Ionizable lipids and nanoparticles containing the same

Abstract

One or more ionizable lipids and lipid nanoparticles containing the same are provided. Also provided is a pharmaceutical composition comprising lipid nanoparticles encapsulating an active agent.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority under 35 USC§119(e) to U.S. Provisional Patent Application No. 63 / 350,540, filed Jun. 9, 2022, and U.S. Provisional Patent Application No. 63 / 437,800, filed Jan. 9, 2023, both entitled "IONIZABLE LIPIDS AND NANOPARTICLES COMPRISING SAME", the contents of which are hereby incorporated by reference in their entirety.

[0002] Field of the Invention The present invention relates to ionizable lipids and lipid nanoparticles comprising the same, and their use in pharmaceutical compositions.

Background Art

[0003] New delivery methods for therapeutic and diagnostic compounds are constantly being developed. Lipid - based nanoparticles are well - known delivery systems, but these agents are also constantly being improved. In particular, the ability of a therapeutic carrier to efficiently load an active agent and then deliver it to a target site is very important for reducing the dosage and improving the therapeutic efficiency.

[0004] Although various ionizable lipids capable of encapsulating hydrophilic agents such as DNA and / or RNA are known, there is a constant need for novel and superior ionizable lipids. In particular, there is a great need for the development of novel and superior ionizable lipids that can enhance drug delivery to specific sites in the body.

Summary of the Invention

[0005] The present invention provides novel compounds suitable for use as ionizable lipids. Further, nanoparticles containing the same are provided. Also provided are compositions containing nanoparticles useful for delivering an active agent to a subject, such as for treating or preventing a disease or disorder in the subject.

[0006] In a first aspect, a compound of formula I:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0007] In one embodiment, any one of R and R1 further includes at least one unsaturated bond.

[0008] In one embodiment, the heteroatom includes O, N, NH, NR1, S, or a phosphate group.

[0009] In one embodiment, each L is independently [Chemical formula] or [Chemical formula] is.

[0010] In one embodiment, each X is independently O or absent.

[0011] In one embodiment, any one of R and R1 is a linear or branched C1-C 24 or C1-C 10 alkyl.

[0012] In one embodiment, the compound has the formula II:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0013] In one embodiment, the compound is characterized by a pKa value of 5-9.

[0014] In one embodiment, the compound includes any one of the compounds of Example 1 or Example 4.

[0015] In another aspect, lipid nanoparticles comprising the compound of the present invention and an active agent are provided.

[0016] In one embodiment, the average size of the lipid nanoparticles ranges from 50 to 300 nm.

[0017] In one embodiment, the active agent includes a polynucleotide.

[0018] In one embodiment, the lipid nanoparticles further comprise a lipid, which includes a helper lipid and optionally a structural lipid, a modified lipid, or any combination thereof.

[0019] In one embodiment, the weight ratio between (i) the total amount of the compound and the lipid and (ii) the polynucleic acid within the lipid nanoparticles is from 0.001:1 to 10:1.

[0020] In one embodiment, the lipid includes a helper lipid, a modified lipid, and a sterol.

[0021] In one embodiment, the ratio of the compound to the total lipid content of the lipid nanoparticles is from 10 to 80 mol%.

[0022] In another aspect, a pharmaceutical composition is provided that includes a plurality of the lipid nanoparticles of the present invention and a pharmaceutically acceptable carrier.

[0023] In one embodiment, a pharmaceutical composition is provided that includes an effective amount of an active agent.

[0024] In one embodiment, the pharmaceutical composition is formulated for systemic administration to a subject, topical administration to a subject, or both.

[0025] In one embodiment, the pharmaceutical composition is for use in the treatment of a disease or disorder in a subject in need thereof.

[0026] In another aspect, a method is provided for delivering an active agent to a tissue of a subject, the method including administering to the subject an effective amount of the pharmaceutical composition of the present invention, thereby delivering the active agent to the tissue.

[0027] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given below. However, the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, and it is to be understood that various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art from this detailed description.

[0028] In this specification, several embodiments of the present invention will be described for illustrative purposes only with reference to the accompanying drawings. Regarding the drawings to which particular reference is made herein, it should be emphasized that the details shown are illustrative and are intended to consider embodiments of the present invention by way of illustration. In this regard, the method of implementing embodiments of the present invention will be apparent to those skilled in the art from the description using the drawings.

Brief Description of the Drawings

[0029]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0030] The compounds disclosed by the present invention were discovered by means of computer screening methods. Candidates for ionizable lipids were generated in silico and ranked based on their predicted activities using machine learning algorithms. After multiple in silico optimization cycles, a chemical library containing several molecules was obtained. The disclosed compounds were selected based on the results obtained from in vitro experiments as shown in this specification (the Examples section).

[0031] Compounds having at least 30% encapsulation efficiency (e.g., at least 80% encapsulation efficiency, etc.) and inducing at least about 10-fold intracellular expression and / or intracellular translocation of RNA sequences compared to gene transfection with Lipofectamine 2000 (see the Examples section) were selected as suitable candidates for ionizable lipids for further in vivo studies.

[0032] In a first aspect, there is provided a compound comprising an ionizable moiety (e.g., a head group) covalently attached to a lipophilic tail (e.g., a hydrocarbon-based chain), the ionizable moiety being represented by any one of the following formulas:

Chemical formula

[0033] In some embodiments, the lipophilic tail comprises 10 to 50 carbon atoms (either a straight-chain, branched, or cyclic hydrocarbon chain), optionally including 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 an aqueous solution.

[0034] In some embodiments, the ionizable moiety can undergo ionization (protonation, or positive ionization) in a solution having a pH value less than the pKa value of the ionizable moiety. In some embodiments, the ionizable moiety can undergo protonation in a solution having a pH value less than 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 having a pH value less than the pKa value of the ionizable moiety.

[0035] 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.

[0036] In some embodiments, the ionizable moiety is attached to the lipophilic tail via a spacer or via a covalent bond.

[0037] In some embodiments, the lipophilic tail has the formula:

Chemical formula

Chemical formula

Chemical formula

[0038] In some embodiments, the compounds of the invention have an MW of 100 to 2,000 Da, 100 to 300 Da, 100 to 500 Da, 100 to 800 Da, 300 to 500 Da, 100 to 1,000 Da, 500 to 800 Da, 500 to 2,000 Da, 500 to 1,000 Da, 800 to 1,000 Da, 800 to 1,500, 1,000 to 2,000 (including any range therebetween).

[0039] In another aspect, a compound and / or a salt thereof is provided, the compound having the formula 1:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0040] In some embodiments, any one of R and R1 further includes at least one unsaturated bond.

[0041] In some embodiments, the compound of the present invention has the formula: [Chemical formula] represented by, where each L1 and L2 is independently R1, [Chemical formula] or [Chemical formula] wherein; X, Z, L, R, R1, p, n, m are as described herein; at least one of L and L1 is [Chemical formula] or [Chemical formula] or includes this.

[0042] In some embodiments, each n and p is independently 0 - 5, 0 - 3, 0 - 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 1 - 3 (e.g., 1, 2 or 3), including any combination of these.

[0043] In some embodiments, R is of the formula: [Chemical formula] or of the formula [Chemical formula] represented by, wherein [Chemical formula] represents a single bond, triple bond or double bond; k and n are each independently an integer from 1 to 10, or from 1 to 24 (including any range therebetween); Z independently represents -OH or -SH; y is from 1 to 3; each Y is absent or independently, to the extent permitted by valence, CH2, 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; each T independently represents an optionally substituted C5-C 30 alkyl or an optionally substituted C5-C 30 alkenyl. In some embodiments, each R’ is independently H or an optionally substituted C1-C 10 alkyl, C1-C 10 alkyl-aryl, C1-C 10 alkyl-cycloalkyl, an optionally substituted C3-C 10 cycloalkyl, an optionally substituted C3-C 10 heterocyclyl, an optionally substituted heteroaryl, an optionally substituted aryl or combinations thereof, or R’ is absent.

[0044] In some embodiments, the heteroatom includes O, N, NH, NR1, or S. In some embodiments, each X is independently O or absent.

[0045] In some embodiments, one of R and R1 each independently represents a straight-chain or branched alkyl.

[0046] In some embodiments, L is

Chemical formula

[0047] In some embodiments, L is

Chemical formula

[0048] In some embodiments, L is

Chemical formula

[0049] In some embodiments, L is

Chemical formula

[0050] As used herein, the term "alkyl" represents aliphatic hydrocarbons including straight-chain and branched-chain groups. In some embodiments, the alkyl group has 1 to 10 carbon atoms, 1 to 30 carbon atoms, 1 to 24 carbon atoms, or 5 to 30 carbon atoms. In some embodiments, the alkyl group is C1-C6 alkyl. In some embodiments, the alkyl group is C1-C6 alkyl, C1-C 10 alkyl, C1-C8 alkyl, C5-C 30 alkyl, C5-C 24 alkyl, C5-C 20 alkyl, C5-C 10 alkyl, C8-C 30 alkyl, C8-C 24 alkyl, C8-C15 Alkyl, C8-C 20 Alkyl, C8-C 12 is alkyl and includes any range therebetween. When a numerical range, e.g., "5-30", is recited herein it is always understood that the base, in this case the alkyl group, can have 5 carbon atoms, 6 carbon atoms, 10 carbon atoms, 5-20, 5-25, 5-30, 10-20, 10-25, 10-30 carbon atoms (up to 30 carbon atoms and any range therebetween). Alkyl, as defined herein, may be substituted or unsubstituted.

[0051] The term "alkyl" as used herein also includes saturated or unsaturated hydrocarbons and thus further includes alkenyl and alkynyl.

[0052] The term "alkenyl" as defined herein represents an unsaturated alkyl having 2-30 carbon atoms and at least one carbon-carbon double bond. Alkenyl may or may not be substituted with one or more substituents as described herein.

[0053] The term "alkynyl" as defined herein is an unsaturated alkyl having 2-30 carbon atoms and at least one carbon-carbon triple bond. Alkynyl may or may not be substituted with one or more substituents as described herein.

[0054] In some embodiments, the alkyl group is C1-C 10 alkyl or C1-C6 alkyl.

[0055] As used herein, any C1-C 10 The term "C1-C 10"Alkyl" refers to any straight-chain 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, or 9 to 10 carbon atoms (including any range therebetween). In some embodiments, C1-C 10 Alkyl includes any one of methyl, ethyl, propyl, butyl, pentyl, isopentyl, hexyl, heptyl, octyl, nonyl, decyl, and tert-butyl, or any combination thereof. In some embodiments, the C1-C 10 Alkyl further includes an unsaturated bond, and the unsaturated bond is located at the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, or 10th position of the C1-C 10 alkyl.

[0056] As used herein, the term "C1-C6 alkyl" including any C1-C6 alkyl-related compound refers to any straight-chain or branched alkyl chain containing 1 to 6, 1 to 2, 2 to 3, 3 to 4, 4 to 5, or 5 to 6 carbon atoms (including any range therebetween). In some embodiments, C1-C6 alkyl includes any one of 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, and the unsaturated bond is located at the 1st, 2nd, 3rd, 4th, 5th, or 6th position of the C1-C6 alkyl.

[0057] In some embodiments, the compounds of the present invention have the formula II:

Chemical formula

Chemical formula

Chemical formula

[0058] In some embodiments, the compound of the present invention has the formula III:

Chemical formula

Chemical formula

Chemical formula

[0059] In some embodiments, the compound of the present invention is represented by formulas I to III, and the sum of all n in the molecule is 2 to 15, 2 to 10, 2 to 8, including any range therebetween.

[0060] In some embodiments, the compound of the present invention has the formula IIA:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0061] In some embodiments, the compounds of the invention are of Formula IIIA:

Chemical formula

Chemical formula

Chemical formula

[0062] In some embodiments, the compound of the invention is of formula IIIB: [Chemical formula] , where each r independently represents an integer from 2 to 15, an integer from 2 to 10, an integer from 2 to 8, including any range therebetween, and each L and R2 are independently as described herein.

[0063] In some embodiments, the compound of the invention is represented by formula IIIA, where each X is O; each n1 and n'1 are independently 0 to 3, n2, n'2, n3 and n'3 are independently 0 to 3, and the sum of n1, n2 and n3 is 2 to 15, 2 to 10, 2 to 8 (including any range therebetween); the sum of n'1, n'2, and n'3 is 2 to 15, 2 to 10, 2 to 8 (including any range therebetween); and each L is [Chemical formula] or [Chemical formula] , where R is as described herein.

[0064] In some embodiments, the compound of the invention is represented by any one of the formulas disclosed herein, and at least one L is [Chemical formula] or

Chem.

Chem.

Chem.

[0065] In some embodiments, the compounds of the invention include any one of the compounds of Example 1, including any salt, any tautomer, and / or any stereoisomer (e.g., enantiomer, and / or diastereomer) thereof.

[0066] As used herein, the term "substituted" or the term "substituent" relates to one or more (e.g., 2, 3, 4, 5, or 6) substituents, and the substituents are as described herein.

[0067] As used herein, the term substituent includes halogen, -NO2, -CN, -OH, -CONH2, -CONR’2, -CNNR’2, -CSNR’2, -CONH-OH, -CONH-NH2, -NHCOR, -NHCSR, -NHCNR, -NC(=O)OR, -NC(=O)NR’, -NC(=S)OR’, -NC(=S)NR’, -SO2R’, -SOR’, -SR’, -SO2OR’, -SO2N(R’)2, -NHNR’2, -NNR’, C1-C6 haloalkyl, optionally substituted C1-C6 alkyl, -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy(C1-C6 alkyl), hydroxy(C1-C6 alkoxy), alkoxy(C1-C6 alkyl), alkoxy(C1-C6 alkoxy), C1-C6 alkyl, -NR’2, C1-C6 alkyl, -SR’, -CONH(C1-C6 alkyl), -CON(C1-C6 alkyl)2, -CO2H, -CO2R’, -OCOR, -OCOR’, -OC(=O)OR’, -OC(=O)NR’, -OC(=S)OR’, -OC(=S)NR’, or combinations thereof; each R’ independently represents hydrogen or optionally substituted C1-C 10 alkyl, optionally substituted C3-C 10 cycloalkyl, optionally substituted C3-C 10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or combinations thereof.

[0068] As used herein, the term “C1-C6 haloalkyl” refers to C1-C6 alkyl as described herein substituted with one, two, three, four, five, six, seven, eight, nine, ten fluoride, bromide, chloride, and iodide atoms, or combinations thereof, selected from F, Br, Cl, and I.

[0069] As used herein, the term “(C3-C 10 ) cycloalkyl” refers to an optionally substituted C3, C4, C5, C6, C7, C8, C9 or C10 ring. In some embodiments, (C3-C 10) The ring includes optionally substituted cyclopropane, cyclobutene, cyclopentane, cyclohexane, or cycloheptane.

[0070] As used herein, the term "C3-C 10 "heterocyclyl" refers to an optionally substituted C3, C4, C5, C6, C7, C8, C9 or C10 heterocyclic aromatic ring and / or aliphatic ring, or unsaturated ring.

[0071] In some embodiments, the terms "hydroxy(C1-C6 alkyl)" and "C1-C6 alkoxy" are used interchangeably herein and refer to a C1-C6 alkyl as described herein substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hydroxy groups, the hydroxy groups being located at the 1-, 2-, 3-, 4-, 5-, or 6-position of the C1-C6 alkyl and including any combination thereof.

[0072] In some embodiments, the compounds of the present invention substantially comprise any single enantiomer of the compounds described herein, substantially being at least 70 wt%, at least 75 wt%, at least 80 wt%, at least 85 wt%, at least 90 wt%, at least 92 wt%, at least 93 wt%, at least 95 wt%, at least 97 wt%, at least 98 wt%, at least 99 wt%, including any value therebetween.

[0073] In some embodiments, the compounds of the present invention further include any structurally similar functional derivatives of the compounds disclosed herein, where structurally similar is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% structural similarity, including any range therebetween.

[0074] In some embodiments, the functional derivative refers to an ionizable lipid that has a pKa value of 6.2 to 6.8 and is capable of self-organizing in water to stably bind and / or encapsulate polynucleic acids. In some embodiments, the functional derivative is further configured to effect intracellular translocation of the polynucleic acid (e.g., by forming the lipid nanoparticles described herein). Intracellular translocation can be determined as described below.

[0075] In some embodiments, the term "structural similarity" refers to the fingerprint similarity between two molecules. The term "fingerprint similarity" is well understood by those skilled in the art. In some embodiments, the fingerprint similarity is calculated based on circular fingerprints, substructure key-based fingerprints, and / or topological or path-based fingerprints.

[0076] 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 the Morgan fingerprint.

[0077] carrier In another aspect, a carrier for an active agent is provided, the carrier being in the form of nanoparticles. In some embodiments, the carrier encapsulates the active agent within the core. In some embodiments, the active agent is a small molecule and / or a biological molecule such as a polypeptide, 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 of 20 to 30 °C). In some embodiments, the active agent is selected from therapeutic agents, prophylactic agents, and diagnostic agents including any combination thereof. In some embodiments, the one or more active agents are selected from the group consisting of proteins, peptides, nucleic acids, small molecules, and antibodies.

[0078] In some embodiments, the carrier is in the form of lipid nanoparticles comprising a compound of the invention and an active agent. In some embodiments, the lipid nanoparticles comprise a shell containing the active agent and an aqueous core. In some embodiments, the shell of the lipid nanoparticles contains a compound of the invention. In some embodiments, the shell of the lipid nanoparticles further contains 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 invention, lipids, and an active agent. In some embodiments, the lipid nanoparticles are in the form of core-shell nanoparticles, and the shell of the nanoparticles contains lipids and at least one compound of the invention. In some embodiments, the compound of the invention is bound (e.g., via electrostatic interactions) to an active agent (e.g., a polynucleotide).

[0079] In some embodiments, under appropriate conditions, at least one compound of the invention, and optionally lipids (and optionally an active agent), spontaneously self-assemble in an aqueous solution to form lipid nanoparticles. In some embodiments, the term "lipid nanoparticles" refers to nanoparticles (e.g., substantially spherical particles), and the shell of the nanoparticles contains one or more compounds of the invention and optionally one or more lipids (e.g., helper lipids such as cationic lipids, non-cationic lipids; and optionally sterols and / or PEG-modified lipids). Preferably, the lipid nanoparticles are formulated to deliver one or more drugs to one or more target cells.

[0080] In some embodiments, the nanoparticles have a spherical dimension or shape. In some embodiments, the nanoparticles have an expanded or contracted shape. In some embodiments, the plurality of core-shell particles lack any characteristic dimension or shape. In some embodiments, the nanoparticles have a spherical, quasi-spherical, quasi-ellipsoidal, contracted, concave, irregular shape, or any combination thereof.

[0081] In some embodiments, the plurality of core-shell particles are substantially spherical, substantially as described herein. In some embodiments, the plurality of core-shell particles are substantially ellipsoidal, substantially as described herein. One of ordinary skill in the art will understand that the exact shape of each of the plurality of core-shell particles can vary from particle to particle. Further, the exact shape of the nanoparticles may be derived from any of the geometric forms listed above, such that the shape of the particles does not perfectly conform to a particular geometric shape. One of ordinary skill in the art will understand that the exact shape of the nanoparticles can have a substantial deviation (such as at least 5%, at least 10%, at least 20% deviation, etc.) from a particular geometric shape (e.g., a sphere or an ellipse).

[0082] In some embodiments, the lipid comprises a helper lipid. In some embodiments, the lipid comprises a structural lipid, a PEG lipid, or both. In some embodiments, the lipid comprises a helper lipid and optionally a structural lipid and / or a PEG lipid. In some embodiments, the term "structural lipid" encompasses non-liposome-forming lipids as described herein. In some embodiments, the structural lipid is a sterol or comprises a sterol.

[0083] In some embodiments, the helper lipid is a phospholipid or comprises a phospholipid. In some embodiments, the helper lipid is a liposome-forming lipid or comprises a liposome-forming lipid. As used herein, the term "liposome-forming lipid" encompasses lipids (e.g., phospholipids) that self-assemble to form stable vesicles (e.g., lipid nanoparticles) when dispersed or dissolved in an aqueous solution at a temperature above the transition temperature (T m ). As used herein, the terms "liposome-forming lipid" and "lipid nanoparticle-forming lipid" are used interchangeably. As used herein, the term T m refers to the temperature at which a lipid undergoes a phase transition from a solid (also called an ordered phase, a gel phase) to a fluid (also called a disordered phase, a fluid crystalline phase). T mIt also refers to the temperature (or temperature range) at which the maximum change in heat capacity occurs during the phase transition.

[0084] In some embodiments, the phospholipid comprises a single phospholipid species or a plurality of chemically different phospholipids.

[0085] In some embodiments, the liposome-forming lipid has one or two C 12 ~C 24 hydrocarbon tails (typically acyl, alkyl or alkenyl chains), and has various degrees of unsaturation of lipids that are fully saturated to fully, partially hydrogenated or non-hydrogenated (the level of saturation can affect the rigidity of the liposomes thus formed, and typically liposomes formed from lipids with saturated chains are more rigid than liposomes formed from lipids of the same chain length with unsaturated chains, especially cis double bonds). In some embodiments, at least one of the liposome-forming lipids is a phospholipid having one or two C 12 ~C 20 , C 16 ~C 20 , or C 16 ~C 18 (including any values and ranges therebetween) hydrocarbon tails. In some embodiments, the liposome-forming lipid is fully saturated, straight-chain or branched.

[0086] Furthermore, the phospholipid may be of natural origin (e.g., natural origin phospholipids), semi-synthetic or fully synthetic lipids, and electrically neutral (e.g., zwitterionic), negatively or positively charged.

[0087] Non-limiting examples of neutral lipids include, but are not limited to, diacyl phosphatidylcholine, dialkyl phosphatidylcholine, sphingomyelin, and diacyl phosphatidylethanolamine. Phosphatidylcholine (PC) includes those obtained from eggs, soybeans, or other plant sources, or partially or fully synthetic, or of various lipid chain lengths and degrees of unsaturation, and is suitable for use in the present compositions. Synthetic, semi-synthetic, and natural phosphatidylcholines, including but not limited to POPC, DOPC, DMPC, distearoyl phosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), soy phosphatidylcholine (soy PC), egg phosphatidylcholine (egg PC), hydrogenated egg phosphatidylcholine (HEPC), and dipalmitoyl phosphatidylcholine (DPPC), are phosphatidylcholines suitable for use in the preparation of liposomes. Charged lipids can include phosphatidylglycerol, cardiolipin, or headgroup-modified lipids such as N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine, and PEG-derivatized phosphatidylethanolamine.

[0088] Non-limiting examples of cationic lipids or ionizable cationic lipids include, but are not limited to, 5-carboxyspermine dioctadecylamide or "DOGS", N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or "DOTMA", 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-pr-opanaminium or "DOSPA", 1,2-dioleoyl-3-dimethylammonium-propane or "DODAP", 1,2-dioleoyl-3-trimethylammonium-propane or "DOTAP".Intended cationic lipids 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-dilinolenyloxy-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 "DMRIE", 3-dimethylamino-2-(cholest-5-en-3-β-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane or "CLinDMA", 2-[5'-(cholest-5-en-3-β-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9,12-octadecadienoxy)propane or "CpLinDMA", N,N-dimethyl-3,4-dioleyloxybenzylamine or "DMOBA", 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane or "DOcarbDAP", 2,3-dilinoleoyloxy-N,N-dimethylpropylamine or "DLinDAP", 1,2-N,N'-dilinoleyloylcarbamyl-3-dimethylaminopropane or "DLincarbDAP", 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane or "DLinCDAP", 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or "DLin-K-DMA", 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or "DLin-K-XTC2-DMA", and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLin-KC2-DMA).

[0089] In some embodiments, the helper lipid is a non-cationic lipid or comprises a non-cationic lipid. As used herein, the term "non-cationic lipid" refers to any neutral or zwitterionic lipid. Non-cationic lipids include, but are not limited to, dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), or mixtures thereof.

[0090] In some embodiments, the helper lipid is DOPE, DSPC, POPE, or any combination thereof or comprises any of these.

[0091] In some embodiments, the helper lipid is a cationic lipid. In some embodiments, the cationic lipid is any of DOTAP, DDAB, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, DPPC ethyl (EPC 16:0, or 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine), or any combination thereof or comprises any of these.

[0092] In some embodiments, the PEG lipid comprises a single PEG moiety covalently attached to the head group of the lipid. In some embodiments, the PEG lipid comprises multiple PEG moieties covalently attached to the head group of the lipid. In some embodiments, the PEG moiety comprises an alkylated PEG such as methoxypoly(ethylene glycol) (mPEG). The PEG moiety can have a head group molecular weight of from about 750 Da to about 20,000 Da, sometimes from about 750 Da to about 12,000 Da, and typically from about 1,000 Da to about 5,000 Da (including any range therebetween).

[0093] In some embodiments, the term “non-vesicle-forming lipid” should be understood to refer to a lipid that does not spontaneously form vesicles when brought into an aqueous medium.

[0094] There are various types of lipids that do not spontaneously vesiculate but can be used in vesicles or incorporated into vesicles. In some embodiments, the non-vesicle-forming lipid is a sterol or comprises a sterol.

[0095] Non-limiting examples of sterols include, but are not limited to, β-sitosterol, β-sitostanol, stigmasterol, stigmasterol, campesterol, campestanol, ergosterol, avenasterol, brassicasterol, fucosterol, cholesterol (Chol), cholesteryl hemisuccinate, and cholesteryl sulfate (including any salts or any combination thereof).

[0096] In some embodiments, the structural lipid is any one of betulin, brassicasterol, calcipotriol, campesterol, cholesterol, daucosterol, DC-cholesterol, dehydroergosterol, DMAPC-Chol, DMHAPC-Chol, ergosterol, fucosterol, HAPC-Chol, lupeol, MHAPC-Chol, OH-C-Chol, OH-Chol, 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 a steroid (including any salt thereof or any combination thereof).

[0097] In some embodiments, the molar concentration of one or more compounds of the present invention within the nanoparticles is 10 to 80 mol%, 15 to 55 mol%, 10 to 20 mol%, 20 to 60 mol%, 10 to 60 mol%, 20 to 40 mol%, 40 to 60 mol%, 60 to 80 mol%, including any range therebetween. As used herein, the terms "concentration" or "molar concentration" refer to the molar ratio relative to the total lipid content of the nanoparticles. In some embodiments, the total lipid content refers to the combined content of the compounds and lipids of the present invention, and the lipids include liposome-forming lipids, modified lipids (e.g., PEG lipids), and non-liposome-forming lipids. Those skilled in the art will understand that the molar ratios of the essential components within the LNPs and within the compositions of the present invention (i.e., the compounds of the present invention, helper lipids, structural lipids, and modified lipids) are the same. Thus, for example, the molar concentrations and molar ratios disclosed herein with respect to LNPs also encompass the corresponding molar concentrations and molar ratios within the compositions of the present invention, and vice versa.

[0098] In some embodiments, the molar concentration of the helper lipid in the nanoparticles is 5 to 60 mol%, 5 to 10 mol%, 5 to 15 mol%, 10 to 40 mol%, 10 to 30 mol%, 5 to 20 mol%, 20 to 40 mol%, including any range therebetween.

[0099] In some embodiments, the molar concentration of the structural lipid in the nanoparticles is 5 to 60 mol%, 20 to 60 mol%, 10 to 50 mol%, 20 to 50 mol%, 5 to 40 mol%, 20 to 40 mol%, 30 to 40 mol%, including any range therebetween.

[0100] In some embodiments, the molar concentration of the modified lipid (e.g., PEG lipid) in the nanoparticles is 0.5 to 10 mol%, 0.1 to 10 mol%, 0.1 to 0.5 mol%, 0.5 to 1 mol%, 1 to 5 mol%, 0.5 to 2 mol%, 5 to 10 mol%, 5 to 7 mol%, 7 to 10 mol%, including any range therebetween.

[0101] In some embodiments, the molar ratio of the helper lipid to the modified lipid is in the range of 1:0.2 to 1:0.01, 1:0.15 to 1:0.01, 1:0.1 to 1:0.01, 1:0.05 to 1:0.01, including any range therebetween.

[0102] In some embodiments, the molar ratio of the compound to the helper lipid is in the range of 1:0.5 to 5:1, 1:0.5 to 4:1, 1:0.5 to 3:1, 1:0.5 to 2:1, 1:0.5 to 1:1, 1:0.1 to 5:1, 1:1 to 1:2, 1:1 to 1:5, 1:0.25 to 5:1, 1:0.5 to 2:1, including any range therebetween.

[0103] In some embodiments, the molar ratio of the structural lipid to the modified lipid is in the range of 200:1 to 2:1, 100:1 to 5:1, 100:1 to 10:1, 100:1 to 30:1, 100:1 to 50:1, 100:1 to 70:1, 100:1 to 90:1, 100:1 to 100:3, 100:1 to 20:1, 150:1 to 20:1, 200:1 to 50:1, 200:1 to 10:1, including any range therebetween.

[0104] In some embodiments, the weight ratio between the compound of the present invention (or the total lipid content, the total amount of the compound of the present invention and lipid within and / or in the composition containing the nanoparticles) and the polynucleic acid within the nanoparticles (or in the composition containing the same) is 0.001:1 to 10:1, 0.001:1 to 0.1:1, 0.1:1 to 1:1, 1:1 to 10:1, including any range therebetween.

[0105] In some embodiments, the N:P ratio within the lipid nanoparticles or in the composition of the present invention ranges from 3 to 20, 3 to 5, 4 to 8, 6 to 8, 8 to 10, 10 to 12, 10 to 20, 8 to 20, 8 to 15, and 12 to 14 (including any range therebetween). The term "N:P ratio" refers to the ratio between the N atoms of the compound of the present invention and the P atoms of the polynucleotide within the lipid nanoparticles or in the composition of the present invention.

[0106] In some embodiments, the N:P ratio is about 12. In some embodiments, the N:P ratio within the lipid nanoparticles or in the composition of the present invention ranges from 3 to 12.

[0107] In one embodiment, the nanoparticles in the composition are characterized by an average particle size of less than 500 nm to facilitate their entry through the extracellular matrix into cells. In one embodiment, the carrier is characterized by an average particle size of less than 300 nm to facilitate its entry through the extracellular matrix into cells.

[0108] In one embodiment, the nanoparticles in 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).

[0109] In another embodiment, the nanoparticles in the composition are characterized by an average particle size of 50 - 300 nm, 50 - 250 nm, 50 - 200 nm, 100 - 300 nm, 50 - 100 nm, 100 - 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 particle size distribution (polydispersity index, PDI) of 0.05 - 0.4, 0.05 - about 0.3, about 0.1 - 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.

[0110] In another embodiment, the nanoparticles are characterized by a negative or positive zeta potential (e.g., measured at a pH of about 7, e.g., a pH of about 6.5 - about 7.5). In some embodiments, the nanoparticles are characterized by a zeta potential in the range of -40 to +40 mV (including any range therebetween). In some embodiments, the nanoparticles are characterized by a zeta potential in the range of -20 to +20 mV (including any range therebetween). In some embodiments, the nanoparticles are characterized by a negative zeta potential in the range of -0.1 to -40 mV (including any range therebetween). In some embodiments, the nanoparticles are characterized by a positive zeta potential in the range of +0.1 to +40 mV (including any range therebetween). In some embodiments, the nanoparticles are characterized by a neutral zeta potential at physiological pH (e.g., a pH of 6.5 - 7.5 (including any range therebetween)).

[0111] In some embodiments, the nanoparticles are stable over a period of 1 day to 1 year, or longer, or 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 loading 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 an aqueous solution.

[0112] In some embodiments, the terms "polynucleic acid" and "polynucleotide" are used interchangeably herein. In some embodiments, the polynucleotide consists of 60 to 15,000 nucleic acid bases, 15,000 to 10,000 nucleic acid bases, 10,000 to 4,700 nucleic acid bases, 200 to 5,000 nucleic acid bases, 300 to 5,000 nucleic acid bases, 400 to 5,000 nucleic acid bases, 400 to 2,500 nucleic acid bases, 200 to 3,000 nucleic acid bases, 400 to 2,000 nucleic acid bases, 400 to 1,000 nucleic acid bases, including any range therebetween.

[0113] In some embodiments, the polynucleotide comprises at least 20 nucleic acid bases, at least 250 nucleic acid bases, at least 300 nucleic acid bases, at least 350 nucleic acid bases, at least 400 nucleic acid bases, at least 450 nucleic acid bases, at least 475 nucleic acid bases, or at least 500 nucleic acid bases. Each possibility represents a separate embodiment of the invention.

[0114] In some embodiments, the polynucleotide comprises at most 500 nucleic acid bases, at most 750 nucleic acid bases, at most 1,000 nucleic acid bases, at most 1,250 nucleic acid bases, at most 1,750 nucleic acid bases, at most 2,500 nucleic acid bases, at most 3,000 nucleic acid bases, at most 4,000 nucleic acid bases, or at most 5,000 nucleic acid bases. Each possibility represents a separate embodiment of the invention.

[0115] 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, and each type of nanoparticle comprises a specific polynucleotide.

[0116] In some embodiments, a particular polynucleotide comprises a plurality of polynucleotide molecules having the same or identical nucleic acid sequences. In some embodiments, a particular polynucleotide comprises a plurality of polynucleotide molecules having substantially the same nucleic acid sequences.

[0117] As used herein, the term "plurality" encompasses any integer greater than or equal to 2. In some embodiments, the plurality is 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 invention.

[0118] As used herein, the term "polynucleotide type" refers to a plurality of polynucleotides, wherein each of the plurality of polynucleotides contains a nucleic acid sequence that differs from any one of the other polynucleotides of the plurality of polynucleotides by at least 1 nucleic acid base, at least 1 nucleic acid base, at least 1 nucleic acid base, at least 1 nucleic acid base, at least 1 nucleic acid base, at least 10 nucleic acid bases, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0119] 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 nanoparticles of the invention comprise a polynucleotide selected from RNA, DNA, synthetic analogs of RNA, synthetic analogs of DNA, DNA / RNA hybrids, or any combination thereof.

[0120] In some embodiments, the polynucleotide comprises or consists of RNA. The polynucleotide comprises or consists of messenger RNA (mRNA). “Messenger RNA” (mRNA) refers to any polynucleotide that encodes a (at least one) polypeptide (a polymer of naturally occurring, non-naturally occurring, or modified amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ, or ex vivo. The basic components of an mRNA molecule typically include at least one coding region, a 5′ untranslated region (UTR), a 3′ UTR, a 5′ cap, and a polyA tail. The polynucleotide can function as mRNA, but can be distinguished from wild-type mRNA in those functional and / or structural design features that help overcome existing problems of effective polypeptide expression using nucleic acid-based therapeutics.

[0121] The mRNA provided herein comprises at least one (one or more) ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one polypeptide of interest. In some embodiments, the RNA polynucleotide of the mRNA encodes 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 polypeptides. In some embodiments, the RNA polynucleotide of the mRNA encodes at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 polypeptides. In some embodiments, the RNA polynucleotide of the mRNA encodes at least 100 or at least 200 polypeptides.

[0122] In some embodiments, the nucleic acid is a therapeutic mRNA. As used herein, the term "therapeutic mRNA" refers to an mRNA that encodes a therapeutic protein. Therapeutic proteins mediate various effects in host cells or subjects to treat a disease or to improve the signs and symptoms of a disease. For example, a therapeutic protein can replace a defective or abnormal protein, enhance the function of an endogenous protein, and provide a new function to a cell (e.g., inhibit or activate an endogenous cellular activity, function as a delivery agent for another therapeutic compound (e.g., an antibody-drug conjugate)). Therapeutic mRNAs are useful for the treatment of the following diseases and conditions: bacterial infections, viral infections, parasitic infections, cell proliferation disorders, genetic disorders, autoimmune diseases.

[0123] Accordingly, the constructs of the present invention can be used as therapeutic or prophylactic agents. They are provided for use in medicine. For example, the mRNAs of the constructs described herein can be administered to a subject, and the polynucleotide is translated in vivo to produce a therapeutic peptide.

[0124] In some embodiments, the polynucleotide comprises an inhibitory nucleic acid. In some embodiments, the polynucleotide comprises an antisense oligonucleotide.

[0125] As used herein, "antisense oligonucleotide" refers to a nucleic acid sequence that is reverse and complementary to a DNA or RNA sequence.

[0126] As used herein, an "inverted complementary nucleic acid sequence" is a nucleic acid sequence that can hybridize with another nucleic acid sequence containing complementary nucleotide bases. "Hybridize" means forming a double-stranded molecule between complementary nucleotide bases under appropriate stringency conditions (e.g., adenine (A) forms a base pair with thymine (T) (or uracil (U) in the case of RNA), and guanine (G) forms a base pair with cytosine (C)); see, e.g., Wahl, G.M., and S.L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507). For the purposes of the present method, the inhibitory nucleic acid need not be complementary to the entire sequence, as long as it is a sequence sufficient to provide specific inhibition; for example, in some embodiments, the sequence is at least nucleotides (nt) 2-7 or 2-8 (e.g., the "seed sequence") at the 5' end of the microRNA itself, e.g., nt2-7 or 20 and 100% complementary.

[0127] In some embodiments, the inhibitory nucleic acid has one or more chemical modifications in the backbone or side chain. In some embodiments, the inhibitory nucleic acid has at least one locked nucleotide and / or a phosphorothioate backbone.

[0128] Non-limiting examples of useful inhibitory nucleic acids 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 bases / locked nucleic acids (LNA), antagomirs, peptide nucleic acids (PNA), ribozymes (catalytic RNA molecules capable of cleaving other specific sequences of RNA molecules), and other oligomeric compounds or oligonucleotide mimetics that hybridize to at least a portion of the target nucleic acid and regulate its function. In some embodiments, the inhibitory nucleic acid is antisense RNA, antisense DNA, chimeric antisense oligonucleotide, antisense oligonucleotide containing modified linkages, interfering RNA (RNAi), small interfering RNA (siRNA); microRNA (miRNA); small temporal RNA (stRNA); or small hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNA (saRNA), or combinations thereof.

[0129] 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).

[0130] As used herein, "interfering RNA" refers to any double-stranded or single-stranded RNA sequence that can inhibit or downregulate gene expression - directly or indirectly (i.e., upon conversion) - by mediating RNA interference. 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-complementary messenger RNA transcripts.

[0131] In some embodiments, the polynucleotide is chemically modified. In some embodiments, the chemical modification is a modification of the backbone of the polynucleotide. 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 nucleobase of the polynucleotide. In some embodiments, the chemical modification enhances the stability of the polynucleotide in a cell. In some embodiments, the chemical modification enhances the stability of the polynucleotide in vivo. In some embodiments, the chemical modification enhances the stability of the polynucleotide in vitro, such as in open air, in the field, on a surface exposed to air. In some embodiments, the chemical modification increases the ability of the polynucleotide to induce silencing of a target gene or sequence, 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 the following: ribose phosphate backbone, deoxyribose phosphate backbone, phosphorothioate deoxyribose backbone, 2′-O-methyl-phosphorothioate backbone, phosphorodiamidate morpholino backbone, peptide nucleic acid backbone, 2-methoxyethyl phosphorothioate backbone, constrained ethyl backbone, alternating locked nucleic acid backbone, phosphorothioate backbone, N3′-P5′ phosphoramidate, 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid, cyclohexene nucleic acid backbone nucleic acid, tricyclo-DNA (tcDNA) nucleic acid backbone, ligand-binding antisense, and combinations thereof.

[0132] In some embodiments, the carrier (e.g., lipid nanoparticle) is prepared by mixing an aqueous phase optionally containing an active agent with an organic phase containing one or more lipid components and a compound of the invention. The selection of specific lipids (such as cationic lipids, non-cationic lipids, sterols and / or PEG-modified lipids) including lipid nanoparticles, as well as the relative molar ratios of such lipids to each other and / or the molar ratio between the lipid and the compound of the invention, are based on the properties of the selected lipids and the properties of the agent to be delivered. Additional considerations include, for example, the degree of saturation of the alkyl chain, the size of the selected lipid, T minclude charge, pH, pKa, membrane fusibility, and toxicity.

[0133] In another aspect, there is provided a pharmaceutical composition comprising the lipid nanoparticles of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is also referred to as an excipient or an adjuvant. As used herein, the terms "carrier", "excipient" or "adjuvant" refer to any component of a pharmaceutical composition that is not an active agent. As used herein, the term "pharmaceutically acceptable carrier" 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 physiological saline. Examples of materials that can function as pharmaceutically acceptable carriers include saccharides such as lactose, glucose and sucrose; starches such as corn starch and potato starch; celluloses and their derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter, suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, polyethylene glycol; esters such as ethyl oleate, ethyl laurate; buffers such as agar, magnesium hydroxide, aluminum hydroxide; alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, phosphate buffer, and other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances that can function as carriers herein include sugar, starch, cellulose and its derivatives, powdered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oil, polyol, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer, cocoa butter (suppository base), emulsifier, and other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, 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 and are, for example, those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the “Inactive Ingredient Guide,” U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the entire contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers, and diluents useful in the present composition include distilled water, physiological saline, Hartmann's solution, Ringer's solution, glucose solution, Hank's solution, and DMSO. These additional inactive ingredients, as well as effective formulation 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 these is hereby incorporated by reference in its entirety.The compositions described herein may also be contained in artificially produced structures such as liposomes, ISCOMs, sustained release particles, and other vehicles that increase the half-life of peptides or polypeptides in serum. Liposomes for use with the peptides described herein are formed from standard vesicle-forming lipids that generally include neutral and negatively charged phospholipids and sterols such as cholesterol. The choice of lipids is generally determined in consideration of, for example, the size of the liposomes and their stability in the blood. Various methods are available for the preparation of liposomes, as outlined, for example, in Coligan, J.E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York. See also U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369. The carrier may in total comprise from about 0.1% to about 99.99999% by weight of the pharmaceutical composition presented herein.

[0134] 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 an experimental animal. Examples of experimental 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.

[0135]

[0136] ​In some embodiments, the pharmaceutical composition is for use in a therapeutic method. In some embodiments, the therapeutic method is a method of treatment. In some embodiments, the pharmaceutical composition is for use in a diagnostic method. In some embodiments, the method comprises administering the composition of the invention to a subject. In some embodiments, the pharmaceutical composition is for use in the treatment or prevention of a disease or condition in humans and other mammals. The active therapeutic agent of the invention comprises a nanoparticle or a polypeptide translated from a polynucleotide contained in the nanoparticle.

[0137] As used herein, terms such as "administering," "administration," etc. refer to any method of delivering a composition comprising an active agent to a subject in a manner that provides a therapeutic effect in the context of sound medical practice. One aspect of the subject matter provides for intravenous administration of a therapeutically effective amount of the composition of the subject matter to a patient in need thereof. Other suitable routes of administration may include parenteral, intravenous, subcutaneous, oral, intramuscular, intrathecal, inhalation, intraventricular, intravenous, transdermal, or intraperitoneal.

[0138] The amount administered depends on the age, health, and weight of the recipient, and, if any, the type of concurrent treatment, the frequency of treatment, and the nature of the desired effect.

[0139] In another aspect, a method of treating a subject in need thereof is provided, the method comprising administering the therapeutic composition of the invention to the subject.

[0140] In some embodiments, a method for delivering an active agent into a target cell is provided, the method comprising contacting each nanoparticle of the present invention comprising the active agent (e.g., the active agent is encapsulated within the nanoparticle) with the cell. In some embodiments, the active agent is as described hereinabove. In some embodiments, the active agent is cell-impermeable. Cellular delivery (i.e., intracellular delivery) can be measured by quantifying the amount of the active agent within the cell (e.g., by fluorescence labeling of the compound or, in the case of a polynucleotide, by determining the expression level of the polynucleotide).

[0141] In some embodiments, the delivery is performed to obtain an increased concentration of the active agent within the cell as compared to a control formulation comprising Lipofectamine as the same active agent and intracellular trafficking 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 increased concentration refers to a concentration that is at least 10-fold, at least 50-fold, at least 100-fold, at least 200-fold greater than the control.

[0142] In some embodiments, the cell is a tissue cell. In some embodiments, the method is for delivering the active agent to a target tissue. In some embodiments, the delivery is to obtain a therapeutically effective concentration of the active agent within the tissue. In some embodiments, the delivery is to obtain an increased amount of the active agent within the tissue, where the increased amount is as compared to a control as disclosed herein.

[0143] Definitions As used herein, the term "consisting essentially of" means that a composition, method or structure may include additional components, steps and / or parts only if the additional components, steps and / or parts do not substantially change the basic and novel features of the claimed composition, method or structure.

[0144] 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 60 - 99.9%, 70 - 80%, 70 - 90%, 80 - 90%, 90 - 95%, 95 - 99.9% (including any range or value therebetween).

[0145] As used herein, the term "substituent" includes hydrogen, halogen, -NO2, -CN, -OH, oxo, imino, -CONH2, -CONR’2, -CNNR’2, -CSNR’2, -CONH-OH, -CONH-NH2, -NHCOR, -NHCSR, -NHCNR, -NC(=O)OR, -NC(=O)NR’, -NC(=S)OR’,-NC(=S)NR’,-SO2R’, -SOR’, -SR’, -SO2OR’, -SO2N(R’)2, -NHNR’2, -NNR’, C1-C6 haloalkyl, optionally substituted C1-C6 alkyl, -NH2, -NR’2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy(C1-C6 alkyl), hydroxy(C1-C6 alkoxy), alkoxy(C1-C6 alkyl), alkoxy(C1-C6 alkoxy), C1-C6 alkyl-NR’2, C1-C6 alkyl-SR’, -CONH(C1-C6 alkyl), -CON(C1-C6 alkyl)2, -CO2H, -CO2R’, -OCOR, -OCOR’, -OC(=O)OR’, -OC(=O)NR’, -OC(=S)OR’, -OC(=S)NR’, or combinations thereof; wherein each R’ independently represents hydrogen or optionally substituted C1-C 10 alkyl, optionally substituted C3-C 10 cycloalkyl, optionally substituted C3-C 10It is selected from the group consisting of heterocycloalkyl, optionally substituted heteroaryl, optionally substituted aryl, hydroxy, amino, -NH2, -NR’2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy(C1-C6 alkyl), hydroxy(C1-C6 alkoxy), alkoxy(C1-C6 alkyl), alkoxy(C1-C6 alkoxy), C1-C6 alkyl-NR’2, C1-C6 alkyl-SR’, or combinations thereof.

[0146] As used herein, the term "alkyl" represents aliphatic hydrocarbons including straight-chain and branched-chain groups. The term "alkyl" also, as used herein, encompasses saturated or unsaturated hydrocarbons and thus this term further includes alkenyl and alkynyl.

[0147] The term "alkenyl", as defined herein, represents unsaturated alkyl having at least 2 carbon atoms and at least 1 carbon-carbon double bond. Alkenyl may or may not be substituted by one or more substituents as described herein.

[0148] The term "alkynyl" is, as defined herein, unsaturated alkyl having at least 2 carbon atoms and at least 1 carbon-carbon triple bond. Alkynyl may or may not be substituted by one or more substituents as described herein.

[0149] The term "cycloalkyl" represents all-carbon monocyclic or fused-ring (i.e., rings sharing adjacent pairs of carbon atoms) groups having no more than one completely conjugated π-electron system in the ring. Cycloalkyl groups may or may not be substituted as shown herein.

[0150] The term "aryl" represents a monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group of all carbon atoms having a fully conjugated π electron system. The aryl group may or may not be substituted as shown herein.

[0151] The term "alkoxy" represents both O-alkyl groups and -O-cycloalkyl groups as defined herein. The term "aryloxy" represents -O-aryl as defined herein.

[0152] Each of the alkyl, cycloalkyl, and aryl groups in the general formulas herein may be substituted with one or more substituents, whereby each substituent is independently, depending on the substituent and its position in the molecule, for example, a halide, alkyl group, alkoxy group, cycloalkyl group, nitro group, amino group, hydroxyl group, thiol group, thioalkoxy group, carboxy group, amide group, aryl group, and aryloxy group. Additional substituents are also contemplated.

[0153] The terms "halide", "halogen", or "halo" represent fluorine, chlorine, bromine, or iodine. The term "haloalkyl" represents an alkyl group as defined herein further substituted with one or more halides. The term "haloalkoxy" represents an alkoxy group as defined herein further substituted with one or more halides. The term "hydroxyl" or "hydroxy" represents an -OH group. The term "mercapto" or "thiol" represents an -SH group. The term "thioalkoxy" represents both -S-alkyl and -S-cycloalkyl groups as defined herein. The term "thioaryloxy" represents both -S-aryl and -S-heteroaryl groups as defined herein. The term "amino" represents an -NR'R'' group having R' and R'' as described herein, or a salt thereof.

[0154] The term "heterocyclyl" refers to a monocyclic or fused cyclic 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 are piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholino, etc.

[0155] The term "carboxy" refers to a -C(O)OR' group, or its carboxylate salt, where R' is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl (bonded through a ring carbon), or heterocyclyl (bonded through a ring carbon) as defined herein.

[0156] The term "carbonyl" refers to a -C(O)R' group, where R' is as defined herein. The above term also includes its thio - derivatives (thiocarboxy and thiocarbonyl).

[0157] The term "thiocarbonyl" refers to a -C(S)R' group, where R' is as defined herein. The "thiocarboxy" group refers to a -C(S)OR' group, where R' is as defined herein. The "sulfinyl" group refers to a -S(O)R' group, where R' is as defined herein. The "sulfonyl" group or "sulfonate" group refers to a -S(O)2R' group, where R' is as defined herein.

[0158] The "carbamyl" group or "carbamate" group represents an -OC(O)NR’R’’ group, where R’ is as defined herein and R’’ is as defined with respect to R’. The "nitro" group refers to a -NO2 group. As used herein, the term "amide" encompasses C-amides and N-amides. The term "C-amide" represents a -C(O)NR’R” terminal group or a -C(O)NR’- linking group, these terms being as defined above herein, and R’ and R” being as defined herein. The term "N-amide" represents a -NR”C(O)R’ terminal group or a -NR’C(O)- linking group, these terms being as defined above, and R’ and R” being as defined herein.

[0159] The "cyano" or "nitrile" group refers to a -CN group. The terms "azo" or "diazo" represent a -N=NR’ terminal group or a -N=N- linking group, these terms being as defined herein, and R’ being as defined above. The term "guanidine" represents a -R’NC(N)NR”R’” terminal group or a -R’NC(N)NR”- linking group, these terms being as defined above herein, and R’, R” and R’” being as defined herein. As used herein, the term "azide" refers to a -N3 group. The term "sulfonamide" refers to a -S(O)2NR’R’’ group, where R’ and R’’ are as defined herein.

[0160] The term "phosphonyl" or "phosphonate" represents an -OP(O)-(OR’)2 group, where R’ is as defined herein. The term "phosphinyl" represents a -PR’R’’ group, where R’ and R’’ are as defined above herein. The term "alkylaryl" represents an alkyl as defined herein, which is substituted by an aryl as described herein. An exemplary alkylaryl is benzyl.

[0161] The term "heteroaryl" refers to a monocyclic or fused-ring (i.e., rings sharing a pair of adjacent atoms) group that has in the ring one or more atoms such as, for example, nitrogen, oxygen, and sulfur, and further has a fully conjugated π electron system. As used herein, the term "heteroaryl" refers to an aromatic ring in which at least one atom forming the aromatic ring is a heteroatom. Heteroaryl rings can be formed by 3, 4, 5, 6, 7, 8, 9, and more than 9 atoms. Heteroaryl groups may be optionally substituted. Examples of heteroaryl groups include aromatic C containing one oxygen atom or sulfur atom, or two oxygen atoms, or two sulfur atoms, or up to four nitrogen atoms, or a combination of one oxygen atom or sulfur atom and up to two nitrogen atoms 3-8 heterocyclic groups, and their substituents, and benzo-fused derivatives and pyrido-fused derivatives linked, for example, through one of the ring-forming carbon atoms, but are not limited thereto. In certain embodiments, heteroaryl is selected from oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolyl, benzimidazolyl, quinolinyl, isoquinolinyl, quinazolinyl or quinoxalinyl.

[0162] In some embodiments, the heteroaryl group is selected from pyrrolyl, furanyl (furyl), thiophenyl (thienyl), imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3-oxazolyl (oxazolyl), 1,2-oxazolyl (isoxazolyl), oxadiazolyl, 1,3-thiazolyl (thiazolyl), 1,2-thiazolyl (isothiazolyl), tetrazolyl, pyridinyl (pyridyl), pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, indazolyl, indolyl, benzothiophenyl, benzofuranyl, benzothiazolyl, benzimidazolyl, benzodioxolyl, acridinyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, thieno[2,3-b]thiophenyl, 1,8-naphthyridinyl, other naphthyridinyl, pteridinyl or phenothiazinyl. When the heteroaryl group contains two or more rings, each additional ring is of the saturated type (perhydro type) or partially unsaturated type (e.g., dihydro type or tetrahydro type) or maximally unsaturated (non-aromatic) type. The term heteroaryl thus includes bicyclic radicals in which two rings are aromatic and bicyclic radicals in which only one ring is aromatic. Such examples of heteroaryl include 3H-indolinyl, 2(1H)-quinolinonyl, 4-oxo-1,4-dihydroquinolinyl, 2H-1-oxoisoquinolyl, 1,2-dihydroquinolinyl, (2H)quinolinyl N-oxide, 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-tetrahydroisoquinolinyl, 1H-2,3-dihydroisoindolyl, 2,3-dihydrobenzo[f]isoindolyl, 1,2,3,4-tetrahydrobenzo[g]isoquinolinyl, 1,2,3,4-tetrahydro-benzo[g]isoquinolinyl, chromanyl, isochromanonyl, 2,3-dihydrochromonyl, 1,4-benzodioxanyl, 1,2,3,4-Tetrahydro-quinoxalinyl, 5,6-dihydro-quinolyl, 5,6-dihydro-iso-quinolyl, 5,6-dihydroquinoxalinyl, 5,6-dihydroquinazolinyl, 4,5-dihydro-1H-benzimidazolyl, 4,5-dihydro-benzoxazolyl, 1,4-naphthoquinolyl, 5,6,7,8-tetrahydro-quinolinyl, 5,6,7,8-tetrahydro-isoquinolinyl, 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-onyl, 1,3-dihydroimidazolo-[4,5]-pyridin-2-onyl, 2,3-dihydro-1,4-dinaphtho-quinonyl, 2,3-dihydro-1H-pyrrolo[3,4-b]quinolinyl, 1,2,3,4-tetrahydrobenzob-[1,7]naphthyridinyl, 1,2,3,4-tetra-hydrobenzob[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-azepino[3,4-b]indolyl, 1H-2,3,4,5-tetrahydroazepino-[4,3-b]indolyl, 1H-2,3,4,5-tetrahydro-azepino[4,5-b]indolyl, 5,6,7,8-tetrahydro[1,7]naphthyridinyl, 1,2,3,4-tetrahydro-[2,7]-naphthyridyl, 2,3-dihydro[1,4]dioxino[2,3-b]pyridyl, 2,3-dihydro[1,4]-dioxino[2,3-b]puridyl, 3,4-dihydro-2H-1-oxa[4,6]diazanaphthalenyl, 4,5,6,7-tetrahydro-3H-imidazo-[4,5-c]pyridyl, 6,7-dihydro[5,8]diazanaphthalenyl, 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) Napthyridinyl is included. In some embodiments, the heteroaryl group may be optionally substituted. In one embodiment, each of one or more substituents is independently halo, 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.

[0163] Examples of heteroaryl groups include 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, phenoxazole, oxadiazole, benzopyrazole, quinolidine, cinnoline, phthalazine, quinazoline and quinoxaline unsubstituted and mono - or di - substituted derivatives, but are not limited thereto. In some embodiments, the substituents are halo, hydroxy, cyano, O - C 1-6 alkyl, C 1-6 alkyl, hydroxy - C 1-6 alkyl, and amino - C 1-6 alkyl.

[0164] As used herein, the terms "halo" and "halide" are referred to interchangeably herein and represent an atom of a halogen, i.e., fluorine, chlorine, bromine, or iodine, and are also referred to herein as fluoride, chloride, bromide, and iodide.

[0165] General As used herein, the term "about" when combined with a 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.

[0166] It should be noted that as used in this specification and 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, reference to "the polypeptide" includes reference to one or more polypeptides known to those skilled in the art and their equivalents, and so on. It should further be noted that the claims can be drafted to exclude any element. Thus, this specification is intended to serve as antecedent basis for the use of exclusive terms such as "alone", "only", etc. in connection with the recitation of claim elements or the use of "negative" limitations.

[0167] In instances where a convention similar to "at least one of A, B, and C, etc." is used, generally such a construction is intended in the sense that one of ordinary skill in the art would understand the convention (e.g., a system having at least one of A, B, and C would include, but not be limited to, a system having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It should also be understood by one of ordinary skill in the art that, regardless of the description in this specification or the claims, substantially any conjunctive word and / or phrase presenting two or more alternative terms contemplates the possibility of including either one of the terms, any one of the terms, or both terms. For example, the phrase "A or B" is understood to include the possibilities of "A" or "B" or "A and B".

[0168] It is understood that certain features of the invention that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, for brevity, the various features of the invention that are described as a single embodiment may also be provided separately or in any suitable sub-combination. All combinations of embodiments related to the invention are specifically encompassed by the invention and are disclosed herein as if each combination were individually and explicitly disclosed. Furthermore, all partial combinations of the various embodiments and their elements are also specifically encompassed by the invention and are disclosed herein as if each such partial combination were individually and explicitly disclosed herein.

[0169] Additional objects, advantages, and novel features of the invention will become apparent to those skilled in the art by considering the following examples, which are not intended to be limiting. Furthermore, each of the various embodiments and aspects of the invention described above and claimed in the claims section below is experimentally verified in the following examples.

[0170] The various embodiments and aspects of the invention described above and claimed in the claims below are experimentally verified in the following examples.

[0171] Examples Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are well explained in the literature. For example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R.M., 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); the methodologies described in 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, J.E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J.E., 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). Please refer to these. All of these are incorporated herein by reference. Other general references are provided throughout this specification.

[0172] Example 1 Exemplary compounds of the present invention were synthesized according to the synthetic scheme shown below. The inventors successfully utilized the exemplary compounds of the present invention for the preparation of stable LNPs and tested them in cell-based assays to examine their expression efficiency. Exemplary compounds (ionizable lipids) of the present invention are shown below. Most of these compounds below were tested and found to be able to form RNA-encapsulated LNPs. Additional compound / LNP formulations are currently being screened in various in vivo / in vitro assays.

Chemical Structure

[0173] Some of the exemplary LNPs of the present invention were characterized by excellent cell permeability as measured in cell-based tests. For example, LNPs containing MB-77, 208, 212, and 205 (exemplary compounds of the present invention) showed excellent internalization efficiency compared to similar compositions containing Dlin-MC3-DMA as the ionizable lipid.

[0174] General synthetic schemes for some of the exemplary compounds of the present invention are shown herein. Other possible synthetic strategies are well known to those skilled in the art. [Chemical formula] TIFF2025521244000068.tif90159

[0175] An exemplary composition of the LNP is as follows: about 5-15 mol% of helper lipid (e.g., DOPE); about 30-45 mol% of structural lipid (e.g., cholesterol); 0.5-5 mol% of PEG lipid (e.g., DMG-PEG2000); and about 40-60 mol% of the compound of the present invention. The LNP prepared by the inventors was characterized by an average particle size in the range of about 60 to about 300 nm.

[0176] The LNP was prepared as follows: The lipids were weighed and dissolved in ethanol (ETOH) at 55-60 °C. The mRNA was added to the citrate buffer at a pH of 5.0 (range 4.5-5.5). The mixing of the lipids including PEG lipid, helper lipid, cholesterol, and ionizable lipid (compound) was carried out by ethanol injection of the lipids into the mRNA-containing citrate buffer under microfluidic mixing or under certain mixing conditions. The pH of the mixture was then raised using PBS diluent, and the residual EtOH was removed before injection using dialysis such as FloatAlyzer or a standard dialysis membrane with a cut-off of 3 kDa, 8 kDa, 12-14 kDa, or 100 kDa according to the separation requirements.

[0177] The encapsulation efficiency was calculated using the Ribogreen assay, Quant-iT™ RiboGreen® RNA Reagent and Kit (Invitrogen) according to the manufacturer's instructions. Here, the particles were diluted with Tris-EDTA buffer, supplemented with RiboGreen (ThermoFisher) diluted 1:2000, and fluorescence was read using a plate reader (mRNA signal not encapsulated in the LNP). Triton x100 was diluted with TE and added to each well, and incubated at 37 °C for 10 minutes to allow perturbation of the LNP. Fluorescence was measured again (mRNA signal of total mRNA in the formulation). The encapsulation efficiency was calculated using %EE = 100*(total amount - amount not encapsulated) / total amount.

[0178] The inventors measured the cellular permeability of the exemplary LNP and the intracellular delivery of the mRNA.

[0179] More specifically, the particles were incubated on cells at 37 °C for 16 - 24 hours under 5% CO2, and the expression levels were measured. The formulation was compared to a Lipofectamine control, an MC3 control, and an untreated control.

[0180] Additional exemplary formulations were investigated, and stable LNP were obtained with an average particle size of 60 - 210 nm and a low PDI (0.1 - 0.3). The results are summarized in Table 1.

Table 1

[0181] Example 2 Some of the exemplary compositions of the present invention are characterized by enhanced specificity for lung cells as determined by cell-based assays. For example, the LNPs of the present 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 the ionizable lipid (Figure 1). Surprisingly, the inventors found that exemplary LNPs containing from about 15 to about 50% ionizable lipid (e.g., MB-212 or MB-222), from about 30 to about 50% helper lipid (e.g., DOTAP), from about 20 to about 50% structural lipid (cholesterol), and from about 0.5 to about 5% modified lipid (e.g., PEG lipid) result in enhanced lung specificity of the LNPs as evaluated by in vivo expression analysis. The exemplary LNP compositions of the present invention exhibited high lung specificity resulting in significantly enhanced expression of encapsulated polynucleic acid (mRNA F-LUC) in the lung compared to commercially available controls (Figure 1). Furthermore, a specific LNP composition of the present invention (FMB-1143) containing from about 15 to about 30% ionizable lipid exhibited excellent lung specificity (see Figure 1).

[0182] Preparation of FMB-745 40% DOTAP, 22.5% cholesterol, 2.5% DMG-PEG2000 and 35% commercially available ionizable lipid (Dlin-MC3-DMA) were dissolved in ethanol (EtOH) at 55-60 °C. mRNA, F-LUC, was added to citrate buffer at a pH of 5.0 (range 4.5 - 5.5). The lipid lipid mixture was performed by microfluidic mixing or by ethanol injection of the lipids into the citrate buffer containing mRNA firefly luciferase (F-LUC) under constant mixing conditions. The pH of the mixture was then raised using PBS dilution and residual EtOH was removed prior to injection using dialysis prepared by the inventors and characterized by an average particle size in the range of about 60 - about 130 nm.

[0183] Preparation of FMB-748 40% DOTAP, approximately 22% cholesterol, approximately 2 - 2.5% DMG PEG2000 and 35% ionized MB - 222 were dissolved in ethanol (EtOH) at 55 - 60 °C. mRNA, F - LUC was added to citrate buffer at a pH of 5.0 (range 4.5 - 5.5). Lipid mixing was carried out by microfluidic mixing or by EtOH injection of the lipids into the mRNA, F - LUC - containing citrate buffer under constant mixing conditions. The pH of the mixture was then raised using PBS dilution, and residual EtOH was removed prior to injection using dialysis prepared by the inventors, and was characterized by an average particle size in the range of approximately 60 - approximately 130 nm.

[0184] Preparation of lung - specific formulation (FMB - 1143) 40% DOTAP, approximately 38% cholesterol, 1.5% DMG - PEG2000 and 20% ionized MB - 212 were dissolved in ethanol (EtOH) at 55 - 60 °C. mRNA, F - LUC was added to citrate buffer at a pH of 5.0 (range 4.5 - 5.5). Lipid mixing was carried out by microfluidic mixing or by EtOH injection of the lipids into the mRNA, F - LUC - containing citrate buffer under constant mixing conditions. The pH of the mixture was then raised using PBS dilution, and residual EtOH was removed prior to injection using dialysis prepared by the inventors, and was characterized by an average particle size in the range of approximately 60 - approximately 130 nm.

[0185] The inventors determined the in - vivo expression distribution of exemplary LNPs 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 was performed on the lung, heart, spleen, kidney, and liver using IVIS. Histological evaluation was determined by H&E staining, and a pathologist reported on the toxicity. The histological evaluation concluded that there were no treatment - related pathological changes and the morphology was normal.

[0186] The expression profile shown in Fig. 1 shows a luciferase signal more than 100-fold higher in the lung compared to the heart, liver, spleen, and kidney, indicating the excellent lung specificity of the exemplary LNP of the present invention.

[0187] Example 3 The inventors successfully prepared LNP formulations (FMB-428 and FMB-389) based on the exemplary ionizable lipids of the present invention. FMB-428 and FMB-389 showed greater liver expression in vivo compared to similar LNP formulations based on commercially available ionizable lipids (Dlin-MC3-DMA) (see Fig. 2).

[0188] Preparation of FMB-386, FMB-428, FMB-389, FMB-1050 10% DOPE, 38.5% cholesterol, 1.5% DMG-PEG2000, and 50% each of ionizable MB-205, MB-222, MB-208, Dlin-MC3-DMA were dissolved in ethanol (EtOH) at 55 - 60 °C. mRNA, F-LUC was added to citrate buffer at a pH of 5.0 (range 4.5 - 5.5). Lipid mixing was carried out by microfluidic mixing or by injection of the lipid EtOH into the mRNA, F-LUC-containing citrate buffer under constant mixing conditions. The pH of the mixture was then raised using PBS dilution, and residual EtOH was removed prior to injection using dialysis prepared by the inventors, and was characterized by an average particle size in the range of about 60 to about 180 nm.

[0189] The inventors determined the in vivo expression profile of an exemplary LNP by evaluating the expression of encapsulated mRNA (mRNA F-LUC). The LNP of the present invention encapsulating mRNA F-LUC was 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 was performed on the lung, heart, spleen, kidney, and liver using IVIS. Histological evaluation was determined by H&E staining and a pathologist reported on the toxicity. The histological evaluation concluded that there were no treatment-related pathological changes and the morphology was normal.

[0190] The results of this experiment are summarized in Figure 2, showing enhanced liver expression in liver tissue compared to a control LNP composition containing a commercially available "standard reference" ionizable lipid.

[0191] Although the invention has been described in connection with its particular embodiments, it will be apparent to those skilled in the art that many alternatives, modifications, and variations are possible. Accordingly, the invention is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0192] Example 4 Exemplary thioether-based compounds of the present invention were synthesized according to the synthetic scheme shown above in this specification (Example 1).

Chemical formula

[0193] Based on in silico predictions, the inventors hypothesized that it is preferable to utilize more than 10 mol% (e.g., 15 - 60 mol%, or 20 - 55 mol%) of the thioether-based compounds of the present invention to obtain the LNP of the present invention having an average particle size of 50 - 180 nm with a low PDI. The predictions demonstrated that less than 10 mol% of the ionizable lipid of the present invention in the formulation results in LNP having a high PDI value.

[0194] The present inventors successfully utilized the exemplary thiol compounds of the present invention, MB-2036 and MB-2037, for the preparation of stable LNPs (FMB-1723, FMB-1724, FMB-1725, FMB-1726) having a low PDI and an average particle diameter of 80 to 110 nm.

[0195] Preparation of FMB-1723, FMB-1724, FMB-1725, FMB-1726 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 citrate buffer at a pH of 5.0 (range 4.5 - 5.5). Lipid mixing was performed under microfluidic mixing or by ethanol injection of lipids into the mRNA-containing citrate buffer under constant mixing conditions maintaining an N:P of 8 or 12. The LNPs were characterized by an average particle size in the range of about 80 to about 92 nm.

Claims

1. Formula I: 【Chemistry 1】 Represented by [wherein each L is independently, R １ , 【Chemistry 2】 ,or 【Transformation 3】 And; Each L １ R is independent, １ , 【Chemistry 4】 ;or 【Transformation 5】 And, 【Transformation 6】 represents a single bond, triple bond, or double bond; Each Z independently represents -OH or -SH; Each k is independently between 0 and 10; Each Y is independent, insofar as it does not exist or is permitted by its valence, CH ２ ----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 containing phosphate; Each T is independently optionally substituted C ５ - C ３０ alkyl or optionally substituted C ５ - C ３０ represents alkenyl; Each R' is independently H or can be substituted with any C. １ ~C １０ Alkyl, C １ ~C １０ Alkylaryl, C １ ~C １０ Alkylcycloalkyl, optionally substituted C ３ ~C １０ Cycloalkyl, optionally substituted C ３ ~C １０ This includes heterocyclines, optionally substituted heteroaryls, optionally substituted aryls, or combinations thereof; Each X is independently a heteroatom, CH ２ , C which can be arbitrarily substituted １ ~C １０ The alkyl group, or X, is not present; 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 can be arbitrarily substituted with C. ５ -C ３０ Contains alkyl; Each R １ C is arbitrarily substituted. １ -C ２４ Alkyl and at least one L or L １ teeth, 【Transformation 7】 or 【Transformation 8】 The compound, a salt of the compound, or both.

2. R and R １ The compound according to claim 1, wherein one of the compounds further comprises at least one unsaturated bond.

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

4. Each L is independent, 【Chemistry 9】 or 【Chemistry 10】 The compound according to claim 1.

5. The compound according to claim 1, wherein each X is independently O or absent.

6. R and R １ One of these is a linear or branched C １ -C ２４ The compound according to claim 1, representing an alkyl group.

7. Formula II: 【Chemistry 11】 The compound according to claim 1, represented by [the given expression].

8. Each L is independent, R １ , 【Chemistry 12】 The compound according to claim 7, wherein Z represents -OH.

9. Lipid nanoparticles comprising the compound described in claim 1 and an activator, wherein the activator comprises a polynucleic acid.

10. The lipid nanoparticles according to claim 9, further comprising lipids, wherein the lipids comprise helper lipids, structural lipids, and modified lipids, and the average size of the lipid nanoparticles is in the range of 50 to 300 nm.

11. (i) The weight ratio of the total amount of the compound and the lipid to (ii) the weight ratio of the polynucleic acid in the lipid nanoparticles is 0.001:1 to 10:1, and the ratio of the compound to the total lipid content of the lipid nanoparticles is 10 to 80 mol%, as described in claim 9.

12. The lipid nanoparticles according to claim 9, wherein the lipid nanoparticles contain 20 to 60 mol% of the compound, 5 to 50 mol% of the structural lipid, 10 to 50 mol% of the helper lipid, and 0.5 to 5 mol% of the modified lipid, based on the total lipid content of the lipid nanoparticles.

13. A pharmaceutical composition comprising a plurality of lipid nanoparticles according to claim 9 and a pharmaceutically acceptable carrier.

14. The pharmaceutical composition according to claim 13, for use in the preparation of a pharmaceutical for the treatment of a disease or disorder in a subject that requires it.

15. The pharmaceutical composition according to claim 13 for delivering an activator into a target tissue.

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