Lipids for nanoparticle delivery platform

Abstract

The present disclosure includes ionizable lipids suitable for lipid nanoparticle compositions and pharmaceutical formulations thereof. The lipids have general formula (I).

Description

LIPIDS FOR NANOPARTICLE DELIVERY PLATFORMSUMMARY OF THE INVENTION

[0001] Other aspects, features and advantages of the invention will be apparent from the following disclosure, including the detailed description of the invention and its preferred embodiments and the appended claims.

[0002] Some embodiments include a compound represented by formula (I):whereinA is -(CH2)nX(CH2)nY;X is O, C3-C6 cycloalkyl, alkenyl, or aromatic;Y is OH, NR’R’;n is and integer of 0, 1 or 2;R’ is C1-C4 alkyl or together form a 4-7-membered ring;Ri and R2are independently a Cs-Cs alkyl optionally substituted by one or more O, C2- alkenyl, cycloalkyl;Li and L2are independently selected from -C(=O)O-, -OC(=O)-; -NR”C(=O)O-; - C(=O)OZ-; a moiety of formula (A) or a moiety of formula (B);Xi and X2are optionally substituted Cs-Cis alkyl where one or more carbon may be substituted by one or more O, C2-alkenyl, cycloalkyl.

[0003] Some embodiments include a compound represented by formula (II):(II), whereinA is -(CH2)nX(CH2)nY;X is CH2, O, C3-C4 cycloalkyl, alkenyl, Y is OH, NR’R’;n is and integer of 0, 1 or 2;R’ is C1-C4 alkyl or together form a 4-7-membered ring;Ri and R2are independently a Cs-Cs alkyl optionally substituted by one or more O, C2- alkenyl, cycloalkyl;Li and L2are independently selected from -C(=O)O-, -OC(=O)-; -NR”C(=O)O-; - C(=O)O-; a moiety of formula (A) or a moiety of formula (B);Xi and X2are a substituted Cs-Cis alkyl where one or more carbon may be substituted by one or more O, C2-alkenyl, cycloalkyl.

[0004] Some embodiments include a compound represented by formula (III):whereinA is -(CH2)nX(CH2)nY;X is CH2, O, C3-C4 cycloalkyl, alkenyl, Y is OH, NR’R’;n is and integer of 0, 1 or 2;R’ is C1-C4 alkyl or together form a 4-7-membered ring;Ri and R2are independently a Cs-Cs alkyl optionally substituted by one or more O, C2- alkenyl, cycloalkyl;Li and L2are independently selected from O, -C(=O)O-, -OC(=O)-; -NR”C(=O)O-; - C(=O)OZ-;Ls and L4 are independently a bond or -C(=O)-;R4 and R5 are optionally substituted Cs-Cis alkyl where one or more carbon may be substituted by an O, wherein at least one of is an optionally substituted C14-C18 alkyl where one or more carbon may be substituted by one or more O, C2-alkenyl, cycloalkyl.

[0005] Some embodiments include a compound represented by formula (IV):whereinRais an optionally substituted alkyl;Rxis a C2-C8 alkyl optionally substituted by one or more O, C2-alkenyl, cycloalkylRi and R2 are independently a Cs-Cs alkyl optionally substituted by one or more O, C2- alkenyl, cycloalkyl;Li and L2 are independently selected from -C(=O)O-, -OC(=O)-; -NR”C(=O)O-; - C(=O)O-; a moiety of formula (A) or a moiety of formula (B);Xi and X2 are a substituted C14-C18 alkyl where one or more carbon may be substituted by one or more O, C2-alkenyl, cycloalkyl.

[0006] In some embodiments, X is O. In some embodiments, X is cycloalkyl, such as a C3, C4,C5, or Ce cycloalkyl. In some embodiments, A is -(CH2)nX’, and X’ isR' . In some embodiments, Ri and R2 are independently a Cs-Cs alkyl. In some embodiments, Ri and R2 are independently a Cs-Cs alkyl substituted by one or more O, C2-alkenyl, cycloalkyl. In some embodiments, Li and L2 are a moiety of formula (A):whereinR3 is independently H, halo, or OR’”;R’” is in each instance independently H or an optionally substituted C1-3 alkyl;Z is O, S or C(R”’)2. In some embodiments, Xi and X2 are independently an optionally substituted linear Cs-Cu and / or branched C12-C24 alkyl. In some embodiments, Xi and X2 comprise at least one O or cycloalkyl. In some embodiments, Xi and X2 comprise atthe compound is selected from the compounds of Table 1. Some embodiments include a lipid nanoparticle comprising a compound of any of the preceding embodiments.BRIEF DESCRIPTION OF THE DRAWING

[0007] FIGURE 1 shows a bar graph depicting red firefly luciferase (rFF) expression encoded by small activating (sa) RNA in BHK-21 cells transduced with increasing doses of experimental lipid nanoparticles (LNP) used to formulate LNP-saRNA-rFF compounds. RLUlog = log of relative light units.DETAILED DESCRIPTION OF THE INVENTION

[0008] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in itsentirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

[0009] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set in the specification. All patents, published patent applications, and publications cited herein are incorporated by reference as if set forth fully herein.

[0010] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0011] Unless otherwise stated, any numerical value herein to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, a dosage of 10 mg includes 9 mg to 11 mg. As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

[0012] As used herein, the conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or.”

[0013] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention.

[0014] As used herein, unless otherwise noted, the term “alkyl” refers to a straight- or branched-chain alkyl group having from 1 to 26 carbon atoms in the chain. Examples of alkyl groups include methyl, ethyl (Et), n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl (tBu), pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, and groups that in light of the ordinary skill in the art and the teachings provided herein would be considered equivalent to any one of the foregoing examples. As used herein, alkyl may include one or more cycloalkyl moiety.

[0015] As used herein, unless otherwise noted, the term “Cx-ycycloalkyl”, wherein X and Y are integers, shall mean any stable X- to Y-membered monocyclic, bicyclic, polycyclic, bridged, or spiro-cyclic saturated ring system, preferably a monocyclic, bicyclic, bridged or spiro-cyclic saturated ring system. For example, “C3-7cycloalkyl” refers to a cycloalkyl having from 3 to 7 carbon atoms in the ring(s), “Cs-ecycloalkyl” refers to a cycloalkyl having from 3 to 6 carbon atoms in the ring, “C3-4cycloalkyl” refers to a cycloalkyl having from 3 to 4 carbon atoms in the ring. Illustrative examples of cycloalkyl groups include the following entities, in the form of properly bonded

[0016] As used herein, the term “heteroaryl” refers to an aromatic monocyclic or multicyclic ring system comprising 5 to 14 ring atoms, wherein from 1 to 4 of the ring atoms is independently O, N or S and the remaining ring atoms are carbon atoms. In one embodiment, a heteroaryl group has 5 to 10 ring atoms. In another embodiment, a heteroaryl group is monocyclic and has 5 or 6 ring atoms. In another embodiment, a heteroaryl group is multicyclic and has 6 or 14 ring atoms and at least one nitrogen ring atom. A heteroaryl group is joined via a ring carbon atom and any nitrogen atom of a heteroaryl can be optionally oxidized to the corresponding N-oxide.

[0017] Those skilled in the art will recognize that if more than one such substituent is present for a given ring, the bonding of each substituent is independent of all of the others. The groups listed or illustrated above are not exhaustive.

[0018] As used herein, the term “aryl” refers to a Ce-i4 monocyclic or polycyclic aromatic group, preferably a Ce-io monocyclic or bicyclic aromatic group, or Cio-14 polycyclic aromaticgroup. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, pyrenyl, biphenyl, and terphenyl.

[0019] As used herein, the term “alkenyl” refers to linear or branched radicals having at least one carbon-carbon double bond of two to about six carbon atoms. Most preferred alkenyl radicals are radicals having two to about four carbon atoms. Examples of alkenyl radicals include ethenyl, propenyl, allyl, propenyl, butenyl and 4- methylbutenyl. The terms “alkenyl” and “lower alkenyl”, embrace radicals having “cis” and “trans” orientations, or alternatively, “E” and “Z” orientations. In some embodiments, other moieties, such as alkyl or cycloalkyl, will include one or more alkenyl moiety within the carbon chain.

[0020] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having.”

[0021] When used herein “consisting of’ excludes any element, step, or ingredient not specified in the claim element. When used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the aforementioned terms of “comprising,” “containing,” “including,” and “having,” whenever used herein in the context of an aspect or embodiment of the invention can be replaced with the term “consisting of’ or “consisting essentially of’ to vary scopes of the disclosure.

[0022] As used herein, “subject” means any animal, preferably a mammal, most preferably a human, to whom will be or has been treated by a method according to an embodiment of the application. The term “mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) such as monkeys or apes, humans, etc., more preferably a human. A human subject can include a patient.

[0023] In an attempt to help the reader of the application, the description has been separated in various paragraphs or sections or is directed to various embodiments of the application. Theseseparations should not be considered as disconnecting the substance of a paragraph or section or embodiments from the substance of another paragraph or section or embodiments. To the contrary, one skilled in the art will understand that the description has broad application and encompasses all the combinations of the various sections, paragraphs and sentences that can be contemplated. The discussion of any embodiment is meant only to be exemplary and is not intended to suggest that the scope of the disclosure, including the claims, is limited to these examples. The application contemplates use of any of the applicable components in any combination having any sequence that can be used in ribonucleic acid molecules of the application, whether or not a particular combination is expressly described.

[0024] In a general aspect, the application provides a cationic lipid suitable for use in a lipid particle that includes, e.g., a neutral lipid, a lipid capable of reducing aggregation, a cationic lipid, and optionally a sterol. In certain embodiments, the lipid particle further includes an active agent. Various exemplary embodiments of these cationic lipids, lipid particles and compositions comprising the same, and their use to deliver therapeutic agents and modulate gene and protein expression are described in further detail below.Cationic Ionizable Lipids

[0025] In a general aspect, the application provides a cationic ionizable lipid, such as a compound represented by compound represented by formula (I):whereinA is -(CH2)nX(CH2)nY;X is O, C3-C6 cycloalkyl, alkenyl, or aromatic;Y is OH, NR’R’;n is and integer of 0, 1 or 2;R’ is C1-C4 alkyl or together form a 4-7-membered ring;Ri and R2are independently a Cs-Cs alkyl optionally substituted by one or more O, C2- alkenyl, cycloalkyl;Li and L2 are independently selected from -C(=O)O-, -OC(=O)-; -NR”C(=O)O-; - C(=O)OZ-; a moiety of formula (A) or a moiety of formula (B);Xi and X2 are optionally substituted Cs-Cis alkyl where one or more carbon may be substituted by one or more O, C2-alkenyl, cycloalkyl.

[0026] Other embodiments include a compound represented by formula (II):whereinA is -(CH2)nX(CH2)nY;X is CH2, O, C3-C4 cycloalkyl, alkenyl,Y is OH, NR’R’;n is and integer of 0, 1 or 2;R’ is C1-C4 alkyl or together form a 4-7-membered ring;Ri and R2 are independently a Cs-Cs alkyl optionally substituted by one or more O, C2- alkenyl, cycloalkyl;Li and L2 are independently selected from -C(=O)O-, -OC(=O)-; -NR”C(=O)O-; - C(=O)O-; a moiety of formula (A) or a moiety of formula (B);Xi and X2 are a substituted Cs-Cis alkyl where one or more carbon may be substituted by one or more O, C2-alkenyl, cycloalkyl.

[0027] Other embodiments include a compound represented by formula (III):R)LIR3A R2 (III), whereinA is -(CH2)nX(CH2)nY;X is CH2, O, C3-C4 cycloalkyl, alkenyl, Y is OH, NR’R’;n is and integer of 0, 1 or 2;R’ is C1-C4 alkyl or together form a 4-7-membered ring;Ri and R2 are independently a Cs-Cs alkyl optionally substituted by one or more O, C2- alkenyl, cycloalkyl;Li and L2 are independently selected from O, -C(=O)O-, -OC(=O)-; -NR”C(=O)O-; -L3 and L4 are independently a bond or -C(=O)-;R4 and R5 are optionally substituted Cs-Cis alkyl where one or more carbon may be substituted by an O, wherein at least one of is an optionally substituted C14-C18 alkyl where one or more carbon may be substituted by one or more O, C2-alkenyl, cycloalkyl.

[0028] Other embodiments include a compound represented by formula (IV):whereinRais an optionally substituted alkyl;Rxis a C2-C8 alkyl optionally substituted by one or more O, C2-alkenyl, cycloalkylRi and R2 are independently a Cs-Cs alkyl optionally substituted by one or more O, C2- alkenyl, cycloalkyl;Li and L2 are independently selected from -C(=O)O-, -OC(=O)-; -NR”C(=O)O-; - C(=O)O-; a moiety of formula (A);Xi and X2 are a substituted C14-C18 alkyl where one or more carbon may be substituted by one or more O, C2-alkenyl, cycloalkyl.

[0029] In some embodiments, Formula (IV) is symmetrical, e.g., eachis identical. In other embodiments, Formula (IV) is not symmetrical, e.g., eachisindependently selected from Formula (IV).

[0030] In some embodiments, X is O. In other embodiments, X is cycloalkyl, such as a C3, C4, C5, or Ce cycloalkyl. In some embodiments, X is a C2 alkenyl. IN other embodiments, X is an aromatic moiety, such as an aryl (e.g., a phenyl), or a heteroaryl (e.g., a monocyclic heteroaryl).

[0031] In some embodiments, Y is OH, OR’ or NR’R’. Examples of Y include OMe, OH,

[0033] In some embodiments, Ri and R2 are independently a Cs-Cs alkyl. In other embodiments, Ri and R2 are independently a Cs-Cs alkyl substituted by one or more O, C2- alkenyl, cycloalkyl. The cycloalkyl can form a ring between Ri and R2, for example, with the N that Ri and R2 are bound to. Examples include:

[0034] In other embodiments, Ri and R2 are independently a Cs-Cs alkyl that together form a ringsome embodiments, Ri and R2 are substituted by one or more O, C2-alkenyl, cycloalkyl.

[0035] In some embodiments, the moiety of formula (A) is represented by:whereinR3 is independently H, halo, or OR’”;R’” is in each instance independently H or an optionally substituted C1-3 alkyl;Z is O, S or C(R’”)2.

[0036] In some embodiments, the moiety of formula (A) is represented by:

[0037] In some embodiments, Xi and X2 are independently optionally substituted C8-C24 alkyl. For example, each may be selected from a Cs, C9, C10, Cn, C12, C13, C14, C15, Ci6, C17, Cis, C19, C20, C21, C22, C23 or C24 alkyl, which may be optionally substituted. In some embodiments, Xi and X2 are independently an optionally substituted linear Cs-Ci4 and / or branched C12-C24 alkyl.In some embodiments, the optional substitution is one or more O, C2-alkenyl, cycloalkyl.Examples of Xi and X2 include:

[0038] Additional embodiments of the present disclosure include the compounds of Table 1.Table 1Compositions

[0039] The application also relates to compositions and pharmaceutical compositions comprising one or more polynucleotide. The application provides, for example, a pharmaceutical composition comprising any nucleic acid molecule and / or vector described herein, together with a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is non-toxic and should not interfere with the efficacy of the active ingredient. Pharmaceutically acceptable carriers can include one or more excipients such as binders, disintegrants, swelling agents, suspending agents, emulsifying agents, wetting agents, lubricants, flavorants, sweeteners, preservatives, dyes, solubilizers and coatings. The precise nature of the carrier or other material can depend on the route of administration, e.g., intramuscular, intradermal, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., gut), intranasal or intraperitoneal routes. For liquid injectable preparations, for example, suspensions and solutions, suitable carriers and additives include water, glycols, oils, alcohols, preservatives, coloring agents and the like. For solid oral preparations, for example, powders, capsules, caplets, gelcaps and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. For nasal sprays / inhalant mixtures, the aqueous solution / suspension cancomprise water, glycols, oils, emollients, stabilizers, wetting agents, preservatives, aromatics, flavors, and the like as suitable carriers and additives.

[0040] Pharmaceutical compositions of the application can be formulated in any matter suitable for administration to a subject to facilitate administration and improve efficacy, including, but not limited to, oral (enteral) administration and parenteral injections. The parenteral injections include intravenous injection or infusion, subcutaneous injection, intradermal injection, and intramuscular injection. Pharmaceutical compositions of the application can also be formulated for other routes of administration including transmucosal, ocular, rectal, long acting implantation, sublingual administration, under the tongue, from oral mucosa bypassing the portal circulation, inhalation, or intranasal.

[0041] In a preferred embodiment of the application, pharmaceutical compositions of the application are formulated for parental injection, preferably subcutaneous, intradermal injection, or intramuscular injection, more preferably intramuscular injection.

[0042] According to embodiments of the application, pharmaceutical compositions for administration will typically comprise a buffered solution in a pharmaceutically acceptable carrier, e.g., an aqueous carrier such as buffered saline and the like, e.g., phosphate buffered saline (PBS). The compositions can also contain pharmaceutically acceptable substances as required to approximate physiological conditions such as pH adjusting and buffering agents. For example, a pharmaceutical composition of the application comprising a ribonucleic acid molecule can contain phosphate buffered saline (PBS) as the pharmaceutically acceptable carrier. The ribonucleic acid molecule can be administered at 1-1000 pg / dose, e.g., 1 pg / dose, 10 pg / dose, 20 pg / dose, 30 pg / dose, 40 pg / dose, 50 pg / dose, 60 pg / dose, 70 pg / dose, 80 pg / dose, 90 pg / dose, 100 pg / dose, 200 pg / dose, 300 pg / dose, 400 pg / dose, 500 pg / dose, 600 pg / dose, 700 pg / dose, 800 pg / dose, 900 pg / dose, 1000 pg / dose, or any number in between.Pharmaceutically Acceptable LNP Carrier

[0043] In another general aspect, the application relates to a pharmaceutical composition comprising an active compound and a cationic ionizable lipid of the application in encapsulating carrier, such as a lipid nanoparticle (LNP) encapsulating an active compound.

[0044] In some embodiments, the RNA or combination of nucleic acid molecules is fully encapsulated within the lipid portion of the lipid formulation such that the nucleic acid or combination of nucleic acid molecules in the lipid formulation is resistant in aqueous solution tonuclease degradation. The term “fully encapsulated” means that the nucleic acid (in the nucleic acid-lipid particle is not significantly degraded after exposure to serum or a nuclease assay that would significantly degrade free nucleic acid. When fully encapsulated, preferably less than 25% of the nucleic acid in the particle is degraded in a treatment that would normally degrade 100% of free nucleic acid, more preferably less than 10%, and most preferably less than 5% of the nucleic acid in the particle is degraded. “Fully encapsulated” as used herein also means that the nucleic acid-lipid particles do not rapidly decompose into their component parts upon in vivo administration. In other embodiments, the lipid formulations described herein are substantially non-toxic to mammals such as humans. In some embodiments, the combination of nucleic acids is encapsulated within the same lipid nanoparticle. In some embodiments, each nucleic acid molecule in the combination of nucleic acid molecules is independently encapsulated in individual lipid nanoparticles.

[0045] The lipid formulations of the present disclosure typically have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, or about 150 nm, and are substantially non-toxic. The diameter may be any value or subvalue within the recited ranges, including endpoints. In addition, nucleic acids, when present in the lipid nanoparticles of the present disclosure, are resistant in aqueous solution to degradation with a nuclease.

[0046] In preferred embodiments, the lipid formulations comprise a nucleic acid molecule, a cationic ionizable lipid of the application (e.g., one or more cationic lipids or salts thereof described herein), a phospholipid, and a conjugated lipid that inhibits aggregation of the particles (e.g., one or more PEG-lipid conjugates). The lipid formulations can also include cholesterol. The term “lipid conjugate” means a conjugated lipid that inhibits aggregation of lipid particles. Such lipid conjugates include, but are not limited to, PEG-lipid conjugates such as, e.g., PEG coupled to dialkyloxypropyls (e.g., PEG-DAA conjugates), PEG coupled to diacylglycerols (e.g.,PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, and PEG conjugated to ceramides, cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates, polyamide oligomers, and mixtures thereof. PEG or POZ can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG or the POZ to a lipid can be used including, e.g., non-ester-containing linker moieties and ester-containing linker moieties. In certain preferred embodiments, non-ester-containing linker moieties, such as amides or carbamates, are used. In certain preferred embodiments, the PEG- lipid conjugate is 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (i.e., ALC-0159).

[0047] The term “anionic lipid” as used herein refers to a lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N- glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[0048] In the nucleic acid-lipid formulations, the nucleic acid molecules may be fully encapsulated within the lipid portion of the formulation, thereby protecting the nucleic acid from nuclease degradation. In preferred embodiments, a lipid formulation comprising an RNA or combination of nucleic acid molecules is fully encapsulated within the lipid portion of the lipid formulation, thereby protecting the nucleic acid from nuclease degradation. In certain instances, the RNA or combination of nucleic acid molecules in the lipid formulation is not substantially degraded after exposure of the particle to a nuclease at 37 °C for at least 20, 30, 45, or 60 minutes. In certain other instances, the RNA or combination of nucleic acid molecules in the lipid formulation is not substantially degraded after incubation of the formulation in serum at 37 °C for at least 30, 45, or 60 minutes or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments, the RNA or combination of nucleic acid molecules is complexed with the lipid portion of the formulation.

[0049] In the context of nucleic acids, full encapsulation may be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that has enhanced fluorescence when associated with a nucleic acid. Encapsulation is determined by adding the dye to a lipid formulation, measuring the resulting fluorescence, and comparing it to the fluorescenceobserved upon addition of a small amount of nonionic detergent. Detergent-mediated disruption of the lipid layer releases the encapsulated nucleic acid, allowing it to interact with the membrane-impermeable dye. Nucleic acid encapsulation may be calculated as E = (Io - I) / Io, where I and Io refer to the fluorescence intensities before and after the addition of detergent.

[0050] Preferably, the lipid nanoparticle encapsulating nucleic acid molecules comprises a cationic ionizable lipid of the present disclosure and at least one other lipid selected from the group consisting of anionic lipids, zwitterionic lipids, neutral lipids, steroids, polymer conjugated lipids, phospholipids, glycolipids, and combinations thereof.

[0051] In some embodiments, the lipid nanoparticle formulation consists of (i) at least one cationic ionizable lipid of the present disclosure; (ii) a helper lipid; (iii) a sterol (e.g. , cholesterol); and (iv) a PEG-lipid, in a molar ratio of about 30% to about 60% cationic ionizable lipid: about 5% to about 20% helper lipid: about 35% to about 50% sterol: about 0.5-5% PEG- lipid.

[0052] The selection of specific lipids and their relative % compositions depends on several factors including the desired therapeutic effect, the intended in vivo delivery target, and the planned dosing regimen and frequency. Generally, lipids that correspond to both high potency (i.e., therapeutic effect such as knockdown activity or translation efficiency) and biodegradability resulting in rapid tissue clearance are most preferred. However, biodegradability may be less important for formulations that are intended for only one or two administrations within the subject. In addition, the lipid composition may require careful engineering so that the lipid formulation preserves its morphology during in vivo administration and its journey to the intended target, but will then be able to release the active agent upon uptake into target cells. Thus, several formulations typically need to be evaluated in order to find the best possible combination of lipids in the best possible molar ratio of lipids as well as the ratio of total lipid to active ingredient.Helper Lipids and Sterols

[0053] The lipid formulations of the present disclosure can comprise a helper lipid, which can be referred to as a neutral lipid, a neutral helper lipid, non-cationic lipid, non-cationic helper lipid, anionic lipid, anionic helper lipid, or a zwitterionic lipid. It has been found that lipid formulations, particularly cationic liposomes and lipid nanoparticles have increased cellular uptake if helper lipids are present in the formulation. (Curr. Drug Metab. 2014; 15(9): 882-92).For example, some studies have indicated that neutral and zwitterionic lipids such as 1,2- dioleoyl-sn-glycero-3 -phosphatidylcholine (DOPC), Di-Oleoyl -Phosphatidyl-Ethanoalamine (DOPE) and l,2-DiStearoyl-sn-glycero-3-PhosphoCholine (DSPC), being more fusogenic (i.e., facilitating fusion) than cationic lipids, can affect the polymorphic features of lipid-nucleic acid complexes, promoting the transition from a lamellar to a hexagonal phase, and thus inducing fusion and a disruption of the cellular membrane. (Nanomedicine (Lond). 2014 Jan; 9(1): 105- 20). In addition, the use of helper lipids can help to reduce any potential detrimental effects from using many prevalent cationic lipids such as toxicity and immunogenicity.

[0054] Non-limiting examples of non-cationic lipids suitable for lipid formulations of the present disclosure include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), di oleoylphosphatidyl ethanolamine (DOPE), palmitoyl oleoyl -phosphatidylcholine (POPC), palmitoyloleoyl -phosphatidylethanolamine (POPE), palmitoyloleyol -phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl -phosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoyl -phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having Cio- C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0055] Additional examples of non-cationic lipids include sterols such as cholesterol and derivatives thereof. One study concluded that as a helper lipid, cholesterol increases the spacing of the charges of the lipid layer interfacing with the nucleic acid making the charge distribution match that of the nucleic acid more closely. (J. R. Soc. Interface. 2012 Mar 7; 9(68): 548-561). Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-cholestanol,5a-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'- hydroxy)-butyl ether, and 6- ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5a- cholestanone, and cholesteryl decanoate; and mixtures thereof. In preferred embodiments, the cholesterol derivative is a polar analogue such as cholesteryl-(4'-hydroxy)-butyl ether.

[0056] In some embodiments, the helper lipid present in the lipid formulation comprises or consists of a mixture of one or more phospholipids and cholesterol or a derivative thereof. In other embodiments, the helper lipid present in the lipid formulation comprises or consists of one or more phospholipids, e.g., a cholesterol-free lipid formulation. In yet other embodiments, the helper lipid present in the lipid formulation comprises or consists of cholesterol or a derivative thereof, e.g., a phospholipid-free lipid formulation.

[0057] Other examples of helper lipids include nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkylaryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, and sphingomyelin.

[0058] In some embodiments, the helper lipid comprises from about 30 mol% to about 60 mol%, from about 32 mol% to about 58 mol%, from about 34 mol% to about 56 mol%, about 35 mol% to about 54 mol%, from about 36 mol% to about 52 mol%, from about 37 mol% to about 51 mol%, from about 38 mol% to about 50 mol%, or about 39 mol%, about 50 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, or about 49 mol% (or any fraction thereof or the range therein) of the total lipid present in the lipid formulation.

[0059] In some embodiments, the total of helper lipid in the formulation comprises two or more helper lipids and the total amount of helper lipid comprises from about 30 mol% to about 60 mol%, from about 32 mol% to about 58 mol%, from about 34 mol% to about 56 mol%, about 35 mol% to about 54 mol%, from about 36 mol% to about 52 mol%, from about 37 mol% to about 51 mol%, from about 38 mol% to about 50 mol%, or about 39 mol%, about 50 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, or about 49 mol% (or any fraction thereof or the range therein) of the total lipid present in the lipid formulation. In some embodiments, the helper lipids are acombination of DSPC and DOTAP. In some embodiments, the helper lipids are a combination of DSPC and DOTMA.

[0060] The cholesterol or cholesterol derivative in the lipid formulation may comprise up to about 50 mol%, about 35 mol%, about 40 mol%, about 45 mol%, or about 50 mol% of the total lipid present in the lipid formulation. In some embodiments, the cholesterol or cholesterol derivative comprises about 15 mol% to about 45 mol%, about 20 mol% to about 45 mol%, about 30 mol% to about 45 mol%, or about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, or about 45 mol% of the total lipid present in the lipid formulation.

[0061] The percentage of helper lipid present in the lipid formulation is a target amount, and the actual amount of helper lipid present in the formulation may vary, for example, by ± 5 mol%.

[0062] A lipid formulation containing a cationic lipid compound or ionizable cationic lipid compound may be on a molar basis about 30-60% cationic lipid compound, about 35-50 % cholesterol, about 5-20% helper lipid, and about 0.5-5% of a polyethylene glycol (PEG) lipid, wherein the percent is of the total lipid present in the formulation. In some embodiments, the composition is about 40-50% cationic lipid compound, about 35-45% cholesterol, about 5-15% helper lipid, and about 0.5-3% of a PEG-lipid, wherein the percent is of the total lipid present in the formulation.Lipid Conjugates

[0063] The lipid formulations described herein may further comprise a lipid conjugate. The conjugated lipid is useful for preventing the aggregation of particles. Suitable conjugated lipids include, but are not limited to, PEG-lipid conjugates, cationic-polymer-lipid conjugates, and mixtures thereof. Furthermore, lipid delivery vehicles can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to its surface or to the terminal end of the attached PEG chains (Front. Pharmacol. 2015 Dec 1; 6:286).

[0064] In a preferred embodiment, the lipid conjugate is a PEG-lipid. The inclusion of polyethylene glycol (PEG) in a lipid formulation as a coating or surface ligand, a technique referred to as PEGylation, helps protect nanoparticles from the immune system and their escape from RES uptake (Nanomedicine (Lond). 2011 Jun; 6(4): 715 -28). PEGylation has been widely used to stabilize lipid formulations and their payloads through physical, chemical, and biological mechanisms. Detergent-like PEG lipids (e.g., PEG-DSPE) can enter the lipid formulation to forma hydrated layer and steric barrier on the surface. Based on the degree of PEGylation, the surface layer can be generally divided into two types, brush-like and mushroom-like layers. For PEG- DSPE-stabilized formulations, PEG will take on the mushroom conformation at a low degree of PEGylation (usually less than 5 mol%) and will shift to brush conformation as the content of PEG-DSPE is increased past a certain level (J. Nanomaterials. 2011 ;2011 : 12). It has been shown that increased PEGylation leads to a significant increase in the circulation half-life of lipid formulations (Annu. Rev. Biomed. Eng. 2011 Aug 15; 13:507-30; J. Control Release. 2010 Aug 3; 145(3):178-81).

[0065] Suitable examples of PEG-lipids include, but are not limited to, PEG coupled to dialkyloxypropyls (PEG-DAA), PEG coupled to di acylglycerol (PEG-DAG), PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides, PEG conjugated to cholesterol or a derivative thereof, and mixtures thereof.

[0066] PEG is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified by their molecular weights and include the following: monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycolsuccinate (MePEG-S), monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S- NHS), monomethoxypolyethylene glycol-amine (MePEG-NFE), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM), as well as such compounds containing a terminal hydroxyl group instead of a terminal methoxy group (e g., HO-PEG-S, HO-PEG-S-NHS, HO-PEG-NH2).

[0067] The PEG moiety of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain instances, the PEG moiety has an average molecular weight of from about 750 daltons to about 5,000 daltons (e.g., from about 1,000 daltons to about 5,000 daltons, from about 1,500 daltons to about 3,000 daltons, from about 750 daltons to about 3,000 daltons, from about 750 daltons to about 2,000 daltons). In preferred embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons or about 750 daltons. The average molecular weight may be any value or subvalue within the recited ranges, including endpoints.

[0068] In certain instances, the PEG monomers can be optionally substituted by an alkyl, alkoxy, acyl, or aryl group. The PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG to a lipid can beused including, e.g., non-ester-containing linker moieties and ester-containing linker moieties. In a preferred embodiment, the linker moiety is a non-ester-containing linker moiety. Suitable non- ester-containing linker moieties include, but are not limited to, amido (-C(O)NH-), amino (-NR- ), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-S-S-), ether (-0- ), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), ether, as well as combinations thereof (such as a linker containing both a carbamate linker moiety and an amido linker moiety). In a preferred embodiment, a carbamate linker is used to couple the PEG to the lipid.

[0069] In other embodiments, an ester-containing linker moiety is used to couple the PEG to the lipid. Suitable ester-containing linker moieties include, e.g., carbonate (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonate esters, and combinations thereof.

[0070] Phosphatidylethanolamines having a variety of acyl chain groups of varying chain lengths and degrees of saturation can be conjugated to PEG to form the lipid conjugate. Such phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those of skill in the art. Phosphatidylethanolamines containing saturated or unsaturated fatty acids with carbon chain lengths in the range of Cio to C20 are preferred. Phosphatidylethanolamines with mono- or di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl- phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), di oleoyl -phosphatidylethanolamine (DOPE), and distearoylphosphatidylethanolamine (DSPE).

[0071] In some embodiments, the PEG-DAA conjugate is a PEG-di decyl oxypropyl (Cio) conjugate, a PEG-dilauryloxypropyl (C12) conjugate, a PEG-dimyristyloxypropyl (C14) conjugate, a PEG-dipalmityloxypropyl (Cie) conjugate, or a PEG-di stearyloxypropyl (Cis) conjugate. In these embodiments, the PEG preferably has an average molecular weight of about 750 to about 2,000 daltons. In particular embodiments, the terminal hydroxyl group of the PEG is substituted with a methyl group.

[0072] In addition to the foregoing, other hydrophilic polymers can be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl,methacrylamide, polymethacrylamide, and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0073] In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprises from about 0.1 mol% to about 2 mol%, from about 0.5 mol% to about 2 mol%, from about 1 mol% to about 2 mol%, from about 0.6 mol% to about 1.9 mol%, from about 0.7 mol% to about 1.8 mol%, from about 0.8 mol% to about 1.7 mol%, from about 0.9 mol% to about 1.6 mol%, from about 0.9 mol% to about 1.8 mol%, from about 1 mol% to about 1.8 mol%, from about 1 mol% to about 1.7 mol%, from about 1.2 mol% to about 1.8 mol%, from about 1.2 mol% to about 1.7 mol%, from about 1.3 mol% to about 1.6 mol%, or from about 1.4 mol% to about 1.6 mol% (or any fraction thereof or range therein) of the total lipid present in the lipid formulation. In other embodiments, the lipid conjugate (e.g., PEG-lipid) comprises about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or 5%, (or any fraction thereof or range therein) of the total lipid present in the lipid formulation. The amount may be any value or subvalue within the recited ranges, including endpoints.

[0074] In some embodiments, the PEG-lipid is PEG550-PE. In some embodiments, the PEG- lipid is PEG750-PE. In some embodiments, the PEG-lipid is PEG2000-DMG. In some preferred embodiments, the PEG-lipid is 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (also known as ALC-0159).

[0075] The percentage of lipid conjugate (e.g., PEG-lipid) present in the lipid formulations of the disclosure is a target amount, and the actual amount of lipid conjugate present in the formulation may vary, for example, by ± 0.5 mol%. One of ordinary skill in the art will appreciate that the concentration of the lipid conjugate can be varied depending on the lipid conjugate employed and the rate at which the lipid formulation is to become fusogenic.Lipid Formulation Manufacture

[0076] There are many different methods for the preparation of lipid formulations comprising a nucleic acid, e.g. RNA or combination of nucleic acid molecules. (Curr. Drug Metabol. 2014, 15, 882-892; Chem. Phys. Lipids 2014, 177, 8-18; Int. J. Pharm. Stud. Res. 2012, 3, 14-20). The techniques of thin film hydration, double emulsion, reverse phase evaporation, microfluidic preparation, dual asymmetric centrifugation, ethanol injection, detergent dialysis, spontaneousvesicle formation by ethanol dilution, and encapsulation in preformed liposomes are briefly described herein.Thin Film Hydration

[0077] In Thin Film Hydration (TFH) or the Bangham method, the lipids are dissolved in an organic solvent, then evaporated through the use of a rotary evaporator leading to a thin lipid layer formation. After the layer hydration by an aqueous buffer solution containing the compound to be loaded, Multilamellar Vesicles (MLVs) are formed, which can be reduced in size to produce Small or Large Unilamellar vesicles (LUV and SUV) by extrusion through membranes or by the sonication of the starting MLV.Double Emulsion

[0078] Lipid formulations can also be prepared through the Double Emulsion technique, which involves lipids dissolution in a water / organic solvent mixture. The organic solution, containing water droplets, is mixed with an excess of aqueous medium, leading to a water-in-oil-in-water (W / O / W) double emulsion formation. After mechanical vigorous shaking, part of the water droplets collapse, giving Large Unilamellar Vesicles (LUVs).Reverse Phase Evaporation

[0079] The Reverse Phase Evaporation (REV) method also allows one to achieve LUVs loaded with nucleic acid. In this technique a two-phase system is formed by phospholipids dissolution in organic solvents and aqueous buffer. The resulting suspension is then sonicated briefly until the mixture becomes a clear one-phase dispersion. The lipid formulation is achieved after the organic solvent evaporation under reduced pressure. This technique has been used to encapsulate different large and small hydrophilic molecules including nucleic acids.Microfluidic Preparation

[0080] The Microfluidic method, unlike other bulk techniques, gives the possibility of controlling the lipid hydration process. The method can be classified in continuous-flow microfluidic and droplet-based microfluidic, according to the way in which the flow is manipulated. In the microfluidic hydrodynamic focusing (MHF) method, which operates in a continuous flow mode, lipids are dissolved in isopropyl alcohol which is hydrodynamically focused in a microchannel cross junction between two aqueous buffer streams. Vesicles size can be controlled by modulating the flow rates, thus controlling the lipids solution / buffer dilutionprocess. The method can be used for producing oligonucleotide (ON) lipid formulations by using a microfluidic device consisting of three-inlet and one-outlet ports.Dual Asymmetric Centrifugation

[0081] Dual Asymmetric Centrifugation (DAC) differs from more common centrifugation as it uses an additional rotation around its own vertical axis. An efficient homogenization is achieved due to the two overlaying movements generated: the sample is pushed outwards, as in a normal centrifuge, and then it is pushed towards the center of the vial due to the additional rotation. By mixing lipids and an NaCl-solution a viscous vesicular phospholipid gel (VPC) is achieved, which is then diluted to obtain a lipid formulation dispersion. The lipid formulation size can be regulated by optimizing DAC speed, lipid concentration and homogenization time.Ethanol Injection

[0082] The Ethanol Injection (El) method can be used for nucleic acid encapsulation. This method provides the rapid injection of an ethanolic solution, in which lipids are dissolved, into an aqueous medium containing nucleic acids to be encapsulated, through the use of a needle. Vesicles are spontaneously formed when the phospholipids are dispersed throughout the medium.Detergent Dialysis

[0083] The Detergent dialysis method can be used to encapsulate nucleic acids. Briefly lipid and plasmid are solubilized in a detergent solution of appropriate ionic strength, after removing the detergent by dialysis, a stabilized lipid formulation is formed. Unencapsulated nucleic acid is then removed by ion-exchange chromatography and empty vesicles by sucrose density gradient centrifugation. The technique is highly sensitive to the cationic lipid content and to the salt concentration of the dialysis buffer, and the method is also difficult to scale.Spontaneous Vesicle Formation by Ethanol Dilution

[0084] Stable lipid formulations can also be produced through the Spontaneous Vesicle Formation by Ethanol Dilution method in which a stepwise or dropwise ethanol dilution provides the instantaneous formation of vesicles loaded with nucleic acid by the controlled addition of lipid dissolved in ethanol to a rapidly mixing aqueous buffer containing the nucleic acid.Methods of Delivery

[0085] Pharmaceutical compositions of the application can be administered to a subject by any method known in the art in view of the present disclosure, including, but not limited to, parenteral administration (e.g., intramuscular, subcutaneous, intravenous, or intradermal injection), oral administration, transdermal administration, and nasal administration. Preferably, pharmaceutical compositions are administered parenterally (e.g., by intramuscular injection or intradermal injection).

[0086] In some embodiments of the application in which a pharmaceutical composition comprises one or more ribonucleic acid molecules, administration can be by injection through the skin, e.g., intramuscular or intradermal injection, preferably intramuscular injection. Intramuscular injection can be combined with electroporation, i.e., application of an electric field to facilitate delivery of the ribonucleic acid molecules to cells. As used herein, the term “electroporation” refers to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane. During in vivo electroporation, electrical fields of appropriate magnitude and duration are applied to cells, inducing a transient state of enhanced cell membrane permeability, thus enabling the cellular uptake of molecules unable to cross cell membranes on their own. Creation of such pores by electroporation facilitates passage of biomolecules from one side of a cellular membrane to the other. In vivo electroporation for the delivery of DNA vaccines has been shown to significantly increase plasmid uptake by host cells, while also leading to mild-to-moderate inflammation at the injection site. As a result, transfection efficiency and immune response are significantly improved (e.g., up to 1,000 fold and 100 fold respectively) with intradermal or intramuscular electroporation, in comparison to conventional injection.

[0087] Methods of delivery are not limited to the above described embodiments, and any means for intracellular delivery can be used. Other methods of intracellular delivery contemplated by the methods of the application include, but are not limited to, liposome encapsulation, lipoplexes, nanoparticles, etc. For example, a ribonucleic acid molecule of the application can be formulated in a composition that comprises one or more lipid molecules, preferably positively charged lipid molecules. In some embodiments, a ribonucleic acid molecule of the disclosure can be formulated using one or more liposomes, lipoplexes, and / or lipid nanoparticles. In some embodiments, liposome or lipid nanoparticle formulations described herein can comprise apolycationic composition. In some embodiments, the formulations comprising a polycationic composition can be used for the delivery of the ribonucleic acid molecule described herein in vivo and / or ex vitro.EXAMPLES

[0088] The following examples are offered to illustrate but not to limit the invention(s) of the present disclosure. One of skill in the art will recognize that the following procedures may be modified using methods known to one of ordinary skill in the art.Example 1: Representative synthesis of Compound 1

[0089] Compound 1 was synthesized according to the following scheme 1. In analogous manner compound 3 and 27 were be made.Scheme 167 %)001-2(1 eq.), NaH(1.5 eq), DMF(10ml / g), 0°C - 40°C, 30min) 001-1(44 g, 1.2 eq), Nal(0.2 eq)0°C - 25°C, 1 h48% 38 g45%silica column purification (DCM / MeOH) Compound 1

[0090] Compound 2 was synthesized according to the following scheme 2. In analogous manner compounds 4 and 5 were made.Scheme 265%Silica column purification Silica column purificationSilica column purification used at next step without further purificationSilica column purification Silica column purificationCompound 2

[0091] Compound 8s was synthesized according to the following scheme 3. In analogous manner compounds 7s, 7r, 8sl-8s7, 8r, 18x2, and 22x3 were made.Scheme 345% column purification (DCM / MeOH)column purification (DCM / MeOH) Unstable; used for the next step immediately

[0092] Compound 9 was synthesized according to the following scheme 4. In analogous manner compounds 9x1 -9x4 were made.Scheme 4Used for next step without purificationColumn purification Column purificationColumn purificationCompound 9

[0093] Compound 13b was synthesized according to the following scheme 5. In analogous manner compounds 12a and 12b were made.Scheme 5used for the next step used for the next without purification step without purification60% over two steps silica column purification used for the next step without purificationsilica column purificationSilica column purificationSilica column purification Compound 13b

[0094] Compound 18 was synthesized according to the following scheme 6. In analogous manner compounds 14, 15, 16, and 17 were made.Scheme 646%Prep-HPLCCompound 18

[0095] Compound 22 was synthesized according to the following scheme 7. In analogous manner compounds 6, 20, 21, 23, and 24 were made.Scheme 7Compound 22

[0096] Compound 32 was synthesized according to the following scheme 8. In analogous manner compounds 18x1, 22x1, 22x2, 28, 29, 30, 31, 34x, 35x, and 37x were made.Scheme 8silica column purificationsilica column purificationCompound 32

[0097] Mass spectrometry data was collected for compounds of Table 1 synthesized based on one or more of the above schemes.Example 2: Formulation of LNPs

[0098] Lipid nanoparticles were formulated using cationic lipids described herein. saRNA formulations were prepared on Nanoassembler (microfluidic device from Precision Nanosystems Inc.) by mixing lipids in ethanol with saRNA dissolved in acidic buffer (e.g. acetate, citrate etc.) with pH 3-5 at various concentrations (lOmM to lOOmM). The lipid constituents in the formulation are ionizable lipid:DSPC:cholesterol:DMG-PEG2000 at a molar ratio of , e.g., 50:8.5:40: 1.5 (may vary where needed). The ethanolic solution containing lipids was mixed with the buffer stream containing RNA at a flow rate ratio of 3 : 1 (buffer: ethanol) using microfluidic device with the flow rate of 6-12 mL / min. Ethanol concentration in the lipid / RNA mixture was-25% (v / v). The mixed material was immediately diluted with 1-4 volumes of the process buffer (e.g. 20mM Tris.HCl, 10% sucrose, pH 7.5) followed by the dialysis against the same buffer system using dialysis cassettes (100 KD MWCO) at 4 °C for overnight. Dialyzed formulations were concentrated using AMICON ultra centrifugal filters at 4 °C at 2000-3500 rpm. Lipid nanoparticles size and PDI (polydispersity index) were analyzed by Zetasizer at each step of the process from preparation to concentration step. Concentrated formulation was filtered through 0.2 pm syringe filter and RNA concentration in the formulation was measured by Ribogreen assay (PerkinElmer’s Envision 2105). The concentration was adjusted accordingly with the final buffer followed by the Endotoxin analysis (Charles river’s Nexgen-mcs using Limulus Amebocyte Lysate (LAL) cartridges). The aliquoted formulations were frozen and stored at -80 °C. Prior to final submission, the size, PDI and encapsulation efficiency of the formulations was analyzed to ensure stability.Example 3: Protocol describing the methods used to measure in vitro rFF and GFP expression

[0099] BHK-21 cells were seeded at 25,000 cells / well in 96-well flat-bottom plates. The following day, cells were transduced with increasing doses of experimental LNP used to formulate saRNA encoding either red firefly luciferase (rFF) or green fluorescent protein (GFP), and incubated at 37°C with 5% CO2. After 24 hours, luciferase expression in cells transduced with LNP-saRNA-rFF formulations was measured using the Promega ONE-Glo luciferase detection kit. Cells transduced with LNP-saRNA-GFP formulations were analyzed for GFP fluorescence using Agilent eSIGHT real-time imaging analysis with 2-hour recording intervals. Additionally, GFP expression was assessed by flow cytometry at 24 hours post -transduction to quantify the frequency of GFP-expressing cells. Normalized fold change in rFF expression was determined by dividing the raw luminescence values for experimental LNPs by the raw luminescence values of the average benchmark ALC-315 formulations at a given dose. For normalized GFP expression, area under the curve (AUC) calculations for total integrated GFP intensity served as a surrogate readout of total GFP expression over the imaging period.Normalized fold change in GFP expression was then computed by dividing the AUC values for experimental LNPs by the AUC values of the average benchmark ALC-315 formulations at a given dose. rFF. SaRNA

[0100] Other embodiments are set forth in the following claims.

Claims

ClaimsWe claim:

1. A compound represented by formula (I):whereinA is -(CH2)nX(CH2)nY;X is O, C3-C6 cycloalkyl, alkenyl, or aromatic;Y is OH, NR’R’;n is and integer of 0, 1 or 2;R’ is C1-C4 alkyl or together form a 4-7-membered ring;Ri and R2are independently a Cs-Cs alkyl optionally substituted by one or more O, C2- alkenyl, cycloalkyl;Li and L2are independently selected from -C(=O)O-, -OC(=O)-; -NR”C(=O)O-; - C(=O)OZ-; a moiety of formula (A) or a moiety of formula (B);Xi and X2are optionally substituted Cs-Cis alkyl where one or more carbon may be substituted by one or more O, C2-alkenyl, cycloalkyl.

2. A compound represented by formula (II):(II), whereinA is -(CH2)nX(CH2)nY;X is CH2, O, C3-C4 cycloalkyl, alkenyl,Y is OH, NR’R’;n is and integer of 0, 1 or 2;R’ is C1-C4 alkyl or together form a 4-7-membered ring;Ri and R2are independently a Cs-Cs alkyl optionally substituted by one or more O, C2- alkenyl, cycloalkyl;Li and L2are independently selected from -C(=O)O-, -OC(=O)-; -NR”C(=O)O-; - C(=O)O-; a moiety of formula (A) or a moiety of formula (B);Xi and X2are a substituted Cs-Cis alkyl where one or more carbon may be substituted by one or more O, C2-alkenyl, cycloalkyl.

3. A compound represented by formula (III):whereinA is -(CH2)nX(CH2)nY;X is CH2, O, C3-C4 cycloalkyl, alkenyl, Y is OH, NR’R’;n is and integer of 0, 1 or 2;R’ is C1-C4 alkyl or together form a 4-7-membered ring;Ri and R2are independently a Cs-Cs alkyl optionally substituted by one or more O, C2- alkenyl, cycloalkyl;Li and L2are independently selected from O, -C(=O)O-, -OC(=O)-; -NR”C(=O)O-; - C(=O)OZ-;L3 and L4 are independently a bond or -C(=O)-;R.4 and Rs are optionally substituted Cs-Cis alkyl where one or more carbon may be substituted by an O, wherein at least one of is an optionally substituted C14-C18 alkyl where one or more carbon may be substituted by one or more O, C2-alkenyl, cycloalkyl.

4. A compound represented by formula (IV):whereinRais an optionally substituted alkyl;Rxis a C2-C8 alkyl optionally substituted by one or more O, C2-alkenyl, cycloalkylRi and R2 are independently a Cs-Cs alkyl optionally substituted by one or more O, C2- alkenyl, cycloalkyl;Li and L2 are independently selected from -C(=O)O-, -OC(=O)-; -NR”C(=O)O-; - C(=O)O-; a moiety of formula (A) or a moiety of formula (B);Xi and X2 are a substituted C14-C18 alkyl where one or more carbon may be substituted by one or more O, C2-alkenyl, cycloalkyl.

5. The compound of any of the preceding claims, wherein X is O.

6. The compound of any of the preceding claims, wherein X is cycloalkyl, such as a C3, C4, C5, or Ce cycloalkyl.

7. The compound of any of the preceding claims, wherein A is -(CH2)nX’, and X’ is8. The compound of any of the preceding claims, wherein Ri and R2 are independently a C5-C8alkyl.

9. The compound of any of the preceding claims, wherein Ri and R2 are independently a Cs-Cs alkyl substituted by one or more O, C2-alkenyl, cycloalkyl.

10. The compound of any of the preceding claims, wherein Li and L2 are a moiety of formula (A):whereinRs is independently H, halo, or OR’”;R’” is in each instance independently H or an optionally substituted C1-3 alkyl;Z is O, S or C(R’”)2.

11. The compound of any of the preceding claims, wherein Xi and X2are independently an optionally substituted linear Cs-Ci4 and / or branched Ci2-C24 alkyl.

12. The compound of any of the preceding claims, wherein Xi and X2comprise at least one O or cycloalkyl.

13. The compound of any of the preceding claims, wherein Xi and X2comprise at least14. The compound of any of the preceding claims, wherein the compound is selected from the compounds of Table 1.

15. A lipid nanoparticle comprising a compound of any of the preceding claims.

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