has a value of typically +100 to -100 mV. Zeta potential values ​​less than negative 20 mV or greater than positive 20 mV are typically required for electrostatically stable suspensions. Zeta potential is affected by culture medium pH. Other factors include ionic strength, additive concentration, and temperature.

[0198] In some embodiments, the zeta potential of the pharmaceutical composition comprising 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.

[0199] 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 titratable particle size is required to determine the ability of the compound or pharmaceutical composition or lipid aggregate to penetrate in vivo from the site of administration, but is not limited thereto.

[0200] In some embodiments, the properties of the culture medium in which the compounds, pharmaceutical compositions, or lipid aggregates according to the present disclosure are formed are used to control the Z-average particle size.

[0201] In an embodiment, the ionic strength of the culture medium in which the compounds, pharmaceutical compositions or lipid aggregates according to the present disclosure are formed is used to control the Z-average particle size.

[0202] In embodiments, the particle size is determined by controlling the concentration of any one or more solutes (such as but not limited to sodium chloride, or calcium chloride, or potassium chloride, or sodium phosphate) in the medium in which the lipid aggregates are formed. In embodiments, the lipid aggregates are formed in deionized water without added solutes, which may optionally be used to control the particle size.

[0203] In embodiments, the ionic strength of the medium in which the compounds, pharmaceutical compositions, or lipid aggregates according to the disclosure are formed is used to maintain a particle size of less than or equal to 200 nm.

[0204] In an embodiment, the pH of the culture medium in which the compounds, pharmaceutical compositions or lipid aggregates according to the present disclosure are formed is used to control the Z-average particle size.

[0205] In embodiments, the compounds, pharmaceutical compositions, or lipid aggregates according to the present disclosure comprise a stable particle dispersion. In some embodiments, the stable dispersion has a pH of about 7.0 to about 8.0, or about 7.4.

[0206] In some embodiments, the complexing of nucleic acids (e.g., small interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), long non-coding RNA (lncRNA), plasmid DNA, etc.) with lipid nanoparticles confers nuclease resistance. In some embodiments, the nuclease is an RNase, optionally RNase A. In some embodiments, the RNase is naturally present in vivo.

[0207] The ratio of nucleic acid to lipid refers to the amount of nucleic acid in a formulation of a specified volume divided by the amount of lipid in the same volume. This can be based on mol / mol, or based on weight / weight, or based on weight / mol, or based on mol / weight. For final ready-to-use formulations, the nucleic acid:lipid ratio can be optionally calculated after dialysis, and chromatography and / or enzyme (e.g., nuclease) digestion has been used to remove as much external nucleic acid as possible.

[0208] Encapsulation efficiency refers to the ratio of the drug (including nucleic acid) of the starting mixture to the lipid divided by the ratio of the drug (including nucleic acid) to the lipid of the final competent preparation. This is a measure of relative efficiency. For the measure of absolute efficiency, the total amount of nucleic acid added to the starting mixture in the competent preparation can also be calculated. The amount of lipid lost during the preparation can also be calculated. Efficiency is a measure of waste and expense of the preparation.

[0209] Transfection

[0210] In embodiments, the compounds of the invention (e.g., compounds of Formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers are useful in lipid aggregates for delivery of macromolecules and other compounds into cells. In embodiments, the compounds of the invention (e.g., compounds of Formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers can be used to deliver nucleic acids into cells.

[0211] In an embodiment, a method of transfecting a cell with a nucleic acid is provided, comprising contacting the cell with a complex of a nucleic acid and a compound of the invention (e.g., a compound of Formula I-XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier. In an embodiment, a complex of a nucleic acid and a compound of the invention (e.g., a compound of Formula I-XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier is formed prior to contacting the cell.

[0212] In an embodiment, the compounds of the present invention (e.g., compounds of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers efficiently encapsulate nucleic acids, and / or have a high drug: lipid ratio, and / or protect the encapsulated nucleic acids from degradation and / or removal in serum, and / or are suitable for systemic delivery, and / or provide intracellular delivery of encapsulated nucleic acids. In addition, the compounds of the present invention (e.g., compounds of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers have good tolerability and provide sufficient therapeutic index, so that the treatment of patients with effective doses of nucleic acids does not produce significant toxicity and / or risk to the patients.

[0213] In embodiments, the compounds of the invention (e.g., compounds of Formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers are polycationic. In embodiments, the compounds of the invention (e.g., compounds of Formula 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., compounds of Formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers have the property of forming lipid aggregates when dispersed in water in multiple embodiments, and the lipid aggregates are strongly and advantageously bound to the polyanions via their cationic moieties. By using an excess of cationic charge relative to the anionic compound, the polyanion-lipid complex can be adsorbed on the cell membrane, thereby promoting the cell's uptake of the desired compound.

[0214] In an embodiment, the compounds of the invention (e.g., compounds of Formulae 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) without wishing to be bound by theory, compresses the nucleic acid payload to be delivered, protecting it from nuclease degradation and enhancing receptor-mediated uptake, (ii) without wishing to be bound by theory, improves association with negatively charged cell membranes by imparting a positive charge to the complex, (iii) without wishing to be bound by theory, promotes fusion with endosomal membranes, facilitating release of the complex from endosomal compartments, and (iv) enhances trafficking from the cytoplasm to the nucleus.

[0215] In embodiments, the present invention relates to compounds of the invention (e.g., compounds of formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers or transfection methods with high transfection efficiency for transfection. In embodiments, transfection efficiency is measured by determining the percentage of cells transfected compared to the entire population during the transfection protocol. In multiple embodiments, the transfection efficiency of the present composition and method 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 multiple embodiments, the transfection efficiency of the present composition and method is higher than the transfection efficiency 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 invention is about 5 times, or 10 times, or 15 times, or 20 times, or 30 times the transfection efficiency of commercially available products (e.g., LIPOFECTIN, LIPOFECTAMINE, LIPOFECTAMINE 2000, LIPOFECTAMINE 3000 (Life Technologies)).

[0216] In embodiments, the invention relates to compounds of the invention (e.g., compounds of Formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers for transfection or transfection methods that allow high levels of endosomal escape. In various embodiments, the endosomal escape of the compositions and methods of the invention is higher than the endosomal escape 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 invention is about 5 times, or 10 times, or 15 times, or 20 times, or 30 times the endosomal escape of commercially available products (e.g., LIPOFECTIN, LIPOFECTAMINE, LIPOFECTAMINE 2000, LIPOFECTAMINE 3000 (Life Technologies)).

[0217] In embodiments, the compounds of the invention (e.g., compounds of formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers are serum-resistant. In embodiments, the compounds of the invention (e.g., compounds of formula 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 invention (e.g., compounds of formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers are serum-resistant. In embodiments, the transfection methods of the invention can work in the presence of serum and / or do not require serum inactivation and / or culture medium changes. In embodiments, stability in serum and / or antiserum can be measured via in vitro assays. The ability to transfect macromolecules (e.g., but not limited to DNA or RNA) can be assessed using transfection efficiency in different amounts of serum, optionally compared with commercially available products (e.g., LIPOFECTIN, LIPOFECTAMINE, LIPOFECTAMINE 2000, LIPOFECTAMINE 3000 (Life Technologies)).

[0218] In embodiments, the present invention relates to compounds of the invention (e.g., compounds of Formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers for transfection or methods of transfection with low or reduced toxic effects. In embodiments, the present invention relates to compounds of the invention (e.g., compounds of Formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers for transfection or methods of transfection with reduced toxic effects compared to commercially available products (e.g., LIPOFECTIN, LIPOFECTAMINE, LIPOFECTAMINE 2000, LIPOFECTAMINE 3000 (Life Technologies)). In various embodiments, the compositions and methods of the present invention provide cells with greater than about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95% viability after transfection. In various embodiments, the compositions and methods of the invention provide cells with 5-fold, or 10-fold, or 15-fold, or 20-fold, or 30-fold greater viability after transfection than 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.

[0219] In embodiments, the present invention relates to compounds of the invention (e.g., compounds of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers for transfection or transfection methods that allow high levels of protein to be expressed by transfected nucleic acids (e.g., DNA or RNA). In various embodiments, the protein expression of the compositions and methods of the present invention is about 30% higher than that of non-transfected, or about 40% higher, or about 50% higher, or about 60% higher, or about 70% higher, or about 80% higher, or about 90% higher, or about 95% higher. In various embodiments, the resulting protein expression of the compositions and methods of the present invention is higher than that of commercially available products (e.g., LIPOFECTIN, LIPOFECTAMINE, LIPOFECTAMINE 2000, LIPOFECTAMINE 3000 (Life Technologies)). In various embodiments, the resulting protein expression of the compositions and methods of the invention is about 5-fold, or 10-fold, or 15-fold, or 20-fold, or 30-fold higher than the protein expression of commercially available products (e.g., LIPOFECTIN, LIPOFECTAMINE, LIPOFECTAMINE 2000, LIPOFECTAMINE 3000 (Life Technologies)).

[0220] In embodiments, the invention relates to compounds of the invention (e.g., compounds of Formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers for transfection, or transfection methods that allow transfection in various cell types, including efficient transfection as described herein. In embodiments, the invention relates to compounds of the invention (e.g., compounds of Formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers for transfection, or transfection methods that allow transfection in established cell lines, difficult-to-transfect cells, primary cells, stem cells, and blood cells, including efficient transfection as described herein. In embodiments, the cell type is a keratinocyte, a fibroblast, or a PBMC.

[0221] In embodiments, the compounds of the invention (eg, compounds of Formulae I-XVI), pharmaceutical compositions, and / or lipid aggregates and / or lipid carriers are suitable for in vitro or in vivo transfection or delivery of compounds to target cells.

[0222] In an embodiment, the invention relates to compounds of the invention (e.g., compounds of Formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers for use in transfection, or methods of transfection that do not require additional transfection reagents such as LipofectAMINE PLUS reagent (Life Technologies).

[0223] In an embodiment, the compound of the invention is a component of a pharmaceutical composition and / or lipid aggregate and / or lipid carrier that does not require additional or auxiliary lipids (e.g., for effective transfection). For example, in an embodiment, one or more of DOPE, DOPC, cholesterol, and polyethylene glycol (PEG)-modified lipids (including but not limited to, or PEGylation of DOPE, DOPC, and / or cholesterol) are not required (e.g., for effective transfection).

[0224] In an embodiment, the compound of the invention is a component of a pharmaceutical composition and / or lipid aggregate and / or lipid carrier further comprising an additional or helper lipid.

[0225] In embodiments, the additional or auxiliary lipids are selected from one or more of the following categories: cationic lipids; anionic lipids; neutral lipids; multivalent charged lipids; and zwitterionic lipids. In some cases, cationic lipids can be used to promote charge-charge interactions with nucleic acids.

[0226] In embodiments, the additional or auxiliary 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.

[0227] In embodiments, the additional or auxiliary lipid is another 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-(trimethylammonium)propane (DOTAP) or 1,2-dioleoyl-3-dimethylammonium-propane (DODAP).

[0228] In embodiments, phospholipids 18:0PC, 18:1PC, 18:2PC, 18:2PE, DSPE, DOPE, 18:2PE, DMPE or combinations thereof are used as helper lipids. In embodiments, additional or helper lipids are DOTMA and DOPE, optionally in a ratio of about 1:1. In embodiments, additional or helper lipids are DHDOS and DOPE, optionally in a ratio of about 1:1.

[0229] In an embodiment, the additional or helper lipid is a commercially available product (eg, LIPOFECTIN, LIPOFECTAMINE, LIPOFECTAMINE 2000, LIPOFECTAMINE 3000 Life Technologies).

[0230] In an embodiment, the additional or helper lipid is a compound having formula (A):

[0231]

[0232] wherein R1 and R4 are straight chain alkenyl groups having 17 carbon atoms; R2 and R5 are -(CH2)p-NH2, wherein p is 1-4; l is 1-10; and Xa is a physiologically acceptable anion.

[0233] In embodiments, the additional or auxiliary lipid is a PEGylated lipid. In embodiments, the PEGylated lipid has a PEG molecule covalently attached thereto, wherein the PEG has an average molecular weight of about 10 kDa to about 400 kDa. In embodiments, the polyethylene glycol suitable for use in the present invention is those having an average molecular weight of at least 10,000 daltons to 40,000 daltons. In an embodiment, the average molecular weight of PEG is 20,000 Daltons, such as in the range of 20,000 to 700,000 Daltons, for example in the range of 20,000 to 600,000 Daltons, such as in the range of 35,000 to 500,000 Daltons, for example in the range of 35,000 to 400,000 Daltons, such as in the range of 35,000 to 350,000 Daltons, for example in the range of 50,000 to 350,000 Daltons, such as in the range of 100,000 to 300,000 Daltons, for example in the range of 150,000 to 350,000 Daltons, such as in the range of 200,000 to 300,000 Daltons. In certain embodiments, polyethylene glycols suitable for use with the compositions and methods described herein are those having an average molecular weight selected from the group consisting of 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, when referring to the average molecular weight of polyethylene glycol, "about" means + / - 30%. In embodiments, the average molecular weight of PEG covalently attached to the compositions and methods described herein is 10 kDa, 20 kDa, or 40 kDa. In embodiments, PEG is a branched PEG, a star PEG, or a comb PEG.

[0234] In one embodiment, the compounds of the invention (e.g., compounds of Formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers include one or more polyethylene glycol (PEG) chains, optionally selected from PEG200, PEG300, PEG400, PEG600, PEG800, PEG1000, PEG1500, PEG2000, PEG3000, and PEG4000. In an embodiment, PEG is PEG2000. In an embodiment, the compounds of the invention (e.g., compounds of Formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers comprise 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) or a derivative thereof. In one embodiment, the compounds of the invention (e.g., compounds of Formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers comprise the PEGylated lipid 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG); in another embodiment, the compounds of the invention (e.g., compounds of Formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers comprise 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG); in yet another embodiment, the compounds of the invention (e.g., compounds of Formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers comprise 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG). In other embodiments, the compounds (eg, compounds of Formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers of the invention comprise a mixture of PEGylated lipids or free PEG chains.

[0235] In an embodiment, a compound of the invention (e.g., a compound of Formula I-XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier comprises N-(carbonyl-ethoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG2000-DSPE), perhydrophosphatidylcholine, cholesterol, LIPOFECTAMINE 3000, a cationic lipid, a polycationic lipid, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folic acid (polyethylene glycol)-5000] (FA-MPEG5000-DSPE).

[0236] In some embodiments, one or more PEGylated helper lipids are incorporated into pharmaceutical compositions and / or lipid aggregates and / or lipid carriers and / or liposomes and / or lipid nanoparticles comprising a compound of the invention (e.g., a compound of Formula 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 invention (e.g., a compound of Formula I-XVI), or the ratio of the PEGylated helper lipid to the nucleic acid is used to influence the particle size. In some embodiments, the particle size is determined by other factors (e.g., but not limited to, the ionic strength of the solution containing the particles, the addition of salts to the solution containing the particles, the addition of other small molecules to the solution containing the particles, and the adjustment of the pH of the solution containing the particles). In some embodiments, particle size control by other factors is used in combination with one or more PEGylated helper lipids, or is used instead of one or more PEGylated helper lipids.

[0237] In one embodiment, the compound of the invention (e.g., a compound of Formula I-XVI) and / or pharmaceutical composition and / or lipid aggregate and / or lipid carrier comprises about 3.2 mg / mL N-(carbonyl-ethoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG2000-DSPE), about 9.6 mg / mL fully hydrogenated phosphatidylcholine, about 3.2 mg / mL cholesterol, about 2 mg / mL ammonium sulfate and histidine as a buffer, and about 0.27 mg / mL 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate (polyethylene glycol)-5000] (FA-MPEG5000-DSPE) added to the lipid mixture. In another embodiment, the nucleic acid is complexed by combining 1 μL LIPOFECTAMINE3000 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 comprising lipids at a concentration of about 1 mg / mL. In an embodiment, nucleic acids are encapsulated by combining about 10 μg of a compound of the invention (e.g., a compound of Formula I-XVI) and / or a pharmaceutical composition and / or lipid aggregates and / or a lipid carrier per about 1 μg of nucleic acid and incubating at room temperature for about 5 minutes.

[0238] In an embodiment, the compounds of the invention (e.g., compounds of formula 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 multiple embodiments, the compounds of the invention (e.g., compounds of formula 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 multiple embodiments, the compounds of the invention (e.g., compounds of formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers comprise one or more of polymer nanoparticles, the polymer nanoparticles comprising polyethylene glycol (PEG)-modified polylactic acid (PLA) diblock copolymers (PLA-PEG), PEG-polypropylene glycol-PEG-modified PLA-tetrablock copolymers (PLA-PEG-PPG-PEG) and poly (lactic acid-co-glycolic acid) copolymers. In another embodiment, the compounds of the invention (e.g., compounds of Formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers comprise statistical, alternating, or periodic copolymers, or any other type of polymer.

[0239] In an embodiment, the compounds of the invention (e.g., compounds of Formula 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 contents of which are incorporated herein by reference.

[0240] In an embodiment, the compound of the invention (e.g., a compound of Formula I-XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier comprises a Polybrene as described in U.S. Pat. No. 5,627,159. TM (hexylenediamine bromide), the entire contents of which are incorporated herein by reference.

[0241] In various embodiments, the compounds of the invention (e.g., compounds of Formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers comprise one or more polymers. Examples of polymers include hexadimethrine bromide (Polybrene TM ), DEAE-dextran, protamine, protamine sulfate, poly-L-lysine or poly-D-lysine. These polymers can be used in combination with cationic lipids to produce synergistic effects on cellular uptake, stability of the compounds of the invention (e.g., compounds of Formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers, including serum stability (e.g., in vivo serum stability), endosomal escape, cell viability and protein expression.

[0242] In an embodiment, the compound of the invention is a component of a pharmaceutical composition and / or lipid aggregate and / or lipid carrier further comprising one or more additional lipids or polymers selected from Table 1. In other embodiments, the nucleic acid of the invention is a component of a pharmaceutical composition and / or lipid aggregate and / or lipid carrier further comprising one or more lipids or polymers selected from Table 1.

[0243] 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 combined with a compound of the invention in a formulation and are a component of a pharmaceutical composition and / or lipid aggregate and / or lipid carrier.

[0244] Table 1. Illustrative biocompatible lipids and polymers

[0245]

[0246]

[0247]

[0248] Additional components that may be present in the pharmaceutical compositions and / or lipid aggregates and / or lipid carriers and / or lipid nucleic acid complexes and / or liposomes and / or lipid nanoparticles of the invention include bilayer stabilizing components such as polyamide oligomers (see, e.g., U.S. Pat. No. 6,320,017), peptides, proteins, detergents, lipid derivatives such as PEG coupled to phosphatidylethanolamine and PEG conjugated to ceramide (see, U.S. Pat. No. 5,885,613).

[0249] In embodiments, lipid of the present invention includes other cationic lipid, neutral lipid, sterol and lipid, and the lipid is selected to reduce the aggregation of lipid particles in the formation process, which may be caused by the steric stability of the particles preventing the aggregation induced by charge in the formation process. The example of lipid reducing particle aggregation in the formation process includes lipid, monosialoganglioside Gm1 and polyamide oligomer ("PAO") modified by polyethylene glycol (PEG), such as (polyamide oligomer described in U.S. Patent No. 6,320,017). With uncharged, hydrophilic, steric hindrance part, other compounds that prevent aggregation during formulation, such as PEG, Gm1 or ATTA, can also be coupled with lipid for use in the methods and compositions of the present invention. ATTA-lipid is described in, for example, U.S. Patent No. 6,320,017, and PEG-lipid conjugate is described in, for example, U.S. Patent No. 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% (by mole percent of lipid). If the particles are stable after formulation, the PEG or ATTA can be dialyzed away prior to administration to a subject.

[0250] In an embodiment, it is desirable to target a compound of the invention (e.g., a compound of Formula I-XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier using a targeting moiety specific for a cell type or tissue. It has been previously described that a variety of targeting moieties, such as ligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin), and monoclonal antibodies are used to target lipid particles (see, e.g., U.S. Patent Nos. 4,957,773 and 4,603,044). The targeting moiety may comprise the entire protein or a fragment thereof. The targeting mechanism generally requires that the targeting agent be positioned on the surface of the lipid particle in a manner such that the targeting moiety can be used to interact with a target, such as a cell surface receptor. A variety of different targeting agents and methods are known and available in the art, including those described in, e.g., 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 coupling targeting agents can be used. For example, phosphatidylethanolamine that can be activated to connect to the targeting agent, or a derived lipophilic compound, such as lipid-derived bleomycin, can be used. Antibody-targeted liposomes can be constructed using, for example, 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 conjugation are disclosed in U.S. Patent No. 6,027,726, the teachings of which are incorporated herein by reference. Examples of targeting moieties may also include other proteins that are specific to cellular components, including antigens associated with neoplasms or tumors. Proteins used as targeting moieties can be attached to liposomes via covalent bonds (see, Heath, Covalent Attachment of Proteins to Liposomes, 149 Methods in Enzymology 111-119 (Academic Press, Inc. 1987)). Other targeting methods include the biotin-avidin system.

[0251] In embodiments, the compositions and methods of the present invention employ complex culture medium. In one embodiment, the pH of the complex culture medium is 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 culture medium comprises transferrin. In another embodiment, the complex culture medium comprises DMEM. In yet another embodiment, the complex culture medium comprises DMEM / F12.

[0252] Nucleic Acids

[0253] In an embodiment, the compound of the invention (eg, a compound of Formula I-XVI) and / or pharmaceutical composition and / or lipid aggregate and / or lipid carrier is suitable for association with a nucleic acid, including, for example, any oligonucleotide or polynucleotide.

[0254] In an embodiment, the nucleic acid is RNA, small interfering RNA (siRNA), micro RNA (miRNA), messenger RNA (mRNA), long noncoding RNA (lncRNA), antisense oligonucleotide, ribozyme, plasmid, immunostimulatory nucleic acid, antisense, microRNA antagonist (antagomir), antisense microRNA (antimer), microRNA mimic, super microRNA (supermir), U1 adapter or aptamer.

[0255] In an embodiment, the nucleic acid is completely encapsulated in the compounds of this invention (e.g., compounds of formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers. In other embodiments, the nucleic acid is partially encapsulated in the compounds of this invention (e.g., compounds of formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers. In yet other embodiments, nucleic acid and compounds of this invention (e.g., compounds of formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers are present and the nucleic acid is not encapsulated in the compounds of this invention (e.g., compounds of formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers.

[0256] Complete encapsulation means that nucleic acids in the compounds of the invention (e.g., compounds of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers are not significantly degraded after exposure to serum or nuclease assays that would significantly degrade free nucleic acids. In a fully encapsulated system, in embodiments, less than about 25% of the particle nucleic acids are degraded in a treatment that would normally degrade about 100% of the free nucleic acids. In embodiments, less than about 10% or less than about 5% of the particle nucleic acids are degraded.

[0257] The extent of encapsulation can be determined by the Oligreen assay. Oligreen is an ultrasensitive fluorescent nucleic acid stain for quantification of oligonucleotides and single-stranded DNA in solution (available from Invitrogen Corporation, Carlsbad, CA).

[0258] Complete encapsulation also indicates that the compound of the invention (eg, compound of Formula I-XVI) and / or pharmaceutical composition and / or lipid aggregate and / or lipid carrier is serum stable, that is, it does not rapidly break down into its component parts upon in vivo administration.

[0259] In an embodiment, the compounds of the invention (e.g., compounds of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers of the invention are complexed with nucleic acids (e.g., DNA or RNA) at different ratios depending on the target cell type, typically ranging from about 1:16 to about 25:1 ng lipid: ng RNA. Illustrative lipid: RNA ratios are from about 1:1 to about 10:1, such as, but not limited to, about 2.5:1 or about 5:1.

[0260] Additional parameters such as nucleic acid concentration, buffer type and concentration, etc. will have an impact on transfection efficiency and can be varied by one of ordinary skill in the art through routine experimentation.

[0261] In an embodiment, the nucleic acid is selected from RNA or DNA.

[0262] In embodiments, DNA is a plasmid, cosmid, phage, recombinant virus or other vector. In embodiments, a vector (or plasmid) refers to a discrete element for, for example, introducing heterologous nucleic acid into a cell for its expression or replication. The vector can remain free or can be designed to realize the integration of a gene or part thereof into the chromosome of a genome. The vectors of artificial chromosomes (such as yeast artificial chromosomes and mammalian artificial chromosomes) are also contemplated. The selection and use of such vectors are well known to those skilled in the art. Including vectors capable of expressing DNA operably connected to regulatory sequences such as promoter regions, the regulatory sequences capable of realizing the expression of such DNA fragments (such as expression vectors). Therefore, a vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus or other vector, which causes the expression of DNA after being introduced into a suitable host cell. Suitable vectors are well known to those skilled in the art and include those vectors that can be replicated in eukaryotic cells and / or prokaryotic cells and those vectors that remain free or those vectors that are integrated into the host cell genome.

[0263] In an embodiment, the RNA is a synthetic RNA. In an embodiment, the RNA is a chemically synthesized RNA. In an embodiment, the RNA is an in vitro transcribed RNA.

[0264] In an embodiment, the RNA is selected from the group consisting of siRNA, lncRNA, antisense oligonucleotide, microRNA, antagomir, aptamer, ribozyme and mRNA.

[0265] In an embodiment, the synthetic RNA (including but not limited to mRNA) does not comprise atypical nucleotides.

[0266] In embodiments, the synthetic RNA (including but not limited to mRNA) comprises one or more atypical nucleotides.

[0267] In embodiments, one or more atypical nucleotides avoid substantial cytotoxicity. In embodiments, one or more atypical nucleotides substantially avoid in vivo cytotoxicity. In embodiments, one or more atypical nucleotides substantially avoid an immune response in a human subject. For example, an 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 embodiments, an effective dose eliminates the need to treat a human subject with an immunosuppressant (e.g., B18R) for alleviating residual toxicity.

[0268] 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 greater than about 99.9% 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 greater than about 99.9% compared to the upregulation of one or more immune response markers induced by the corresponding unmodified nucleic acid. In an embodiment, the immune response marker comprises mRNA or protein products of interferon genes, including interferon alpha genes, IFNB1, TLR3, RARRES3, EIF2AK2, STAT1, STAT2, IFIT1, IFIT2, IFIT3, IFIT5, OAS1, OAS2, OAS3, OASL, ISG20, or fragments, variants, analogs or family members thereof. In an embodiment, the immune response marker comprises mRNA or protein products of TNF genes, which include the TNFα 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 its fragments, variants, analogs or family members. In an embodiment, the immune response marker comprises mRNA or protein products of interleukin genes, which include 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 fragments, variants, analogs or family members thereof.

[0269] In embodiments, cell death is about 10%, about 25%, about 50%, about 75%, about 85%, about 90%, about 95%, or more than about 95% less than cell death observed with the corresponding unmodified nucleic acid. In addition, cell death can affect 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 cells contacted with the modified nucleic acid.

[0270] When incorporated into RNA molecules, some atypical nucleotides can partially reduce the toxicity of RNA molecules by interfering with the binding of proteins (e.g., proteins of the protein kinase R, Rig-1, and oligoadenylate synthetase family) that detect exogenous nucleic acids, but do not wish to be bound by theory. Atypical nucleotides that have been reported to reduce the toxicity of RNA molecules when incorporated therein include: pseudouridine, 5-methyluridine, 2-thiouridine, 5-methylcytidine, N6-methyladenosine, and some combinations thereof. However, the chemical properties of atypical nucleotides that can enable them to achieve the reduction of the in vivo toxicity of synthetic RNA molecules are still unknown until this time. In addition, the incorporation of a large amount of most atypical nucleotides (e.g., 5-methyluridine, 2-thiouridine, 5-methylcytidine, and N6-methyladenosine) can reduce the efficiency with which RNA molecules can be translated into proteins, limiting the utility of RNA molecules containing these nucleotides in applications requiring protein expression. Additionally, although 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 circumstances, such as when frequent repeated transfections are performed, synthetic RNA molecules containing only adenosine, guanosine, cytidine, and pseudouridine can exhibit excessive toxicity.

[0271] In an embodiment, the atypical nucleotide has one or more substitutions at a position selected from the 2C, 4C and 5C positions of a pyrimidine or selected from the 6C, 7N and 8C positions of a purine.

[0272] In an embodiment, the atypical nucleotides comprise one or more of 5-hydroxycytidine, 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, optionally accounting for at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100% of the amount of atypical nucleotides.

[0273] In an embodiment, at least about 50% of the cytidine residues are atypical nucleotides, and the atypical nucleotides are selected from 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, and 5-methoxycytidine.

[0274] In embodiments, at least about 75% or at least about 90% of the cytidine residues are atypical nucleotides, and the atypical nucleotides are selected from 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, and 5-methoxycytidine.

[0275] In an embodiment, at least about 20% of the uridines, or at least about 40%, or at least about 50%, or at least about 75%, or at least about 90% of the uridine residues are atypical nucleotides, and the atypical 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.

[0276] In an embodiment, at least about 40%, or at least about 50%, or at least about 75%, or at least about 90% of the uridine residues are atypical nucleotides and the atypical 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.

[0277] In embodiments, at least about 10% of the guanine residues are atypical nucleotides, and the atypical nucleotides are optionally 7-deazaguanosine.

[0278] In embodiments, the synthetic RNA comprises no more than about 50% 7-deazaguanosine in place of guanosine residues.

[0279] In an embodiment, the synthetic RNA does not comprise atypical nucleotides in place of adenosine residues.

[0280] In embodiments, synthetic RNA molecules containing one or more atypical nucleotides including one or more substitutions at the 2C and / or 4C and / or 5C positions in the case of pyrimidines or at the 6C and / or 7N and / or 8C positions in the case of purines can be less toxic than synthetic RNA molecules containing only typical nucleotides, in part because substitutions at these positions interfere with the ability of the synthetic RNA molecules to be recognized by proteins that detect exogenous nucleic acids, and further, substitutions at these positions can have minimal effects on the efficiency with which the synthetic RNA molecules can be translated into protein, in part due to the lack of interference of substitutions at these positions with base pairing interactions and base stacking interactions.

[0281]

[0282] Examples of atypical nucleotides comprising one or more substitutions at the 2C and / or 4C and / or 5C positions in the case of pyrimidines or at the 6C and / or 7N and / or 8C positions in the case of purines include, but are not limited to, 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-azaur ... 4-thio-5-azauridine, 4-thio-pseudouridine, 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-hydroxycytidine, 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-hydroxycytidine, 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-hydroxycytidine, 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-hydroxycytidine, 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-hydroxycytidine, 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-methyl-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-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.

[0283] In an embodiment, the present invention relates to one or more atypical nucleotides selected from the group consisting of 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 an embodiment, 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 atypical 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, 5-methoxypseudouridine.

[0284] In an embodiment, 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 atypical nucleotides selected from the group consisting of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine.

[0285] 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 atypical nucleotides selected from the group consisting 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.

[0286] In an embodiment, at least about 10% (e.g., 10%, or about 20%, or about 30%, or about 40%, or about 50%) of the guanosine residues are atypical nucleotides, and the atypical nucleotides are optionally 7-deazaguanosine. In an embodiment, the RNA contains no more than about 50% 7-deazaguanosine instead of guanosine residues.

[0287] In an embodiment, the RNA does not contain atypical nucleotides in place of adenosine residues.

[0288] Note that there are alternative naming schemes for certain atypical nucleotides. For example, in some cases, 5-methylpseudouridine may be referred to as "3-methylpseudouridine" or "N3-methylpseudouridine" or "1-methylpseudouridine" or "N1-methylpseudouridine".

[0289] A nucleotide containing the prefix "amino" may refer to any nucleotide containing a nitrogen atom bound to an atom at a specified position of the nucleotide, for example, 5-aminocytidine may refer to 5-aminocytidine, 5-methylaminocytidine, and 5-nitrocytidine. Similarly, a nucleotide containing the prefix "methyl" may refer to any nucleotide containing a carbon atom bound to an atom at a specified 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 bound to an atom at a given position of the nucleotide, and a nucleotide containing the prefix "hydroxy" may refer to any nucleotide containing an oxygen atom bound to an atom at a given position of the nucleotide, for example, 5-hydroxyuridine may refer to 5-hydroxyuridine and uridine with a methyl group bound to an oxygen atom, wherein the oxygen atom is bound to the atom at the 5C position of the uridine.

[0290] Accordingly, certain embodiments relate to an RNA comprising one or more atypical nucleotides, wherein the RNA molecule contains one or more atypical nucleotides comprising one or more substitutions at the 2C and / or 4C and / or 5C positions in the case of pyrimidines or at the 6C and / or 7N and / or 8C positions in the case of purines.

[0291] In an embodiment, the atypical nucleotides include at least one of pseudouridine, 2-thiouridine, 4-thiouridine, 5-azauridine, 5-hydroxyuridine, 5-methyluridine, 5-aminouridine, 2-thiouridine, 4-thiouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-aminopseudouridine, pseudoisocytidine, N4-methylcytidine, 2-thiouridine, 5-azacytidine, 5-hydroxycytidine, 5-aminocytidine, 5-methylcytidine, N4-methylpseudoisocytidine, 2-thiouridine, 5-hydroxypseudoisocytidine, 5-aminopseudoisocytidine, 5-methylpseudoisocytidine, 7-deazaadenosine, 7-deazaguanosine, 6-thiouridine, and 6-thio-7-deazaguanosine. In another embodiment, the one or more nucleotides include at least one of pseudouridine, 2-thiouridine, 4-thiouridine, 5-azauridine, 5-hydroxyuridine, 5-methyluridine, 5-aminouridine, 2-thiouridine, 4-thiouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, and 5-aminopseudouridine; and at least one of pseudoisocytidine, N4-methylcytidine, 2-thiouridine, 5-azacytidine, 5-hydroxycytidine, 5-aminocytidine, 5-methylcytidine, N4-methylpseudoisocytidine, 2-thiouridine, 5-hydroxypseudoisocytidine, 5-aminopseudoisocytidine, and 5-methylpseudoisocytidine. In yet another embodiment, the one or more nucleotides include at least one of pseudouridine, 2-thiouridine, 4-thiouridine, 5-azauridine, 5-hydroxyuridine, 5-methyluridine, 5-aminouridine, 2-thiouridine, 4-thiouridine, 5-hydroxypseudouridine and 5-methylpseudouridine, 5-aminopseudouridine and at least one of pseudoisocytidine, N4-methylcytidine, 2-thiouridine, 5-azacytidine, 5-hydroxycytidine, 5-aminocytidine, 5-methylcytidine, N4-methylpseudoisocytidine, 2-thiouridine, 5-hydroxypseudoisocytidine, 5-aminopseudoisocytidine and 5-methylpseudoisocytidine and at least one of 7-deazaguanosine, 6-thiouridine, 6-thio-7-deazaguanosine and 5-methoxyuridine. In yet another embodiment, the one or more nucleotides include 5-methylcytidine and 7-deazaguanosine. In another embodiment, the one or more nucleotides further include pseudouridine or 4-thiouridine or 5-methyluridine or 5-aminouridine or 4-thiouridine or 5-methylpseudouridine or 5-aminopseudouridine. In yet another embodiment, the one or more nucleotides further include 7-deazaadenosine. In another embodiment, the one or more nucleotides include pseudoisocytidine and 7-deazaguanosine and 4-thiouridine. In yet another embodiment, the one or more nucleotides include pseudoisocytidine or 7-deazaguanosine and pseudouridine. In yet another embodiment, the one or more nucleotides include 5-methyluridine and 5-methylcytidine and 7-deazaguanosine.In another embodiment, the one or more nucleotides include pseudouridine or 5-methylpseudouridine and 5-methylcytidine and 7-deazaguanosine. In another embodiment, the one or more nucleotides include pseudoisocytidine and 7-deazaguanosine and pseudouridine. In one embodiment, RNA comprising one or more atypical nucleotides exists in vivo.

[0292] By RNA polymerases commonly used for in vitro transcription, certain atypical nucleotides can be more effectively incorporated into RNA molecules than other atypical nucleotides, partly due to the tendency of these specific atypical nucleotides to participate in standard base pairing interactions and base stacking interactions, and to interact with RNA polymerase in a manner similar to that of the corresponding typical nucleotides. Therefore, certain nucleotide mixtures containing one or more atypical nucleotides can be beneficial, partly because the in vitro transcription reactions containing these nucleotide mixtures can produce a large amount of RNA. Therefore, some embodiments relate to nucleotide mixtures, which contain one or more nucleotides including one or more substitutions at 2C and / or 4C and / or 5C positions in the case of pyrimidines or at 6C and / or 7N and / or 8C positions in the case of purines.Nucleotide mixtures include, but are not limited to (the number preceding each nucleotide indicates an illustrative portion of the atypical nucleoside triphosphate 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, wherein pseudoisocytidine-5'-triphosphate is present in the reaction in an amount approximately equal to 0.2 times the total amount of pseudoisocytidine-5'-triphosphate + cytidine-5'-triphosphate present in the reaction, wherein amounts are measured on a molar or mass basis, and wherein more than one number preceding a nucleotide indicates an illustrative range of portions): 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-thiouridine pseudouridine, 0.1-1.0 4-thiouridine pseudouridine, 0.1-1.0 5-hydroxypseudouridine, 0.2-1 5-methylpseudouridine, 0.2-1.0 5-methoxypseudouridine, 0.1-1.0 5-aminopseudouridine, 0.2-1.0 2-thiouridine, 0.1-0.8 pseudoisocytidine, 0.2-1.0 5-methylcytidine, 0.2-1.0 5-hydroxycytidine, 0.2-1.0 5-hydroxymethylcytidine, 0.2-1.0 5-methoxycytidine, 0.1-1.0 5-aminocytidine, 0.2-1.0 4-methylcytidine, 0.2-1.0 5-methylpseudoisocytidine, 0.2-1.0 5-hydroxypseudoisocytidine, 0.2-1.0 5-aminopseudoisocytidine, 0.2-1.0N4-methylpseudoisocytidine, 0.2-1.0 2-thiopseudoisocytidine, 0.2-1.0 7-deazaguanosine, 0.2-1.0 6-thioguanosine, 0.2-1.06-thio-7-deazaguanosine, 0.2-1.0 8-azaguanosine, 0.2-1.0 7-deaza-8-azaguanosine, 0.2-1.0 6-thio-8-azaguanosine, 0.1-0.5 7-deazaadenosine and 0.1-0.5N6-methyladenosine.

[0293] In embodiments, RNAs (e.g., which can be prepared by in vitro transcription) comprising one or more atypical nucleotide compositions or synthetic polynucleotide compositions contain substantially or completely typical nucleotides at positions with adenine or "A" in the genetic code. In this context, the term "substantially" refers to at least 90%. In these embodiments, the RNA composition or synthetic polynucleotide composition may also contain (e.g., consist of) 7-deazaguanosine and the corresponding typical nucleotide "G" at positions with "G" in the genetic code, and the typical and atypical nucleotides at positions with G may be in the range of 5:1 to 1:5, or in embodiments in the range of 2:1 to 1:2. In these embodiments, the RNA composition or synthetic polynucleotide composition may also contain (e.g., consist of) one or more (e.g., two, three, or four) 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, and the canonical nucleotide "C" at the position with "C" in the genetic code, and the canonical and atypical nucleotides at the position with C may be in the range of 5:1 to 1:5, or in embodiments in the range of 2:1 to 1:2. In embodiments, the level of atypical nucleotides at the "C" position is as described in the previous paragraph. In these embodiments, the RNA composition or synthetic polynucleotide composition may also contain (e.g., consist of) one or more (e.g., two, three, or four) 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyluridine, 5-formyluridine, 5-methoxyuridine, pseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxylpseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine and the typical nucleotide "U" at the position with "U" in the genetic code, and the typical and atypical nucleotides at the position with "U" may 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 atypical nucleotides at the "U" position is as described in the previous paragraph.

[0294] Incorporation of certain atypical nucleotides can be beneficial, in part because the contribution of atypical nucleotides to reducing the toxicity of RNA molecules is additive. Thus, embodiments relate to nucleotide mixtures, wherein the nucleotide mixture contains more than one of the atypical nucleotides listed above, for example, the nucleotide mixture contains pseudoisocytidine and 7-deazaguanosine, or the nucleotide mixture contains N4-methylcytidine and 7-deazaguanosine, etc. In one embodiment, the nucleotide mixture contains more than one of the atypical nucleotides listed above, and each atypical nucleotide is present in the mixture in the above-listed portions, for example, the nucleotide mixture contains 0.1-0.8 pseudoisocytidine and 0.2-1.0 7-deazaguanosine, or the nucleotide mixture contains 0.2-1.0N4-methylcytidine and 0.2-1.0 7-deazaguanosine, etc.

[0295] In some cases, for example, when it is not necessary or desirable to maximize the productive rate of in vitro transcription reaction, nucleotide moieties can be used instead of those given above. The illustrative parts listed above and the scope of parts relate to the nucleotide-triphosphate solution with typical purity (greater than 90% purity). The larger part of these and other nucleotides can be used by using the nucleotide-triphosphate solution with greater purity, the greater purity is for example greater than about 95% purity or greater than about 98% purity or greater than about 99% purity or greater than about 99.5% purity, the purity can for example be realized by using existing chemical purification technology such as high pressure liquid chromatography (HPLC) or by other means purifying nucleoside triphosphate solution. In one embodiment, the nucleotide with multiple isomer is purified with the isomer required for enrichment.

[0296] Other embodiments relate to methods for inducing in vivo cell expression of target protein by contacting cells with compounds of the present invention (e.g., compounds of formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers and RNA molecules, wherein the RNA molecules contain one or more atypical nucleotides including one or more substitutions at 2C and / or 4C and / or 5C positions in the case of pyrimidines or at 6C and / or 7N and / or 8C positions in the case of purines. Still other embodiments relate to methods for transfecting, reprogramming and / or gene editing in vivo cells by contacting cells with compounds of the present invention (e.g., compounds of formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers and RNA molecules, wherein the synthetic RNA molecules contain one or more atypical nucleotides including one or more substitutions at 2C and / or 4C and / or 5C positions in the case of pyrimidines or at 6C and / or 7N and / or 8C positions in the case of purines. In one embodiment, RNA molecules are produced by in vitro transcription. In one embodiment, RNA molecules encode one or more reprogramming factors. In another embodiment, the one or more reprogramming factors include Oct4 protein. In another embodiment, the cell is also contacted with an RNA molecule encoding Sox2 protein. In another embodiment, the cell is also contacted with an RNA molecule encoding Klf4 protein. In another embodiment, the cell is also contacted with an RNA molecule encoding c-Myc protein. In another embodiment, the cell is also contacted with an RNA molecule encoding Lin28 protein.

[0297] In an in vitro transcription reaction containing typical nucleotides and atypical nucleotides, an enzyme such as T7 RNA polymerase may preferentially incorporate typical nucleotides. Thus, an in vitro transcription reaction containing a certain fraction of atypical nucleotides may produce RNA containing a different (usually lower) fraction of atypical nucleotides than the fraction of atypical nucleotides present in the reaction. In certain embodiments, reference to a nucleotide incorporation fraction (e.g., "synthetic RNA molecules containing 50% pseudoisocytidine" or "0.1-0.8 pseudoisocytidine") may therefore refer to RNA molecules containing nucleotides of a specified fraction and RNA molecules synthesized in a reaction containing nucleotides (or nucleotide derivatives, such as nucleotide-triphosphates) of a specified fraction, even though such reactions may produce RNA containing nucleotides of a different fraction than the fraction of atypical nucleotides present in the reaction.

[0298] Different nucleotide sequences can encode the same protein by using alternative codons. In certain embodiments, reference to a nucleotide incorporation fraction can therefore refer to an RNA molecule containing a specified fraction of nucleotides and an RNA molecule encoding the same protein as a different RNA molecule, wherein the different RNA molecule contains a specified fraction of nucleotides.

[0299] When incorporated into synthetic RNA, atypical nucleotide members of the 5-methylcytidine demethylation pathway can increase the efficiency with which the synthetic RNA can be translated into protein in vivo, and can reduce the in vivo toxicity of the synthetic RNA. These atypical nucleotides include, for example, 5-methylcytidine, 5-hydroxymethylcytidine, 5-formylcytidine, and 5-carboxycytidine (also known as "cytidine-5-carboxylic acid"). Therefore, certain embodiments relate 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 atypical nucleotides. In one embodiment, the nucleic acid comprises one or more atypical nucleotide members in 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 a derivative thereof. In another 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 a derivative thereof.

[0300] 5-Methylcytidine demethylation pathway

[0301]

[0302] Certain combinations of atypical nucleotides can be particularly effective in increasing the efficiency with which the synthetic RNA can be translated into protein in vivo, and reducing the in vivo toxicity of the synthetic RNA, such as 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. Certain embodiments therefore relate 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 relate 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 relate to nucleic acids comprising 5-methyluridine, 5-methylcytidine, 5-hydroxymethylcytidine and 7-deazaguanosine, all 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.

[0303] Synthetic RNA molecules containing certain parts of certain atypical nucleotides and their combinations can show particularly high translation efficiency and low toxicity in vivo. Certain embodiments therefore relate to nucleic acids comprising at least one of one or more uridine residues, one or more cytidine residues and one or more guanosine residues and comprising one or more atypical 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 another 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 another 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 another 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 residues and / or N4-acetylcytidine residues. In another embodiment, each cytidine residue is a 5-methylcytidine residue. In another 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 residues 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 another embodiment, 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 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 embodiments, 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 in 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 another 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.

[0304] Other atypical nucleotides that can be used instead of 5-methyluridine 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 atypical 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 only a single transfection, injection or delivery is performed, 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 fraction of atypical nucleotides can be reduced. Reducing the fraction of atypical nucleotides may be beneficial, in part because reducing the fraction of atypical nucleotides can reduce the cost of nucleic acids. In some cases, for example, when minimal immunogenicity of nucleic acids is desired, the fraction of atypical nucleotides can be increased.

[0305] In an in vitro transcription reaction containing typical nucleotides and atypical nucleotides, an enzyme such as T7 RNA polymerase may preferentially incorporate typical nucleotides. Thus, an in vitro transcription reaction containing a certain fraction of atypical nucleotides may produce RNA containing a different (usually lower) fraction of atypical nucleotides than the fraction of atypical nucleotides present in the reaction. In certain embodiments, reference to a nucleotide incorporation fraction (e.g., "50% 5-methyluridine") may therefore refer to nucleic acids containing nucleotides of a specified fraction and nucleic acids synthesized in a reaction containing nucleotides of a specified fraction (or nucleotide derivatives, such as nucleotide-triphosphates), even if such reactions may produce nucleic acids containing nucleotides of a different fraction than the fraction of non-canonical nucleotides present in the reaction. In addition, different nucleotide sequences may encode the same protein by using alternative codons. In certain embodiments, reference to a nucleotide incorporation fraction may therefore refer to nucleic acids containing nucleotides of a specified fraction and nucleic acids encoding the same protein as different nucleic acids, wherein the different nucleic acids contain nucleotides of a specified fraction.

[0306] Certain embodiments relate to nucleic acids comprising a 5'-cap structure selected from Cap 0, Cap 1, Cap 2 and Cap 3, or a derivative thereof. In one embodiment, the nucleic acid comprises one or more UTRs. In another embodiment, one or more UTRs increase the stability of the nucleic acid. In another embodiment, one or more UTRs comprise α-globin or β-globin 5'-UTR. In yet another embodiment, one or more UTRs comprise α-globin or β-globin 3'-UTR. In yet another embodiment, the synthetic RNA molecule comprises α-globin or β-globin 5'-UTR and α-globin or β-globin 3'-UTR. Certain embodiments relate to nucleic acids comprising post-transcriptional regulatory elements. In one embodiment, the post-transcriptional regulatory element is selected from woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE), hepatitis B virus post-transcriptional regulatory element (HPRE), chicken lysozyme nuclear matrix binding region (cMAR) and 5'-DNA enzyme I-hypersensitive site 4 (cHS4). In another embodiment, one or more UTRs include WPRE. In another embodiment, the synthetic RNA molecule comprises an α-globin or β-globin 5'-UTR and a 3'-UTR comprising a WPRE. In yet another embodiment, the synthetic RNA molecule comprises one or more copies of a WPRE in addition to an α-globin or β-globin 5'-UTR and an α-globin or β-globin 3'-UTR. Illustrative 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.

[0307] In one embodiment, the 5'-UTR comprises a Kozak sequence substantially similar to a Kozak consensus sequence. In another embodiment, the nucleic acid comprises a 3'-poly (A) tail. In another embodiment, the length of the 3'-poly (A) tail is between about 20nt and about 250nt or between about 120nt and about 150nt. In another 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.

[0308] The length of the poly (A) tail produced by poly (A) polymerase can vary according to reaction conditions (including reaction time and enzyme activity), and the enzymatic tailing reaction can produce a mixture of RNA molecules with poly (A) tails of different lengths. Some embodiments relate to synthetic RNA molecules containing 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 nucleotide tails. In one embodiment, the tail is a poly (A) tail. Other embodiments relate to tails containing less than about 10 nucleotides.

[0309] The template of using the coding tail to synthesize RNA can increase the control to the tail length, and reduce the variability in the reaction or between the reactions. Some embodiments therefore relate to the template of the coding 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 relate to the synthetic RNA molecules synthesized using the template of the coding tail.

[0310] Including nucleotides other than adenosine in the tail can enhance the stability of the synthetic RNA molecule and / or the translation efficiency, and improve the fidelity of template DNA production in bacteria. Some embodiments therefore relate to synthetic RNA molecules comprising a tail, wherein the tail comprises adenosine nucleotides and one or more other nucleotides. Other embodiments relate to templates encoding the tail, wherein 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 another embodiment, the tail comprises uridine nucleotides. In yet another embodiment, the tail comprises one or more chemically modified nucleotides and / or atypical nucleotides. In multiple embodiments, other nucleotides are incorporated with adjacent nucleotides separated by one or more adenosine nucleotides at regular intervals, or at random intervals, or in pairs or groups. In one embodiment, the tail comprises deoxyguanosine nucleotides. In another embodiment, the tail comprises deoxycytosine nucleotides. In another embodiment, the tail comprises deoxyuridine nucleotides. In multiple embodiments, other nucleotides are incorporated with adjacent nucleotides separated by one or more deoxyadenosine nucleotides at regular intervals, or at random intervals, or in pairs or groups. In embodiments, the tail region consists of about 2%-10% non-uridine nucleotides, about 10%-20% non-uridine nucleotides, about 20%-35% non-uridine nucleotides.

[0311] Including a stem-loop structure before or after the tail can enhance the stability and / or translation efficiency of the synthetic RNA molecule. Some embodiments therefore relate to a synthetic RNA molecule comprising a tail and a stem-loop structure. In a plurality of embodiments, the stem-loop structure occurs before the tail, after the tail, or before or 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.

[0312] One aspect of the invention is a composition comprising a DNA template comprising: (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.

[0313] In embodiments, the length of the tail region is between about 80 base pairs and about 120 base pairs, between about 120 base pairs and about 160 base pairs, between about 160 base pairs and about 200 base pairs, between about 200 base pairs and about 240 base pairs, between about 240 base pairs and about 280 base pairs, or between about 280 base pairs and about 320 base pairs.

[0314] In an embodiment, the tail region is greater than 320 base pairs in length.

[0315] 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.

[0316] 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% cytidine residues.

[0317] 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% uridine residues.

[0318] In any of the foregoing embodiments and aspects, the tail region comprises 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.

[0319] In embodiments, the synthetic RNA comprises from about 200 nucleotides to about 5000 nucleotides.

[0320] In embodiments, the synthetic RNA comprises about 500 to about 2000 nucleotides, or about 500 to about 1500 nucleotides, or about 500 to about 1000 nucleotides.

[0321] Target protein

[0322] In an embodiment, a compound, pharmaceutical composition, or lipid aggregate described herein is complexed or associated with a nucleic acid (eg, DNA or RNA, such as mRNA), and the nucleic acid encodes a recombinant protein of interest.

[0323] In an embodiment, the recombinant protein of interest is a soluble protein.

[0324] In an embodiment, the protein of interest is selected from Table 2B.

[0325] 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.

[0326] In embodiments, the target recombinant protein is a gene-editable protein. In embodiments, the gene-editing protein is 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.

[0327] In an embodiment, the gene editing protein comprises a DNA binding domain comprising a plurality of repeat sequences, and at least one repeat sequence comprises the amino acid sequence LTPvQVVAIAwxyzα ​​(SEQ ID NO: 819), wherein 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 empty; “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 four consecutive amino acids or empty. In embodiments, "v" is Q, D or E, "w" is S or N, "x" is I, H, N or I, and "y" is D, A, I, N, H, K, S, G or null. In embodiments, the length of the repeat sequence is between 36 and 39 amino acids (e.g., 36, or 37, or 38, or 39 amino acids in length).

[0328] In an embodiment, α comprises at least one glycine (G) residue. In an embodiment, α comprises at least one histidine (H) residue. In an embodiment, α comprises at least one histidine (H) residue at any of positions 33, 34 or 35. In an embodiment, α comprises at least one aspartic acid (D) residue. In an embodiment, α comprises at least one, two or three of a glycine (G) residue, a histidine (H) residue and an aspartic acid (D) residue.

[0329] In an embodiment, α comprises one or more hydrophilic residues, optionally selected from: polar and positively charged hydrophilic amino acids, optionally selected from arginine (R) and lysine (K); polar and neutrally charged hydrophilic amino acids, optionally selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C); polar and negatively charged hydrophilic amino acids, optionally selected from aspartic acid (D) and glutamic acid (E), and aromatic, polar and positively charged hydrophilic amino acids, optionally selected from histidine (H).

[0330] In some embodiments, α comprises one or more polar and positively charged hydrophilic amino acids selected from arginine (R) and lysine (K). In some embodiments, α comprises one or more polar and neutrally charged hydrophilic amino acids selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In some embodiments, α comprises one or more polar and negatively charged hydrophilic amino acids selected from aspartic acid (D) and glutamic acid (E). In some embodiments, α comprises one or more aromatic, polar and positively charged hydrophilic amino acids selected from histidine (H).

[0331] In embodiments, α comprises one or more hydrophobic residues, optionally selected from: hydrophobic aliphatic amino acids, optionally selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V), and hydrophobic aromatic amino acids, optionally 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).

[0332] In an embodiment, α is defined by GabG, wherein "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 void.

[0333] In embodiments, alpha is selected from the group consisting of 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:835), 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), HRGE (SEQ ID NO: 847), RHGD (SEQ ID NO: 848), HRGD (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).

[0334] In an embodiment, the gene editing protein comprises a DNA binding domain comprising a plurality of repeat sequences, and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ ID No: 629) and is between 36 and 39 amino acids in length, wherein: “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 empty, and “z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 630) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 631).

[0335] In an embodiment, the gene editing protein further comprises a nuclease domain comprising a nuclease catalytic domain.

[0336] In embodiments, the gene editing protein is capable of creating single-stranded or double-stranded breaks in a gene.

[0337] In embodiments, single-strand or double-strand breaks result in persistently altered gene splicing.

[0338] It has been found that incorporating microRNA binding sites into synthetic RNA molecule sequences can render the encoded proteins immune tolerant. Certain embodiments therefore relate to synthetic RNA molecules comprising microRNA binding sites. 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 a sequence of SEQ ID NO: 810). In some embodiments, the microRNA binding site is present in the 3'-UTR of the synthetic RNA molecule. In other embodiments, the 3'-UTR of the synthetic RNA molecule comprises a plurality of microRNA binding sites. In other embodiments, the 3'-UTR of the synthetic RNA molecule comprises a plurality of miR142 microRNA binding sites. In certain embodiments, the 3'-UTR of the synthetic RNA molecule comprises a plurality of 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 comprises four copies of the miR142 microRNA binding site (SEQ ID NO: 812). In another embodiment, the microRNA binding site is separated by two or more nucleotides. In one embodiment, the encoded protein is a gene editing protein. In another embodiment, the encoded protein is preferably expressed in non-hematopoietic cells. In certain embodiments, the encoded protein is immune-tolerant. In other embodiments, the encoded protein is absent in the subject or exists at a level below normal. In other embodiments, the encoded protein is a gene editing protein, and a vector for repairing or inserting a gene is delivered to the cell. In some embodiments, the gene product is immune-tolerant.

[0339] Some embodiments relate to the excitation and / or stimulation and / or propagation of an immune response. In some embodiments, the target recombinant protein is an antigen. In some embodiments, the target recombinant protein provides acquired immunity to one or more diseases, optionally infectious diseases. In some embodiments, the immune system reacts to the target recombinant protein. In some embodiments, the target recombinant protein is derived from a pathogen or toxin; in some other embodiments, the target recombinant protein is a synthetic mimetic of a pathogen or toxin. In some embodiments, the target recombinant protein is a fusion protein, which optionally independently comprises a sequence derived from or simulating a pathogen or toxin, and / or a sequence known to stimulate an immune response. In some embodiments, after administering a synthetic RNA encoding a target antigen, the subject is made immune or resistant to certain pathogens or toxins.

[0340] In various embodiments, the pathogen is a coronavirus.Coronaviruses (CoV) are members of the Coronaviridae family, which includes betacoronavirus and alphacoronavirus respiratory pathogens.

[0341] 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.

[0342] 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 embodiments, the alpha coronavirus is selected from HCoV-NL63 and HCoV-229E. In embodiments, the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0343] In some embodiments, the target recombinant protein is a coronavirus protein. In multiple embodiments, the coronavirus protein is a protein from SARS-CoV-2, such as, for example, the SARS-CoV-2 spike protein. In embodiments, the SARS-CoV-2 protein is selected from the spike surface glycoprotein, the membrane glycoprotein M, the envelope protein E, and the nucleocapsid phosphoprotein N, or an antigenic fragment thereof. In embodiments, the SARS-CoV-2 protein selected from the spike surface glycoprotein includes S1, S2, and S2'.

[0344] In some embodiments, the SARS-CoV-2 spike protein comprises the following amino acid sequence:

[0345] MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQ LSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVP AQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGIN ASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT(SEQ ID NO:100).

[0346] In some embodiments, the envelope protein comprises the following amino acid sequence:

[0347] MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPSFYVYSRVKNLNSSRVPDLLV (SEQ ID NO: 101).

[0348] In some embodiments, the membrane protein comprises the following amino acid sequence: MADSNGTITVEELKKLLEQWNLVIGFLFLTWICLLQFAYANRNRFLYIIKLIFLWLLWPVTLACFVLAAVYRINWITGGIAIAMACLVGLMWLSYFIASFRLFARTRSMWSFNPETNILLNVPLHGTILTRPLLESELVIGAVILRGHLRIAGHHLGRCDIKDLPKEITVATSRTLSYYKLGASQRVAGDSGFAAYSRYRIGNYKLNTDHSSSSDNIALLVQ (SEQ ID NO: 102).

[0349] In some embodiments, the nucleocapsid phosphoprotein N comprises the following amino acid sequence:

[0350] MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMA GNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGM SRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA(SEQ ID NO: 103).

[0351] 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 that is at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identical to any of the foregoing, or an antigenic fragment of any of the foregoing.

[0352] In various embodiments, the SARS-CoV-2 protein can comprise a SARS-CoV-2 protein having 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% similarity to any known wild-type amino acid sequence of a SARS-CoV-2 protein or a SARS-CoV-2 amino acid sequence disclosed herein (e.g., relative to any of SEQ ID NOs: 100, 101, 102, 103). , 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%, or at least about 100%, or at least about 101%, or at least about 102%, or at least about 103%, or at least about 104%, or at least about 105%, or at least about 106%, or at least about 107%, or at least about 108%, or at least about 109%, or at least about 110%, or at least about 111%, or at least about 112%, or at least about 113%, or at least about 114%, or at least about 115%, or at least about 116%, or at least about 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% sequence identity. , 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), or an antigenic fragment thereof.

[0353] In an embodiment, a nucleic acid encoding a coronavirus protein or an antigenic fragment thereof is provided. The coronavirus protein can be any of the coronavirus proteins, including a beta coronavirus protein or an alpha coronavirus protein. Nucleic acids include, but are not limited to, RNA, such as, but not limited to, mRNA. In some embodiments, nucleic acids include, but are not limited to, DNA. The DNA can be associated with an AAV encoding a coronavirus protein or an antigenic fragment thereof.

[0354] In some embodiments, the nucleic acid comprises a vector, which can comprise any type of nucleotides, including but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and which in exemplary aspects contains natural, non-natural or altered nucleotides.

[0355] In embodiments, vaccines for one or more diseases, pathogens or toxins are provided. In some embodiments, vaccines according to embodiments of the present disclosure produce protective antibody titers and / or T cell responses to encoded antigens. Encoded antigens may be, for example, infectious agent antigens, such as SARS-CoV-2 antigens. In some embodiments, vaccines according to embodiments of the present disclosure produce antigen-specific antibody titers (e.g., IgG, IgM and / or IgA) specific to encoded antigens, which may be infectious agent antigens, such as SARS-CoV-2 antigens.

[0356] It has been found that certain factors expressed together with proteins can make the encoded proteins immune tolerant. In embodiments, the present invention relates to delivering synthetic RNA molecules capable of inducing immune tolerance to encoded proteins by co-delivering factors that induce tolerance. In certain embodiments, the co-delivered factors are expressed by synthetic RNA molecules. 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) (for example, but not limited to 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).

[0357] Gene Editing

[0358] In various aspects, the present invention relates to a cell transfected with a complex of one or more synthetic RNA molecules and a compound described herein (e.g., a compound of Formulae I-XVI), wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more gene editing proteins, wherein the gene editing proteins are selected from nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases, large-range nucleases, 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.

[0359] In various aspects, the present invention relates to a method for gene editing cells, comprising transfecting cells with a complex of one or more synthetic RNA molecules and a compound described herein (e.g., a compound of Formulae I-XVI), wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more gene editing proteins, wherein the gene editing proteins are selected from nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases, large-range nucleases, 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.

[0360] Several naturally occurring proteins contain DNA binding domains that recognize specific DNA sequences, such as zinc fingers (ZFs) and transcription activator-like effectors (TALEs). Fusion proteins containing one or more of these DNA binding domains and a FokI endonuclease cleavage domain can be used to generate double-strand breaks in desired regions of DNA in cells (see, e.g., U.S. Patent Application Publication No. US2012 / 0064620, U.S. Patent Application Publication No. US2011 / 0239315, U.S. Patent No. 8,470,973, U.S. Patent Application Publication No. US2013 / 0217119, U.S. Patent No. 8,420,782, U.S. Patent Application Publication No. US2011 / 0301073, U.S. Patent Application Publication No. US2011 / 0145940, U.S. Patent No. 8,450,471, U.S. Patent No. 8,440,431, U.S. Patent No. 8,440,432, and U.S. Patent Application Publication No. 2013 / 0122581, the contents of all of which are hereby incorporated by reference). Other gene editing proteins include clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins. However, current methods for gene editing cells are inefficient and carry the risk of uncontrolled mutagenesis, making them unsuitable for research, therapeutic or cosmetic use.

[0361] Some embodiments relate to methods of gene editing and / or gene correction using compounds of the invention (e.g., compounds of Formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers. For example, in embodiments, compounds of the invention (e.g., compounds of Formula I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers are associated with synthetic RNA encoding gene editing proteins, and the resulting compositions are used, for example, for ex vivo or in vivo gene editing and / or gene correction cells.

[0362] Some embodiments encompass synthetic RNA-based gene editing and / or gene correction, for example, with RNA containing atypical nucleotides, such as RNA encoding one or more of the following: nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases, meganucleases, nickases, clustered regularly spaced short palindromic repeats (CRISPR)-related proteins, DNA repair proteins, DNA modification proteins, base modification proteins, DNA methyltransferases, proteins that cause DNA demethylation, enzymes with DNA as substrates, or natural or engineered variants, family members, orthologs, fragments or fusion constructs thereof. In embodiments, gene editing and / or gene correction are efficient, for example, higher than DNA-based gene editing and / or gene correction. In embodiments, the gene editing and / or gene correction methods of the present invention are sufficiently effective for in vivo applications. In embodiments, the gene editing and / or gene correction methods of the present invention are sufficiently effective to not require cell selection (e.g., selecting cells that have been edited).

[0363] In embodiments, the gene editing efficiency of the present method 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 multiple embodiments, the gene correction efficiency of the present method 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%.

[0364] Some embodiments relate to efficient gene editing proteins comprising engineered nuclease cleavage or DNA modification domains. Other embodiments relate to high-fidelity gene editing proteins comprising engineered nuclease cleavage or DNA modification domains. Multiple embodiments relate to efficient gene editing proteins comprising engineered DNA binding domains. Other embodiments relate to high-fidelity gene editing proteins comprising engineered DNA binding domains. Other embodiments relate to gene editing proteins comprising engineered repetitive sequences. Some embodiments relate to gene editing proteins comprising one or more CRISPR-related family members. Some embodiments relate to methods for changing the DNA sequence of cells by transfecting cells with gene editing proteins or inducing cells to express gene editing proteins. Other embodiments relate to methods for changing the DNA sequence of cells present in in vitro cultures. Still other embodiments relate to methods for changing the DNA sequence of cells present in vivo.

[0365] Gene editing proteins containing the StsI nuclease cleavage domain (SEQ ID NO: 1) exhibit significantly 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 have also been discovered that, when used as nuclease domains of gene editing 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)), they can exhibit high on-target activity in vivo, low off-target activity in vivo, small size, solubility, and other desirable properties. StsI-HA, StsI-HA2 (high activity), StsI-UHA, and StsI-UHA2 (ultra-high activity) exhibited higher on-target activity in vivo than wild-type StsI and wild-type FokI, partly due to specific amino acid substitutions at positions 34 and 61 in the N-terminal region, while StsI-HF (high fidelity) and StsI-UHF (ultra-high fidelity) exhibited lower off-target activity in vivo than wild-type StsI and wild-type FokI, partly due to specific amino acid substitutions at positions 141 and 152 in the C-terminal region.

[0366] Certain embodiments therefore relate to proteins. In embodiments, the protein is present in vivo. In other embodiments, the protein comprises a nuclease domain. In one embodiment, the nuclease domain comprises one or more of the cleavage domain of FokI endonuclease (SEQ ID NO: 53), the cleavage domain of 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 biologically active fragments or variants thereof.

[0367] Engineered gene editing proteins containing DNA binding domains containing certain novel repetitive sequences show lower off-target activity in vivo than previously disclosed gene editing proteins, while maintaining high levels of on-target activity in vivo. Some of these engineered gene editing proteins can provide several advantages over previously disclosed gene editing proteins, including, for example, increased flexibility in the linker region connecting the repetitive sequences, which can cause increased binding efficiency. Certain embodiments therefore relate to proteins comprising multiple repetitive sequences. In one embodiment, at least one of the repetitive sequences contains the amino acid sequence: GabG, wherein "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 repetitive sequences are present in the DNA binding domain. In another embodiment, "a" and "b" are each independently selected from the following groups: 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 at the C-terminus of the repetitive sequence. In another embodiment, the amino acid sequence is present at the C-terminus of the repetitive sequence. In an embodiment, one or more Gs in the amino acid sequence GabG are replaced by one or more amino acids other than G (e.g., A, H, or GG). In one embodiment, the length of the repetitive sequence is between about 32 and about 40 amino acids, or between about 33 and about 39 amino acids, or between about 34 and 38 amino acids, or between about 35 and 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 relate to proteins comprising one or more transcription activator-like effector domains. In one embodiment, at least one of the transcription activator-like effector domains comprises a repetitive sequence. Other embodiments relate to a protein comprising a plurality of repetitive sequences produced by inserting one or more amino acids between at least two of the repetitive sequences of the transcription activator-like effector domain. In one embodiment, one or more amino acids are inserted from the C-terminus of at least one repetitive sequence by about 1 or about 2 or about 3 or about 4 or about 5 amino acids. Other embodiments relate to proteins comprising a plurality of repetitive sequences, wherein about every other repetitive sequence has a different length from the repetitive sequence immediately before or after the repetitive sequence. In one embodiment, the length of every other repetitive sequence is about 36 amino acids. In another embodiment, the length of every other repetitive sequence is 36 amino acids. Other embodiments relate to proteins comprising a plurality of repeat sequences, wherein the plurality of repeat sequences comprises at least two repeat sequences, each repeat sequence being at least 36 amino acids in length, and wherein at least two 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 relate to proteins comprising one or more sequences selected from, for example, 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.

[0368] Other embodiments relate to proteins comprising DNA binding domains. In an embodiment, the DNA binding domain comprises a plurality of repetitive sequences. In one embodiment, a plurality of repetitive sequences can highly specifically identify a binding site in a target DNA molecule. In another embodiment, at least two repetitive 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 another embodiment, at least one of the repetitive sequences comprises one or more regions capable of binding to a binding site in a target DNA molecule. In yet another embodiment, the binding site comprises a determined sequence having a length between about 1 and about 5 bases. 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 another embodiment, a plurality of repetitive sequences include at least one repetitive 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 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 joint. In another embodiment, the joint connects the DNA binding domain to the nuclease domain. In another embodiment, the length of the joint is between about 1 and about 10 amino acids. In an embodiment, the length of the joint is about 1, 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 one embodiment, the gene editing protein is capable of producing a nick or double-strand break in a target DNA molecule.

[0369] In an embodiment, the gene editing protein comprises: (a) a DNA binding domain comprising a plurality of repeat sequences, and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 629), wherein "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 empty, 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 a catalytic domain of a protein comprising the amino acid sequence SEQID NO: 632. In an embodiment, at least one of the repeated sequences comprising the amino acid sequence LTPvQVVAIAwxyzGHGG (SEQ ID NO: 629) is between 36 and 39 amino acids in length.

[0370] Certain embodiments relate to methods for modifying the genome of a cell in vivo, the method comprising introducing a nucleic acid molecule encoding a non-naturally occurring fusion protein into a cell in vivo, the fusion protein comprising an artificial transcription activator-like (TAL) effector repeat domain comprising one or more repeat units of 36 amino acids in length and an endonuclease domain, wherein the repeat domain is engineered to recognize a predetermined nucleotide sequence, and wherein the fusion protein recognizes a predetermined nucleotide sequence. In one embodiment, the cell is a eukaryotic cell. In another embodiment, the cell is an animal cell. In another 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 endonucleolytic cleavage in the nucleic acid of the cell, thereby modifying the genome of the cell.

[0371] Certain embodiments relate to compositions comprising nucleic acids for changing the DNA sequence of cells in vivo, wherein the nucleic acids encode gene editing proteins. Other embodiments relate to compositions comprising a mixture of nucleic acids for changing the DNA sequence of cells in vivo, wherein the mixture of nucleic acids comprises: a first nucleic acid encoding a first gene editing protein, and a second nucleic acid encoding 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 in the same target DNA molecule. In another embodiment, the binding site of the first gene editing protein is less than about 50 bases, or less than about 40 bases, or less than about 30 bases, or less than about 20 bases, or less than about 10 bases, or about 10 bases to about 25 bases or about 15 bases apart from the binding site of the second gene editing protein. In one embodiment, the nuclease domain of the first gene editing protein and the nuclease domain of the second gene editing protein can form a dimer. In another embodiment, the dimer can produce a nick or double-strand break in the target DNA molecule.

[0372] Certain embodiments relate to therapeutic compositions. Other embodiments relate to cosmetic compositions. In an embodiment, the composition comprises a repair template. In another embodiment, the repair template is a single-stranded DNA molecule or a double-stranded DNA molecule.

[0373] Other embodiments relate to products for synthesizing proteins or nucleic acids encoding proteins. In one embodiment, the product is a nucleic acid. In another embodiment, the protein comprises a DNA binding domain. In another 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 repetitive sequences. In another embodiment, the nucleic acid encodes a plurality of repetitive sequences. In another embodiment, the nuclease domain is selected from FokI, StsI, StsI-HA, StsI-HA2, StsI-UHA, StsI-UHA2, StsI-HF and StsI-UHF or its natural or engineered variants or biologically active fragments. 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 another 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 an in vitro transcription template.

[0374] Certain embodiments relate to methods for inducing cells to express proteins in vivo. Other embodiments relate to methods for changing the DNA sequence of cells in vivo, comprising transfecting cells in vivo with gene editing proteins or inducing cells to express gene editing proteins in vivo. Other embodiments relate to methods for reducing the expression of target proteins in cells in vivo. In one embodiment, cells are induced to express gene editing proteins, wherein the gene editing proteins are capable of producing nicks or double-strand breaks in target DNA molecules. In another embodiment, the nicks or double-strand breaks lead to gene inactivation. Other embodiments relate to methods for producing inactive, reduced activity, or dominant negative forms of proteins in vivo. In one embodiment, the protein is a survival protein. Other embodiments relate to methods for repairing one or more mutations in cells in vivo. In one embodiment, cells are contacted with a repair template. In another embodiment, the repair template is a DNA molecule. In another embodiment, the repair template does not contain a binding site for a gene editing protein. In yet another embodiment, the repair template encodes an amino acid sequence encoded by a DNA sequence comprising a gene editing protein binding site.

[0375] 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.

[0376] In various embodiments, the mass ratio of RNA (e.g., synthetic RNA encoding a gene editing protein) to 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.

[0377] In various embodiments, the molar ratio of RNA (e.g., synthetic RNA encoding a gene editing protein) to 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.

[0378] In multiple embodiments, the repair template has a dual function, causing repair of the target sequence for gene editing and preventing further binding of the gene editing protein, thereby reducing or eliminating further gene editing (e.g., causing repair via the repair template so that the gene editing protein binding site is no longer suitable for gene editing protein binding). Therefore, in some embodiments, the gene editing method of the present invention is adjustable to ensure single gene editing of each target site.

[0379] Reprogramming

[0380] In various aspects, the present invention relates to a method for reprogramming differentiated cells to a less differentiated state, comprising (a) providing differentiated cells or non-pluripotent cells; (b) culturing differentiated cells or non-pluripotent cells; and (c) transfecting differentiated cells or non-pluripotent cells with a complex of one or more synthetic RNA molecules and a compound described herein (e.g., a compound of Formula I-XVI), wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors and wherein the transfection causes the cell to express one or more reprogramming factors, so that the cell is reprogrammed to a less differentiated state. In embodiments, step (c) is performed in the presence of a culture medium containing a component that supports the reprogramming of differentiated cells to a less differentiated state. In embodiments, step (c) is also included in repeating step (c) at least twice within 5 consecutive days. In embodiments, the amount of one or more synthetic RNA molecules transfected in one or more later transfections is greater than the amount transfected in one or more earlier transfections. In embodiments, steps (a)-(c) are performed without using feeder cells in the presence of feeder cell conditioned medium. In embodiments, step (c) is performed without using irradiated human neonatal fibroblast feeder cells in the presence of feeder cell conditioned medium. In an embodiment, the synthetic RNA molecule encodes one or more reprogramming factors selected from the group consisting of 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.

[0381] In embodiments, the differentiated or non-pluripotent cells are derived from a biopsy. In embodiments, the differentiated or non-pluripotent cells are derived from a human subject. In embodiments, the differentiated or non-pluripotent cells are derived from a skin punch biopsy sample. In embodiments, the differentiated or non-pluripotent cells are keratinocytes, fibroblasts, or PBMCs.

[0382] In an embodiment, the method for reprogramming further comprises contacting the cell with at least one member of the following group: poly-L-lysine, poly-L-ornithine, RGD peptide, fibronectin, vitronectin, collagen, and laminin.

[0383] In embodiments, the method for reprogramming uses a culture medium that is substantially free of immunosuppressants.

[0384] Cells can be reprogrammed by exposing them to specific extracellular signals (cue) and / or by ectopic expression of specific proteins, microRNAs, etc. Although several reprogramming methods have been described previously, most methods relying on ectopic expression require the introduction of exogenous DNA, which can have a risk of mutation. A DNA-free reprogramming method based on direct delivery of reprogramming proteins has been reported. However, these methods are too inefficient and unreliable for commercial use. In addition, RNA-based reprogramming methods have been described (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; The contents of all the documents are incorporated herein by reference). However, existing RNA-based reprogramming methods are slow, unreliable, and inefficient when performed on adult cells, require multiple transfections (leading to significant expense and opportunities for error), can only reprogram a limited number of cell types, can only reprogram cells into a limited number of cell types, require the use of immunosuppressants, and require the use of multiple human-derived components, including blood-derived HSA and human fibroblast feeder layers. The many shortcomings of previously disclosed RNA-based reprogramming methods make them unsuitable for research, therapeutic, or cosmetic purposes.

[0385] Reprogramming can be performed by transfecting cells 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 its biologically active fragments, analogs, variants and family members. Certain embodiments therefore relate to methods for reprogramming cells in vivo. In one embodiment, cells in vivo are reprogrammed by transfecting cells with one or more nucleic acids encoding one or more reprogramming factors. In one embodiment, one or more nucleic acids include RNA molecules encoding Oct4 protein. In another embodiment, 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, one or more nucleic acids also include RNA molecules encoding Lin28 protein. In one embodiment, the cell is a human skin cell, and the human skin cell is reprogrammed into a pluripotent stem cell. In another embodiment, the cell is a human skin cell, and the human skin cell is reprogrammed to a glucose-responsive insulin-producing cell. Other cells and cells that 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, cardiac cells, airway epithelial cells, neural stem cells, neurons, glial cells, osteocytes, blood cells, and dental stem cells. In one embodiment, the cell contacts the culture medium that supports the reprogrammed cells. In one embodiment, the culture medium also supports the cells.

[0386] Importantly, it has been reported that infection of skin cells with viruses encoding Oct4, Sox2, Klf4, and c-Myc, combined with culturing cells in a medium that supports the growth of cardiomyocytes, can cause skin cells to be reprogrammed into cardiomyocytes without first reprogramming the skin cells into pluripotent stem cells (see Efs et al., Nat Cell Biol. 2011; 13: 215-22, the contents of which are incorporated herein by reference). In some cases, direct reprogramming (reprogramming one 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, additional processing involved in establishing and characterizing stable pluripotent stem cell lines can present an increased risk of contamination, and additional time in culture associated with first generating pluripotent stem cells can present an increased risk of genomic instability and acquisition of mutations (including point mutations, copy number variations, and karyotype abnormalities). Certain embodiments therefore relate to methods for reprogramming somatic cells in vivo, wherein the cells are reprogrammed into somatic cells, and wherein no characterized pluripotent stem cell lines are generated.

[0387] In some cases, the total transfection required according to the method reprogramming cells of the present invention may be less than that required according to other methods. Some embodiments therefore relate to a method for reprogramming cells in vivo, wherein a transfection between about 1 and about 12 times is performed during a continuous period of about 20 days, or a transfection between about 4 and about 10 times is performed during a continuous period of about 15 days, or a transfection between about 4 and about 8 times is performed during a continuous period of about 10 days. It is recognized that when a cell contacts a culture medium containing a nucleic acid molecule, the cell may contact and / or internalize more than one nucleic acid molecule simultaneously or at different times. Even when a cell contacts a culture medium containing a nucleic acid only once, the cell may therefore contact more than one nucleic acid, such as repeatedly contacting.

[0388] It is noteworthy that the nucleic acid may contain one or more atypical or "modified" residues as described herein. For example, any of the atypical nucleotides described herein can be used in the reprogramming method of the present invention. In one embodiment, pseudouridine-5'-triphosphate can replace uridine-5'-triphosphate in an in vitro transcription reaction to produce a synthetic RNA, wherein up to 100% of the uridine residues of the synthetic RNA can be replaced by pseudouridine residues. Even when pseudouridine and 5-methylcytidine completely replace uridine and cytidine, respectively, in vitro transcription can also produce RNA with residual immunogenicity (see, for example, Angel Reprogramming Human Somatic Cells to Pluripotency Using RNA [Doctoral Thesis]. Cambridge, MA: MIT; 2011, the contents of the document are incorporated herein by reference). For this reason, when cells are transfected with RNA, immunosuppressants are usually added to the transfection medium. In some cases, it may not be desirable to add an immunosuppressant to the transfection medium, in part because the most commonly used recombinant immunosuppressant B18R for this purpose is expensive and difficult to manufacture. In vivo cells can be transfected and / or reprogrammed according to the methods of the present invention without the use of B18R or any other immunosuppressant. Reprogramming in vivo cells according to the methods of the present invention without the use of immunosuppressants can be fast, effective and reliable. Therefore, certain embodiments relate to methods for transfecting in vivo cells, wherein the transfection medium does not contain an immunosuppressant. Other embodiments relate to methods for reprogramming in vivo cells, wherein the transfection medium does not contain an immunosuppressant. In some cases, such as when high cell density is used, it can be beneficial to add an immunosuppressant to the transfection medium. Therefore, certain embodiments relate to methods for transfecting in vivo cells, wherein the transfection medium contains an immunosuppressant. Other embodiments relate to methods for reprogramming in vivo cells, wherein the transfection medium contains an immunosuppressant. In one embodiment, the immunosuppressant 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 immunosuppressant, and the nucleic acid dose is selected to prevent excessive toxicity. In another embodiment, the nucleic acid dose is less than about 1 mg / cm 2 Tissue or less than about 1 mg / 100,000 cells or less than about 10 mg / kg.

[0389] The reprogrammed cells produced according to certain embodiments of the present invention are suitable for treatment and / or cosmetic applications because they do not contain undesirable exogenous DNA sequences, and they are not exposed to animal-derived or human-derived products, which may be undefined, and which may contain toxic pollutants and / or pathogenic pollutants. In addition, the high speed, high efficiency and reliability of certain embodiments of the present invention can reduce the risk of obtaining and accumulating mutations and other chromosomal abnormalities. Certain embodiments of the present invention can therefore be used to produce cells with safety features suitable for use in treatment and / or cosmetic applications. For example, using RNA and the culture medium of the present invention to reprogram cells, wherein the culture medium does not contain animal or human-derived components, cells that are not exposed to allogeneic materials can be produced. Certain embodiments therefore relate to reprogrammed cells with desired safety features. In one embodiment, the reprogrammed cells have a normal karyotype. In another embodiment, the reprogrammed cells have less than about 5 copy number variations (CNVs) relative to the patient's genome, such as less than about 3 copy number variations relative to the patient's genome, or there is no copy number variation relative to the patient's genome. In yet another embodiment, the reprogrammed cell has a normal karyotype and less than about 100 single nucleotide variants in the coding region relative to the patient's genome, or less than about 50 single nucleotide variants in the coding region relative to the patient's genome, or less than about 10 single nucleotide variants in the coding region relative to the patient's genome.

[0390] Endotoxins and nucleases can be co-purified and / or become associated with other proteins (such as serum albumin). Specifically, recombinant proteins can often have high levels of associated endotoxins and nucleases, partly due to cell lysis that occurs during their production. Endotoxins and nucleases can be reduced, removed, replaced or otherwise inactivated by many methods of the present invention, including, for example, by acetylation; by adding stabilizers such as sodium octanoate, followed by heat treatment; by adding nuclease inhibitors to albumin solutions and / or culture media; by crystallization; by contact with one or more ion exchange resins; by contact with charcoal; by preparative electrophoresis or by affinity chromatography. Partially or completely reducing, removing, replacing or otherwise inactivating endotoxins and / or nucleases from culture media and / or from one or more components of culture media can increase the efficiency of cells that can be transfected and reprogrammed. Certain embodiments therefore relate to methods for transfecting cells in vivo using one or more nucleic acids, wherein the transfection culture media is treated to partially or completely reduce, remove, replace or otherwise inactivate one or more endotoxins and / or nucleases. Other embodiments relate to culture media that cause minimal degradation of nucleic acids. In one embodiment, the culture medium contains less than about 1 EU / mL, or less than about 0.1 EU / mL, or less than about 0.01 EU / mL.

[0391] In some cases, protein-based lipid carriers such as serum albumin can be replaced by non-protein-based lipid carriers such as methyl-β-cyclodextrin. The culture medium of the present invention can also be used without a lipid carrier, for example, when using a method that may not need or may not benefit from the presence of a lipid carrier, for example, using one or more lipid-based transfection reagents, polymer-based transfection reagents, or peptide-based transfection reagents or using electroporation to transfect. Many protein-associated molecules (such as metals) can be highly toxic to cells in vivo. This toxicity can cause a decrease in vigor and obtain mutations. Some embodiments therefore have the additional benefit of producing cells that do not contain toxic molecules.

[0392] The associated molecular components of a protein can be measured by suspending the protein in a solution and measuring the conductivity of the solution. Certain embodiments therefore relate to a culture medium containing a protein wherein about 10% of the protein in water has a conductivity of less than about 500 μmho / cm. In one embodiment, the conductivity of the solution is less than about 50 μmho / cm. In another embodiment, less than about 0.65% of the dry weight of the protein comprises lipids and / or less than about 0.35% of the dry weight of the protein comprises free fatty acids.

[0393] The amount of nucleic acid delivered to cells in vivo can be increased to increase the required effect of nucleic acid. However, increasing the amount of nucleic acid delivered to cells in vivo will cause the viability of cells to decrease beyond a certain point, partly due to the toxicity of transfection reagents. When nucleic acid is delivered to a cell group (e.g., cells in a tissue region) in a fixed volume, the amount of nucleic acid delivered to each cell can depend on the total amount of nucleic acid delivered to the cell group and the density of cells, wherein higher cell density causes the nucleic acid delivered to each cell to be less. In certain embodiments, cells in vivo are transfected with one or more nucleic acids more than once. Under certain conditions, for example, when cells are proliferating, cell density may change between a transfection to the next transfection. Therefore, some embodiments relate to a method for transfecting cells in vivo with nucleic acids, wherein transfected cells are more than once, and wherein the amount of nucleic acid delivered to cells is different for two transfections. In one embodiment, cells proliferate between two transfections, and the amount of nucleic acid delivered to cells for the second time in two transfections is larger than for the first time in two transfections. In another embodiment, the cell is transfected more than twice, and the amount of nucleic acid delivered to the cell for the second of three transfections is greater than for the first of the same three transfections, and the amount of nucleic acid delivered to the cell for the third of the same three transfections is greater than for the second of the same three transfections. In yet another embodiment, the cell is transfected more than once, and for at least two consecutive transfections, the maximum amount of nucleic acid delivered to the cell during each transfection is low enough to produce at least about 80% viability.

[0394] The amount of nucleic acid delivered to the in vivo proliferating cell group in a series of transfections is regulated to cause the effect of nucleic acid to increase and the viability of cells to increase. In some cases, when in vivo cells are contacted with one or more nucleic acids encoding one or more reprogramming factors in a series of transfections, for at least part of a series of transfections, when the amount of nucleic acid delivered in the transfection behind is greater than the amount of nucleic acid delivered in the transfection ahead, the efficiency of reprogramming can be increased. Some embodiments are therefore related to a method for reprogramming in vivo cells, wherein one or more nucleic acids are repeatedly delivered to cells in a series of transfections, and the amount of nucleic acid delivered to cells for at least one subsequent transfection is greater than for at least one preceding transfection. In one embodiment, transfected cells are between about 2 and about 10 times, or between about 3 and about 8 times, or between about 4 and about 6 times. In another embodiment, one or more nucleic acids include at least one RNA molecule, transfected cells are between about 2 and about 10 times, and the amount of nucleic acid delivered to cells in each transfection is the same or greater than the amount of nucleic acid delivered to cells in the previous most recent transfection. In yet another embodiment, the amount of nucleic acid delivered to the cell in the first transfection is between about 20 ng / cm 2 About 250ng / cm 2 , or 100ng / cm 2 and 600ng / cm 2 In yet another embodiment, the cells are transfected about 5 times at intervals between about 12 hours and about 48 hours, and the amount of nucleic acid delivered to the cells for the first transfection is about 25 ng / cm 2 , about 50 ng / cm for the second transfection 2 , about 100 ng / cm for the third transfection 2 , about 200 ng / cm for the fourth transfection 2 , and about 400 ng / cm for the fifth transfection 2 In yet another embodiment, the cells are further transfected at least once after the fifth transfection, and the amount of nucleic acid delivered to the cells is about 400 ng / cm 2 .

[0395] Certain embodiments relate to methods for transfecting cells in vivo with nucleic acids, wherein the amount of nucleic acid is determined by measuring cell density and selecting the amount of nucleic acid to transfect based on the measured value of cell density. In one embodiment, the cell density is measured optically. In another embodiment, cells are repeatedly transfected, the cell density increases between two transfections, and the amount of nucleic acid transfected for the second of the two transfections is greater than for the first of the two transfections.

[0396] By administering nucleic acid to a patient at multiple administration sites, the amount of circulating protein produced in the patient can be increased. In certain embodiments, the amount of circulating protein is increased relative to the amount of circulating protein produced in the patient by administering nucleic acid to the patient at a single injection site. In one embodiment, administration is by injection. In another embodiment, the injection is intradermal injection. In yet another embodiment, the injection is subcutaneous or intramuscular injection. In embodiments, multiple administration sites include administration sites in the skin. In other embodiments, multiple administration sites are 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, 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 a circulating protein is increased by at least about 10%, or at least about 20%, or at least about 50%, or at least about 100%, 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 greater than 1000-fold.

[0397] In some cases, the in vivo transfection efficiency and viability of cells contacted with the culture medium of the present invention can be improved by adjusting the culture medium. Certain embodiments therefore relate to methods for adjusting the culture medium. Other embodiments relate to regulated culture medium. In one embodiment, the feeder layer is a fibroblast, and the culture medium is adjusted for about 24 hours. Other embodiments relate to methods for transfecting cells in vivo, wherein the transfection culture medium is adjusted. Other embodiments relate to methods for reprogramming and / or gene editing cells in vivo, wherein the culture medium is adjusted. In one embodiment, the mitotic inactivation feeder layer is, for example, by exposure to chemicals such as mitomycin-C or by exposure to gamma radiation. In certain embodiments, it may be beneficial to use only autologous materials to partially (for example and not wishing to be bound by theory) avoid the risk of disease transmission from the feeder layer to cells or patients. Certain embodiments therefore relate to methods for transfecting cells in vivo, wherein the transfection culture medium is adjusted, and wherein the feeder layer is derived from the same individual as the transfected cell. Other embodiments relate to methods for reprogramming and / or gene editing cells in vivo, wherein the culture medium is adjusted, and wherein the feeder layer is derived from the same individual as the reprogrammed and / or gene edited cell.

[0398] Can be added to culture medium by adjusting some molecules.Some embodiments therefore relate to culture medium supplemented with one or more molecules present in conditioned medium.In one embodiment, culture medium is supplemented with Wnt1, Wnt2, Wnt3, Wnt3a or its biologically active fragment, analog, variant, agonist or family member.In another embodiment, culture medium is supplemented with TGF-β or its biologically active fragment, analog, variant, agonist or family member.In another embodiment, according to the method for reprogramming in vivo cells of the present invention, wherein culture medium is not supplemented with TGF-β between about 1 day and about 5 days, and then supplements TGF-β for at least about 2 days.In another embodiment, culture medium is supplemented with IL-6, IL-6R or its biologically active fragment, analog, variant, agonist or family member.In another embodiment, culture medium is supplemented with sphingolipid or fatty acid.In another embodiment, sphingolipid is lysophosphatidic acid, lyso-nerve sphingomyelin, sphingosine-1 phosphate or its biologically active analog, variant or derivative.

[0399] In addition to mitotic inactivated cells, under certain conditions, radiation can change the gene expression of cells, causing cells to produce less certain proteins and more certain other proteins than non-radiated cells, such as members of the Wnt protein family. In addition, some members of the Wnt protein family can promote the growth and transformation of cells. In some cases, by contacting the culture medium regulated by the feeder layer treated with radiation feeder layer instead of mitomycin-c in vivo, the efficiency of reprogramming can be greatly increased. The increase in reprogramming efficiency observed when using the feeder layer of radiation is caused in part by the Wnt protein secreted by the feeder layer. Some embodiments therefore relate to a method for reprogramming cells in vivo, wherein cells are contacted with Wnt1, Wnt2, Wnt3, Wnt3a or its biologically active fragments, analogs, variants, family members or agonists (including agonists of downstream targets of Wnt proteins and / or agents (such as 2-amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyl)pyrimidine)) of simulating one or more biological effects of Wnt proteins.

[0400] Because many DNA-based reprogramming methods are inefficient, these methods may be difficult or impossible to use with cells derived from patient samples, and the patient samples may contain only a small amount of cells. In contrast, the high efficiency of certain embodiments of the present invention allows a small amount of cells (including single cells) to be reliably reprogrammed. Certain embodiments relate to methods for reprogramming a small amount of cells. Other embodiments relate to methods for reprogramming single cells. In one embodiment, cells are contacted with one or more enzymes. In another embodiment, the enzyme is collagenase. In another embodiment, collagenase does not contain animal components. In one embodiment, collagenase is present in a concentration between about 0.1 mg / mL and about 10 mg / mL or about 0.5 mg / mL and about 5 mg / mL. In another embodiment, cells are blood cells. In another embodiment, cells are contacted with a culture medium containing one or more proteins derived from patient blood. In yet another embodiment, the cells are contacted with a culture medium comprising: DMEM / F12 + 2 mM L-alanyl-L-glutamine + between about 5% and about 25% patient-derived serum, or between about 10% and about 20% patient-derived serum, or about 20% patient-derived serum.

[0401] In some cases, using the culture medium of the present invention to transfect cells in vivo with a mixture of RNA encoding Oct4, Sox2, Klf4 and c-Myc can cause an increase in cell proliferation rate. When the amount of the RNA delivered to the cell is too low to ensure that all cells are transfected, only a portion of the cells can show an increased proliferation rate. In some cases, such as when producing a personalized therapeutic agent, it may be desirable to increase the proliferation rate of the cell, partly because doing so can reduce the time required for producing the therapeutic agent, and therefore the cost of the therapeutic agent can be reduced. Some embodiments therefore relate to a method for transfecting cells in vivo with a mixture of RNA encoding Oct4, Sox2, Klf4 and c-Myc. In one embodiment, the cell shows an increased proliferation rate. In another embodiment, the cell is reprogrammed.

[0402] Also provided are methods for simultaneously or sequentially gene editing and reprogramming somatic cells as described herein.

[0403] Treatment

[0404] In embodiments, the invention relates to methods of treating a disease or condition by delivering a therapeutic agent with a compound of the invention (eg, a compound of Formula I-XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier.

[0405] In embodiments, the invention relates to methods of treating a disease or condition by delivering a nucleic acid therapeutic with a compound of the invention (e.g., a compound of Formula I-XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier.

[0406] In an embodiment, the invention relates to a method of treating a disease or condition by delivering an RNA (e.g., synthetic RNA) therapeutic agent with a compound of the invention (e.g., a compound of Formula I-XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier.

[0407] In an embodiment, the invention relates to a method of treating a disease or condition by delivering a therapeutic agent (e.g., a nucleic acid, e.g., an RNA (e.g., a synthetic RNA) therapeutic agent) having a compound of the invention (e.g., a compound of Formula I-XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier in a manner that expresses a target protein with a therapeutic effect.

[0408] In an embodiment, the invention relates to a method of treating a disease or condition by delivering a therapeutic agent (e.g., a nucleic acid, e.g., an RNA (e.g., a synthetic RNA) therapeutic agent) having a compound of the invention (e.g., a compound of Formula I-XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier in a method of expressing a gene editing protein with a therapeutic effect (e.g., gene editing or gene correction, e.g., in vivo and / or ex vivo).

[0409] In an embodiment, the invention relates to a method of treating a disease or condition by delivering a therapeutic agent (e.g., a nucleic acid, e.g., an RNA (e.g., a synthetic RNA) therapeutic agent) having a compound of the invention (e.g., a compound of Formula I-XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier in a method of expressing reprogramming factors having a therapeutic effect (e.g., ex vivo).

[0410] In an embodiment, the invention relates to a method of treating a disease or condition by delivering a therapeutic agent (e.g., a nucleic acid, e.g., an RNA (e.g., a synthetic RNA) therapeutic agent) having a compound of the invention (e.g., a compound of Formula I-XVI) and / or a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier in a method of expressing a gene editing protein with a therapeutic effect (e.g., gene editing or gene correction, e.g., in vivo and / or ex vivo) and expressing a reprogramming factor with a therapeutic effect (e.g., ex vivo).

[0411] Certain embodiments relate to methods for treating patients, comprising: a. inducing cells to express target proteins by transfecting cells in vivo with nucleic acids encoding target proteins and / or b. reprogramming cells in vivo. In one embodiment, cells are reprogrammed to a state with a lower degree of differentiation. In another embodiment, cells are reprogrammed by transfecting cells with one or more synthetic RNA molecules encoding one or more reprogramming proteins. In another embodiment, cells are differentiated. In yet another embodiment, cells differentiate into skin cells, glucose-responsive insulin-producing cells, hematopoietic cells, heart cells, retinal cells, kidney cells, neural cells, stromal cells, adipocytes, bone cells, muscle cells, oocytes, and sperm cells. Other embodiments relate to methods for treating patients, comprising: a. inducing cells to express gene editing proteins by transfecting cells in vivo with nucleic acids encoding gene editing proteins and / or b. reprogramming cells in vivo.

[0412] In embodiments, treatment results in improvement of one or more symptoms in the patient.

[0413] Certain embodiments relate to methods and compositions for treating rare diseases. In embodiments, the rare disease is one or more of the following: rare metabolic diseases, rare cardiovascular diseases, rare skin diseases, rare nervous system diseases, rare developmental diseases, rare genetic diseases, rare lung diseases, rare liver diseases, rare kidney diseases, rare psychiatric diseases, rare reproductive system diseases, rare musculoskeletal diseases, rare orthopedic diseases, inborn errors of metabolism, lysosomal storage diseases, and rare ophthalmic diseases.

[0414] Examples of diseases that can be treated with the present invention include, but are not limited to, Alzheimer's disease, spinal cord injury, amyotrophic lateral sclerosis, cystic fibrosis, heart disease, 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.

[0415] Other examples of diseases that can be treated with the present invention include, but are not limited to, type 1 diabetes, heart disease, 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, including hepatitis and HIV / AIDS.

[0416] In embodiments, examples of diseases that can be treated with the present invention include infectious diseases. In some embodiments, infectious diseases are pathogens, and the pathogens are optionally selected from bacteria, viruses, fungi or parasites. In some embodiments, the virus is: (a) influenza virus, optionally selected from influenza A, B, C and D, or (b) a member of the Coronavirus family, optionally selected from beta coronavirus, optionally 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 coronavirus, optionally selected from HCoV-NL63 and HCoV-229E. In some embodiments, the virus is SARS-CoV-2. In some embodiments, as described elsewhere herein, the target protein is an antigen such as a 2019-nCoV protein, an antigenic fragment thereof, or a nucleic acid encoding it, and the target protein is optionally selected from a spike surface glycoprotein, a membrane glycoprotein M, an envelope protein E, and a nucleocapsid phosphoprotein N. In some embodiments, as described elsewhere herein, the protein of interest is an antigen such as the S1 or S2 subunit of the spike surface glycoprotein, or an antigenic fragment thereof.

[0417] In various embodiments, the subject suffers from coronavirus disease 2019 (COVID-19). In further embodiments, the subject is elderly and / or suffers from one or more comorbidities, including but not limited to hypertension and / or diabetes. Subjects suffering from coronavirus infection may experience symptoms, including but not limited to fever, fatigue, dry cough, pain, shortness of breath and other breathing difficulties, diarrhea, upper respiratory tract symptoms (e.g., sneezing, runny nose, nasal congestion, cough, sore throat), pneumonia, pneumonitis respiratory failure, liver and kidney dysfunction, acute respiratory distress syndrome (ARDS), and cytokine imbalance.

[0418] In some embodiments, the virus is an influenza virus. In some embodiments, as described elsewhere herein, the target protein is an antigen such as an influenza virus antigen, optionally selected from hemagglutinin (HA) protein, matrix 2 (M2) protein and neuraminidase, or antigenic fragments thereof, or nucleic acids encoding them.

[0419] In embodiments, the disease or condition 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, varicella (chickenpox), rotavirus, and herpes zoster. In some embodiments, the invention relates to the treatment of hepatitis. Illustrative hepatitis that can be treated include, but are not limited to, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, autoimmune hepatitis, alcoholic hepatitis, acute hepatitis, and chronic hepatitis.

[0420] In embodiments, the disease or disorder is a metabolic disorder. In embodiments, the metabolic disorder is selected from a carbohydrate metabolism disorder, an amino acid metabolism disorder, a urea cycle disorder, a fatty acid metabolism disorder, a porphyrin metabolism disorder, a lysosomal storage disorder, a peroxisome biogenesis disorder, and a purine or pyrimidine metabolism disorder.

[0421] In embodiments, the metabolic disorder is a carbohydrate metabolism disorder and wherein the disease is galactosemia and the defective gene is optionally GALT, GALK1 or GALE; wherein the disease is primary fructosuria and the defective gene is optionally KHK; wherein the disease is hereditary fructose intolerance and the defective gene is optionally ALDOB; wherein the disease is glycogen storage disease type I and the defective gene is optionally G6PC, SLC37A4 or SLC17A3; wherein the disease is glycogen storage disease type II and the defective gene is optionally GAA; wherein the disease is glycogen storage disease type I wherein the disease is glycogen storage disease type II and the defective gene is optionally AGL; wherein the disease is glycogen storage disease type IV and the defective gene is optionally GBE1; wherein the disease is glycogen storage disease type V and the defective gene is optionally PYGM; wherein the disease is glycogen storage disease type VI and the defective gene is optionally PYGL; wherein the disease is glycogen storage disease type VII and the defective gene is optionally PYGM; wherein the disease is glycogen storage disease type IX and the defective gene is optionally PHKA1, PHKA2, PHKB, PHKG1 or PHKG2; wherein the disease is glycogen storage disease XI wherein the disease is glycogen storage disease type XII and the defective gene is optionally ALDOA; wherein the disease is glycogen storage disease type XIII and the defective gene is optionally ENO1, ENO2 or ENO3; wherein the disease is glycogen storage disease type 0 and the defective gene is optionally GYS1 or GYS2; wherein the disease is pyruvate carboxylase deficiency and the defective gene is optionally PC; wherein the disease is pyruvate kinase deficiency and the defective gene is optionally PKLR; wherein the disease is transaldolase deficiency and the defective gene Optionally, it is TALDO1; wherein the disease is triosephosphate isomerase deficiency and the defective gene is optionally TPI1; wherein the disease is fructose bisphosphatase deficiency and the defective gene is optionally FBP1; wherein the disease is hyperoxaluria and the defective gene is optionally AGXT or GRHPR; wherein the disease is hexokinase deficiency and the defective gene is optionally HK1; wherein the disease is glucose-galactose malabsorption and the defective gene is optionally SLC5A1; or wherein the disease is glucose-6-phosphate dehydrogenase deficiency and the defective gene is optionally G6PD.

[0422] In embodiments, the metabolic disorder is an amino acid metabolism disorder, wherein the disease is alkaptonuria and the defective gene is optionally HGD; wherein the disease is aspartylglucosaminuria and the defective gene is optionally AGA; wherein the disease is methylmalonic acidemia and the defective gene is optionally MUT, MCEE, MMAA, MMAB, MMACHC, MMADHC, or LMBRD1; wherein the disease is maple syrup urine disease and the defective gene is optionally BCKDHA, BCKDHB, DBT, or DLD; wherein the disease is homocystinuria and the defective gene is optionally CBS; wherein the disease is tyrosinemia and the defective gene is optionally FAH, TAT, or HPD; wherein the disease is Trimethylaminuria and the defective gene is optionally FMO3; wherein the disease is Hartnup disease disease) and the defective gene is optionally SLC6A19; wherein the disease is biotinidase deficiency and the defective gene is optionally BTD; wherein the disease is ornithine carbamoyltransferase deficiency and the defective gene is optionally OTC; wherein the disease is carbamoyl phosphate synthase I deficiency and the defective gene is optionally CPS1; wherein the disease is citrullinemia and the defective gene is optionally ASS or SLC25A13; wherein the disease is hyperargininemia and the defective gene is optionally ARG1; wherein the disease is hyperhomocysteinemia and the defective gene is optionally MTHFR; wherein the disease is hypermethioninemia and the defective gene is optionally MAT1A, GNMT or AHCY; wherein the disease is hyperlysinemia and the defective gene is optionally AASS wherein the disease is nonketotic hyperglycinemia and the defective gene is optionally GLDC, AMT or GCSH; wherein the disease is propionic acidemia and the defective gene is optionally PCCA or PCCB; wherein the disease is hyperprolinemia and the defective gene is optionally ALDH4A1 or PRODH; wherein the disease is cystinuria and the defective gene is optionally SLC3A1 or SLC7A9; wherein the disease is dicarboxylate aminoaciduria and the defective gene is optionally SLC1A1; wherein the disease is glutaric acidemia type 2 and the defective gene is optionally ETFA, ETFB or ETFDH; wherein the disease is isovaleric acidemia and the defective gene is optionally IVD; or wherein the disease is 2-hydroxyglutaric aciduria and the defective gene is optionally L2HGDH or D2HGDH.

[0423] In embodiments, the metabolic disorder is a urea cycle disorder, wherein the disease is N-acetylglutamate synthetase deficiency and the defective gene is optionally NAGS; wherein the disease is argininosuccinic aciduria and the defective gene is optionally ASL; or wherein the disease is argininemia and the defective gene is optionally ARG1.

[0424] In an embodiment, the metabolic disorder is a fatty acid metabolism disorder, wherein the disease is very long chain acyl-CoA dehydrogenase deficiency and the defective gene is optionally ACADVL; wherein the disease is long chain 3-hydroxyacyl-CoA dehydrogenase deficiency and the defective gene is optionally HADHA; wherein the disease is medium chain acyl-CoA dehydrogenase deficiency and the defective gene is optionally ACADM; wherein the disease is short chain acyl-CoA dehydrogenase deficiency and the defective gene is optionally ACADS; wherein the disease is 3-hydroxyacyl-CoA dehydrogenase deficiency and the defective gene is optionally HADH; wherein the disease is 2,4 dienoyl-CoA reductase deficiency and the defective gene is optionally NADK2; wherein the disease is 3-hydroxy-3-methylglutaryl-CoA wherein the disease is carnitine palmitoyltransferase deficiency and the defective gene is optionally HMGCL; wherein the disease is malonyl-CoA decarboxylase deficiency and the defective gene is optionally MLYCD; wherein the disease is systemic primary carnitine deficiency and the defective gene is optionally SLC22A5; wherein the disease is carnitine-acylcarnitine translocase deficiency and the defective gene is optionally SLC25A20; wherein the disease is carnitine palmitoyltransferase I deficiency and the defective gene is optionally CPT1A; wherein the disease is carnitine palmitoyltransferase II deficiency and the defective gene is optionally CPT2; wherein the disease is lysosomal acid lipase deficiency and the defective gene is optionally LIPA; or wherein the disease is Gaucher's disease and the defective gene is optionally GBA.

[0425] In embodiments, the metabolic disorder is a porphyrin metabolism disorder, wherein the disease is acute intermittent porphyria and the defective gene is optionally HMBS; wherein the disease is Gunther disease and the defective gene is optionally UROS; wherein the disease is porphyria cutanea tarda and the defective gene is optionally UROD; wherein the disease is hepatoerythropoietic porphyria and the defective gene is optionally UROD; wherein the disease is hereditary coproporphyria and the defective gene is optionally CPOX; wherein the disease is variegated porphyria and the defective gene is optionally PPOX; wherein the disease is erythropoietic protoporphyria and the defective gene is optionally FECH; or wherein the disease is aminolevulinic acid dehydratase deficiency porphyria and the defective gene is optionally ALAD.

[0426] In embodiments, the metabolic disorder is a lysosomal storage disorder, wherein the disease is Farber disease and the defective gene is optionally ASAH1; wherein the disease is Krabbe disease and the defective gene is optionally GALC; wherein the disease is galactosialidosis and the defective gene is optionally CTSA; wherein the disease is fabry disease and the defective gene is optionally GLA; wherein the disease is Schindler disease and the defective gene is optionally NAGA; wherein the disease is GM1 gangliosidosis and the defective gene is optionally GLB1; wherein the disease is Tay-Sachs disease and the defective gene is optionally HEXA; wherein the disease is Sandhoff disease and the defective gene is optionally HEXB; wherein the disease is GM2-gangliosidosis, AB variant and the defective gene is optionally GM2A; wherein the disease is Niemann-Pick disease disease) and the defective gene is optionally SMPD1, NPC1 or NPC2; wherein the disease is metachromatic leukodystrophy and the defective gene is optionally ARSA or PSAP; wherein the disease is polysulfatase deficiency and the defective gene is optionally SUMF1; wherein the disease is Hurler syndrome and the defective gene is optionally IDUA; wherein the disease is Hunter syndrome and the defective gene is optionally IDS; wherein the disease is Sanfilippo syndrome and the defective gene is optionally SGSH, NAGLU, HGSNAT or GNS; wherein the disease is Morquio syndrome and the defective gene is optionally GALNS or GLB1; wherein the disease is Maroteaux–Lamy syndrome and the defective gene is optionally ARSB; wherein the disease is Sly syndrome syndrome) and the defective gene is optionally GUSB; wherein the disease is sialic acidosis and the defective gene is optionally NEU1, NEU2, NEU3 or NEU4; wherein the disease is an I-cell disease and the defective gene is optionally GNPTAB or GNPTG; wherein the disease is mucolipidosis type IV and the defective gene is optionally MCOLN1; wherein the disease is infantile neuronal ceroid lipofuscinosis and the defective gene is optionally PPT1 or PPT2; wherein the disease is Jansky–Bielschowsky disease and the defective gene is optionally TPP1;wherein the disease is Batten disease and the defective gene is optionally CLN1, CLN2, CLN3, CLN5, CLN6, MFSD8, CLN8 or CTSD; wherein the disease is Kufs disease, type A and the defective gene is optionally CLN6 or PPT1; wherein the disease is Kufs disease, type B and the defective gene is optionally DNAJC5 or CTSF; wherein the disease is alpha-mannosidosis and the defective gene is optionally MAN2B1, MAN2B2 or MAN2C1; wherein the disease is beta-mannosidosis and the defective gene is optionally MANBA; wherein the disease is fucosidosis and the defective gene is optionally FUCA1; wherein the disease is cystinosis and the defective gene is optionally CTNS; wherein the disease is pycnodystrophy and the defective gene is optionally CTSK; wherein the disease is Salla disease disease) and the defective gene is optionally SLC17A5; wherein the disease is infantile free sialic acid storage disease and the defective gene is optionally SLC17A5; or wherein the disease is Danon disease and the defective gene is optionally LAMP2. ;

[0427] In embodiments, the metabolic disorder is a peroxisome biogenesis disorder, wherein the disease is Zellweger syndrom and the defective gene is optionally PEX1, PEX2, PEX3, PEX5, PEX6, PEX12, PEX14, or PEX26; wherein the disease is Infantile Refsum disease and the defective gene is optionally PEX1, PEX2, or PEX26; wherein the disease is neonatal adrenoleukodystrophy and the defective gene is optionally PEX5, PEX1, PEX10, PEX13, or PEX26; wherein the disease is RCDP wherein the disease is acyl-CoA oxidase deficiency and the defective gene is optionally ACOX1; wherein the disease is D-bifunctional protein deficiency and the defective gene is optionally HSD17B4; wherein the disease is dihydroxyacetone phosphate acyltransferase deficiency and the defective gene is optionally GNPAT; wherein the disease is X-linked adrenoleukodystrophy and the defective gene is optionally ABCD1; wherein the disease is alpha-methylacyl-CoA racemase deficiency and the defective gene is optionally AMACR; wherein the disease is RCDP type 2 and the defective gene is optionally DHAPAT; wherein the disease is RCDP Type 3 and the defective gene is optionally AGPS; wherein the disease is adult Refsum disease-1 and the defective gene is optionally PHYH; or wherein the disease is mulibrey dwarfism and the defective gene is optionally TRIM37.

[0428] In embodiments, the metabolic disorder is a purine or pyrimidine metabolism disorder, wherein the disease is Lesch–Nyhan syndrome and the defective gene is optionally HPRT; wherein the disease is adenine phosphoribosyltransferase deficiency and the defective gene is optionally APRT; wherein the disease is adenosine deaminase deficiency and the defective gene is optionally ADA; wherein the disease is adenosine monophosphate deaminase deficiency type 1 and the defective gene is optionally AMPD1; wherein the disease is adenylate succinate lyase deficiency and the defective gene is optionally ADSL; wherein the disease is dihydropyrimidine dehydrogenase deficiency and the defective gene is optionally DPYD; wherein the disease is Miller syndrome and the defective gene is optionally DHODH; wherein the disease is orotic aciduria and the defective gene is optionally UMPS; wherein the disease is purine nucleoside phosphorylase deficiency and the defective gene is optionally PNP; or wherein the disease is xanthinuria and the defective gene is optionally XDH, MOCS1 or MOCS2, GEPH.

[0429] In an embodiment, the present invention relates to a method for regulating transthyretin (TTR). The method comprises the step of administering to a subject an effective amount of a synthetic RNA encoding TTR, wherein the synthetic RNA comprises one or more atypical nucleotides that avoid substantial cytotoxicity.

[0430] In embodiments, the modulation results in an increase in the amount of TTR in the subject.

[0431] In embodiments, the modulation results in a decrease in the amount of TTR in the subject.

[0432] In embodiments, modulation results in treatment of one or more of an amyloid disease, senile systemic amyloidosis (SSA), familial amyloid polyneuropathy (FAP), and familial amyloid cardiomyopathy (FAC).

[0433] In an embodiment, the atypical nucleotide has one or more substitutions at a position selected from the 2C, 4C and 5C positions of a pyrimidine or selected from the 6C, 7N and 8C positions of a purine.

[0434] In an embodiment, the present invention treats or reduces pain, such as postoperative pain and / or chronic pain, by administering to a subject in need thereof an effective amount of a synthetic RNA encoding a gene editing protein capable of producing double-strand breaks in a voltage-gated sodium channel type 1 (NaV1) gene (e.g., NaV1.3, NaV1.7, NaV1.8 and / or NaV1.9).

[0435] In embodiments, administration is directed to the central nervous system (CNS) or peripheral nervous system (PNS), eg, to neurons and glial cells of the CNS or PNS.

[0436] In embodiments, the disease or condition is a lung disease or condition. In embodiments, the disease or condition is an inflammation associated with a lung disease or condition. In embodiments, the lung disease or condition is selected from Asbestosis, Asthma, Bronchiectasis, Bronchitis, Chronic Cough, Chronic Obstructive Pulmonary Disease (COPD), Common Cold, Croup, Cystic Fibrosis, Hantavirus, Idiopathic Pulmonary Fibrosis, Influenza, Lung Cancer, Pandemic Influenza, Whooping Cough, Pleurisy, Pneumonia, Pulmonary Embolism, Pulmonary Hypertension, Respiratory Syncytial Virus (RSV), Sarcoidosis, Sleep Apnea, Spirometry, Sudden Infant Death Syndrome (SIDS) and Tuberculosis.

[0437] In addition, in some embodiments, the compositions of the present invention can be used to treat, control or prevent diseases, conditions and / or disorders and / or can change, modify or alter the appearance of members of the integumentary system in a subject suffering from a disease, condition and / or disorder such as, but not limited to, acne vulgaris, summer acne, acne conglobata, acne cosmetica, acne fulminans, acne keloidalis on the neck, acne mechanica, acne medica, miliary necrotizing acne, acne necrotizing, acne rosacea, actinic keratosis, acne vulgaris, acne vulgaris, acne vulgaris, acne conglobata, acne cosmetica, acne fulminans, acne keloidalis on the neck, acne mechanica, acne medica Acne vulgaris, miliary necrotizing acne, necrotizing acne, rosacea, acute urticaria, allergic contact dermatitis, alopecia areata, angioedema, athlete's foot, atopic dermatitis, autoeczema, infantile acne, baldness, blastomycosis, blackheads, birthmarks and other skin pigmentation problems, boils, bruises, insect bites and stings, burns, cellulitis, chiggers, chloracne, cholinergic or stress urticaria, chronic urticaria, cold urticaria, confluent and reticular papillomatosis, corns, cysts, dandruff, dermatitis herpetiformis, dermatographism, hyperhidrotic eczema, diaper rash, dry skin, excessive sweating, ectodermal dysplasia (such as hyperhidrotic ectodermal dysplasia dysplasia, epidermodysplasia verruciformis, and X-linked hyperhidrotic ectodermal dysplasia), eczema, epidermodysplasia verruciformis, erythema nodosum, exfoliative acne, exercise-induced allergic folliculitis, excessive oiliness of the skin, folliculitis, freckles, frostbite, fungal nails, hair density, hair growth rate, halogen acne, alopecia, prickly heat, hematoma, herpes simplex infection (e.g., nongenital herpes simplex infection), hidradenitis suppurativa, urticaria, hyperhidrosis, hyperpigmentation, hypohidrotic ectodermal dysplasia, hypopigmentation, impetigo, ingrown hairs, heat urticaria, ingrown toenails, infantile or neonatal acne, pruritus, irritant contact dermatitis, tinea cruris , keloids, keratosis pilaris, lichen planus, lichen sclerosus, lupus facie, melasma, moles, molluscum contagiosum, nail growth rate, nail health, neurodermatitis, nummular eczema, occupational acne, oily acne, onychomycosis, physical urticaria, pilonidal cyst, pityriasis rosea, tinea versicolor, poison ivy, oily acne, pseudofolliculitis barbae or keloidal acne on the neck, psoriasis, psoriatic arthritis, pressure or delayed pressure urticaria, puncture wounds (such as cuts and scrapes), rash, rare or watery urticaria, rhinoplasty, ringworm, rosacea, rothmund-thomson syndromesyndrome), sagging skin, scabies, scars, seborrhea, seborrheic dermatitis, hinges, skin cancer, skin tags, solar urticaria, spider bites, stretch marks, sunburn, tar acne, tropical acne, thinning skin, thrush, tinea versicolor, transient acantholytic dermatosis, mogul's cap or necrotizing miliary acne, uneven skin tone, varicose veins, venous eczema, vibration angioedema, vitiligo, warts, Weber-Christian disease, wrinkles, X-linked hypohidrotic ectodermal dysplasia, eczema sicca, yeast infections and general signs of aging.

[0438] Illustrative cancers and / or tumors of the present invention include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intraepithelial neoplasia; kidney cancer; laryngeal cancer; leukemia; liver cancer (including pancreatic cancer); pancreatic cancer (including pancreatic cancer); pancreatic cancer (including pancreatic cancer); leukemia; ovarian cancer (including pancreatic cancer); ovarian cancer (including pancreatic 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; laryngeal cancer; leukemia; liver cancer (including pancreatic cancer); cancer); lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; colorectal cancer; respiratory cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary tract cancer; vulvar cancer; lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma The invention relates to non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate grade / follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphocytic NHL, high grade small non-lytic cell NHL, massive NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's Macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with nevus hamartomatosis, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0439] In an embodiment, one or more rare diseases are treated, managed or prevented with the compositions of the invention, including, for example, erythropoietic protoporphyria, Hailey-Hailey Disease, epidermolysis bullosa (EB), xeroderma pigmentosum, Ehlers-Danlos Syndrome, Cutis Laxa, Protein C & Protein S Deficiency, Alport Syndrome, palmoplantar keratoderma, lethal acantholytic EB, pseudoxanthoma elasticum (PXE), ichthyosis vulgaris, pemphigus vulgaris, and basal cell nevus syndrome.

[0440] In an embodiment, the compositions of the invention are used to treat, control or prevent 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, inflammatory eye diseases, celiac disease, Leigh Syndrome, glycerol kinase deficiency, familial eosinophilia (FE), autosomal recessive spastic ataxia, inflammatory diseases of the larynx; tuberculosis, chronic cholecystitis, bronchiectasis, silicosis and other pneumoconiosis.

[0441] In an embodiment, the compositions of the invention are used to treat, control or prevent one or more autoimmune diseases or conditions such as multiple sclerosis, diabetes mellitus, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome, scleroderma, Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, primary biliary cirrhosis, sclerosing cholangitis, autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, fibromyalgia, Menier's syndrome, syndrome); transplant rejection (e.g., prevention of allogeneic transplant rejection); pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjögren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, Grave's disease, and other autoimmune diseases.

[0442] In an embodiment, the composition of the present invention is used to treat, control or prevent one or more nervous system diseases, including ADHD, AIDS-neurological complications, septum pellucidum absence, acquired epileptiform aphasia, acute disseminated encephalomyelitis, adrenoleukodystrophy, corpus callosum agenesis, agnosia, Aicardi Syndrome, Alexander Disease, Alpers' Disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis, anencephaly, aneurysm, Angelman Syndrome, angiomatosis, hypoxia, aphasia, apraxia, arachnoid cyst, arachnoiditis, Arnold-ChiariMalformation, arteriovenous malformation, aspartame, Asperger's syndrome, Syndrome), Ataxia-telangiectasia, Ataxia, Attention Deficit Hyperactivity Disorder, Autism, Dysautonomia, Back Pain, Barth Syndrome, Batten Disease, Behcet's Disease, Bell's Palsy, Benign Essential Blepharospasm, Benign Focal Muscular Atrophy, Benign Intracranial Hypertension, Bernhardt-Roth Syndrome, Binswanger's Disease, Blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus Birth Trauma, Brachial Plexus Injury, Budd-Ellison Syndrome, Brain Aneurysm, Brain Injury, Brain and Spinal Cord Tumors, Brown-Sequard Syndrome, Bulbous Spinal Muscular Atrophy, Canavan Disease Disease), carpal tunnel syndrome, causalgia, cavernous tumor, cavernous hemangioma, cavernous malformation, central cervical cord syndrome, central spinal cord syndrome, central pain syndrome, head disease, cerebellar degeneration, cerebellar hypoplasia, cerebral aneurysm, cerebral arteriosclerosis, brain atrophy, cerebral beriberi, cerebral gigantism, cerebral hypoxia, cerebral palsy, cerebro-oculofacial-skeletal syndrome, Charcot-Marie-ToothDisorder, Chiari Malformation, chorea, chorea acanthocytosis, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic orthostatic intolerance, chronic pain, Cockayne Syndrome Type II, Coffin-Lowry SyndromeSyndrome), Coma (including persistent vegetative state), Complex Regional Pain Syndrome, Congenital Facial Diplegia, Congenital Myasthenia, Congenital Myopathy, Congenital Cavernous Malformation, Corticobasal Degeneration, Cranial Arteritis, Craniosynostosis, Creutzfeldt-Jakob Disease, Cumulative Trauma Disorders, Cushing's Syndrome, Cytomegalovirus Infection, Dandy-Walker Syndrome, Dawson Disease, De Morsier's Syndrome, Dejerine-Klumpke Palsy, Multi-Infarct Dementia, Subcortical Dementia, Dementia With Lewy Bodies, Dermatomyositis, Developmental Dyspraxia, Devic's Syndrome, Diabetic Neuropathy, Diffuse Sclerosis, Dravet's Syndrome), autonomic dysfunction, dysgraphia, dyslexia, dysphagia, dyskinesia, dystonia, epileptic encephalopathy of early infancy, empty sella syndrome, encephalitis lethargica, encephalitis and meningitis, encephalocele, encephalopathy, cerebral trigeminal angiomatosis, epilepsy, Erb's Palsy, Erb-Duchenne and Dejerine-Klumpke Palsies, Fabry's Disease, Fahr's Syndrome, syncope, familial autonomic dysfunction, familial angiomatosis, familial idiopathic basal ganglia calcification, familial spastic palsies, febrile seizures (e.g., GEFS and GEFS plus), Fisher Syndrome, floppy infantile syndrome, Friedreich's Ataxia, Gaucher's Disease), Gerstmann's Syndrome, Gerstmann-Straussler-Scheinker Disease, Giant Cell Arteritis, Giant Cell Inclusion Disease, Globoid Cell Leukodystrophy, Glossopharyngeal Neuralgia, Guillain-Barre Syndrome, HTLV-1-Associated Myelopathy, Hallervorden-Spatz DiseaseDisease), Head Injury, Headache, Continuous Migraine, Hemifacial Spasm, Alternating Hemiplegia, Hereditary Neuropathy, Hereditary Spastic Paraplegia, Amorphous Hereditary Disease, Herpes Zoster Oticus, Herpes Zoster, Hirayama Syndrome, Prosencephaly, Huntington's Disease, Hydrocephalus, Normal Pressure Hydrocephalus, Hydrocephalus, Hydromyelia, Hypercortisolism, Narcolepsy, Hypertonia, Hypotonia, Hypoxia, Immune-Mediated Encephalomyelitis, Inclusion Body Myositis, Incontinence Pigmentis, Hypotonia Infantile, Phytanic Acid Storage Disease Infantile, Vomiting Disease Infantile, Infantile Spasms, Inflammatory Myopathy, Intestinal Lipodystrophy, Intracranial Cyst, Intracranial Hypertension, Isaac's Syndrome, Joubert Syndrome, Kearns-Sayre Syndrome, Kennedy's Disease, Kinsbourne Syndrome, syndrome), Kleine-Levin syndrome, Kinsbourne syndrome, Klippel-Trenaunay Syndrome (KTS), Klüver-Bucy Syndrome, Korsakoffs Amnesic Syndrome, Krabbe Disease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton Myasthenic Syndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous Nerve Entrapment, Lateral Bulbar Syndrome, Learning Disabilities, Leigh's Disease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome Syndrome, Leukodystrophy, Levine-Critchley Syndrome, Lewy Body Dementia, Anencephaly, Locked-in Syndrome, Lou Gehrig's Disease, Neurological Sequelae of Lupus, Neurological Complications of Lyme Disease—NeurologicalComplications, Machado-Joseph Disease, megalencephaly, megalencephaly, Machado-Joseph Disease, meningitis, Menkes Disease, hindlimb paresthesia, metachromatic leukodystrophy, microcephaly, migraine, Miller Fisher syndrome Syndrome), mini-stroke, mitochondrial myopathy, Mobius Syndrome, Monosomic muscular dystrophy, motor neuron disease, moyamoya disease, mucolipidosis, mucopolysaccharidosis, multi-infarct dementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy with orthostatic hypotension, multiple system atrophy, muscular dystrophy, congenital myasthenia gravis, diffuse myelin sclerosis, infantile myoclonic encephalopathy, myoclonus, congenital myopathy, thyrotoxic myopathy, myopathy, congenital myotonia, myotonia, narcolepsy, neuroacanthocytosis, neurodegeneration with brain iron accumulation, neurofibromatosis, neuroleptic malignant syndrome, neurologic complications of AIDS, Pompe disease Disease, neuromyelitis optica, neuromyotonia, neuronal ceroid lipofuscinosis, neuronal migration abnormalities, neurogenetic diseases, neurosarcoidosis, neurotoxicity, cavernous nevus, Niemann-Pick Disease, O'Sullivan-McLeod Syndrome, occipital neuralgia, latent spinal dysfunction sequence, Ohtahara Syndrome, olivopontocerebellar atrophy, opsoclonus-myoclonus, orthostatic hypotension, overuse syndrome, chronic pain, paraneoplastic syndrome, paresthesia, Parkinson's disease, myotonia congenita, paroxysmal choreoathetosis, paroxysmal migraine, Parry-Romberg disease, Pelizaeus-Merzbacher disease, Pena Shokeir II syndrome, II Syndrome), perineural cysts, periodic paralysis, peripheral neuropathy, periventricular leukomalacia, persistent vegetative state, pervasive developmental disorder, phytanic acid storage disease, Pick's diseaseDisease), piriformis syndrome, pituitary tumor, polymyositis, Pompe disease, brain abscess, postpoliomyelitis syndrome, postherpetic neuralgia, postinfectious encephalomyelitis, postural hypotension, postural orthostatic tachycardia syndrome, postural tachycardia syndrome, primary lateral sclerosis, prion disease, progressive hemifacial atrophy, progressive motor ataxia, progressive multifocal leukoencephalopathy, progressive sclerosing poliomyelitis, progressive supranuclear palsy, pseudotumor cerebri, pyridoxine-dependent and pyridoxine-responsive epilepsy, Ramsay Hunt Syndrome Type I, Ramsay Hunt Syndrome Type II, Rasmussen's encephalitis Encephalitis and other autoimmune epilepsy, Reflex Sympathetic Dystrophy Syndrome, Infantile Refsum's Disease, Refsum's Disease, Repetitive Movement Disorder, Repetitive Stress Injury, Restless Legs Syndrome, Retroviral Myelopathy, Rett Syndrome, Reye's Syndrome, Riley-Day Syndrome, SUNCT Headache, Sacral Root Cyst, Saint Vitus Dance, Salivary Gland Disease, Sandhoff's Disease, Schilder's Disease, Schizophrenia, Epilepsy, Optic Nerve Hypoplasia, Severe Myoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome, Herpes Zoster, Shy-Drager Syndrome Syndrome), Sjögren's Syndrome, Sleep Apnea, Sleeping Sickness, Soto's Syndrome, Spasticity, Spina Bifida, Spinal Cord Infarction, Spinal Cord Injury, Spinal Cord Tumor, 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, Syncope, Syphilitic Myelosclerosis, Syringomyelia, Syringomyelia, Systemic Lupus Erythematosus, Tabes Dorsalis, Tardive Dyskinesia, Tarlov Cysts, Tay-Sachs Disease, Temporal Arteritis, Tethered Cord Syndrome, ThomsenDisease), Thoracic Outlet Syndrome, Thyrotoxic Myopathy, Tic Douloureux, Todd's Paralysis, Tourette Syndrome, Transient Ischemic Attack, Transmissible Spongiform Encephalopathy, Transverse Myelitis, Traumatic Brain Injury, Tremor, Trigeminal Neuralgia, Tropical Spastic Paraplegia, Tuberous Sclerosis Complex, Vascular Erection Neoplasm, Vasculitis (including Temporal Arteritis), Von Economo's Disease, Von Hippel-Lindau disease (VHL), Von Recklinghausen's Disease, Wallenberg's Syndrome, Werdnig-Hoffman Disease, Wernicke-Korsakoff Syndrome, West Syndrome, Whipple's Disease), Williams Syndrome, Wilson's Disease, X-linked spinal and bulbar muscular atrophy, and Zellweger syndrome.

[0443] In an embodiment, the compositions of the invention are used to treat and reduce pain, such as post-operative pain or chronic pain.

[0444] In an embodiment, the compositions of the invention are used to treat Huntington's disease.

[0445] In an embodiment, the compositions of the invention are used to treat sickle cell anemia or thalassemia.

[0446] In an embodiment, the compositions of the invention alter RNA splicing of an exon associated with a disease or condition. In an embodiment, the disease or condition is selected from Alport syndrome, Alzheimer's disease, Bethlemmyopathy and Ullrich scleroatonic muscular dystrophy, Duchenne muscular dystrophy, dystrophic epidermolysis bullosa, Friedreich's ataxia, Huntington's disease, junctional epidermolysis bullosa, Leber's congenital amaurosis (LCA), and various myopathies and dystrophies.

[0447] In an embodiment, the compositions of the invention are used to treat one or more respiratory diseases, such as asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis, allergic rhinitis, sinusitis, pulmonary vasoconstriction, inflammation, allergies, breathing disorders, respiratory distress syndrome, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, Hantavirus Pulmonary Syndrome (HPS), Loeffler's syndrome, Goodpasture's syndrome, pleurisy, pneumonia, pulmonary edema, pulmonary fibrosis, sarcoidosis, complications associated with respiratory syncytial virus infection, and other respiratory diseases.

[0448] In an embodiment, the compositions of the invention are used to treat, control or prevent cardiovascular disease, such as diseases or disorders 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, arrhythmias, valvular disease and / or congestive heart failure.

[0449] In embodiments, the compositions of the invention are used to treat, manage or prevent one or more metabolic related disorders. In embodiments, the invention can be used to treat, manage or prevent diabetes, including type 1 and type 2 diabetes and diabetes associated with obesity. The compositions and methods of the invention can be used to treat or prevent diabetes-related conditions, including, but not limited to, diabetic nephropathy, hyperglycemia, impaired glucose tolerance, insulin resistance, obesity, lipid disorders, dyslipidemia, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, low HDL levels, high LDL levels, atherosclerosis and its sequelae, vascular restenosis, irritable bowel syndrome, inflammatory bowel disease, including Crohn's disease and ulcerative colitis, other inflammatory disorders, pancreatitis, abdominal obesity, neurodegenerative diseases, retinopathy, neoplastic diseases, adipocyte tumors, adipocyte 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 suffers from three or more of the following conditions: abdominal obesity, hypertriglyceridemia, low HDL cholesterol, hypertension, and high fasting blood glucose), ovarian hyperandrogenism (polycystic ovary syndrome), and other diseases in which insulin resistance is a factor, such as sleep apnea. The compositions and methods of the present invention can be used to treat, control or prevent obesity, including genetic obesity or environmental obesity, and obesity-related disorders. Obesity-related disorders herein are associated with obesity, caused by obesity or caused by obesity. Examples of obesity-related disorders include obesity, diabetes, overeating, binge eating and bulimia, hypertension, elevated plasma insulin concentrations and insulin resistance, dyslipidemia, hyperlipidemia, endometrial, breast, prostate, kidney and colon cancers, osteoarthritis, obstructive sleep apnea, gallstones, heart disease, arrhythmias and arrhythmias, myocardial infarction, congestive heart failure, coronary heart disease, sudden death, stroke, polycystic ovary disease, craniopharyngioma, Prader-Willi Syndrome, Frohlich's syndrome, subjects with GH deficiency, normal variant short stature, Turner's syndrome, and other pathological conditions showing reduced metabolic activity or reduced resting energy expenditure as a percentage of total fat-free mass, such as children with acute lymphoblastic leukemia.Further examples of obesity-related disorders are metabolic syndrome, insulin resistance syndrome, reproductive hormone abnormalities, sexual and reproductive dysfunction, such as impaired fertility, infertility, male hypogonadism and female hirsutism, fetal defects associated with maternal obesity, gastrointestinal motility disorders, such as obesity-related gastroesophageal reflux, respiratory disorders, such as obesity-hypoventilation syndrome (Pickwick syndrome (Pickwickian syndrome)), dyspnea, cardiovascular disease, inflammation, such as vascular systemic inflammation, arteriosclerosis, hypercholesterolemia, low back pain, gallbladder disease, hyperuricemia, gout and renal cancer, and increased risk of anesthesia. The compositions and methods of the present invention can also be used to treat Alzheimer's disease.

[0450] Nucleic acids, including liposome preparations containing nucleic acids, can accumulate in the liver and / or spleen when delivered in vivo. It has been found that nucleic acids encoding proteins can regulate protein expression in the liver and spleen, and nucleic acids used in this way can constitute effective therapeutic agents for treating liver and spleen diseases. Certain embodiments therefore relate to methods for treating liver and / or spleen diseases by delivering nucleic acids encoding target proteins to patients. Other embodiments relate to therapeutic compositions comprising nucleic acids encoding target proteins for treating liver and / or spleen diseases. Treatable liver and / or spleen diseases and disorders include, but are not limited to, hepatitis, alcoholic 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 liver and / or spleen cancer.

[0451] In some embodiments, the compositions and methods of the invention relate to the treatment of type 1 diabetes, heart disease, 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, including hepatitis and HIV / AIDS.

[0452] In embodiments, the methods and compositions of the present invention can be used to treat or prevent one or more metabolic diseases or disorders. In embodiments, the methods and compositions of the present invention can be used to treat or prevent one or more carbohydrate metabolism diseases or disorders, amino acid metabolism diseases or disorders, urea cycle diseases or disorders, fatty acid metabolism diseases or disorders, porphyrin metabolism diseases or disorders, lysosomal storage disorders, peroxisome biogenesis disorders, and purine or pyrimidine metabolism diseases or disorders.

[0453] In embodiments, the methods and compositions of the invention are useful for treating or preventing one or more ophthalmic diseases or conditions, including, but not limited to, diabetic retinopathy, dry eye, cataracts, retinal vein occlusion, macular edema, macular degeneration (wet & dry), refractive and accommodative disorders, keratoconus, amblyopia, glaucoma, Stargardt disease, endophthalmitis, conjunctivitis, uveitis, retinal detachment, corneal ulcer, dacryocystitis, Duane retraction syndrome, and optic neuritis.

[0454] In embodiments, the ophthalmic disease or condition is central serous retinopathy (CSR), adult vitelliform disease, uveitis, which is primary and secondary to systemic diseases (e.g., as non-limiting examples, sarcoma, rheumatoid disease, arthritis, etc.), white dot syndrome (including MEWDS (multiple transient white dot syndrome)), serpiginous choroidopathy, AMPPE (acute multifocal posterior papillary epitheliopathy), POHS (pseudo-ocular histoplasmosis syndrome) or serpiginous chorioretinopathy. In some embodiments, the disease may be maculopathy or cone dystrophy, such as, as a non-limiting example, Stargardt's disease. In some embodiments, the disease may be an inherited degenerative disease, such as retinitis pigmentosa (RP). In some embodiments, the ocular disease may be an ocular melanoma, an ocular tumor, or an invasive tumor.

[0455] In embodiments, the ophthalmic disease or disorder is Fuch's corenal dystrophy. In embodiments, the ophthalmic disease or disorder is Leber's congenital amaurosis.

[0456] In embodiments, the methods and compositions of the invention can be used to target any of the proteins in Table 2A, Table 2B, and / or Table 2C or to treat any of the diseases or conditions in Table 2A, Table 2B, and / or Table 2C.

[0457] In various embodiments, the methods and compositions of the present invention include the use of nucleic acid drugs, including synthetic RNA, in diagnosing, treating, preventing or ameliorating diseases, disorders and / or conditions described herein. In various embodiments, the methods and compositions of the present invention include the use of nucleic acid drugs, including synthetic RNA, in altering, modifying and / or modifying tissues (e.g., cosmetic aspects).

[0458] Generally speaking, in various embodiments, a synthetic RNA as described herein is administered to a human at a specific dose as described herein, and the synthetic RNA comprises a sequence encoding a protein of interest, sometimes referred to as a target sequence, which may be a therapeutic protein.

[0459] Synthetic RNAs containing only typical nucleotides can bind to pattern recognition receptors, can be recognized as pathogen-associated molecular patterns, and can trigger effective immune responses in cells, resulting in translational arrest, secretion of inflammatory cytokines, and cell death. Synthetic RNAs containing certain atypical nucleotides can escape detection by the innate immune system and can be efficiently translated into proteins, including in humans. Synthetic RNAs containing at least one of the atypical nucleotides described herein, including, for example, members of the following groups: 5-methylcytidine, 5-hydroxycytidine, 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 escape detection by the innate immune system and can be efficiently translated into proteins, including in humans. Some embodiments therefore relate to a method for inducing cells to express a target protein, comprising contacting the cell with a synthetic RNA. Other embodiments relate to a method for transfecting cells with synthetic RNA, comprising contacting the cell with a solution comprising one or more synthetic RNA molecules. Other embodiments relate to a method for treating a patient, comprising administering synthetic RNA to the patient. In one embodiment, the synthetic RNA comprises at least one of the atypical nucleotides described herein, including members of the following groups such as 5-methylcytidine, 5-hydroxycytidine, 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 target protein. Illustrative RNAs may contain atypical and combinations and levels of atypical nucleotides as described elsewhere herein. In embodiments, the methods result in expression of a protein of interest. In embodiments, the methods result in expression of a protein of interest in the skin of a patient.

[0460] Other embodiments relate to methods for delivering nucleic acids to cells in vivo. Other embodiments relate to methods for inducing cells in vivo to express a target protein. Other embodiments relate to methods for treating patients. In one embodiment, the method comprises destroying the stratum corneum. In another embodiment, the method comprises contacting a cell with a nucleic acid. In yet another embodiment, the method causes the cell to internalize the nucleic acid. In another embodiment, the method causes the cell to express the target protein. In yet another embodiment, the method causes the target protein to be expressed in the patient. In yet another embodiment, the method causes one or more improvements in the patient's symptoms. In yet another embodiment, the patient needs the target protein. In yet another embodiment, the patient lacks the target protein.

[0461] Other embodiments relate to methods for treating patients, comprising delivering a composition to the patient. In another embodiment, the composition comprises one or more nucleic acid molecules. In yet another embodiment, at least one of the one or more nucleic acid molecules encodes a target protein. In an embodiment, the nucleic acid is a synthetic RNA. In other embodiments, the method causes one or more improvements in the patient's symptoms. Other embodiments relate to methods for treating indications by delivering nucleic acids encoding proteins or peptides to cells or patients. Other embodiments relate to compositions comprising nucleic acids encoding proteins or peptides. Indications for treatment using the methods and compositions of the present invention and proteins and peptides that can be encoded by the compositions of the present invention are listed in Table 2A, Table 2B, and / or Table 2C, and are given as examples, not limitations. In one embodiment, indications are selected from Table 2A, Table 2B, and / or Table 2C. In another embodiment, proteins or peptides are selected from Table 2A, Table 2B, and / or Table 2C. In yet another embodiment, indications and proteins or peptides 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 yet another embodiment, the gene editing protein targets genes that at least partially cause the disease phenotype. In yet another embodiment, the gene editing protein targets a gene encoding a protein selected from Table 2A, Table 2B, and / or Table 2C. In yet another embodiment, the gene editing protein alone or in combination with one or more other molecules or gene editing proteins corrects or eliminates a mutation that at least partially causes the disease phenotype.

[0462] In various embodiments, the present invention relates to targeting any of the proteins disclosed in Table 2A, Table 2B and / or Table 2C and precursor forms and / or mature forms and / or isoforms and / or mutants of such proteins. In embodiments, any of the precursor forms and / or mature forms and / or isoforms and / or mutants has enhanced secretion relative to the corresponding wild-type protein. In embodiments, any of the precursor forms and / or mature forms and / or isoforms and / or mutants has altered half-life (e.g., serum, plasma, intracellular) - for example, longer or shorter half-life. In embodiments, this is relative to wild-type.

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508] In various embodiments, the methods and compositions of the present invention can be used to treat or prevent one or more of the diseases or conditions in the following table. In various embodiments, the methods and compositions of the present invention can be used to treat or prevent one or more of the diseases or conditions in the following table, for example, by regulating genes associated with the diseases in the following table. In embodiments, the methods and compositions of the present invention can be used to gene edit the genes described in Table 2C below using the compositions of the present invention.

[0509] Table 2C

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517] The Entrez items listed in the table above are hereby incorporated by reference in their entirety.

[0518] Additional illustrative targets of the invention include the cosmetic targets listed in Table 6 of International Patent Publication No. WO 2013 / 151671, the contents of which are hereby incorporated by reference in their entirety.

[0519] In addition, in some embodiments, the methods and compositions of the present invention can be used to target any of the proteins in Table 2A, Table 2B, and / or Table 2C or treat any of the diseases or conditions in Table 2A, Table 2B, and / or Table 2C. In various embodiments, the present invention relates to targeting full-length and / or truncated forms of any of the proteins disclosed in Table 2B. In various embodiments, the present invention relates to targeting precursor forms and / or mature forms and / or isoforms of the proteins disclosed in Table 2A, Table 2B, and / or Table 2C.

[0520] In various embodiments, the present invention relates to targeting any of the protein sequences disclosed herein (e.g., any of the protein sequences in Table 2A, Table 2B, and / or Table 2C) that has about 60% (e.g., about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%). , about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%) sequence identity.

[0521] In various embodiments, the present invention relates to a protein targeting an amino acid sequence comprising one or more amino acid mutations relative to any of the protein sequences described herein (e.g., any of the protein sequences in Table 2A, Table 2B, and / or Table 2C). For example, the present invention relates to a protein targeting an amino acid sequence comprising 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12 amino acid mutations relative to any of the protein sequences described herein (e.g., any of the protein sequences in Table 2A, Table 2B, and / or Table 2C). In embodiments, one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.

[0522] In an embodiment, the amino acid mutation is an amino acid substitution, and may include conservative and / or non-conservative substitutions.

[0523] For example, "conservative substitutions" can be made based on similarities in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be divided 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 affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0524] As used herein, "conservative substitution" is defined as an amino acid exchanged by another amino acid listed in the same group of the six standard amino acid groups shown above. For example, exchanging Asp by Glu retains a negative charge in the polypeptide so modified. In addition, glycine and proline can replace each other based on their ability to destroy alpha-helices.

[0525] As used herein, "non-conservative substitution" is defined as the exchange of an amino acid with another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.

[0526] 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, sarcosine, citrulline, homocitrulline, cysteic acid, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs in general).

[0527] Manufacturing method

[0528] Methods for the chemical synthesis of the compounds of the invention (eg, compounds of Formulae I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers are described elsewhere herein.

[0529] The methods and compositions of the present invention utilize certain cationic lipids, the synthesis, preparation and characterization of which are described elsewhere herein and in the accompanying Examples. In addition, the present invention provides methods for preparing lipid aggregates and / or lipid carriers, including those lipid aggregates and / or lipid carriers associated with therapeutic agents such as nucleic acids.

[0530] In embodiments, lipid aggregates and / or lipid carriers of the present invention, such as liposomes, are produced using microfluidics. In embodiments, lipid aggregates and / or lipid carriers of the present invention are manufactured using Nanoassemblr instruments (PrecisionNanosystems). In embodiments, a syringe pump is used to mix organic solutions and aqueous solutions at a specified flow rate. Optionally, the flow rate of the aqueous 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 comprises one or more of ethanol, acetone, acetonitrile, dimethyl sulfoxide, toluene and chloroform, or mixtures thereof. In other embodiments, other solvents are used.

[0531] In embodiments, lipid aggregates and / or lipid carriers of the invention are manufactured by dropwise mixing of one solution into another solution. In embodiments, lipid aggregates and / or lipid carriers of the invention are manufactured using a spray mechanism, or by solvent evaporation, or by sonication, or by extrusion of one or more membranes, or by a self-assembly process, or by a combination of methods.

[0532] In the methods described herein, a mixture of lipids is combined with a buffered aqueous solution of nucleic acids to produce an intermediate mixture containing nucleic acids encapsulated in lipid particles, wherein the encapsulated nucleic acids are present in a nucleic acid / lipid ratio of about 3% by weight to about 25% by weight, for example, 5% by weight to 15% by weight. The intermediate mixture can optionally be sized to obtain lipid-encapsulated nucleic acid particles, wherein the lipid portion is, for example, a single-layer vesicle with a diameter of 30 to 150nm, for example, about 40 to 90nm. The pH is then raised to neutralize at least a portion of the surface charge on the lipid-nucleic acid particles, thereby providing a nucleic acid composition of at least partially neutralized lipid encapsulation.

[0533] Some cationic lipids are amino lipids that are charged at a pH lower than the pKa of the amino group and are substantially neutral at a pH higher than the pKa. These cationic lipids are referred to as titratable cationic lipids and can be used in the compounds of the present invention (e.g., compounds of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers using a two-step method. First, lipid vesicles can be formed at a relatively low pH with titratable cationic lipids and other vesicle components in the presence of nucleic acids. In this way, the vesicles will encapsulate and embed the nucleic acids. Secondly, the surface charge of the newly formed vesicles can be neutralized by increasing the pH of the culture medium to a level (i.e., physiological pH or higher) higher than the pKa of the titratable cationic lipids present. Particularly advantageous aspects of this method include easy removal of any surface-adsorbed nucleic acids and the resulting nucleic acid delivery vehicle with a neutral surface. It is expected that the compounds of the present invention (e.g., compounds of Formulas I-XVI) and / or pharmaceutical compositions and / or lipid aggregates and / or lipid carriers and / or liposomes and / or lipid particles with a neutral surface can avoid rapid clearance from the circulation and avoid certain toxicities associated with cationic liposome preparations. Additional details regarding these uses of such titratable cationic lipids in the formulation of nucleic acid-lipid particles are provided in US Pat. No. 6,287,591 and US Pat. No. 6,858,225, which are incorporated herein by reference.

[0534] The vesicles formed in this manner provide preparations of uniform vesicle size with high nucleic acid content. In addition, the size range of the vesicles is from about 30 to about 150 nm, for example, from about 30 to about 90 nm.

[0535] Without intending to be constrained by any particular theory, it is believed that very efficient nucleic acid encapsulation is the result of electrostatic interaction under low pH. Under acidic pH (e.g., pH 4.0), the vesicle surface is charged and a part of nucleic acid is bound by electrostatic interaction. When the external acidic buffer is replaced with a more neutral buffer (e.g., pH 7.5), the surface of lipid particles or liposomes is neutralized, allowing any external nucleic acid to be removed. More detailed information about the preparation process is provided in various publications (e.g., U.S. Patent No. 6,287,591 and U.S. Patent No. 6,858,225).

[0536] The present invention provides a method for preparing lipid / nucleic acid formulations in an embodiment. 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 acids encapsulated in lipid particles, for example, wherein the encapsulated nucleic acid is present in a nucleic acid / lipid ratio of about 10% by weight to about 50% by weight. The intermediate mixture can be optionally sized to obtain lipid-encapsulated nucleic acid particles, wherein the lipid portion is, for example, a single-layer vesicle with a diameter of 30 to 150nm or about 40 to 90nm. The pH is then raised to neutralize at least a portion of the surface charge on the lipid-nucleic acid particles, thereby providing a nucleic acid composition of at least partially neutralized lipid encapsulation.

[0537] In an embodiment, the mixture of lipids comprises at least two lipid components: a first lipid component of the 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 an embodiment, the amino lipid is a novel cationic lipid of the invention.

[0538] When preparing nucleic acid-lipid particles of the present invention, the mixture of lipids is generally a solution of lipids in an organic solvent. This mixture of lipids can then be dried to form a film or lyophilized to form a powder, and then hydrated with an aqueous buffer to form liposomes. Alternatively, in a method, the lipid mixture can be dissolved in a water-soluble alcohol (such as ethanol), and this ethanol solution is added to an aqueous buffer, causing liposomes to form spontaneously. In an embodiment, the alcohol is used in a form that is commercially available. For example, ethanol can be used as anhydrous ethanol (100%) or 95% ethanol, and the rest is water. This method is described in more detail in U.S. Patent No. 5,976,567, the full content of which is incorporated herein by reference.

[0539] In embodiments, the mixture of lipids is a mixture of cationic lipids, neutral lipids (except cationic lipids), sterols (e.g., cholesterol) and PEG-modified lipids (e.g., PEG-DMG or PEG-DMA) in an alcohol solvent. In embodiments, the lipid mixture is substantially composed of cationic lipids, neutral lipids, cholesterol and PEG-modified lipids in alcohols such as ethanol. In embodiments, the first solution is composed of the above-mentioned lipid mixture of about 20-70% cationic lipids: 5-45% neutral lipids: 20-55% cholesterol: 0.5-15% PEG-modified lipids in a molar ratio. In embodiments, the first solution is substantially composed of lipids selected from Table 1, DSPC, Chol and PEG-DMG or PEG-DMA, for example, a molar ratio of about 20-60% cationic lipids: 5-25% DSPC: 25-55% Chol: 0.5-15% PEG-DMG or PEG-DMA. In embodiments, the lipid molar ratio is about 40 / 10 / 40 / 10 (mol% cationic lipid / DSPC / Chol / PEG-DMG or PEG-DMA), 35 / 15 / 40 / 10 (mol% cationic lipid / DSPC / Chol / PEG-DMG or PEG-DMA) or 52 / 13 / 30 / 5 (mol% cationic lipid / DSPC / Chol / PEG-DMG or PEG-DMA). In embodiments, the neutral lipids in these compositions are replaced with POPC, DPPC, DOPE or SM.

[0540] In embodiments, the lipid mixture is combined with a buffered aqueous solution that may contain nucleic acids. The buffered aqueous solution is typically a solution in which the pH of the buffer in the solution is less than the pKa of the protonatable lipid in the lipid mixture. Examples of suitable buffers include citrate, phosphate, acetate, and MES. The buffer will be in the anionic range of 1-1000mM, depending on the chemical properties of the encapsulated nucleic acid, and the optimization of the buffer concentration may be important for achieving high load levels (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 appropriate to add 5% glucose or another nonionic solute, which will balance the osmotic potential across the particle membrane when the particles are dialyzed to remove ethanol, increase pH, or mix with a pharmaceutically acceptable carrier such as saline. The amount of nucleic acid in the buffer may vary, but is typically about 0.01mg / mL to about 200mg / mL, for example, about 0.5mg / mL to about 50mg / mL.

[0541] The mixture of lipids and the buffered aqueous solution of therapeutic nucleic acid are combined to provide an intermediate mixture. The intermediate mixture is typically a mixture of lipid particles with encapsulated nucleic acids. In addition, due to the ionic attraction of the negatively charged nucleic acid and the positively charged lipid on the lipid particle surface, the intermediate mixture may also contain a part of some nucleic acids, and the part of the nucleic acid is attached to the surface of the lipid particle (liposome or lipid vesicle) (the amino lipid or other lipids constituting the first lipid component that can be protonated are positively charged in a buffer solution having a pH less than the pKa of the protonatable group on the lipid). In an embodiment, the mixture of lipids is an alcoholic solution of lipids, and the volume of each solution is adjusted so that when combined, the resulting alcohol content is recorded as about 20% to about 45% by volume. The method of combining the mixture may include any of a variety of methods, usually depending on the scale of the preparation produced. For example, when the cumulative volume is about 10-20mL or less, the solution can be combined in a test tube and stirred together using a vortex mixer. Large-scale processes can be carried out in glassware of suitable production scale.

[0542] Optionally, the size of the lipid-encapsulated therapeutic agent (e.g., nucleic acid) complex produced by combining a lipid mixture and a buffered aqueous solution of a therapeutic agent (e.g., nucleic acid) can be set to achieve a desired size range and a relatively narrow distribution of lipid particle sizes. In embodiments, the size of the composition provided herein will be set to an average diameter of about 70 to about 200 nm, such as about 90 to about 130 nm. Several techniques can be used to set the size of the liposome to a desired size. A method for setting the size is described in U.S. Patent No. 4,737,323, the entire contents of which are incorporated herein by reference. Ultrasonic treatment of a liposome suspension by a water bath or probe ultrasound method will produce a small unilamellar vesicle (SUV) that is gradually reduced in size to a size less than about 0.05 microns. Homogenization is another method that relies on shear energy to break large liposomes into smaller particles. In a typical homogenization process, multilamellar vesicles are recirculated by a standard emulsion homogenizer until the selected liposome size is observed, typically between about 0.1 and 0.5 microns. In both methods, particle size distribution can be monitored by conventional laser beam particle size determination. For certain methods herein, extrusion is used to obtain uniform vesicle size.

[0543] Extrusion of liposome compositions through small-pore polycarbonate membranes or asymmetric ceramic membranes produces a relatively well-defined size distribution. Typically, the suspension is circulated through the membrane once or multiple times until the desired liposome complex size distribution is achieved. Liposomes can be extruded through membranes whose pores are continuously reduced to achieve a gradual reduction in liposome size. In some cases, the lipid-nucleic acid composition formed can be used without any size setting.

[0544] In embodiments, method of the present invention also comprises the step of at least some in neutralizing the surface charge on the lipid part of lipid-nucleic acid composition.By at least partially neutralizing the surface charge, unencapsulated nucleic acid is released from the lipid particle surface and can use conventional techniques to remove from the composition.In embodiments, unencapsulated and surface adsorbed nucleic acid is removed from the resulting composition by the exchange of buffer solution.For example, replacing citrate buffer (pH about 4.0, for forming composition) with HEPES buffered saline (HBS pH about 7.5) solution will cause neutralization of liposome surface and nucleic acid to release from the surface.Then standard method can be used to remove the nucleic acid released via chromatography, and then it is converted to a buffer having a pH higher than the pKa of the lipid used.

[0545] Optionally, lipid vesicles (i.e. lipid particles) can be formed by hydration in aqueous buffer and set the size using any of the above methods before adding nucleic acid. As mentioned above, the pH of the aqueous buffer should be lower than the pKa of the amino lipid. Then the solution of nucleic acid can be added to these vesicles that have been set to a size and formed in advance. In order to encapsulate nucleic acid in such preformed vesicles, the mixture should contain alcohol, such as ethanol. In the case of ethanol, it should be present in a concentration of about 20% (w / w) to about 45% (w / w). In addition, it may be necessary to warm the mixture of preformed vesicles and nucleic acids in the aqueous buffer-ethanol mixture to a temperature of about 25°C to about 50°C, depending on the composition of the lipid vesicle and the properties of the nucleic acid. It is obvious to those of ordinary skill in the art that optimizing the encapsulation process to achieve the desired nucleic acid level in the lipid vesicle will require manipulated variables, such as ethanol concentration and temperature. Once the nucleic acid is encapsulated in the preformed vesicle, the external pH can be increased to at least partially neutralize the surface charge. Unencapsulated and surface adsorbed nucleic acids can then be removed.

[0546] Vaccines and adjuvants

[0547] In some embodiments, the compounds and compositions of the invention are associated with a protein or nucleic acid antigen composition. For example, a lipid of the invention (e.g., any of Formulas I-XVI) can be associated with a protein or nucleic acid vaccine antigen (e.g., any of the target proteins described herein). For example, the invention can be directed to a method of vaccinating against a disease including any of the infectious diseases described herein by administering a lipid of the invention (e.g., any of Formulas I-XVI) associated with a protein or nucleic acid antigen composition (e.g., any of the target proteins described herein).

[0548] In embodiments, the protein or nucleic acid antigen is a betacoronavirus protein or an alphacoronavirus protein, or an antigenic fragment thereof, or a nucleic acid encoding them (e.g., RNA (e.g., mRNA) or DNA). In some embodiments, the betacoronavirus protein is selected from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, and HCoV-OC43 proteins, or antigenic fragments thereof, or nucleic acids encoding them (e.g., RNA (e.g., mRNA) or DNA). In some embodiments, the alphacoronavirus protein is selected from HCoV-NL63 and HCoV-229E proteins, or antigenic fragments thereof, or nucleic acids encoding them (e.g., RNA (e.g., mRNA) or DNA).

[0549] In some embodiments, the protein or nucleic acid antigen is a coronavirus protein, or an antigenic fragment thereof, or a nucleic acid encoding them (e.g., RNA (e.g., mRNA) or DNA). In multiple embodiments, the coronavirus protein is a protein from SARS-CoV-2, such as, for example, a SARS-CoV-2 spike protein. In embodiments, the SARS-CoV-2 protein is selected from a spike surface glycoprotein, a membrane glycoprotein M, an envelope protein E, and a nucleocapsid phosphoprotein N, or an antigenic fragment thereof, or a nucleic acid encoding them (e.g., RNA (e.g., mRNA) or DNA). In embodiments, the SARS-CoV-2 protein selected from the spike surface glycoprotein includes S1, S2, and S2'.

[0550] 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 embodiments, the alpha coronavirus is selected from HCoV-NL63 and HCoV-229E. In embodiments, the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0551] In an embodiment, the protein or nucleic acid antigen is an influenza virus antigen, optionally selected from hemagglutinin (HA) protein, matrix 2 (M2) protein and neuraminidase, or antigenic fragments thereof, or nucleic acids encoding them (e.g., RNA (e.g., mRNA) or DNA).

[0552] In embodiments, the disease or condition for which the vaccination is directed 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, varicella (chickenpox), rotavirus, and herpes zoster. In some embodiments, the invention relates to the treatment of hepatitis. Illustrative hepatitis that can be treated include, but are not limited to, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, autoimmune hepatitis, alcoholic hepatitis, acute hepatitis, and chronic hepatitis.

[0553] In some embodiments, the compounds, compositions or vaccines of the invention further comprise an adjuvant, optionally selected from an aluminum gel or an aluminum salt. In embodiments, the aluminum gel or aluminum 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 a chimeric protein complex as described herein.

[0554] In some embodiments, the additional adjuvant is selected from an oil-in-water emulsion formulation, a saponin adjuvant, ovalbumin, Freunds Adjuvant, a cytokine, and chitosan. Illustrative additional adjuvants include, but are not limited to: (1) ovalbumin (e.g., ENDOFIT), which is commonly used in biochemical research; (2) oil-in-water emulsion formulations (with or without other specific immunostimulants, such as muramyl peptides or bacterial cell wall components), such as, for example, (a) MF59 (PCT Publication No. WO 90 / 14837), containing 5% squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing varying amounts of MTP-PE), formulated into submicron particles using a microfluidizer such as, for example, a Model HOy microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10% squalane, 0.4% Tween 80, 5% pluronic blocked polymer L121 and thr-MDP, or 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.), containing 2% squalene, 0.2% Tween 80, and optionally one or more bacterial cell wall components selected from the group consisting of monophospholipid A (MPL), trehalose dimycolate (TDM) and cell wall skeleton (CWS), including MPL + CWS (DETOX TM); (d) ADDAVAX (Invitrogen); (3) saponin adjuvants such as STIMULON (Cambridge Bioscience, Worcester, Mass.) or particles produced therefrom, such as ISCOM (immunostimulatory complex); (4) Freund's complete adjuvant (CFA) and Freund's incomplete adjuvant (IFA); (5) cytokines, such as interleukins (as non-limiting examples, 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), tumor necrosis factor (TNF), etc.; (6) chitosan and other chitin or poly-N-acetyl-D-glucosamine derivatives in which a large proportion of the N-acetyl groups have been removed by hydrolysis (see, e.g., European Patent Application EP0460020, which is incorporated herein by reference in its entirety, disclosing pharmaceutical preparations comprising chitosan as a mucosal absorption promoter); 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 aluminum gel, a pattern recognition receptor (PRR) agonist, a CpG ODN, and an imidazoquinoline.In some embodiments, the additional adjuvant is one or more of the following: cyclic [G(3',5')pA(3',5')p] (e.g., 3'3'-cGAMP VACCIGRADE); cyclic [G(2',5')pA(3',5')p]2'3' (e.g., 2'3'cGAMP VACCIGRADE); cyclic [G(2',5')pA(2',5')p] (e.g., 2'2'-cGAMP VACCIGRADE), cyclic di-adenosine monophosphate (e.g., c-di-AMP VACCIGRADE); cyclic di-guanosine monophosphate (e.g., c-di-GMP VACCIGRADE); TLR7 agonist-imidazoquinoline compound (e.g., TLR7 agonist, such as, for example, Gardiquimod VACCIGRADE, Imiquimod VACCIGRADE, R848 coli 0111:B4 strain (e.g., LPS-EB VACCIGRADE); monophosphoryl lipid A (e.g., MPLA-SM VACCIGRADE and MPLA synthetic VACCIGRADE); N-glycolylated 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., ODN 1585 VACCIGRADE, ODN 1826 VACCIGRADE, ODN 2006 VACCIGRADE, ODN 2395 VACCIGRADE, VACCIGRADE), triacylated lipoprotein (e.g. Pam3CSK4VACCIGRADE); polyinosinic-polycytidylic acid (e.g. Poly(I:C)(HMW)VACCIGRADE); and cord factor (i.e. mycobacterial cell wall component trehalose 6,6' dimycolate (TDM)) or its analogs (e.g. TDB VACCIGRADE, TDB-HS15VACCIGRADE). 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, a stimulator of interferon genes (STING) ligand or a related agent.

[0555] In some embodiments, the additional adjuvant is one or more of the following: mineral adjuvants, gel-based adjuvants, tension active agents, bacterial products, oil emulsions, microparticle adjuvants, fusion proteins and lipopeptides. In addition to the aluminum adjuvants described elsewhere, other mineral salt adjuvants include calcium salts (e.g., calcium phosphate), iron salts and zirconium salts. In addition to the aluminum gel-based adjuvants described elsewhere, other gel-based adjuvants include acemannan. Tension active agents include QuilA, a saponin derived from an aqueous extract of a soap tree; saponins, surfactant glycosides containing a triterpene structure with a hydrophobic core and a carbohydrate chain attached to the core, and QS-21. Bacterial products include cell wall peptidoglycan or lipopolysaccharide, N-acetylmuramoyl-L-alanyl-D-isoglutamine (MDP), different compounds derived from MDP (e.g., threonyl-MDP), lipopolysaccharide (LPS) (e.g., cell wall from gram-negative bacteria), trehalose dimycolate (TDM), and DNA containing CpG motifs, from the cell wall of gram-negative bacteria (e.g., from mycobacteria, short corynebacterium, granulomatous corynebacterium, pertussis bordetella, and meningitidis), oil emulsions include oil-based adjuvants and various liposomes of FIA, Montanide, adjuvant 65, Lipovant, montanide family. In particulate and polymer systems, poly (DL-lactide-co-glycolide) microspheres have been extensively studied and can be used herein.

[0556] 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.

[0557] Administration / Dosage / Excipients

[0558] Administration of these compositions according to the present invention may be by any common route, as long as the target tissue can be reached by such route. This includes oral, nasal or buccal administration. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal, intraportal or intravenous injection, or by direct injection into a diseased, e.g., cancerous tissue. The agents disclosed herein may also be administered by a catheter system. Such compositions will normally be administered as pharmaceutically acceptable compositions as described herein.

[0559] In an embodiment, the composition of the invention is administered parenterally, e.g., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In an embodiment, the pharmaceutical composition is administered intravenously or intraperitoneally by bolus injection.

[0560] In some embodiments, the lipid-nucleic acid composition can be inhaled into the lungs in the form of an aerosol (see, Brigham et al., Am. J. Sci. 298 (4): 278-281 (1989)), optionally by using a vibrating mesh nebulizer (e.g., but not limited to, AEROGEN SOLO (Galway, Ireland)). In some embodiments, the lipid-nucleic acid composition can be administered by direct injection at the site of disease (Culver, Human Gene Therapy, Mary Ann Liebert, Inc., Publishers, New York. Pages 70-71 (1994)).

[0561] The administration of the compositions described herein can be, for example, by injection, topical administration, ocular administration, and intranasal administration. Injection can be associated with electricity in some embodiments (e.g., electroporation, including devices that can be used for electrochemotherapy (e.g., CLINI PORATOR, IGEA Srl, Carpi [MO], Italy)). Topical administration can be, but is not limited to, creams, lotions, ointments, gels, sprays, solutions, etc. Topical administration can also include penetration enhancers, such as, but not limited to, surfactants, fatty acids, bile salts, chelating agents, non-chelating non-surfactants, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-hexadecyl ether, fatty acids and / or salts combined with bile acids and / or bile salts, sodium salts combined with lauric acid, capric acid, and UDCA, etc. Topical administration can also include fragrances, colorants, sunscreens, antibacterial agents, and / or moisturizers. The compositions described herein can be applied to at least one site, such as, but not limited to, the forehead, scalp, hair follicles, hair, upper eyelids, lower eyelids, eyebrows, eyelashes, infraorbital area, periorbital area, temples, nose, bridge of the nose, cheeks, tongue, nasolabial folds, lips, periocular area, jawline, ears, neck, breasts, forearms, upper arms, palms, hands, fingers, nails, back, abdomen, flanks, buttocks, thighs, calves, feet, toes, etc.

[0562] Administration routes include, for example, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, intravaginal, transdermal, intraportal, rectal, by inhalation, or topically, particularly to the ear, nose, eye, or skin. In an embodiment, administration is achieved orally or by parenteral injection.

[0563] When formulated, the solution can be administered in a manner compatible with the dosage formulation and in an amount effective for treatment, as described herein. The preparation can be easily administered in a variety of dosage forms, such as injectable solutions, drug release capsules, etc. For parenteral administration in the form of an aqueous solution, for example, the solution is usually appropriately buffered and the liquid diluent is first made isotonic with, for example, sufficient saline or glucose. For example, such aqueous solutions can be used for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Preferably, a sterile aqueous medium known to those skilled in the art, particularly according to the present disclosure, is used.

[0564] In an embodiment, the nucleic acid and lipid of the invention are administered topically, optionally by one or more of subcutaneous injection, intradermal injection, subdermal injection, and intramuscular injection, and an effective dose is administered to about 4 mm 2 To about 150mm 2 (e.g. about or no more than about 4 mm 2 , or about 5mm 2 , or about 6mm 2 , or about 7mm 2 , or about 8mm 2 , or about 10mm 2 , or about 20mm 2 , or about 50mm 2 , or about 100mm 2 , or about 150mm 2 ) surface area. In various embodiments, the nucleic acid and lipid of the invention are administered topically, optionally by one or more of subcutaneous injection, intradermal injection, subdermal injection, and intramuscular injection, and the effective dose is administered to no more than about 4 mm 2 , or about 5mm 2 , or about 6mm 2 , or about 7mm 2 , or about 8mm 2 , or about 10mm 2 , or about 20mm 2 , or about 50mm 2 , or about 100mm 2 , or about 150mm 2 In various embodiments, the nucleic acid and lipid of the invention are administered topically, optionally by one or more of subcutaneous injection, intradermal injection, subdermal injection, and intramuscular injection, and an effective dose is administered to about 4 mm2 , or about 5mm 2 , or about 6mm 2 , or about 7mm 2 , or about 8mm 2 , or about 10mm 2 , or about 20mm 2 , or about 50mm 2 , or about 100mm 2 , or about 150mm 2 surface area.

[0565] In various embodiments, the nucleic acid and the lipid present are administered topically, optionally by one or more of subcutaneous injection, intradermal injection, subdermal injection, and intramuscular injection, and the effective dose (weight RNA / injected surface area) is about 35 ng / cm 2 to about 7000ng / cm 2 In various embodiments, the nucleic acid and lipid of the invention are administered topically, optionally by one or more of subcutaneous injection, intradermal injection, subdermal injection, and intramuscular injection, and the effective dose (weight RNA / injected surface area) does not exceed about 35 ng / cm 2 , or about 50ng / cm 2 , or about 75ng / cm 2 , or about 100ng / cm 2 , or about 125ng / cm 2 , or about 150ng / cm 2 , or about 175ng / cm 2 , or about 200ng / cm 2 , or about 225ng / cm 2 , or about 250ng / cm 2 , or about 500ng / cm 2 , or about 1000ng / cm 2 , or about 2000ng / cm 2 , or about 5000ng / cm 2 , or about 7000ng / cm 2 In various embodiments, the nucleic acid and ...

Claims

1. A method for preparing a compound of formula (IV): wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

2. The method of claim 1, wherein the method comprises contacting a compound of formula (IV-A) with hydrazine to provide a compound of formula (IV), wherein the compound (IV-A) is represented by the following structure: wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

3. The method of claim 2, wherein the method further comprises contacting the compound of formula (IV-B) with N-(2,3-epoxypropyl)-phthalimide to provide a compound of formula (IV-A), wherein the compound (IV-B) is represented by the following structure: wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

4. The method of claim 3, wherein the method further comprises making the structure H2N-(CH2) n The diamine represented by -NH2 is contacted with linoleoyl chloride to provide a compound of formula (IV-B) wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

5. The method of claim 4, wherein the contacting comprises adding triethylamine, a metal hydride, or a combination thereof.

6. The method of claim 5, further comprising extracting the compound of formula (IV-B) from a reaction containing a metal hydride and a first solvent, wherein the extracting comprises: (a) quenching the reaction with water; (b) removing the first solvent; (c) removing excess water; and (d) extracting the compound of formula (IV-B) with a second solvent to obtain an extracted compound of formula (IV-B).

7. The method of claim 1, further comprising purifying the compound of formula (IV), wherein the purifying comprises: (a) suspending a composition comprising a compound of formula (IV) and one or more impurities in a solvent; (b) removing the precipitate containing at least one of the one or more impurities; and (c) removing the solvent to obtain a purified compound of formula (IV).

Images