Ionizable lipids and nanoparticles comprising same

Abstract

The present disclosure provides one or more ionizable lipids and lipid nanoparticles comprising the same. The present disclosure also provides pharmaceutical compositions comprising lipid nanoparticles encapsulating an active agent.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority under 35 USC §119(e) to U.S. Provisional Patent Application No. 63 / 350,540, filed on June 9, 2022, entitled “IONIZABLE LIPIDS AND NANOPARTICLES COMPRISING SAME,” and U.S. Provisional Patent Application No. 63 / 437,800, filed on January 9, 2023, entitled “IONIZABLE LIPIDS AND NANOPARTICLES COMPRISING SAME,” which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to ionizable lipids and lipid nanoparticles containing the same and uses thereof in pharmaceutical compositions. Background Art

[0004] New delivery methods for therapeutic and diagnostic compounds are constantly being developed. Although lipid-based nanoparticles are a well-known mode of delivery, these agents are also constantly being improved. Among other issues, the ability of therapeutic carriers to effectively load and then deliver active agents to the target site is very important for reducing medication and improving treatment efficiency.

[0005] Although various ionizable lipids capable of encapsulating hydrophilic agents such as DNA and / or RNA are known, there is a constant need for new and better ionizable lipids. In particular, there is a great need to develop new and better ionizable lipids capable of enhancing the delivery of drugs to specific locations in the body. Summary of the invention

[0006] The present invention provides novel compounds suitable for use as ionizable lipids. In addition, nanoparticles comprising the same are provided. Compositions comprising the nanoparticles are also provided, which can be used to deliver an active agent to a subject, such as for treating or preventing a disease or disorder in the subject.

[0007] According to a first aspect, a compound represented by the following formula I is provided:

[0008] Where: each L is independently R1, Each L1 is independently R1,

[0009]

[0010] represents a single bond, a triple bond or a double bond; Z independently represents -OH or -SH; each k is independently between 0 and 10 or between 1 and 24, including any range therebetween; each Y independently does not exist or contains CH 2 , CHR' 2 NR' 2 wherein the alkyl groups are selected from the group consisting of: -NH, O, S, -CONH-, -CONR'-, -C(=NH)NR'-, -C(=S)NR'-, -NC(=O)-, -NC(=O)O-, -NC(=O)N-, -NC(=S)O-, -NC(=S)N-, -C(=O)-, -C(=O)O-, -OC(=O)O-, -OC(=O)N-, -OC(=S)O-, -OC(=S)N-, or phosphate, as valency permits; each T independently represents an optionally substituted C5-C30 alkyl or an optionally substituted C5-C30 alkenyl; each R' independently represents H or a C5-C30 alkyl group comprising an optionally substituted C5-C30 alkyl group; 1 -C 10 Alkyl, C 1 -C 10 Alkyl-aryl, C 1 -C 10 Alkyl-cycloalkyl, optionally substituted C 3 -C 10 Cycloalkyl, optionally substituted C 3 -C 10 heterocyclic group, optionally substituted heteroaryl group, optionally substituted aryl group or a combination thereof; each X independently represents a heteroatom, CH 2 , optionally substituted C1-C10 alkyl, or X is absent; each n and p are independently between 0 and 5, and at least one n is not 0; m is between 1 and 3; each R is independently H, or contains optionally substituted C5-C30 alkyl; each R1 is optionally substituted C1-C24 alkyl, and at least one L or L1 is or contains

[0011] In one embodiment, any one of R and R1 further comprises at least one unsaturated bond.

[0012] In one embodiment, the heteroatom comprises O, N, NH, NR1, S or a phosphate group.

[0013] In one embodiment, each L is independently

[0014] In one embodiment, each X is independently O, or absent.

[0015] In one embodiment, any one of R and R1 represents a linear or branched C1-C24 or C1-C10 alkyl group.

[0016] In one embodiment, the compound is represented by Formula II: wherein each L is independently R1, or; and at least one of L is

[0017] In one embodiment, the compound is characterized by a pKa value between 5 and 9.

[0018] In one embodiment, the compound includes any one of Example 1 or Example 4.

[0019] In another aspect, lipid nanoparticles comprising a compound of the invention and an active agent are provided.

[0020] In one embodiment, the average size of the lipid nanoparticles is in the range of 50 to 300 nm.

[0021] In one embodiment, the active agent comprises a polynucleic acid.

[0022] In one embodiment, the lipid nanoparticle further comprises a lipid, wherein the lipid comprises a helper lipid, and optionally comprises a structural lipid, a modified lipid, or any combination thereof.

[0023] In one embodiment, the weight ratio between (i) the total amount of the compound and lipid and (ii) the polynucleic acid within the lipid nanoparticle is between 0.001:1 and 10:1.

[0024] In one embodiment, the lipids include a helper lipid, a modifying lipid, and a sterol.

[0025] In one embodiment, the ratio of the compound relative to the total lipid content of the lipid nanoparticles is between 10 and 80 mol%.

[0026] In another aspect, a pharmaceutical composition is provided, comprising a plurality of lipid nanoparticles of the present invention and a pharmaceutically acceptable carrier.

[0027] In one embodiment, the pharmaceutical composition comprises an effective amount of an active agent.

[0028] In one embodiment, the pharmaceutical composition is formulated for systemic administration to a subject, local administration to a subject, or both.

[0029] In one embodiment, the pharmaceutical composition is used to treat a disease or disorder in a subject in need thereof.

[0030] In another aspect, a method of delivering an active agent to a tissue of a subject is provided, the method comprising administering to the subject an effective amount of a pharmaceutical composition of the present invention, thereby delivering the active agent to the tissue.

[0031] Further embodiments and the full scope of application of the present invention will be apparent based on the detailed description given below. However, it should be understood that although the detailed description and specific examples point out the preferred embodiments of the present invention, they are given only as examples, because based on the detailed description, various changes and modifications within the spirit and scope of the present invention are apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Some embodiments of the present invention are described herein with reference to the accompanying drawings, by way of example only. When referring specifically now to the drawings, it is noted that the details shown are by way of example, and the purpose is to exemplify the embodiments of the present invention. In this regard, the description in conjunction with the drawings makes it apparent to those skilled in the art how the embodiments of the present invention may be practiced.

[0033] Figure 1 . Shows in vivo bar graph expression analysis of three LNP compositions in liver tissue compared to heart tissue, spleen tissue, kidney tissue and lung tissue a. FMB-1050, FMB-428 and FMB-389.

[0034] Figure 2 . Shows in vivo bar graph expression analysis of three LNP compositions a. FMB-1143, FMB-748 and 745 in lung tissue compared to heart tissue, spleen tissue, kidney tissue and liver tissue. DETAILED DESCRIPTION

[0035] Compounds disclosed in the present invention are found by computational screening methods. Ionizable lipid candidates are generated by computers (in-silico) and are ranked based on their activity expected using machine learning algorithms. After several computer optimization cycles, a chemical library containing several molecules is obtained. Disclosed compounds are selected based on the results obtained by in vitro experiments, as shown below (Example section).

[0036] Compounds having at least 30% encapsulation efficiency (such as at least 80% encapsulation efficiency) and inducing at least about 10-fold higher intracellular expression and / or cellular internalization of the RNA sequence compared to transfection with Lipofectamine 2000 (see Examples section) are selected as ionizable lipid candidates suitable for further in vivo studies.

[0037] According to a first aspect, a compound is provided, comprising an ionizable portion (e.g., a head group) covalently bound to a lipophilic tail (e.g., a hydrocarbon-based chain), wherein the ionizable portion is represented by any of the following formulas:

[0038] wherein the wavy bond indicates the point of attachment to H, LI or to the lipophilic tail; wherein each L 1 , X and n are independently as described below, and wherein each R2 independently represents H, or one or more substituents as described herein. In some embodiments, each L and / or X represents the same or different chemical moieties. In some embodiments, each n represents the same or different numerical values ​​or ranges. In some embodiments, at least one wavy bond represents a point of attachment to the lipophilic tail.

[0039] In some embodiments, the lipophilic tail comprises 10 to 50 carbon atoms (straight, branched or cyclic hydrocarbon chains), and optionally comprises one or more unsaturated bonds. In some embodiments, the compound is an amphiphilic compound. In some embodiments, the compound can spontaneously self-assemble to form nanoparticles (e.g., lipid nanoparticles) in aqueous solution.

[0040] In some embodiments, the ionizable moiety is capable of ionizing (protonating or cationizing) in a solution at a pH below the pKa value of the ionizable moiety. In some embodiments, the ionizable moiety is capable of protonating in a solution at a pH below the pKa value of the ionizable moiety. In some embodiments, at least 50 mol% of the ionizable moiety is positively charged (or protonated) in a solution at a pH below the pKa value of the ionizable moiety.

[0041] In some embodiments, the pKa value of the ionizable moiety is between 5 and 9, including any range therebetween. In some embodiments, the pKa value of the ionizable moiety is between 5 and 8, between 6 and 8, between 6 and 7, between 7 and 9, between 6 and 9, including any range therebetween.

[0042] In some embodiments, the ionizable moiety is attached to the lipophilic tail through a spacer or through a covalent bond.

[0043] In some embodiments, the lipophilic tail comprises one or more moieties represented by the formula:

[0044]

[0045] in:

[0046] represents a single bond, a double bond or a triple bond; R2 is as described herein; k and n are integers each independently between 1 and 10, or between 1 and 24, including any range therebetween; and y is between 1 and 3.

[0047] In some embodiments, the MW of the compounds of the invention is between 100 and 2000 Da, between 100 and 300 Da, between 100 and 500 Da, between 100 and 800 Da, between 300 and 500 Da, between 100 and 1,000 Da, between 500 and 800 Da, between 500 and 2000 Da, between 500 and 1,000 Da, between 800 and 1,000 Da, between 800 and 1,500, between 1,000 and 2,000, including any ranges therebetween.

[0048] In another aspect, a compound and / or a salt thereof is provided, wherein the compound is represented by the following Formula 1: Or represented by the following formula 1A:

[0049] Where: each L is independently R1,

[0050]

[0051] Each L1 is independently R1,

[0052] Each X independently represents a heteroatom, CH2, an optionally substituted C1-C10 alkyl, or X is absent (i.e., is a bond); each n and p are independently between 0 and 5, and at least one n is not 0; m is between 1 and 3; Z independently represents -OH or -SH; each R is independently H, or comprises an optionally substituted C5-C30 alkyl, or an optionally substituted C1-C30 alkyl; and each R1 is an optionally substituted C1-C24 alkyl, an optionally substituted C1-C24 alkylhydroxyl, (optionally substituted C1-C10 alkyl)-X(R')-, and at least one of L and L1 is or comprises

[0053] In some embodiments, any one of R and R1 further comprises at least one unsaturated bond.

[0054] In some embodiments, the compounds of the present invention are represented by the following formula: Wherein each L1 and L2 is independently R1, wherein X, Z, L, R, R1, p, n, m are as described herein; and wherein at least one of L and L1 is or comprises

[0055] In some embodiments, each n and p is independently between 0 and 5, between 0 and 3, between 0 and 2 (e.g., 0, 1, 2, 3, 4, or 5), and at least one n is not 0 (e.g., 1, 2, 3, 4, or 5); m is between 1 and 3 (e.g., 1, 2, or 3), including any combination thereof.

[0056] In some embodiments, R is represented by the formula:

[0057] Or it can be expressed as:

[0058] in: represents a single bond, a triple bond or a double bond; k and n are integers, each independently between 1 and 10, or between 1 and 24, including any range therebetween; Z independently represents -OH or -SH; and y is between 1 and 3; and each Y is absent or independently comprises CH2, CHR'2, NR'2, NH, O, S, -CONH-, -CONR'-, -C(=NH)NR'-, -C(=S)NR'-, -NC(= In some embodiments, each R' is independently H or comprises an optionally substituted C1-C10 alkyl, a C1-C10 alkyl-aryl, a C1-C10 alkyl-cycloalkyl, an optionally substituted C3-C10 cycloalkyl, an optionally substituted C3-C10 heterocyclyl, an optionally substituted heteroaryl, an optionally substituted aryl, or a combination thereof, or R' is absent, as valence permits.

[0059] In some embodiments, the heteroatom includes O, N, NH, NR1, or S. In some embodiments, each X is independently O or absent.

[0060] In some embodiments, one of R and R1 each independently represents a linear or branched alkyl group.

[0061] In some embodiments, L is wherein R is as described herein. In some embodiments, each R represents the same or different alkyl groups.

[0062] In some embodiments, L is wherein R is as described herein. In some embodiments, each R represents the same or different alkyl groups.

[0063] In some embodiments, L is wherein R is as described herein. In some embodiments, each R represents the same or different alkyl groups.

[0064] In some embodiments, L is wherein R is as described herein. In some embodiments, each R represents the same or different alkyl groups.

[0065] The term "alkyl" as used herein describes aliphatic hydrocarbons including straight and branched groups. In some embodiments, the alkyl group has 1 to 10 carbon atoms, 1 to 30 carbon atoms, 1 to 24 or 5-30 carbon atoms. In some embodiments, the alkyl group is a C1-C6 alkyl group. In some embodiments, the alkyl group is a C1-C6 alkyl group, a C1-C10 alkyl group, a C1-C8 alkyl group, a C5-C30 alkyl group, a C5-C24 alkyl group, a C5-C20 alkyl group, a C5-C10 alkyl group, a C8-C30 alkyl group, a C8-C24 alkyl group, a C8-C15 alkyl group, a C8-C20 alkyl group, a C8-C12 alkyl group, including any range therebetween. Whenever a numerical range is mentioned herein, such as "5-30", it indicates that the group (in this case, an alkyl group) may contain 5 carbon atoms, 6 carbon atoms, 10 carbon atoms, 5 to 20, 5 to 25, 5 to 30, 10 to 20, 10 to 25, 10 to 30 carbon atoms, including any range therebetween, up to and including 30 carbon atoms. Alkyl groups may be substituted or unsubstituted, as defined herein.

[0066] As used herein, the term "alkyl" also includes saturated or unsaturated hydrocarbons, and thus the term further includes alkenyl and alkynyl groups.

[0067] The term "alkenyl", as defined herein, describes an unsaturated alkyl group having from 2 to 30 carbon atoms and at least one carbon-carbon double bond. An alkenyl group may be substituted with one or more substituents or unsubstituted, as described above.

[0068] The term "alkynyl", as defined herein, is an unsaturated alkyl radical having from 2 to 30 carbon atoms and at least one carbon-carbon triple bond. Alkynyl radicals may be substituted with one or more substituents or unsubstituted, as described above.

[0069] In some embodiments, the alkyl group is a C1-C10 alkyl group or a C1-C6 alkyl group.

[0070] The term "C1-C10 alkyl" as used herein includes any C1-C10 alkyl related compounds, and refers to any straight or branched alkyl chain containing 1 to 6, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10 carbon atoms (including any range therebetween). In some embodiments, the C1-C10 alkyl includes any of the following: methyl, ethyl, propyl, butyl, pentyl, isopentyl, hexyl, heptyl, octyl, nonyl, decyl and tert-butyl, or any combination thereof. In some embodiments, the C1-C10 alkyl described herein further includes an unsaturated bond, wherein the unsaturated bond is located at the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th or 10th position of the C1-C10 alkyl.

[0071] The term "C1-C6 alkyl" as used herein includes any C1-C6 alkyl related compounds, and refers to any straight or branched alkyl chain containing 1 to 6, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6 carbon atoms (including any range therebetween). In some embodiments, the C1-C6 alkyl includes any of the following: methyl, ethyl, propyl, butyl, pentyl, isopentyl, hexyl and tert-butyl, or any combination thereof. In some embodiments, the C1-C6 alkyl described herein further includes an unsaturated bond, wherein the unsaturated bond is located at the 1st, 2nd, 3rd, 4th, 5th or 6th position of the C1-C6 alkyl.

[0072] In some embodiments, the compounds of the present invention are represented by the following Formula II: Wherein L, X, Z and n are each independently as described above, and wherein at least one L is or comprises

[0073] In some embodiments, the compounds of the present invention are represented by the following formula III: wherein L, X and n are each independently as described above, and wherein at least one L is or comprises In some embodiments, the compounds of the invention are represented by any of the formulas disclosed herein, wherein each L is the same or different.

[0074] In some embodiments, the compounds of the present invention are represented by Formula I to III, wherein the sum of all n within the molecule is between 2 and 15, between 2 and 10, between 2 and 8, including any ranges therebetween.

[0075] In some embodiments, the compounds of the present invention are represented by the following Formula IIA: wherein each X is independently 0 or absent; n1, n2 and n3 are each independently between 0 and 3, and wherein the sum of n1, n2 and n3 is between 2 and 15, between 2 and 10, between 2 and 8, including any ranges therebetween, and L is as described herein; and wherein at least one L is or comprises wherein R is as described above, and wherein Z is OH. In some embodiments, the compounds of the invention are represented by Formula IIA, wherein each L is and R is as described above, and wherein Z is OH. In some embodiments, the compounds of the invention are represented by Formula IIA, wherein each X is absent, and the sum of n1, n2, and n3 is between 2 and 15, between 2 and 10, between 2 and 8, including any ranges therebetween. In some embodiments, the compounds of the invention are represented by Formula IIA, wherein each X represents O, n1 is between 1 and 3; and each of n2 and n3 is independently between 1 and 5, between 1 and 3, and wherein the sum of n1, n2, and n3 is between 2 and 15, between 2 and 10, between 2 and 8, including any ranges therebetween.

[0076] In some embodiments, the compounds of the present invention are represented by the following formula IIIA: wherein each X is independently 0 or absent; n1, n'1, n2, n'2, n3 and n'3 are each independently between 0 and 3, wherein the sum of n1, n2 and n3 is between 2 and 15, between 2 and 10, between 2 and 8, including any ranges therebetween; and wherein the sum of n'1, n'2 and n'3 is between 2 and 15, between 2 and 10, between 2 and 8, including any ranges therebetween, and L is as described herein, and wherein at least one L is or comprises wherein R is as described above, and wherein Z is OH. In some embodiments, the compounds of the invention are represented by Formula IIIA, wherein each L is and wherein R is as described above, and wherein Z is OH.

[0077] In some embodiments, the compounds of the present invention are represented by the following Formula IIIB: wherein each r independently represents an integer between 2 and 15, between 2 and 10, between 2 and 8, including any ranges therebetween, and each L and R2 are independently as described herein.

[0078] In some embodiments, the compounds of the invention are represented by Formula IIIA, wherein each X is O; each n1 and n'1 are independently between 0 and 3, n2, n'2, n3 and n'3 are independently between 0 and 3, wherein the sum of n1, n2 and n3 is between 2 and 15, between 2 and 10, between 2 and 8, including any ranges therebetween; and wherein the sum of n'1, n'2 and n'3 is between 2 and 15, between 2 and 10, between 2 and 8, including any ranges therebetween; and wherein each L is and R is as described above.

[0079] In some embodiments, the compounds of the invention are represented by any of the formulas disclosed herein, wherein at least one L is or where each L is and R is as described above.

[0080] In some embodiments, the compound of the invention includes any one of the compounds of Example 1, including any salt, any tautomer and / or any stereoisomer (eg, enantiomer and / or diastereomer) thereof.

[0081] As used herein, the term "substituted" or the term "substituent" refers to one or more (eg, 2, 3, 4, 5, or 6) substituents, wherein the substituents are as described herein.

[0082] As used herein, the term substituent includes halogen, -NO 2 、-CN、-OH、-CONH 2 ,-CONR' 2 ,-CNNR' 2 , -CSNR' 2 , -CONH-OH, -CONH-NH 2 , -NHCOR, -NHCSR, -NHCNR, -NC(=O)OR, -NC(=O)NR', ​​-NC(=S)OR', -NC(=S)NR', ​​-SO 2 R', -SOR', -SR', -SO 2 OR', -SO 2 N(R') 2 、-NHNR' 2 ,-NNR',C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 Alkyl, -NH 2 、-NH(C 1 -C 6 Alkyl), -N(C 1 -C 6 alkyl)2 , C 1 -C 6 Alkoxy, C 1 -C 6 Haloalkoxy, hydroxyl (C 1 -C 6 Alkyl), hydroxyl (C 1 -C 6 Alkoxy), alkoxy (C 1 -C 6 Alkyl), alkoxy (C 1 -C 6 Alkoxy), C 1 -C 6 Alkyl-NR' 2 , C 1 -C 6 Alkyl-SR', -CONH(C 1 -C 6 Alkyl), -CON(C 1 -C 6 alkyl) 2 , -CO 2 H, -CO 2 R', -OCOR, -OCOR', -OC(=O)OR', -OC(=O)NR', ​​-OC(=S)OR', -OC(=S)NR', ​​or a combination thereof; wherein each R' independently represents hydrogen, or is selected from optionally substituted C 1 -C 10 Alkyl, optionally substituted C 3 -C 10 Cycloalkyl, optionally substituted C 3 -C 10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof.

[0083] The term "C 1 -C 6 "Haloalkyl" refers to a C group described herein substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 halogen atoms. 1 -C 6 Alkyl, wherein the halo is selected from F, Br, Cl, and I, or a combination thereof.

[0084] As used herein, the term “(C 3 -C 10 ")cycloalkyl" refers to an optionally substituted C3, C4, C5, C6, C7, C8, C9 or C10 ring. In some embodiments, (C 3 -C 10 ) ring includes optionally substituted cyclopropane, cyclobutene, cyclopentane, cyclohexane or cycloheptane.

[0085] The term "C 3 -C 10 "Heterocyclyl" refers to an optionally substituted C3, C4, C5, C6, C7, C8, C9 or C10 heterocyclic aromatic and / or aliphatic or unsaturated ring.

[0086] In some embodiments, the term "hydroxyl (C 1 -C 6 alkyl)" and the term "C 1 -C 6 "Alkoxy" is used interchangeably herein and refers to a C 2-hydroxyl group as described herein substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hydroxy groups. 1 -C 6 Alkyl, where the hydroxyl group is located at the C 1 -C 6 The 1st, 2nd, 3rd, 4th, 5th or 6th position of the alkyl group, including any combination thereof.

[0087] In some embodiments, the compounds of the invention comprise substantially a single enantiomer of any one of the compounds described herein, wherein substantially is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99% by weight, including any values ​​therebetween.

[0088] In some embodiments, the compounds of the present invention further include any structurally similar functional derivatives of the compounds disclosed herein, wherein the structural similarity is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% structural similarity, including any ranges therebetween.

[0089] In some embodiments, functional derivatives refer to such ionizable lipids: its pKa value is between 6.2 and 6.8, and can be self-assembled in water, thereby stably combining and / or encapsulating polynucleic acids. In some embodiments, functional derivatives are further composed of cells of internalized polynucleic acids (for example, by forming lipid nanoparticles as described herein). Cell internalization can be determined as described below.

[0090] In some embodiments, the term "structural similarity" refers to the fingerprint similarity between two molecules. The term "fingerprint similarity" is well known to those skilled in the art. In some embodiments, the fingerprint similarity is calculated based on a circular fingerprint, a fingerprint based on substructure keypoints, and / or a fingerprint based on topology or path.

[0091] Exemplary circular fingerprints include, but are not limited to, Molprint 2D, ECFP (or Morgan fingerprint), FCFP, etc. In some embodiments, the term "structural similarity" as used herein is calculated by Morgan fingerprint.

[0092] Carrier

[0093] On the other hand, a carrier of an active agent (one or more) is provided, wherein the carrier is in the form of nanoparticles. In some embodiments, the carrier encapsulates the active agent in a core. In some embodiments, the active agent is a small molecule and / or a biomolecule, such as a polypeptide, a polynucleotide, etc. In some embodiments, the active agent is water-soluble (e.g., having a water solubility of at least 0.1 g / L at a temperature between 20 and 30 ° C). In some embodiments, the active agent is selected from a therapeutic agent, a prophylactic agent, and a diagnostic agent, including any combination thereof. In some embodiments, one or more active agents are selected from: proteins, peptides, nucleic acids, small molecules, and antibodies.

[0094] In some embodiments, the carrier is in the form of lipid nanoparticles comprising a compound of the present invention and an active agent. In some embodiments, the lipid nanoparticle includes a shell and an aqueous core (core), and the aqueous core (core) comprises an active agent. In some embodiments, the shell of the lipid nanoparticle comprises a compound of the present invention. In some embodiments, the shell of the lipid nanoparticle also comprises lipids, sterols and / or PEG-lipids, or any combination thereof. In alternative embodiments, the carrier is in the form of lipid nanoparticles comprising a compound of the present invention, a lipid and an active agent. In some embodiments, the lipid nanoparticle is in the form of a core-shell nanoparticle, wherein the shell of the nanoparticle comprises lipids and at least one compound of the present invention. In some embodiments, the compound of the present invention is combined with an active agent (e.g., a polynucleotide) (e.g., by electrostatic interaction).

[0095] In some embodiments, under appropriate conditions, at least one compound of the present invention and optionally lipids (and optionally active agents) spontaneously self-assemble in aqueous solution to form lipid nanoparticles. In some embodiments, the term "lipid nanoparticle" refers to such nanoparticles (e.g., substantially spherical particles): wherein the shell of the nanoparticle comprises one or more compounds of the present invention and optionally one or more lipids (e.g., auxiliary lipids, such as cationic lipids, non-cationic lipids; and optionally sterols, and / or PEG-modified lipids). Preferably, lipid nanoparticles are formulated to deliver one or more agents to one or more target cells.

[0096] In some embodiments, the nanoparticles have a spherical geometry or shape. In some embodiments, the nanoparticles have an inflated or deflated shape. In some embodiments, the plurality of core-shell particles lack any characteristic geometry or shape. In some embodiments, the nanoparticles have a spherical, quasi-spherical, quasi-ellipsoidal, deflated, concave, irregular shape, or any combination thereof.

[0097] In some embodiments, the multiple core-shell particles are substantially spherical, wherein substantially as described herein. In some embodiments, the multiple core-shell particles are substantially elliptical, wherein substantially as described herein. It will be appreciated by those skilled in the art that the exact shape of each of the multiple core-shell particles can vary between particles. In addition, the exact shape of the nanoparticles can be derived from any geometric form listed above, so the shape of the particles does not fully conform to a specific geometric form. It will be appreciated by those skilled in the art that the exact shape of the nanoparticles can have significant deviations (such as at least 5%, at least 10%, at least 20% deviation) from a specific geometry (e.g., spherical or elliptical).

[0098] In some embodiments, lipid includes helper lipid.In some embodiments, lipid includes structured lipid, PEG-lipid or both.In some embodiments, lipid includes helper lipid, and optionally includes structured lipid and / or PEG-lipid.In some embodiments, term "structured lipid" encompasses non-liposome forming lipid, as described herein.In some embodiments, structured lipid is or includes sterol.

[0099] In some embodiments, the helper lipid is or includes a phospholipid. In some embodiments, the helper lipid is or includes a liposome-forming lipid. As used herein, the term "liposome-forming lipid" encompasses lipids (e.g., phospholipids) that have a transition temperature (T m ) or above temperature when dispersed or dissolved in aqueous solution to form stable vesicles (e.g., lipid nanoparticles). The terms "liposome-forming lipids" and "lipid nanoparticle-forming lipids" are used interchangeably herein. The term Tm used herein refers to the temperature at which a lipid undergoes a phase transition from a solid (ordered phase, also referred to as a gel phase) to a fluid (disordered phase, also referred to as a fluid crystalline phase). Tm also refers to the temperature (or temperature range) at which the maximum change in heat capacity occurs during a phase transition.

[0100] In some embodiments, the phospholipids encompass a single phospholipid species or a plurality of chemically distinct phospholipids.

[0101] In some embodiments, the liposome-forming lipids are phospholipids having one or two C12 to C24 hydrocarbon tails (generally acyl, alkyl or alkenyl chains) and have varying degrees of unsaturation, ranging from fully saturated to fully hydrogenated, partially hydrogenated or non-hydrogenated lipids (the level of saturation can affect the rigidity of the liposomes formed thereby (generally, liposomes formed from lipids having saturated chains are more rigid than liposomes formed from lipids of the same chain length in which unsaturated chains (particularly with cis double bonds) are present). In some embodiments, at least one liposome-forming lipid is a phospholipid having one or two C12 to C20, C16 to C20, or C16 to C18 hydrocarbon tails (including any values ​​and ranges therebetween). In some embodiments, the liposome-forming lipids are fully saturated, linear or branched.

[0102] Furthermore, the phospholipids can be of natural origin (eg, naturally occurring phospholipids), semisynthetic, or fully synthetic lipids, and can be neutrally charged (eg, zwitterionic), negatively charged, or positively charged.

[0103] The non-limiting examples of neutral phospholipids include but are not limited to diacylphosphatidylcholine, dialkylphosphatidylcholine, sphingomyelin and diacylphosphatidylethanolamine.Phosphatidylcholine (PC), including those obtained from (chicken) eggs, soybeans or other plant sources or those partially or completely synthesized or with variable lipid chain length and degree of unsaturation, are all suitable for the present composition.Synthesis, semi-synthesis and natural product phosphatidylcholine, including but not limited to POPC, DOPC, DMPC, distearoylphosphatidylcholine (DSPC), hydrogenated soybean phosphatidylcholine (HSPC), soybean phosphatidylcholine (soybean PC), egg phosphatidylcholine (egg PC), hydrogenated egg phosphatidylcholine (HEPC) and dipalmitoylphosphatidylcholine (DPPC), are phosphatidylcholine suitable for preparing liposomes. Charged phospholipids may include phosphatidylglycerol, cardiolipin, or head group modified lipids such as N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, and PEG-derivatized phosphatidylethanolamine.

[0104] Non-limiting examples of cationic lipids or ionizable cationic lipids include, but are not limited to, 5-carboxysperminylglycine dioctadecylamide or "DOGS," N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or "DOTMA," 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propylammonium or "DOSPA," 1,2-dioleoyl-3-dimethylammonium-propane or "DODAP," 1,2-dioleoyl-3-trimethylammonium-propane or "DOTAP." Cationic lipids contemplated also include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane or "DSDMA", 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or "DODMA", 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLinDMA", 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLenDMA", N-dioleyl-N,N-dimethylammonium chloride or "DODAC", N,N-distearyl-N , N-dimethylammonium bromide or "DDAB", N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide or "DMR1E", 3-dimethylamino-2-(cholest-5-ene-3-β-oxobut-4-oxy)-1-(cis,cis-9,12-octadecadienyloxy)propane or "CLinDMA", 2-[5'-(cholest-5-ene-3-β-oxy)-3'-oxapentyloxy)-3-dimethyl-1-(cis,cis-9',1-2' -octadecadienyloxy)propane or "CpLinDMA", N,N-dimethyl-3,4-dioleyloxybenzylamine or "DMOBA", 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane or "DOcarbDAP", 2,3-dilinoleoyloxy-N,N-dimethylpropylamine or "DLinDAP", 1,2-N,N'-dilinoleylcarbamoyl-3-dimethylaminopropane or "DLincarbDAP", 1,2-dilinoleoylcarbamoyl-3-dimethylaminopropane or "DLincarbDAP", 1,3-dioxolane or "DLin-K-DMA", and 2-(2,2-di((9Z,12Z)-octadec-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethylamine (DLin-KC2-DMA)).

[0105] In some embodiments, helper lipid is non-cationic lipid or comprises non-cationic lipid.Term used herein " non-cationic lipid " refers to any neutral or zwitterionic lipid.Non-cationic lipid includes but is not limited to dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), hydrogenated soybean phosphatidylcholine (HSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), or its mixture.

[0106] In some embodiments, the helper lipid is or includes DOPE, DSPC, POPE, or any combination thereof.

[0107] In some embodiments, the helper lipid is a cationic lipid. In some embodiments, the cationic lipid is or includes any of the following: DOTAP, DDAB, 1,2-dioleoyl-sn-glyceryl-3-ethylphosphocholine, DPPC ethyl (EPC16:0, or 1,2-dipalmitoyl-sn-glyceryl-3-ethylphosphocholine) or any combination thereof.

[0108] In some embodiments, the PEG-lipid comprises a single PEG moiety covalently bound to the head group of the lipid. In some embodiments, the PEG-lipid comprises a plurality of PEG moieties covalently bound to the head group of the lipid. In some embodiments, the PEG moiety comprises an alkylated PEG, such as methoxy poly (ethylene glycol) (mPEG). The PEG moiety can have a head group of the following molecular weight: about 750Da to about 20,000Da, sometimes about 750Da to about 12,000Da, and generally about 1,000Da to about 5,000Da, including any range therebetween.

[0109] In some embodiments, the term "non-liposome forming lipids" should be understood to refer to lipids that do not spontaneously form vesicles when introduced into an aqueous medium.

[0110] There are various types of lipids that do not spontaneously form vesicles, but are used in or can be incorporated into vesicles. In some embodiments, the non-liposome forming lipid is or comprises a sterol.

[0111] Non-limiting examples of sterols include, but are not limited to, β-sitosterol, dihydro β-sitosterol (β-sitostanol, β-sitostanol), stigmasterol, stigmasterol, campesterol, campestanol, ergosterol, avenasterol, brassicasterol, fucoxantranol, cholesterol, cholesterol hemisuccinate, and cholesterol sulfate, including any salts or any combination thereof.

[0112] In some embodiments, the structured lipid comprises any of the following: betulin, brassicasterol, calcipotriol, campesterol, cholesterol, carrot sterol, DC-cholesterol, dehydroergosterol, DMAPC-cholesterol, DMHAPC-cholesterol, ergosterol, fuccasterol, HAPC-cholesterol, lupine, MHAPC-cholesterol, OH-C-cholesterol, OH-cholesterol, oleanolic acid, stigmasterol, stigmasterol, ursolic acid, hydrophobic vitamins (e.g., vitamin D2, vitamin D3, vitamin E, etc.), β-sitosterol, β-sitosterol-acetate, β-sitosterol-arginine, β-sitosterol-cysteine, β-sitosterol-glycine, β-sitosterol-histidine, β-sitosterol-serine, or steroids, including any salt or combination thereof.

[0113] In some embodiments, the molar concentration of one or more compounds of the present invention in nanoparticles is between 10 and 80 mol%, between 15 and 55 mol%, between 10 and 20 mol%, between 20 and 60 mol%, between 10 and 60 mol%, between 20 and 40 mol%, between 40 and 60 mol%, between 60 and 80 mol%, including any range therebetween. The term "concentration" or "molar concentration" used herein refers to the molar ratio relative to the total lipid content of the nanoparticle. In some embodiments, the total lipid content refers to the combined content of the compound of the present invention and lipids, wherein the lipids encompass liposome-forming lipids, modified lipids (e.g., PEG-lipids) and non-liposome-forming lipids. It will be understood by those skilled in the art that the molar ratios of the important components (i.e., compounds of the present invention, auxiliary lipids, structural lipids, and modified lipids) in LNP and in the composition of the present invention are the same. Therefore, the molar concentrations and molar ratios disclosed herein (e.g., regarding LNP) also encompass the corresponding molar concentrations and molar ratios in the composition of the present invention, and vice versa.

[0114] In some embodiments, the molar concentration of the helper lipid within the nanoparticle is between 5 and 60 mol%, between 5 and 10 mol%, between 5 and 15 mol%, between 10 and 40 mol%, between 10 and 30 mol%, between 5 and 20 mol%, between 20 and 40 mol%, including any ranges therebetween.

[0115] In some embodiments, the molar concentration of the structural lipids within the nanoparticles is between 5 and 60 mol%, between 20 and 60 mol%, between 10 and 50 mol%, between 20 and 50 mol%, between 5 and 40 mol%, between 20 and 40 mol%, between 30 and 40 mol%, including any ranges therebetween.

[0116] In some embodiments, the molar concentration of the modified lipid (e.g., PEG-lipid) within the nanoparticle is between 0.5 and 10 mol%, between 0.1 and 10 mol%, between 0.1 and 0.5 mol%, between 0.5 and 1 mol%, between 1 and 5 mol%, between 0.5 and 2 mol%, between 5 and 10 mol%, between 5 and 7 mol%, between 7 and 10 mol%, including any ranges therebetween.

[0117] In some embodiments, the molar ratio of the helper lipid to the modifying lipid is in the following ranges: between 1:0.2 and 1:0.01, between 1:0.15 and 1:0.01, between 1:0.1 and 1:0.01, between 1:0.05 and 1:0.01, including any ranges therebetween.

[0118] In some embodiments, the molar ratio of the compound to the helper lipid is in the following ranges: between 1:0.5 and 5:1, between 1:0.5 and 4:1, between 1:0.5 and 3:1, between 1:0.5 and 2:1, between 1:0.5 and 1:1, between 1:0.1 and 5:1, between 1:1 and 1:2, between 1:1 and 1:5, between 1:0.25 and 5:1, between 1:0.5 and 2:1, including any ranges therebetween.

[0119] In some embodiments, the molar ratio of the structural lipid to the modifying lipid is in the following range: between 200: 1 and 2: 1, between 100: 1 and 5: 1, between 100: 1 and 10: 1, between 100: 1 and 30: 1, between 100: 1 and 50: 1, between 100: 1 and 70: 1, between 100: 1 and 90: 1, between 100: 1 and 100: 3, between 100: 1 and 20: 1, between 150: 1 and 20: 1, between 200: 1 and 50: 1, between 200: 1 and 10: 1, including any ranges therebetween.

[0120] In some embodiments, the weight ratio between the compound of the present invention (or the total lipid content, referring to the total amount of the compound of the present invention and lipids in the nanoparticle and / or in the composition comprising the same) and the polynucleic acid in the nanoparticle (or in a composition comprising the same) is between 0.001:1 and 10:1, between 0.001:1 and 0.1:1, between 0.1:1 and 1:1, between 1:1 and 10:1, including any ranges therebetween.

[0121] In some embodiments, the N:P ratio within the lipid nanoparticle or composition of the invention is in the range of between 3 and 20, between 3 and 5, between 4 and 8, between 6 and 8, between 8 and 10, between 10 and 12, between 10 and 20, between 8 and 20, between 8 and 15, and between 12 and 14, including any ranges therebetween. The term "N:P ratio" refers to the ratio between N atoms of the compound of the invention and P atoms of the polynucleotide within the lipid nanoparticle or composition of the invention.

[0122] In some embodiments, the N:P ratio is about 12. In some embodiments, the N:P ratio within the lipid nanoparticle or within the composition of the invention is in the range of between 3-12.

[0123] In one embodiment, the nanoparticles in the composition are characterized by an average particle size of less than 500 nm to facilitate their entry into cells through the extracellular matrix. In one embodiment, the carrier is characterized by an average particle size of less than 300 nm in diameter to facilitate their entry into cells through the extracellular matrix.

[0124] In one embodiment, the nanoparticles within the composition are characterized by an average particle size of less than 300 nm, less than 250 nm, less than 200 nm, less than 150 nm, less than 100 nm, including any range therebetween.

[0125] In another embodiment, the nanoparticles in the composition are characterized by an average particle size between 50 and 300 nm, between 50 and 250 nm, between 50 and 200 nm, between 100 and 300 nm, between 50 and 100 nm, between 100 and 300 nm, including any range therebetween. In another embodiment, the nanoparticles are characterized by an average particle size as disclosed herein, and are further characterized by a size distribution (polydispersity index, PDI) between 0.05 and 0.4, between 0.05 and about 0.3, between about 0.1 and about 0.3, about 0.1, about 0.2, about 0.3, including any range therebetween. In another embodiment, the average particle size and / or PDI are measured by dynamic light scattering.

[0126] In another embodiment, the nanoparticle is characterized by a negative zeta potential or a positive zeta potential (e.g., measured at a pH of about 7, such as between about 6.5 and 7.5). In some embodiments, the nanoparticle is characterized in that its zeta potential is in the range of between -40 and +40 mV, including any range therebetween. In some embodiments, the nanoparticle is characterized in that its zeta potential is in the range of between -20 and +20 mV, including any range therebetween. In some embodiments, the nanoparticle is characterized in that the negative zeta potential is in the range of between -0.1 and -40 mV, including any range therebetween. In some embodiments, the nanoparticle is characterized in that the positive zeta potential is in the range of between +0.1 and +40 mV, including any range therebetween. In some embodiments, the nanoparticle is characterized in that it has a neutral zeta potential at physiological pH values ​​(e.g., at a pH between 6.5 and 7.5, including any range therebetween).

[0127] In some embodiments, the nanoparticles are stable over a period of 1 day to 1 year or longer, including any range therebetween. In some embodiments, the term "stable" refers to the physical and chemical stability of the nanoparticles under appropriate storage conditions (e.g., substantially no phase separation, aggregation, disintegration, and / or substantially retaining the initial load of the active agent). In some embodiments, the term "stable" refers to the physical and chemical stability (e.g., dispersion stability) of the nanoparticles in aqueous solution.

[0128] In some embodiments, the term "polynucleic acid" and the term "polynucleotide" are used interchangeably herein. In some embodiments, the polynucleotide comprises 60 to 15,000 nucleobases, 15,000 to 10,000 nucleobases, 10,000 to 4,700 nucleobases, 200 to 5,000 nucleobases, 300 to 5,000 nucleobases, 400 to 5,000 nucleobases, 400 to 2,500 nucleobases, 200 to 3,000 nucleobases, 400 to 2,000 nucleobases, 400 to 1,000 nucleobases, including any range therebetween.

[0129] In some embodiments, the polynucleotide comprises at least 20 nucleobases, at least 250 nucleobases, at least 300 nucleobases, at least 350 nucleobases, at least 400 nucleobases, at least 450 nucleobases, at least 475 nucleobases, or at least 500 nucleobases. Each possibility represents a separate embodiment of the invention.

[0130] In some embodiments, the polynucleotide comprises at most 500 nucleobases, at most 750 nucleobases, at most 1,000 nucleobases, at most 1,250 nucleobases, at most 1,750 nucleobases, at most 2,500 nucleobases, at most 3000 nucleobases, at most 4000 nucleobases, or at most 5000 nucleobases. Each possibility represents a separate embodiment of the invention.

[0131] In some embodiments, the polynucleotide comprises a plurality of polynucleotide types. In some embodiments, the nanoparticle comprises a plurality of polynucleotide types. In some embodiments, the composition comprises a plurality of nanoparticle types, each type of nanoparticle comprising a specific polynucleotide.

[0132] In some embodiments, a specific polynucleotide comprises a plurality of polynucleotide molecules comprising the same or identical nucleic acid sequence. In some embodiments, a specific polynucleotide comprises a plurality of polynucleotide molecules comprising essentially the same nucleic acid sequence.

[0133] As used herein, the term "plurality" encompasses any integer equal to or greater than 2. In some embodiments, a plurality includes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0134] As used herein, the term "polynucleotide type" refers to a plurality of polynucleotides, each of which comprises a nucleic acid sequence that differs from any of the other polynucleotides in the plurality of polynucleotides by at least 1 nucleobase, at least 1 nucleobase, at least 1 nucleobase, at least 1 nucleobase, or at least 10 nucleobases, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0135] In some embodiments, the polynucleotide comprises RNA, DNA, synthetic analogs of RNA, circular RNA (circRNA), synthetic analogs of DNA, DNA / RNA hybrids, or any combination thereof. In some embodiments, the nanoparticle of the present invention comprises a polynucleotide selected from the group consisting of RNA, DNA, synthetic analogs of RNA, synthetic analogs of DNA, DNA / RNA hybrids, or any combination thereof.

[0136] In some embodiments, polynucleotides include RNA or consist of RNA. Polynucleotides include messenger RNA (mRNA) or consist of messenger RNA (mRNA). "Messenger RNA" (mRNA) refers to any polynucleotide encoding (at least one) polypeptide (naturally occurring, non-naturally occurring or modified amino acid polymer), and can be translated in vitro, in vivo, in situ or in vitro to generate the encoded polypeptide. The basic components of mRNA molecules generally include at least one coding region, 5' untranslated region (UTR), 3'UTR, 5' cap and poly-A tail. Polynucleotides can serve as mRNA, but their function and / or structural design features can be distinguished from wild-type mRNA, which acts on overcoming the existing problems of effectively expressing polypeptides using nucleic acid-based therapies.

[0137] mRNA as provided herein comprises at least one (one or more) ribonucleic acid (RNA) polynucleotide, and it has an open reading frame encoding at least one polypeptide of interest.In some embodiments, the RNA polynucleotide encoding of mRNA is 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9 or 9-10 polypeptide.In some embodiments, the RNA polynucleotide encoding of mRNA is at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 polypeptide. In some embodiments, the RNA polynucleotide of the mRNA encodes at least 100 or at least 200 polypeptides.

[0138] In some embodiments, the nucleic acid is a therapeutic mRNA. The term "therapeutic mRNA" as used herein refers to mRNA encoding a therapeutic protein. Therapeutic proteins mediate a variety of effects in host cells or subjects to treat diseases or improve the signs and symptoms of diseases. For example, therapeutic proteins can replace defective or abnormal proteins, enhance the function of endogenous proteins, provide cells with new functions (e.g., inhibit or activate endogenous cell activity, or serve as a delivery agent for another therapeutic compound (e.g., antibody-drug conjugates). Therapeutic mRNA can be used to treat the following diseases and conditions: bacterial infections, viral infections, parasitic infections, cell proliferation disorders, genetic disorders, and autoimmune disorders.

[0139] Therefore, the structures of the present invention can be used as therapeutic or prophylactic agents. They can be provided for medical treatment (medicine). For example, mRNA of the structures described herein can be administered to a subject, wherein the polynucleotide is translated in vivo to produce a therapeutic peptide.

[0140] In some embodiments, the polynucleotide comprises an inhibitory nucleic acid. In some embodiments, the polynucleotide comprises an antisense oligonucleotide.

[0141] As used herein, "antisense oligonucleotide" refers to a nucleic acid sequence that is reverse and complementary to a DNA or RNA sequence.

[0142] As described herein, "reverse and complementary nucleic acid sequences" are nucleic acid sequences that can hybridize with another nucleic acid sequence consisting of complementary nucleotide bases. "Hybridization" means pairing between complementary nucleotide bases (e.g., adenine (A) and thymine (T) (or uracil (U) in the case of RNA) to form base pairs, and guanine (G) and cytosine (C) to form base pairs) under appropriate stringent conditions to form double-stranded molecules. (See, e.g., Wahl, GM, and SL Berger (1987) Methods Enzymol. 152: 399; Kimmel, AR (1987) Methods Enzymol. 152: 507.) For the purposes of the present method, the inhibitory nucleic acid does not need to be complementary to the entire sequence, as long as it is sufficient to provide specific inhibition; for example, in some embodiments, the sequence is 100% complementary to at least nucleotides (nts) 2-7 or 2-8 (e.g., nts 2-7 or 20) at the 5' end of the microRNA itself (e.g., "seed sequence").

[0143] In some embodiments, the inhibitory nucleic acid has one or more chemical modifications in its backbone or side chains. In some embodiments, the inhibitory nucleic acid has at least one locked nucleotide, and / or has a phosphorothioate backbone.

[0144] Non-limiting examples of inhibitory nucleic acids useful according to the invention disclosed herein include, but are not limited to, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single-stranded or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, modified base / locked nucleic acid (LNA), antagomirs, peptide nucleic acids (PNA), ribozymes (catalytic RNA molecules capable of cleaving other specific RNA molecule sequences), and other oligomeric compounds or oligonucleotide mimetics that hybridize to at least a portion of a target nucleic acid and modulate its function. In some embodiments, the inhibitory nucleic acid includes antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkers, interfering RNA (RNAi), short interfering RNA (siRNA); microRNA (miRNA); small temporary RNA (stRNA); or short hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNA (saRNA) or a combination thereof.

[0145] In some embodiments, the inhibitory nucleic acid is an RNA interference molecule (RNAi). In some embodiments, the RNAi is or comprises double-stranded RNA (dsRNA).

[0146] As used herein, "interfering RNA" refers to any double-stranded or single-stranded RNA sequence that is capable of inhibiting or down-regulating gene expression by mediating RNA interference, either directly or indirectly (i.e., after transformation). Interfering RNA includes, but is not limited to, small interfering RNA ("siRNA") and small hairpin RNA ("shRNA"). "RNA interference" refers to the selective degradation of sequence-compatible messenger RNA transcripts.

[0147] In some embodiments, the polynucleotide is chemically modified. In some embodiments, the chemical modification is a modification of the polynucleotide backbone. In some embodiments, the chemical modification is a modification of the sugar of the polynucleotide. In some embodiments, the chemical modification is a modification of the core base of the polynucleotide. In some embodiments, the chemical modification increases the stability of the polynucleotide in cells. In some embodiments, the chemical modification increases the stability of the polynucleotide in vivo. In some embodiments, the chemical modification increases the stability of the polynucleotide in vitro, such as in the open air, in the wild, on surfaces exposed to air, etc. In some embodiments, the chemical modification increases the ability of the polynucleotide to induce target gene or sequence silencing, including but not limited to, RNA molecules derived from pathogens or RNA derived from plant cells, as described herein. In some embodiments, the chemical modification is selected from: a phosphate-ribose backbone, a phosphate-deoxyribose backbone, a thiophosphate-deoxyribose backbone, a 2'-O-methyl-thiophosphate backbone, a phosphorodiamidate morpholine backbone, a peptide nucleic acid backbone, a 2-methoxyethyl phosphorothioate backbone, a restricted ethyl backbone, an alternating locked nucleic acid backbone, a thiophosphate backbone, an N3'-P5' phosphoamide, a 2'-deoxy-2'-fluoro-β-d-arabinose nucleic acid, a cyclohexene nucleic acid backbone nucleic acid, a tricyclic-DNA (tcDNA) nucleic acid backbone, a ligand-conjugated antisense (nucleic acid) and a combination thereof.

[0148] In some embodiments, carrier (for example, lipid nanoparticle) is prepared by combining aqueous phase (optionally comprising activating agent) with organic phase (comprising one or more lipid components and compound of the present invention). The selection of the specific lipid (such as cationic lipid, non-cationic lipid, sterol (one or more) and / or PEG modified lipid) constituting lipid nanoparticle, and the relative molar ratio of this lipid to each other and / or the molar ratio between lipid (one or more) and the compounds of the present invention, is based on the characteristic of selected lipid (one or more) and the characteristic of the agent to be delivered. Other considerations include, for example, the saturation of alkyl chain, and the size, Tm, charge, pH, pKa, fusibility and toxicity of selected lipid (one or more).

[0149] In another aspect, a pharmaceutical composition is provided, comprising the lipid nanoparticles of the present invention and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutically acceptable carrier is also referred to as an excipient or adjuvant. The terms "carrier", "excipient" or "adjuvant" as used herein refer to any component of an inactive agent in a pharmaceutical composition. The term "pharmaceutically acceptable carrier" as used herein refers to a non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, any type of formulation aid, or simply a sterile aqueous medium, such as saline. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol, polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethanol and phosphate buffer solutions, and other nontoxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of materials that can serve as carriers herein include sugars, starches, cellulose and its derivatives, powdered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffered solution, cocoa butter (suppository base), emulsifiers, and non-toxic pharmaceutically compatible materials used in other pharmaceutical preparations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as colorants, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier can be used to formulate the compositions contemplated herein.Suitable pharmaceutically acceptable carriers, excipients and diluents in this regard are well known to those skilled in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, NJ (2001); CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and "Inactive Ingredient Guide," US Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, all of which are incorporated herein by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers and diluents useful in the compositions of the present invention include distilled water, physiological saline, Hartmann's solution, Ringer's solution, dextrose solution, Hank's solution and DMSO. These additional inactive components and effective formulations and administration procedures are well known in the art and are described in standard textbooks such as Goodman and Gillman's: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated herein by reference in its entirety. The compositions described herein may also be contained in artificially created structures such as liposomes, ISCOMS, sustained-release particles, and other media that increase the half-life of the peptide or polypeptide in serum. Liposomes for the peptides described herein are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and sterols such as cholesterol. The choice of lipid generally depends on considerations such as liposome size and stability in blood.Available methods for preparing liposomes are described, for example, in Coligan, JE et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York; see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0150] Carriers may collectively constitute from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0151] In some embodiments, the pharmaceutical composition is formulated for systemic administration. In some embodiments, the pharmaceutical composition is formulated for administration to a subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a laboratory animal. Examples of laboratory animals include, but are not limited to, mice, rats, rabbits, hamsters, dogs, cats, and monkeys. In some embodiments, the mammal is a mouse or a rat. In some embodiments, the subject is in need of the composition. In some embodiments, the subject is in need of treatment. In some embodiments, the subject is a volunteer for a diagnostic method. In some embodiments, the subject is in need of a diagnosis.

[0152] In some embodiments, the pharmaceutical composition is used in a method of treatment. In some embodiments, the method of treatment is a method of performing treatment. In some embodiments, the pharmaceutical composition is used in a method of diagnosis. In some embodiments, the method comprises administering a composition of the invention to a subject. In some embodiments, the pharmaceutical composition is used to treat or prevent a disease or condition in humans and other mammals. The active therapeutic agents of the invention include nanoparticles, or polypeptides translated from polynucleotides contained in nanoparticles.

[0153] The terms "administering", "administration" and similar terms used herein refer to any method of delivering a composition containing an active agent to a subject in a manner that provides a therapeutic effect in reasonable medical practice. One aspect of the present subject matter provides intravenous administration of a therapeutically effective amount of a composition of the present subject matter to a patient in need thereof. Other suitable routes of administration may include parenteral, intravenous, subcutaneous, oral, intramuscular, intrathecal, inhalation, intracerebroventricular, intravitreal, transdermal or intraperitoneal.

[0154] The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0155] In another aspect, provided is a method of treating a subject in need thereof, the method comprising administering to the subject a therapeutic composition of the invention.

[0156] In some embodiments, a method for delivering an activating agent to a cell of an object is provided, comprising contacting a cell with a nanoparticle (one or more) of the present invention, wherein each nanoparticle comprises an activating agent (e.g., wherein the activating agent is encapsulated in the nanoparticle). In some embodiments, the activating agent is as described above. In some embodiments, the activating agent is cell-impermeable. Cellular delivery (i.e., intracellular delivery) can be determined by the amount of the activating agent in a quantitative cell (e.g., by the fluorescent labeling of a compound, or in the case of a polynucleotide, by determining the expression level of the polynucleotide).

[0157] In some embodiments, such delivery will achieve an increase in the concentration of the active agent in the cell - compared to a control formulation comprising the same active agent and lipofectamine as a cell internalizing agent instead of the lipid nanoparticles of the present invention. In some embodiments, the increased concentration is a therapeutically effective concentration. In some embodiments, the concentration increase refers to a concentration that is at least 10 times, at least 50 times, at least 100 times, at least 200 times higher than the control.

[0158] In some embodiments, the cell is a tissue cell. In some embodiments, the method is for delivering an active agent to a tissue of a subject. In some embodiments, the delivery will result in a therapeutically effective concentration of the active agent in the tissue. In some embodiments, the delivery will achieve an increase in the amount of the active agent in the tissue, wherein the increase is compared to a control, as disclosed herein.

[0159] definition

[0160] As used herein, the term "consisting essentially of" means that a composition, method, or structure may include additional ingredients, steps, and / or parts, but only if the additional ingredients, steps, and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0161] As used herein, the term "substantially" refers to at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or between 60 and 99.9%, between 70 and 80%, between 70 and 90%, between 80 and 90%, between 90 and 95%, between 95 and 99.9%, including any ranges or values ​​therebetween.

[0162] The term "substituent" as used herein encompasses hydrogen, halogen, -NO 2 , -CN, -OH, oxygen, imino, -CONH 2 ,-CONR' 2 ,-CNNR' 2 , -CSNR' 2, -CONH-OH, -CONH-NH 2 , -NHCOR, -NHCSR, -NHCNR, -NC(=O)OR, -NC(=O)NR', ​​-NC(=S)OR', -NC(=S)NR', ​​-SO 2 R', -SOR', -SR', -SO 2 OR', -SO 2 N(R') 2 、-NHNR' 2 ,-NNR',C 1 -C 6 haloalkyl, optionally substituted C 1 -C 6 Alkyl, -NH 2 、-NR' 2 、-NH(C 1 -C 6 Alkyl), -N(C 1 -C 6 alkyl) 2 , C 1 -C 6 Alkoxy, C 1 -C 6 Haloalkoxy, hydroxyl (C 1 -C 6 Alkyl), hydroxyl (C 1 -C 6 Alkoxy), alkoxy (C 1 -C 6 Alkyl), alkoxy (C 1 -C 6 Alkoxy), C 1 -C 6 Alkyl-NR' 2 , C 1 -C 6 Alkyl-SR', -CONH(C 1 -C 6 Alkyl), -CON(C 1 -C 6 alkyl) 2 , -CO 2 H, -CO 2 R', -OCOR, -OCOR', -OC(=O)OR', -OC(=O)NR', ​​-OC(=S)OR', -OC(=S)NR', ​​or a combination thereof; wherein each R' independently represents hydrogen, or is selected from optionally substituted C 1 -C 10 Alkyl, optionally substituted C 3 -C 10 Cycloalkyl, optionally substituted C 3-C 10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, hydroxy, amino, -NH 2 、-NR' 2 -NH(C 1 -C 6 Alkyl), -N(C 1 -C 6 alkyl) 2 , C 1 -C 6 Alkoxy, C 1 -C 6 Haloalkoxy, hydroxyl (C 1 -C 6 Alkyl), hydroxyl (C 1 -C 6 Alkoxy), alkoxy (C 1 -C 6 Alkyl), alkoxy (C 1 -C 6 Alkoxy), C 1 -C 6 Alkyl-NR' 2 , C 1 -C 6 Alkyl-SR', or a combination thereof.

[0163] As used herein, the term "alkyl" describes an aliphatic hydrocarbon, including straight and branched chain groups. As used herein, the term "alkyl" also includes saturated or unsaturated hydrocarbons, thus the term further encompasses alkenyl and alkynyl groups.

[0164] The term "alkenyl" describes an unsaturated alkyl group (as defined herein) having at least two carbon atoms and at least one carbon-carbon double bond. An alkenyl group may be unsubstituted or substituted with one or more substituents, as described above.

[0165] The term "alkynyl" as defined herein is an unsaturated alkyl radical having at least two carbon atoms and at least one carbon-carbon triple bond. Alkynyl radicals may be substituted or unsubstituted with one or more substituents, as described above.

[0166] The term "cycloalkyl" describes an all-carbon monocyclic or fused ring (ie, rings that share a pair of adjacent carbon atoms) group in which one or more of the rings does not have a completely conjugated pi electron system. Cycloalkyl groups may be substituted or unsubstituted as described herein.

[0167] The term "aryl" describes an all-carbon monocyclic or fused-ring polycyclic (ie, rings which share adjacent pairs of carbon atoms) group having a completely conjugated pi-electron system. Aryl groups can be substituted or unsubstituted as described herein.

[0168] The term "alkoxy" describes O-alkyl and -O-cycloalkyl groups, as defined herein. The term "aryloxy" describes -O-aryl groups, as defined herein.

[0169] Each of the alkyl, cycloalkyl and aryl groups in the general formula herein may be substituted with one or more substituents, wherein each substituent may independently be, for example, halo, alkyl, alkoxy, cycloalkyl, nitro, amino, hydroxyl, thiol, thioalkoxy, carboxyl, amide, aryl and aryloxy groups, depending on the substituted group and its position in the molecule. Other substituents are also contemplated.

[0170] The terms "halo", "halogen" or "halo" describe fluorine, chlorine, bromine or iodine. The term "haloalkyl" describes an alkyl group as defined herein further substituted with one or more halogens. The term "haloalkoxy" describes an alkoxy group as defined herein further substituted with one or more halogens. The term "hydroxyl" or "hydroxy" describes an -OH group. The term "mercapto" or "thiol" describes an -SH group. The term "thioalkoxy" describes -S-alkyl and -S-cycloalkyl groups as defined herein. The term "thioaryloxy" describes -S-aryl and -S-heteroaryl groups as defined herein. The term "amino" describes a -NR'R" group or a salt thereof, wherein R' and R" are as described herein.

[0171] The term "heterocyclyl" describes a monocyclic or fused ring group having one or more atoms such as nitrogen, oxygen and sulfur in the ring. The ring may also have one or more double bonds. However, the ring does not have a completely conjugated π electron system. Representative examples include piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholinyl, etc.

[0172] The term "carboxy" describes a -C(O)OR' group or a carboxylate thereof, where R' is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl (bonded through a ring carbon), or heterocyclyl (bonded through a ring carbon), as defined herein. Or a "carboxylic acid group".

[0173] The term "carbonyl" describes a -C(O)R' group, wherein R' is as defined above. The above term also includes the thio derivatives thereof (thiocarboxy and thiocarbonyl).

[0174] The term "thiocarbonyl" describes a -C(S)R' group, where R' is as defined above. A "thiocarboxy" group describes a -C(S)OR' group, where R' is as defined herein. "Sulfinyl" describes a -S(O)R' group, where R' is as defined herein. "Sulfonyl" or "sulfonic acid" describes a -S(O)2R' group, where R' is as defined herein.

[0175] "Carbamoyl" or "carbamate" describes a -OC(O)NR'R" group, where R' is as defined herein and R" is as defined for R'. "Nitro" refers to a -NO2 group. The term "amide" as used herein encompasses C-amides and N-amides. The term "C-amide" describes a -C(O)NR'R" terminal group or a -C(O)NR'- linking group, as these phrases are defined above, where R' and R" are as defined herein. The term "N-amide" describes a -NR"C(O)R' terminal group or a -NR'C(O)- linking group, as these phrases are defined above, where R' and R" are as defined herein.

[0176] A "cyano" or "nitrile" group refers to a -CN group. The term "azo" or "diazo" describes a -N=NR' terminal group or a -N=N- linking group, as these phrases are defined above, where R' is as defined above. The term "guanidine" describes a -R'NC(N)NR"R"' terminal group or a -R'NC(N)NR"- linking group, as these phrases are defined above, where R', R" and R"' are as defined herein. The term "azido" as used herein refers to a -N3 group. The term "sulfonamide" refers to a -S(O)2NR'R" group, where R' and R" are as defined herein.

[0177] The term "phosphono" or "phosphonic acid" describes a -OP(O)-(OR')2 group, where R' is as defined above. The term "phosphinyl" describes a -PR'R" group, where R' and R" are as defined above. The term "alkylaryl" describes an alkyl group, as defined herein, substituted with an aryl group as described herein. An exemplary alkylaryl group is benzyl.

[0178] The term "heteroaryl" describes such a monocyclic or fused ring (i.e., a ring that shares a pair of adjacent atoms) group: it has one or more atoms such as, for example, nitrogen, oxygen and sulfur in the ring, and also has a completely conjugated π electron system. The term "heteroaryl" as used herein refers to an aromatic ring in which at least one atom forming the aromatic ring is a heteroatom. The heteroaryl ring can be formed by three, four, five, six, seven, eight, nine and more than nine atoms. Heteroaryl groups may be optionally substituted. Examples of heteroaryl groups include, but are not limited to, aromatic C containing one oxygen or sulfur atom, or two oxygen atoms, or two sulfur atoms, or up to four nitrogen atoms, or one oxygen or sulfur atom and up to two nitrogen atoms. 3-8 Heterocyclic groups, and substituted derivatives thereof, and benzo and pyrido fused derivatives - for example, connected through one of the ring carbon atoms. In certain embodiments, heteroaryl is selected from oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolyl, benzimidazolyl, quinolyl, isoquinolyl, quinazolinyl or quinoxalinyl.

[0179] In some embodiments, heteroaryl is selected from pyrrolyl, furanyl, thiophenyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3-oxazolyl (oxazolyl), 1,2-oxazolyl (isoxazolyl), oxadiazolyl, 1,3-thiazolyl, 1,2-thiazolyl (isothiazolyl), tetrazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3 1,2,4-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, indazolyl, indolyl, benzothienyl, benzofuranyl, benzothiazolyl, benzimidazolyl, benzodioxolanyl, acridinyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, phthalazinyl, thienothiphenyl, 1,8-naphthyridinyl, other naphthyridinyl, pteridinyl or phenothiazinyl. In the case where the heteroaryl group contains more than one ring, each additional ring is a saturated form (perhydro form) or a partially unsaturated form (e.g., a dihydro form or a tetrahydro form) or a maximum unsaturated (non-aromatic) form. Therefore, the term heteroaryl includes bicyclic radicals in which two rings are aromatic and bicyclic radicals in which only one ring is aromatic.Examples of such heteroaryl groups include 3H-indolyl, 2(1H)-quinolinonyl, 4-oxo-1,4-dihydroquinolinyl, 2H-1-oxoisoquinolinyl, 1,2-dihydroquinolinyl, (2H)quinolinyl N-oxyl, 3,4-dihydroquinolinyl, 1,2-dihydroisoquinolinyl, 3,4-dihydroisoquinolinyl, chromonyl, 3,4-dihydroisoquinoxalinyl, 4-(3H)quinazolinonyl, 4H-chromenyl, 4-chromanonyl, oxindolyl, 1,2,3,4-tetrahydroisoquinolinyl, 1,2,3,4 -tetrahydroquinolinyl, 1H-2,3-dihydroisoindolyl, 2,3-dihydrobenzo[f]isoindolyl, 1,2,3,4-tetrahydrobenzo[g]-isoquinolinyl, 1,2,3,4-tetrahydrobenzo[g]isoquinolinyl, chromanyl, isochromanonyl, 2,3-dihydrochromononyl, 1,4-benzo-dioxanyl, 1.2.3.4-tetrahydroquinoxalinyl, 5,6-dihydroquinolinyl, 5,6-dihydroisoquinolinyl, 5,6-dihydroquinoxalinyl, 5,6-dihydroquinazolinyl, 4,5-dihydro-1H- benzimidazolyl, 4,5-dihydro-benzoxazolyl, 1,4-naphthylquinolyl, 5,6,7,8-tetrahydroquinolyl, 5,6,7,8-tetrahydroisoquinolyl, 5,6,7,8-tetrahydroquinoxalinyl, 5,6,7,8-tetrahydroquinazolinyl, 4,5,6,7-tetrahydro-1H-benzimidazolyl, 4,5,6,7-tetrahydro-benzoxazolyl, 1H-4-oxa-1,5-diaza-naphthalen-2-one, 1,3-dihydroimidazo-[4,5]-pyridin-2-one, 2,3-dihydro-1, 4-Dinaphthoquinolinyl, 2,3-dihydro-1H-pyrrolo[3,4-b]quinolinyl, 1,2,3,4-tetrahydrobenzo[b]-[1,7]naphthyridinyl, 1,2,3,4-tetrahydrobenzo[b]-[1,6]naphthyridinyl, 1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indolyl, 1,2,3,4-tetrahydro-9H-pyrido[4,3-b]indolyl, 2,3-dihydro-1H-pyrrolo[3,4-b]indolyl, 1H-2,3,4,5-tetrahydro-azepine. [3,4-b]indolyl, 1H-2,3,4,5-tetrahydro-aza [4,3-b]indolyl, 1H-2,3,4,5-tetrahydro-aza 1,2,3,4-tetrahydro-[2,7]-naphthyridinyl, 2,3-dihydro-[1,4]-dioxa[2,3-b]pyridinyl, 2,3-dihydro-[1,4]-dioxa[2,3-b]pyridinyl, 3,4-dihydro-2H-1-oxa[4,6]naphthyridinyl, 4,5,6 ,7-tetrahydro-3H-imidazo-[4,5-c]pyridinyl, 6,7-dihydro[5,8]naphthyridinyl, 1,2,3,4-tetrahydro[1,5]-naphthyridinyl, 1,2,3,4-tetrahydro[1,6]naphthyridinyl, 1,2,3,4-tetrahydro[1,7]naphthyridinyl, 1,2,3,4-tetrahydro-[1,8]naphthyridinyl or 1,2,3,4-tetrahydro[2,6]naphthyridinyl. In some embodiments, the heteroaryl is optionally substituted. In one embodiment, the one or more substituents are each independently selected from halogen, hydroxy, amino, cyano, nitro, alkylamide, acyl, C 1-6 -alkyl, C 1-6 -haloalkyl, C 1-6 -Hydroxyalkyl, C 1-6 -aminoalkyl, C 1-6 -alkylamino, alkylsulfenyl, alkylsulfinyl, alkylsulfonyl, sulfamoyl or trifluoromethyl.

[0180] Examples of heteroaryl groups include, but are not limited to, unsubstituted and mono- or di-substituted derivatives of furan, benzofuran, thiophene, benzothiophene, pyrrole, pyridine, indole, oxazole, benzoxazole, isoxazole, benzisoxazole, thiazole, benzothiazole, isothiazole, imidazole, benzimidazole, pyrazole, indazole, tetrazole, quinoline, isoquinoline, pyridazine, pyrimidine, purine, and pyrazine, furazan, 1,2,3-oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, triazole, benzotriazole, pteridine, benzoxazole, oxadiazole, benzopyrazole, quinolizine, cinnoline, phthalazine, quinazoline, and quinoxaline. In some embodiments, the substituents are halogen, hydroxy, cyano, OC 1-6 -alkyl, C 1-6 -alkyl, hydroxy-C 1-6 -alkyl and amino-C 1-6 -alkyl.

[0181] As used herein, the terms "halogen" and "halo," which are used interchangeably herein, describe a halogen (ie, fluorine, chlorine, bromine, or iodine) atom, also referred to herein as fluoro, chloro, bromo, and iodo.

[0182] General

[0183] As used herein, the term "about" when combined with a numerical value refers to plus or minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm±100 nm.

[0184] Please note that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides, and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. Please also note that the claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as antecedent basis for use of such exclusive terminology "solely", "only", or use of a "negative" limitation in connection with reciting claim elements.

[0185] Where a conventional expression similar to "at least one of A, B, and C, etc." is employed, generally such construction is intended to be what one of ordinary skill in the art would understand the conventional expression to be (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, a system having A alone, having B alone, having C alone, both A and B, both A and C, both B and C, and / or both A, B, and C, etc.). One of ordinary skill in the art will also understand that virtually any separating word and / or phrase representing two or more alternative terms, whether in the specification or in the claims, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both (all) of the terms. For example, the phrase "A or B" will be understood to include the possibility of "A" or "B" or "A and B."

[0186] It should be understood that certain features of the invention, which for the sake of clarity are described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which for the sake of brevity are described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of embodiments of the invention are specifically included in the invention and are disclosed herein as if each combination were individually and explicitly disclosed herein. In addition, all subcombinations of various embodiments and elements thereof are also specifically included in the invention and are disclosed herein as if each subcombination were individually and explicitly disclosed herein.

[0187] By studying the following examples (which are not intended to be limiting), other objects, benefits and novel features of the present invention will be apparent to those of ordinary skill in the art. In addition, each of the various embodiments and aspects of the present invention as described above and as defined in the claims section is experimentally supported in the following examples.

[0188] Various embodiments and aspects of the present invention as delineated hereinabove and as defined in the claims section above find experimental support in the following examples.

[0189] Example

[0190] In general, the nomenclature used herein and the laboratory procedures utilized by the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are fully described in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methods as described in the following: U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659; and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994); "Culture of Animal Cells-A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan JE, ed. (1994); Stites et al.(eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization-A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated herein by reference. Other general references are also provided throughout this article. .

[0191] Example 1

[0192] Exemplary compounds of the present invention have been synthesized according to the synthetic scheme proposed below. The inventor successfully used exemplary compounds of the present invention for the preparation of stable LNP, and tested to explore its expression efficiency by cell-based assay. Exemplary compounds of the present invention (ionizable lipids) are shown below. Most of the following compounds have been tested, and found to be able to form RNA encapsulation LNP. Other compounds / LNP preparations are currently being screened in various in vivo / in vitro assays.

[0193]

[0194]

[0195]

[0196]

[0197] Some exemplary LNPs of the present invention have been characterized to have superior cell penetration, as determined by cell-based studies. For example, LNPs containing MB-77, 208, 212, and 205 (exemplary compounds of the present invention) exhibit superior internalization efficiency compared to similar compositions containing Dlin-MC3-DMA as an ionizable lipid.

[0198] Herein, general synthetic schemes for some exemplary compounds of the present invention are presented. Other possible synthetic strategies are well known to those skilled in the art.

[0199]

[0200]

[0201] The composition of exemplary LNP is as follows: auxiliary lipid (e.g., DOPE) about 5-15 mol%; structural lipid (e.g., cholesterol) about 30-45 mol%; PEG-lipid (e.g., DMG-PEG2000) between 0.5 and 5 mol%; and the compound of the present invention about 40-60 mol%. The LNP prepared by the inventors has been characterized as having an average particle size in the range of about 60 to about 300 nm.

[0202] LNP has been prepared as follows: lipids are weighed and dissolved in ethanol (EtOH) at 55-60°C. mRNA is added to a citrate buffer at a pH of 5.0 (range 4.5-5.5). Mixing of lipids containing PEG lipids, auxiliary lipids, cholesterol and ionizable lipids (compounds) is performed under microfluidic mixing or by injecting lipid EtOH into a citrate buffer containing mRNA under constant mixing conditions. The pH of the mixture is then raised using PBS diluent, and the remaining EtOH is removed by dialysis (such as FloatAlyzer or standard dialysis membrane) before injection, with a cutoff of 3kDa, 8kDa, 12-14kDa or 100kDa being used according to the separation requirements.

[0203] Encapsulation efficiency has been determined by R1bogreen, Quant-iT TM RNA reagents and kits (Invitrogen) were calculated according to the manufacturer's instructions. Particles were diluted with TR1s-EDTA buffer and supplemented with 1:2000 dilution of RiboGreen (ThermoFisher), and fluorescence (mRNA signal not encapsulated in LNP) was read using a plate reader. Triton x 100 was diluted in TE and added to each well and incubated at 37°C for 10 minutes to allow perturbation of LNP. Fluorescence (mRNA signal of total mRNA in the preparation) was measured again. Encapsulation efficiency was calculated using the following: %EE=100*(total amount-outside amount) / total amount.

[0204] The inventors determined cell penetration of exemplary LNPs and intracellular delivery of mRNA.

[0205] More specifically, particles were incubated on cells for 16-24 hours at 37°C, 5% CO2, and expression levels were measured. Formulations were compared to a lipofectamine control and to a MC3 control as well as to an untreated control.

[0206] Additional exemplary formulations were investigated to obtain stable LNPs at average particle sizes between 60 and 210 nm and low PDI (between 0.1 and 0.3), and the results are summarized in Table 1.

[0207] Table 1: Formulations of exemplary LNPs

[0208]

[0209]

[0210] Example 2

[0211] Some exemplary compositions of the invention have been characterized as having enhanced specificity for lung cells, as determined in cell-based studies. For example, LNPs of the invention containing MB-212 or MB-222 as ionizable lipids exhibited enhanced specificity for lung cells ( ) compared to similar compositions containing Dlin-MC3-DMA as ionizable lipids. Figure 1 Surprisingly, the inventors found that exemplary LNPs containing about 30 to about 50% of a helper lipid (e.g., DOTAP), about 20 to about 50% of a structural lipid (cholesterol), and about 0.5 to about 5% of a modified lipid (e.g., PEG-lipid), and about 15 and about 50% of an ionizable lipid (e.g., MB-212 or MB-222) resulted in enhanced lung specificity of the LNPs, as assessed by in vivo expression analysis. Exemplary LNP compositions of the invention showed high lung specificity, resulting in significantly enhanced expression of the encapsulated polynucleic acid (mRNA F-LUC) in the lungs compared to commercial controls ( Figure 1 In addition, a specific LNP composition of the invention (FMB-1143) comprising about 15 to about 30% ionizable lipids exhibits superior lung specificity (see Figure 1 ).

[0212] Preparation of FMB-745

[0213] 40% DOTAP, 22.5% cholesterol, 2.5% DMG-PEG2000 and 35% commercial ionizable lipid (Dlin-MC3-DMA) are dissolved in ethanol (EtOH) at 55-60 ℃. Adding mRNA F-LUC to pH is in the citrate buffer of 5.0 (scope 4.5-5.5). Under microfluid mixing or by injecting lipid EtOH into the citrate buffer containing mRNA firefly luciferase (F-LUC) under constant mixing conditions, the mixing of lipid is carried out. Then utilize PBS diluent to raise the pH of the mixture, and utilize dialysis to remove remaining EtOH before injection, the average particle size prepared by the inventor is characterized as between about 60 to about 130nm.

[0214] Preparation of FMB-748

[0215] 40% DOTAP, about 22% cholesterol, about 2-2.5% DMG PEG2000 and 35% ionizable MB-222 were dissolved in ethanol (EtOH) at 55-60°C. mRNAF-LUC was added to a citrate buffer at a pH of 5.0 (range 4.5-5.5). The lipids were mixed under microfluidic mixing or by injecting lipid EtOH into the citrate buffer containing mRNAF-LUC under constant mixing conditions. The pH of the mixture was then raised using PBS diluent, and the remaining EtOH was removed by dialysis before injection, and the inventors prepared the particles characterized as having an average particle size of about 60 to about 130 nm.

[0216] Preparation of lung-specific formulation (FMB-1143)

[0217] 40% DOTAP, about 38% cholesterol, 1.5% DMG-PEG2000 and 20% ionizable MB-212 were dissolved in ethanol (EtOH) at 55-60°C. mRNAF-LUC was added to a citrate buffer at a pH of 5.0 (range 4.5-5.5). The lipids were mixed under microfluidic mixing or by injecting lipid EtOH into a citrate buffer containing mRNAF-LUC under constant mixing conditions. The pH of the mixture was then raised using a PBS diluent, and the remaining EtOH was removed by dialysis before injection, and the inventors prepared the particles characterized as having an average particle size of about 60 to about 130 nm.

[0218] The inventors determined the expression distribution of exemplary LNP in vivo by evaluating the expression of encapsulated mRNA (mRNA F-LUC). The LNPs of the encapsulated mRNA F-LUC of the present invention were intravenously injected into BALB / c mice at a dosage of 13 μg / mouse (0.52-0.65 mg / kg). In vivo imaging was performed by IVIS imaging. In vitro tissue analysis of lungs, heart, spleen, kidneys and liver was performed by IVIS. Histological assessment was determined by H&E staining and pathologist toxicity reports. The histological assessment concluded that the morphology was normal without any treatment-related pathological changes.

[0219] Figure 1 The expression profiles shown in show that the luciferase signal in the lung is more than 100-fold higher than that in the heart, liver, spleen, and kidney, indicating that the exemplary LNPs of the present invention have superior lung specificity.

[0220] Example 3

[0221] The inventors successfully prepared LNP formulations (FMB-428 and FMB-389) based on the exemplary ionizable lipids of the present invention. Compared with similar LNP formulations based on commercial ionizable lipids (Dlin-MC3-DMA), FMB-428 and FMB-389 showed higher liver expression in vivo (see Figure 2 ).

[0222] Preparation of FMB-386, FMB-428, FMB-389, and FMB-1050

[0223] 10% DOPE, 38.5% cholesterol, 1.5% DMG-PEG2000 and 50% ionizable MB-205, MB-222, MB-208 and Dlin-MC3-DMA were dissolved in ethanol (EtOH) at 55-60°C. mRNA F-LUC was added to a citrate buffer at a pH of 5.0 (range 4.5-5.5). The lipids were mixed under microfluidic mixing or by injecting lipid EtOH into a citrate buffer containing mRNA F-LUC under constant mixing conditions. The pH of the mixture was then raised using a PBS diluent, and the remaining EtOH was removed by dialysis before injection. The inventors prepared the , which has been characterized as having an average particle size of about 60 to about 180 nm.

[0224] The inventors determined the expression distribution of exemplary LNP in vivo by evaluating the expression of encapsulated mRNA (mRNA F-LUC). The LNPs of the present invention encapsulating mRNA F-LUC were intravenously injected into BALB / c mice at a dose of 13 μg / mouse (0.52-0.65 mg / kg). In vivo imaging was performed by IVIS imaging. Ex vivo tissue analysis of lungs, heart, spleen, kidneys and liver was performed using IVIS. Histological evaluation was determined by H&E staining and pathologist toxicity reports. The conclusion of the histological evaluation was normal morphology without any treatment-related pathological changes.

[0225] The results of this experiment are summarized in Figure 2 , showing enhanced liver expression in liver tissue compared to a control LNP composition comprising a commercial "gold standard" ionizable lipid.

[0226] Although the present invention has been described in conjunction with its specific embodiments, it is apparent that many alternatives, modifications and variations will be apparent to those skilled in the art. Therefore, it is intended to encompass all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0227] Example 4

[0228] Exemplary thioether-based compounds of the present invention have been synthesized according to the synthesis scheme presented above (Example 1).

[0229]

[0230] Based on computer predictions, the inventors have deduced that it is preferred to use 10 mol% or more (e.g., between 15 and 60 mol%, or between 20 and 55 mol%) of the thioether compounds of the present invention to obtain LNPs of the present invention having an average particle size between 50 and 180 nm and low PDI. The predictions indicate that the ionizable lipids of the present invention in the formulation below 10 mol% will result in LNPs with high PDI values.

[0231] The inventors successfully used the exemplary thiol compounds MB-2036 and MB-2037 of the present invention to prepare stable LNPs (FMB-1723, FMB-1724, FMB-1725, FMB-1726) with low PDI and average particle size between 80 and 110.

[0232] Preparation of FMB-1723, FMB-1724, FMB-1725, and FMB-1726

[0233] 10% DOPE, 38.5% cholesterol, 1.5% DMG-PEG2000 and 50% ionizable MB-2036 or MB-2037 were dissolved in ethanol (EtOH) at 55-60°C. mRNA (F-LUC) was added to a citrate buffer at pH 5.0 (range 4.5-5.5). The lipids were mixed under microfluidic mixing or by injecting lipid EtOH into the citrate buffer containing mRNA to maintain N: P at 8 or 12 under constant mixing conditions. The pH of the mixture was then raised using a PBS diluent. LNPs have been characterized as having an average particle size between about 80 and 92 nm.

Claims

1. A compound, a salt of the compound, or both, wherein the compound is represented by the following formula I: in: Each L is independently R1, Each L1 is independently R1, represents a single, triple, or double bond; Each Z independently represents -OH or -SH; Each k is independently between 0 and 10; each Y independently does not contain or contains CH2, CHR'2, NR'2, NH, O, S, -CONH-, -CONR'-, -C(=NH)NR'-, -C(=S)NR'-, -NC(=O)-, -NC(=O)O-, -NC(=O)N-, -NC(=S)O-, -NC(=S)N-, -C(=O)-, -C(=O)O-, -OC(=O)O-, -OC(=O)N-, -OC(=S)O-, -OC(=S)N-, or a phosphate group, as valency permits; Each T independently represents an optionally substituted C5-C30 alkyl group or an optionally substituted C5-C30 alkenyl group; each R' is independently H or comprises optionally substituted C1-C10 alkyl, C1-C10 alkyl-aryl, C1-C10 alkyl-cycloalkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof; Each X independently represents a heteroatom, CH2, an optionally substituted C1-C10 alkyl group, or X is absent; Each n and p is independently between 0 and 5, and at least one n is not 0; m is between 1 and 3; Each R is independently H, or comprises an optionally substituted C5-C30 alkyl group; Each R1 is an optionally substituted C1-C24 alkyl group, and at least one L or L1 is or comprises 2. The compound according to claim 1, wherein any one of R and R1 further comprises at least one unsaturated bond.

3. The compound of claim 1 or 2, wherein the heteroatom comprises O, N, NH, NR1, S or a phosphate group.

4. A compound according to any one of claims 1 to 3, wherein each L is independently 5. A compound according to any one of claims 1 to 4, wherein each X is independently O, or absent.

6. The compound according to any one of claims 1 to 5, wherein any one of R and R1 represents a linear or branched C1-C24 alkyl group.

7. The compound according to any one of claims 1 to 6, wherein the compound is represented by the following formula II:

8. A compound according to any one of claims 1 to 7, wherein each L is independently R1, and at least one of L is 9. The compound according to any one of claims 1 to 8, wherein the compound is characterized by a pKa value between 5 and 9.

10. A lipid nanoparticle comprising the compound of any one of claims 1 to 9 and an active agent.

11. The lipid nanoparticle of claim 10, wherein the average size of the lipid nanoparticle is in the range of 50 to 300 nm.

12. The lipid nanoparticle of claim 10 or 11, wherein the active agent comprises a polynucleic acid.

13. The lipid nanoparticle according to any one of claims 10 to 12, further comprising a lipid, wherein the lipid comprises a helper lipid, and optionally comprises a structural lipid, a modified lipid, or any combination thereof.

14. The lipid nanoparticle of claim 13, wherein the weight ratio between (i) the total amount of the compound and the lipid and (ii) the polynucleic acid in the lipid nanoparticle is between 0.001:1 and 10:

1.

15. The lipid nanoparticle of claim 13 or 14, wherein the lipid comprises a helper lipid, a modifying lipid and a sterol.

16. The lipid nanoparticle according to any one of claims 10 to 15, wherein the proportion of the compound relative to the total lipid content of the lipid nanoparticle is between 10 and 80 mol%.

17. The lipid nanoparticle of claim 15 or 16, wherein the lipid nanoparticle comprises, relative to the total lipid content of the lipid nanoparticle, between 20 and 60 mol% of the compound, between 5 and 50 mol% of the structural lipid, between 10 and 50 mol% of the auxiliary lipid, and between 0.5 and 5 mol% of the modified lipid.

18. The lipid nanoparticle of claim 17, wherein the lipid nanoparticle comprises, relative to the total lipid content of the lipid nanoparticle, between about 20 to about 50 mol % of the compound, between about 20 to about 45 mol % of the structural lipid, between about 10 to about 50 mol % of the auxiliary lipid, and between about 1 to about 3 mol % of the modified lipid.

19. The lipid nanoparticle according to any one of claims 12 to 18, wherein the lipid nanoparticle consists essentially of: Relative to the total lipid content of the lipid nanoparticles, between about 20 and about 50 mol% of the compound, between about 20 and about 45 mol% of the structural lipid, between about 10 and about 50 mol% of the auxiliary lipid, and between about 1 and about 3 mol% of the modified lipid; and wherein the N:P ratio within the lipid nanoparticles is between 5 and 12.

20. A pharmaceutical composition comprising a plurality of lipid nanoparticles according to any one of claims 10 to 19 and a pharmaceutically acceptable carrier.

21. The pharmaceutical composition of claim 20, comprising an effective amount of the active agent.

22. The pharmaceutical composition of claim 21, wherein the active agent is encapsulated within the lipid nanoparticles.

23. The pharmaceutical composition of any one of claims 20 to 22, formulated for systemic administration, local administration, or both to a subject.

24. The pharmaceutical composition according to any one of claims 20 to 23, for use in treating a disease or disorder in a subject in need thereof.

25. A method of delivering an active agent to a tissue of a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition according to any one of claims 20 to 24, thereby delivering the active agent to the tissue.

26. The method of claim 25, wherein the delivering comprises increasing the concentration of the active agent within the tissue as compared to a control formulation comprising lipofectamine and the active agent without the lipid nanoparticles.

27. The method of claim 26, wherein the increase is at least 10-fold compared to the control formulation.

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