Lipid compositions for nucleic acid delivery

Abstract

The present disclosure provides, among other things, composition and uses of said compositions comprising a cationic lipid, an anionically ionizable lipid, and a nucleic acid, wherein: the cationic lipid comprises a cationic head group, a first bridge group, and a first lipid tail group; the anionically ionizable lipid comprises an anionically ionizable head group, a second bridge group, and a second lipid tail group; the cationic lipid has a lipidic volume from about 700 Å3 to about 1500 Å3, and / or the anionically ionizable lipid has a lipidic volume from about 700 Å3 to about 1500 Å3; or the cationic lipid and the anionically ionizable lipid have a combined lipidic volume greater than 1400 Å3. In some embodiments, the present disclosure provides new cationic lipids and new anionically ionizable lipids.

Description

Attorney Docket No.2013237-1441 Client Ref. No. P1802 LIPID COMPOSITIONS FOR NUCLEIC ACID DELIVERY BACKGROUND

[0001] Nanoparticles, such as those comprising lipids, lipid-like materials, polymers, and the like have been the subject of intense study for the delivery of therapeutic agents, such as nucleic acid agents. Lipid nanoparticles (LNPs), in particular, have been identified as a particular useful vehicle for delivery nucleic acid therapies to cells. See Tenchov, et al., ACS Nano, 2021, 15, 11 16982-17015. SUMMARY

[0002] Nucleic acid therapies face particular challenges when administered to patients. To be effective, the nucleic acid molecule needs to reach the target tissue and produce particular proteins of interest. Nucleic acids, however, can be unstable, are susceptible to degradation after administration, and in isolation have a limited ability to enter the target tissue. As such, nucleic acid particle delivery technologies are needed that encapsulate the nucleic acid and facilitate delivery to the patient and the target of interest within the patient. In particular, nucleic acid particles comprising lipids (e.g., lipid nanoparticles (LNPs)) have emerged as vehicles to facilitate delivery of nucleic acids to a target of interest.

[0003] Lipid nanoparticles (LNPs), generally, comprise a cationically ionizable lipid, a steroid, and certain lipid compounds (e.g., a structural lipid such as a phospholipid, and a grafted lipid, such a polyethylene glycol (PEG)-conjugated lipid) that stabilize the particle, and ensure that the LNP can release the cargo at the right time and place. While LNPs are widely considered to be safe and effective for the delivery of nucleic acid therapies, there are still certain deficiencies with current formulations. For example, it has been noted that intradermal and intramuscular administration of LNPs triggers “inflammation characterized by leukocytic infiltration, activation of different inflammatory pathways, and secretion of a diverse pool of inflammatory cytokines and chemokines.” Ndeupen, et al., iScience, 24, 103479 (Dec. 17, 2021). These responses can be characterized as pain, swelling, fever, and the like. It has been suggested that particular components of the LNP, such as the PEG-conjugated lipid or the cationically ionizable lipid, can contribute to these responses. See Tenchov R, et al., Bioconjug Chem. 2023 Jun 21;34(6):941- Page 1 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 960; Omo-Lamai S, et al. bioRxiv [Preprint]. 2024 Apr 18:2024.04.16.589801. doi: 10.1101 / 2024.04.16.589801.

[0004] Moreover, there remains a need for new LNP formulations that can improve transfection and delivery to the target cells relative to previous formulations. To achieve this end, the present disclosure encompasses an insight, among others, that formulations can be formed from combinations of permanently charged cationic lipids and anionically ionizable lipids together with other lipids such as cholesterol and neutral phospholipids. This is fundamentally different from existing LNP using cationically ionizable lipids together with other lipids such as cholesterol and neutral phospholipids.

[0005] The present disclosure further provides that lipid shape can be modulated by selection of particular cationic lipids, and an anionically ionizable lipid. The present disclosure further encompasses an insight that identification of particular ratios of a cationic lipid and an anionic or anionically ionizable lipid in an LNP formulation can improve transfection and delivery of nucleic acid agents to target cells, and further, in some embodiments, avoid the use of polymer conjugated lipids (such as PEG lipids) to achieve substantially equivalent efficacy.

[0006] Still further, the present disclosure encompasses an insight that use of cationic and anionically ionizable lipids having particular aliphatic tails with prescribed molecular volumes yields particles having desirable shapes useful for improving transfection and delivery of agents, including nucleic acid agents.

[0007] Further, the present disclosure provides new cationic lipids and anionically ionizable lipids that are particularly useful for preparing lipid nanoparticles as described herein, and, in some embodiments, exhibit the improved features described herein relative to previous formulations.

[0008] The present disclosure provides a composition comprising a cationic lipid, an anionically ionizable lipid, and a nucleic acid, wherein: the cationic lipid comprises a cationic head group, a first bridge group, and a first lipid tail group; the anionically ionizable lipid comprises an anionically ionizable head group, a second bridge group, and a second lipid tail group; the cationic lipid has a lipidic volume from about 700 Å3to about 1500 Å3, and / or the anionically ionizable lipid has a lipidic volume from about 700 Å3to about 1500 Å3; or the cationic lipid and the anionically ionizable lipid have a combined lipidic volume greater than 1400 Å3. As described in further detail herein, a lipidic volume refers to a molecular volume of the bridge and tail groups of each lipid. Page 2 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0009] In some embodiments, the present disclosure provides a compound represented by formula ICL:or a pharmaceutically acceptable salt thereof, wherein G1, B1, B2, T2, and T2are as defined in classes and subclasses herein, both singly and in combination.

[0010] In some embodiments, the present disclosure provides compound represented by formula IAL:or a pharmaceutically acceptable salt thereof, wherein G2, B3, B4, T3, and T4, are as described in classes and subclasses herein, both singly and in combination.

[0011] In some embodiments, the present disclosure provides a method of treating a disease, disorder, or condition comprising administering to a subject a composition described herein.

[0012] In some embodiments, the present disclosure provides a composition as described herein, for use in the treatment of a disease, disorder, or condition. BRIEF DESCRIPTION OF THE DRAWING

[0013] Figure 1 is a bar graph showing percent efficiency of transfection of cationic lipids with different headgroups compared to a reference.

[0014] Figure 2A is scatter plot showing the relative activity of lipid compositions comprising inventive cationic lipids and the anionic lipid DOGS in relation to the combined lipidic volume in Å3of the cationic and anionically ionizable lipid. The activity is shown for lipid compositions on HEK293 and HepG2 cells and further in relation to an incubation of the material with buffer or human serum.

[0015] Figure 2B is a bar graph showing the relative activity of lipid compositions comprising inventive cationic lipids and the anionic lipid DOGS in relation to the combined lipidic volume in Page 3 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Å3of the cationic and anionically ionizable lipid. The information is represented as frequency of active formulations using a threshold of 15% of the reference.

[0016] Figure 3A is scatter plot showing the relative activity of lipid compositions comprising inventive cationic lipids and the anionic lipid DOGS in relation to the lipidic volume in Å3of the cationic lipid only. The activity is shown for lipid compositions on HEK293 and HepG2 cells and further in relation to an incubation of the material with buffer or human serum.

[0017] Figure 3B is a bar graph showing the relative activity of lipid compositions comprising inventive cationic lipids and the anionic lipid DOGS in relation to the combined lipidic volume in Å3of the cationic lipid only. The information is represented as frequency of active formulations using a threshold of 15% of the reference.

[0018] Figure 4 shows certain cationic lipids.

[0019] Figure 5 shows certain anionically ionizable lipids.

[0020] Figure 6 is a scatter plot showing the relative activity of lipid compositions comprising inventive anionically ionizable lipids and the cationic lipid PONA in relation to the lipidic volume in Å3of the anionically ionizable lipid. The activity is shown for lipid compositions on HEK293 and HepG2 cells and further in relation to an incubation of the material with buffer or human serum.

[0021] Figure 7 is a line graph showing the relative activity of lipid compositions comprising the cationic lipid PONA and anionically ionizable lipids CHEMS, DMGS, and DOGS in relation to neutral lipids. The activity is shown for lipid compositions on HepG2 cells and further in relation to an incubation of the material with serum.

[0022] Figure 8 is a bar graph showing the relative efficacy of lipid compositions comprising the cationic lipid PONA and anionically ionizable lipids myristic acid, stearic acid, DMGS, compound B-12, and DOGS in relation to their respective lipidic volumes. The activity is shown for lipid compositions on HepG2 cells and further in relation to an incubation of the material with buffer or human serum. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0023] The present disclosure provides, among other things, a composition comprising a cationic lipid, an anionically ionizable lipid, and a nucleic acid, wherein: the cationic lipid comprises a cationic head group, a first bridge group, and a first lipid tail group; the anionically Page 4 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 ionizable lipid comprises an anionically ionizable head group, a second bridge group, and a second lipid tail group; the cationic lipid has a lipidic volume from about 700 Å3to about 1500 Å3, and / or the anionically ionizable lipid has a lipidic volume from about 700 Å3to about 1500 Å3; or the cationic lipid and the anionically ionizable lipid have a combined lipidic volume greater than 1400 Å3. As described in further detail herein, a lipidic volume refers to a molecular volume of the bridge and tail groups of each lipid. In some embodiments, the present disclosure provides new and useful cationic lipids. In some embodiments, the present disclosure provides new and useful anionically ionizable lipids. In some embodiments, the present disclosure provides uses of provided compositions, cationic lipids, and / or anionically ionizable lipids. Compounds and Definitions

[0024] Compounds of this disclosure include those described generally above and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of Elements, CAS version, Handbook of Chemistry and Physics, 75thEd. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March’s Advanced Organic Chemistry”, 5thEd., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0025] Unless otherwise stated, structures depicted herein are meant to include all stereoisomeric (e.g., enantiomeric or diastereomeric) forms of the structure, as well as all geometric or conformational isomeric forms of the structure. For example, the R and S configurations of each stereocenter are contemplated as part of the disclosure. Therefore, single stereochemical isomers, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of provided compounds are within the scope of the disclosure. For example, in some cases, Tables 1, 2A, 2B, 3, and 4 show one or more stereoisomers of a compound, and unless otherwise indicated, represents each stereoisomer alone and / or as a mixture. Unless otherwise stated, all tautomeric forms of provided compounds are within the scope of the disclosure. Page 5 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0026] Unless otherwise indicated, structures depicted herein are meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including replacement of hydrogen by deuterium or tritium, or replacement of a carbon by13C- or14C-enriched carbon are within the scope of this disclosure.

[0027] About or approximately: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In general, those skilled in the art, familiar within the context, will appreciate the relevant degree of variance encompassed by "about" or "approximately" in that context. For example, in some embodiments, the term "approximately" or "about" may encompass a range of values that are within (i.e., ±) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0028] Administering: As used herein, the term "administering" or "administration" typically refers to the administration of a composition to a subject to achieve delivery of an agent that is, or is included in, a composition to a target site or a site to be treated. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may be parenteral. In some embodiments, administration may be oral. In some particular embodiments, administration may be intravenous. In some particular embodiments, administration may be subcutaneous. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for Page 6 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 at least a selected period of time. In some embodiments, administration may comprise a prime- and-boost protocol. A prime-and-boost protocol can include administration of a first dose of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) followed by, after an interval of time, administration of a second or subsequent dose of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine). In the case of an immunogenic composition, a prime-and-boost protocol can result in an increased immune response in a patient.

[0029] Aliphatic: The term “aliphatic” refers to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “cycloaliphatic”), that has a single point or more than one points of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-12 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms (e.g., C1-6). In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms (e.g., C1-5). In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms (e.g., C1-4). In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms (e.g., C1-3), and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms (e.g., C1-2). Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, or alkynyl groups and hybrids thereof. A preferred aliphatic group is C1-6alkyl.

[0030] Alkyl: The term “alkyl”, used alone or as part of a larger moiety, refers to a saturated, optionally substituted straight or branched chain hydrocarbon group having (unless otherwise specified) 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, or 1-2 carbon atoms (e.g., C1-12, C1-10, C1-8, C1-6, C1-4, C1-3, or C1-2). Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, and heptyl.

[0031] Alkylene: The term “alkylene” is refers to a bivalent alkyl group. In some embodiments, “alkylene” is a bivalent straight or branched alkyl group. In some embodiments, an "alkylene chain" is a polymethylene group, i.e., -(CH2)n-, wherein n is a positive integer, e.g., from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. An optionally substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms is optionally replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group and also include those described in the specification herein. It will be appreciated Page 7 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 that two substituents of the alkylene group may be taken together to form a ring system. In certain embodiments, two substituents can be taken together to form a 3- to 7-membered ring. The substituents can be on the same or different atoms. The suffix “-ene” or “-enyl” when appended to certain groups herein are intended to refer to a bifunctional moiety of said group. For example, “-ene” or “-enyl”, when appended to “cyclopropyl” becomes “cyclopropylene” or “cyclopropylenyl” and is intended to refer to a bifunctional cyclopropyl group, e.g., .

[0032] Alkenyl: The term “alkenyl”, used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain or cyclic hydrocarbon group having at least one double bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms(e.g., C2-12, C2-10, C2-8, C2-6, C2-4, or C2-3). Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl. The term “cycloalkenyl” refers to an optionally substituted non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and having about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0033] Alkynyl: The term “alkynyl”, used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain hydrocarbon group having at least one triple bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C2-12, C2-10, C2-8, C2-6, C2-4, or C2-3). Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl.

[0034] Aryl: The term “aryl” refers to monocyclic and bicyclic ring systems having a total of six to fourteen ring members (e.g., C6-C14), wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. In some embodiments, an “aryl” group contains between six and twelve total ring members (e.g., C6-C12). The term “aryl” may be used interchangeably with the term “aryl ring”. In certain embodiments, “aryl” refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Unless otherwise specified, “aryl” groups are hydrocarbons. In some embodiments, an “aryl” ring system is an aromatic ring (e.g., phenyl) that is fused to a non-aromatic ring (e.g., cycloalkyl). Examples of aryl rings include that are fused .8 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0035] Bicyclic: The term “bicyclic ring” or “bicyclic ring system” refers to any bicyclic ring system, i.e., carbocyclic or heterocyclic, saturated or having one or more units of unsaturation, having one or more atoms in common between the two rings of the ring system. Thus, the term includes any permissible ring fusion, such as ortho-fused or spirocyclic. As used herein, the term “heterobicyclic” is a subset of “bicyclic” that requires that one or more heteroatoms are present in one or both rings of the bicycle. Such heteroatoms may be present at ring junctions and are optionally substituted, and may be selected from nitrogen (including N-oxides), oxygen, sulfur (including oxidized forms such as sulfones and sulfonates), phosphorus (including oxidized forms such as phosphates), boron, etc. In some embodiments, a bicyclic group has 7-12 ring members and 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. As used herein, the term “bridged bicyclic” refers to any bicyclic ring system, i.e., carbocyclic or heterocyclic, saturated or partially unsaturated, having at least one bridge. As defined by IUPAC, a “bridge” is an unbranched chain of atoms or an atom or a valence bond connecting two bridgeheads, where a “bridgehead” is any skeletal atom of the ring system which is bonded to three or more skeletal atoms (excluding hydrogen). In some embodiments, a bridged bicyclic group has 7-12 ring members and 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Such bridged bicyclic groups are well known in the art and include those groups set forth below where each group is attached to the rest of the molecule at any substitutable carbon or nitrogen atom. Unless otherwise specified, a bridged bicyclic group is optionally substituted with one or more substituents as set forth for aliphatic groups. Additionally or alternatively, any substitutable nitrogen of a bridged bicyclic group is optionally substituted. Exemplary bicyclic rings include:Page 9 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Exemplary bridged bicyclics include: NH NH NH NH .

[0036] Biological sample: As used herein, the term “biological sample” typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell culture) of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a biological sample is or comprises biological tissue or fluid. In some embodiments, a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and / or excretions; and / or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, obtained cells are or include cells from an individual from whom the sample is obtained. In some embodiments, a sample is a Page 10 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 “primary sample” obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of certain components, etc.

[0037] Carrier: As used herein, the term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered. In some exemplary embodiments, carriers can include sterile liquids, such as, for example, water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil and the like. In some embodiments, carriers are or include one or more solid components.

[0038] Combination therapy: As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents or modality(ies)). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).

[0039] Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate Page 11 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0040] Composition: Those skilled in the art will appreciate that the term “composition” may be used to refer to a discrete physical entity that comprises one or more specified components. In general, unless otherwise specified, a composition may be of any form – e.g., gas, gel, liquid, solid, etc.

[0041] Cycloaliphatic: As used herein, the term “cycloaliphatic” refers to a monocyclic C3-8 hydrocarbon or a bicyclic C6-12hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point or more than one points of attachment to the rest of the molecule.

[0042] Cycloalkyl: As used herein, the term “cycloalkyl” refers to an optionally substituted saturated ring monocyclic or polycyclic system of about 3 to about 10 ring carbon atoms. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0043] Deoxyribonucleic Acid (DNA): As used herein, the term “DNA” refers to a polymeric molecule of nucleotides that are typically double-stranded and comprise adenine, cytosine, guanine and thymine, and a deoxyribose sugar backbone structure as specified in the definition “Nucleic Acid / Polynucleotide.” In some embodiments, DNA is linear DNA, plasmid DNA, minicircle DNA, nanoplasmid DNA, doggybone DNA, or a transposon.

[0044] Deoxyribonucleotide: As used herein, the term “deoxyribonucleotide” refers to unmodified and modified deoxyribonucleotides. For example, unmodified deoxyribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and Page 12 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 thymine (T). Modified deoxyribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g. , replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages.

[0045] Dosage form or unit dosage form: Those skilled in the art will appreciate that the term “dosage form” may be used to refer to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject. Typically, each such unit contains a predetermined quantity of active agent. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen).

[0046] Dosing regimen or therapeutic regimen: Those skilled in the art will appreciate that the terms “dosing regimen” and “therapeutic regimen” may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen). Page 13 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0047] Effective Amount: The term “effective amount” refers to the amount of a compound sufficient to effect beneficial or desired results (e.g., a therapeutic, ameliorative, inhibitory, or preventative result). An effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or administration route.

[0048] Excipient: As used herein, the term “excipient” refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0049] Halogen: The term “halogen” or “halo” means F, Cl, Br, or I.

[0050] Heteroaliphatic: The term “heteroaliphatic” or “heteroaliphatic group”, as used herein, denotes an optionally substituted hydrocarbon moiety having, in addition to carbon atoms, from one to five heteroatoms, that may be straight–chain (i.e., unbranched), branched, or cyclic (“heterocyclic”) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. The term “heteroatom” refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen. The term “nitrogen” also includes a substituted nitrogen. Unless otherwise specified, heteroaliphatic groups contain 1–10 carbon atoms wherein 1–3 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroaliphatic groups contain 1–4 carbon atoms, wherein 1–2 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In yet other embodiments, heteroaliphatic groups contain 1–3 carbon atoms, wherein 1 carbon atom is optionally and independently replaced with a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroaliphatic groups include, but are not limited to, linear or branched, heteroalkyl, heteroalkenyl, and heteroalkynyl groups. For example, a 1- to 10 atom heteroaliphatic group includes the following exemplary groups: -O-CH3, -CH2-O-CH3, -O-CH2- CH2-O-CH2-CH2-O-CH3, and the like.

[0051] Heteroaryl: The terms “heteroaryl” and “heteroar–”, used alone or as part of a larger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refer to monocyclic or bicyclic ring groups Page 14 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 having 5 to 10 ring atoms (e.g., 5- to 6-membered monocyclic heteroaryl or 9- to 10-membered bicyclic heteroaryl); having 6, 10, or 14 π-electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2-a]pyridyl, imidazo[4,5-b]pyridyl, imidazo[4,5-c]pyridyl, pyrrolopyridyl, pyrrolopyrazinyl, thienopyrimidinyl, triazolopyridyl, and benzoisoxazolyl. The terms “heteroaryl” and “heteroar–”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms). Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzotriazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H–quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3–b]–1,4–oxazin–3(4H)–one, 4H-thieno[3,2-b]pyrrole, and benzoisoxazolyl. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of which terms include rings that are optionally substituted.

[0052] Heteroatom: The term “heteroatom” as used herein refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen.

[0053] Heterocycle: As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclic radical”, and “heterocyclic ring” are used interchangeably and refer to a stable 3- to 8-membered monocyclic, a 6- to 10-membered bicyclic, or a 10- to 16-membered polycyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, such as one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0–3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR+(as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any Page 15 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and thiamorpholinyl. A heterocyclyl group may be mono-, bi-, tri-, or polycyclic, preferably mono-, bi-, or tricyclic, more preferably mono- or bicyclic. A bicyclic heterocyclic ring also includes groups in which the heterocyclic ring is fused to one or more aryl rings. Exemplary bicyclic heterocyclic groups include indolinyl, isoindolinyl, benzodioxolyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl, and tetrahydroquinolinyl. A bicyclic heterocyclic ring can also be a spirocyclic ring system (e.g., 7- to 11-membered spirocyclic fused heterocyclic ring having, in addition to carbon atoms, one or more heteroatoms as defined above (e.g., one, two, three or four heteroatoms)). A bicyclic heterocyclic ring can also be a bridged ring system (e.g., 7- to 11-membered bridged heterocyclic ring having one, two, or three bridging atoms.

[0054] Ionizable, Ionized or Non-Ionizable. As used herein, the term “ionizable” refers to compounds or moieties thereof that change their protonation state between pH 4 and pH 8. An example of an ionizable moiety is an anionically ionizable moiety that becomes negatively charged between pH 4 and pH 8. Another example of an ionizable moiety is a cationically ionizable moiety that becomes positively charged between pH 4 and pH 8. As used herein, the term “ionized” refers to compounds or moieties thereof that are charged between pH 4 and pH 8. An example of an ionized moiety is a cationic group having a positive charge state between pH 4 and pH 8. Another example of an ionized moiety is an anionic group having a negative charge state between pH 4 and pH 8. As used herein, the term “non-ionizable” refers to compounds or moieties thereof that do not change their protonation state between pH 4 and pH 8. The lipid structures drawn herein referring to “cationic lipids” can be drawn in protonated or neutral form, but are intended to refer to a lipid that is positively charged between pH 4 and pH 8. For example, the following lipid structures are understood to be representative of “cationic lipids”: Page 16 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 and res areintended to refer to a lipid that is negatively charged between pH 4 and pH 8. For example, the following lipid structures are understood to be representative of “anionic lipids”: andly” as used herein have their art-understood meaning referring to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0056] Partially unsaturated: As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond between ring atoms. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic (e.g., aryl or heteroaryl) moieties, as herein defined.

[0057] Patient or subject: As used herein, the term “patient” or “subject” refers to any organism to which a provided composition is or may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients or subjects Page 17 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, a patient is a human. In some embodiments, a patient or a subject is suffering from or susceptible to one or more disorders or conditions. In some embodiments, a patient or subject displays one or more symptoms of a disorder or condition. In some embodiments, a patient or subject has been diagnosed with one or more disorders or conditions. In some embodiments, a patient or a subject is receiving or has received certain therapy to diagnose and / or to treat a disease, disorder, or condition.

[0058] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in unit dose amount appropriate for administration in a therapeutic or dosing regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0059] Pharmaceutically acceptable: As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0060] Pharmaceutically acceptable salt: The term “pharmaceutically acceptable salt”, as used herein, refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic Page 18 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2– hydroxy–ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2–naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3–phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p–toluenesulfonate, undecanoate, valerate salts, and the like.

[0061] Further, acids which are generally considered suitable for the formation of pharmaceutically useful salts from basic pharmaceutical compounds are discussed, for example, by P. Stahl et al., Camille G. (eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use. (2002) Zurich: Wiley-VCH; S. Berge et al., Journal of Pharmaceutical Sciences 1977, 66(1), 1-19; P. Gould, International J. of Pharmaceutics 1986, 33, 201-217; Anderson et al., The Practice of Medicinal Chemistry (1996), Academic Press, New York; and in The Orange Book (Food & Drug Administration, Washington, D.C. on their website). These disclosures are incorporated herein by reference.

[0062] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1–4alkyl)4salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.

[0063] Polycyclic: As used herein, the term “polycyclic” refers to a saturated or unsaturated ring system having two or more rings (for example, heterocyclyl rings, heteroaryl rings, cycloalkyl Page 19 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 rings, or aryl rings), having between 7 and 20 atoms, in which one or more carbon atoms are common to two adjacent rings. For example, in some embodiments, a polycyclic ring system refers to a saturated or unsaturated ring system having three or more rings (for example, heterocyclyl rings, heteroaryl rings, cycloalkyl rings, or aryl rings), having between 14 and 20 atoms, in which one or more carbon atoms are common to two adjacent rings. The rings in a polycyclic ring system may be fused (i.e., bicyclic or tricyclic), spirocyclic, or a combination thereof. An example polycyclic ring is a steroid.

[0064] Polypeptide: The term “polypeptide” or “peptide”, as used herein, typically has its art- recognized meaning of a polymer of at least three amino acids or more. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional, biologically active, or characteristic fragments, portions or domains (e.g., fragments, portions, or domains retaining at least one activity) of such complete polypeptides. In some embodiments, polypeptides may contain L-amino acids, D-amino acids, or both and / or may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, polypeptides may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof (e.g., may be or comprise peptidomimetics).

[0065] Prevent or prevention: As used herein, the terms “prevent” or “prevention”, when used in connection with the occurrence of a disease, disorder, and / or condition, refer to reducing the risk of developing the disease, disorder and / or condition and / or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.

[0066] Ribonucleotide: As used herein, the term “ribonucleotide” encompasses unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g. , replacement with modified bases, stabilizing Page 20 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. The term “ribonucleotide” also encompasses ribonucleotide triphosphates including modified and non-modified ribonucleotide triphosphates.

[0067] Ribonucleic acid (RNA): As used herein, the term “RNA” refers to a polymer of ribonucleotides. In some embodiments, an RNA is single stranded. In some embodiments, an RNA is double stranded. In some embodiments, an RNA comprises both single and double stranded portions. In some embodiments, an RNA can comprise a backbone structure as described in the definition of “Nucleic acid / Polynucleotide” above. An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA). In some embodiments where an RNA is a mRNA. In some embodiments where an RNA is a mRNA, a RNA typically comprises at its 3’ end a poly(A) region. In some embodiments where an RNA is a mRNA, an RNA typically comprises at its 5’ end an art-recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation. In some embodiments, a RNA is a synthetic RNA. Synthetic RNAs include RNAs that are synthesized in vitro (e.g., by enzymatic synthesis methods and / or by chemical synthesis methods).

[0068] Substituted or optionally substituted: As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. “Substituted” applies to one or more hydrogens that are either explicit or implicit from the structure (e.g refers to at least;orUnless otherwise indicated, an “optionally substituted” group may have a suitablesubstituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, Page 21 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes provided herein. Groups described as being “substituted” preferably have between 1 and 4 substituents, more preferably 1 or 2 substituents. Groups described as being “optionally substituted” may be unsubstituted or be “substituted” as described above.

[0069] Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; –(CH2)0–4R^; –(CH2)0–4OR^; -O(CH2)0-4Ro, –O– (CH2)0–4C(O)OR°; –(CH2)0–4CH(OR^)2; –(CH2)0–4SR^; –(CH2)0–4Ph, which may be substituted with R°; –(CH2)0–4O(CH2)0–1Ph which may be substituted with R°; –CH=CHPh, which may be substituted with R°; –(CH2)0–4O(CH2)0–1-pyridyl which may be substituted with R°; –NO2; –CN; –N3; (CH2)0–4N(R^)2; –(CH2)0–4N(R^)C(O)R^; –N(R^)C(S)R^; –(CH2)0–4N(R^)C(O)NR^2; N(R^)C(S)NR^2; –(CH2)0–4N(R^)C(O)OR^; -N(R^)N(R^)C(O)R^; N(R^)N(R^)C(O)NR^2; N(R^)N(R^)C(O)OR^; –(CH2)0–4C(O)R^; C(S)R^; –(CH2)0–4C(O)OR^; –(CH2)0–4C(O)SR^; (CH2)0–4C(O)OSiR^3; –(CH2)0–4OC(O)R^; –OC(O)(CH2)0–4SR°; –(CH2)0–4SC(O)R^; –(CH2)0–4C(O)NR^2; –C(S)NR^2; –C(S)SR°; –SC(S)SR°, (CH2)0–4OC(O)NR^2; C(O)N(OR^)R^; – C(O)C(O)R^; –C(O)CH2C(O)R^; –C(NOR^)R^; (CH2)0–4SSR^; –(CH2)0–4S(O)2R^; –(CH2)0–4S(O)2OR^; –(CH2)0–4OS(O)2R^; –S(O)2NR^2; (CH2)0–4S(O)R^; N(R^)S(O)2NR^2; – N(R^)S(O)2R^; –N(OR^)R^; –C(NH)NR^2; –P(O)2R^; P(O)R^2; OP(O)R^2; –OP(O)(OR^)2; SiR^3; –(C1–4straight or branched alkylene)O–N(R^)2; or –(C1–4straight or branched alkylene)C(O)O– N(R^)2, wherein each R^ may be substituted as defined below and is independently hydrogen, C1–6 aliphatic, –CH2Ph, –O(CH2)0–1Ph, -CH2-(5- to 6-membered heteroaryl ring), or a 3- to 6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R^, taken together with their intervening atom(s), form a 3- to 12- membered saturated, partially unsaturated, or aryl mono– or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below. Page 22 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0070] Suitable monovalent substituents on R^ (or the ring formed by taking two independent occurrences of R^ together with their intervening atoms), are independently halogen, –(CH2)0–2R^, –(haloR^), –(CH2)0–2OH, –(CH2)0–2OR^, –(CH2)0–2CH(OR^)2, O(haloR^), –CN, –N3, –(CH2)0–2C(O)R^, –(CH2)0–2C(O)OH, –(CH2)0–2C(O)OR^, –(CH2)0–2SR^, –(CH2)0–2SH, –(CH2)0–2NH2, – (CH2)0–2NHR^, –(CH2)0–2NR^ 2, –NO2, –SiR^ 3, –OSiR^ 3, C(O)SR^ , –(C1–4 straight or branched alkylene)C(O)OR^, or –SSR^wherein each R^is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from C1–4 aliphatic, – CH2Ph, –O(CH2)0–1Ph, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R^ include =O and =S.

[0071] Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: =O (“oxo”), =S, =NNR*2, =NNHC(O)R*, =NNHC(O)OR*, =NNHS(O) R*, =NR*, =NOR*, –O(C** 2 (R 2))2–3O–, or –S(C(R 2))2–3S–, wherein each independent *may be substituted as defined below, or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0–4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: –O(CR*2)2–3O–, wherein each independent occurrence of R*is selected from hydrogen, C1–6 aliphatic which may be substituted as defined below, or an unsubstituted 5–6–membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0072] Suitable substituents on the aliphatic group of R*include halogen, –R^, (haloR^), OH, –OR^, –O(haloR^), –CN, –C(O)OH, –C(O)OR^, –NH2, –NHR^, –NR^2, or –NO2, wherein each R^is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1–4 aliphatic, –CH2Ph, –O(CH2)0–1Ph, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0073] Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include –R†, –NR†, –C(O)R†, –C(O)OR†, –C(O)C(O)R†, –C(O)CH C(† †† 2 2 O)R , S(O)2R , S(O)2NR 2, –C(S)N, , ; p y y g , C1–6 aliphatic which may be substituted as defined below, unsubstituted –OPh, or an unsubstituted 3- Page 23 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R†, taken together with their intervening atom(s) form an unsubstituted 3- to 12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0074] Suitable substituents on the aliphatic group of R†are independently halogen, –R^, (haloR^), –OH, –OR^, –O(haloR^), –CN, –C(O)OH, –C(O)OR^, –NH2, –NHR^, –NR^2, or NO2, wherein each R^is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1–4 aliphatic, –CH2Ph, –O(CH2)0–1Ph, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0075] Suffering from: An individual who is “suffering from” a disease, disorder, and / or condition has been diagnosed with and / or displays one or more symptoms of a disease, disorder, and / or condition.

[0076] Small molecule: As used herein, the term “small molecule” means a low molecular weight organic and / or inorganic compound. In general, a “small molecule” is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, a small molecule is not a polymer.

[0077] In some embodiments, a small molecule does not include a polymeric moiety. In some embodiments, a small molecule is not and / or does not comprise a protein or polypeptide (e.g., is not an oligopeptide or peptide). In some embodiments, a small molecule is not and / or does not comprise a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, a small molecule is not and / or does not comprise a polysaccharide; for example, in some embodiments, a small molecule is not a glycoprotein, proteoglycan, glycolipid, etc.). In some embodiments, a small molecule is not a lipid.

[0078] In some embodiments, a small molecule is a modulating agent (e.g., is an inhibiting agent or an activating agent). In some embodiments, a small molecule is biologically active. In Page 24 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 some embodiments, a small molecule is detectable (e.g., comprises at least one detectable moiety). In some embodiments, a small molecule is a therapeutic agent.

[0079] Those of ordinary skill in the art, reading the present disclosure, will appreciate that certain small molecule compounds described herein may be provided and / or utilized in any of a variety of forms such as, for example, crystal forms (e.g., polymorphs, solvates, etc), salt forms, protected forms, pro-drug forms, ester forms, isomeric forms (e.g., optical and / or structural isomers), isotopic forms, etc.

[0080] Those of ordinary skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more stereoisomeric forms. In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in the form of an individual enantiomer, diastereomer or geometric isomer, or may be in the form of a mixture of stereoisomers; in some embodiments, such a small molecule may be utilized in accordance with the present disclosure in a racemic mixture form.

[0081] Those of skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more tautomeric forms. In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in the form of an individual tautomer, or in a form that interconverts between tautomeric forms.

[0082] Those of skill in the art will appreciate that certain small molecule compounds have structures that permit isotopic substitution (e.g.,2H or3H for H;11C,13C or14C for12C;13N or15N for14N;17O or18O for16O;36Cl for35Cl or37Cl;18F for19F;131I for127I; etc.). In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in one or more isotopically modified forms, or mixtures thereof.

[0083] In some embodiments, reference to a particular small molecule compound may relate to a specific form of that compound. In some embodiments, a particular small molecule compound may be provided and / or utilized in a salt form (e.g., in an acid-addition or base-addition salt form, depending on the compound); in some such embodiments, the salt form may be a pharmaceutically acceptable salt form.

[0084] In some embodiments, where a small molecule compound is one that exists or is found in nature, that compound may be provided and / or utilized in accordance in the present disclosure in a form different from that in which it exists or is found in nature. Those of ordinary skill in the art will appreciate that, in some embodiments, a preparation of a particular small molecule Page 25 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 compound that contains an absolute or relative amount of the compound, or of a particular form thereof, that is different from the absolute or relative (with respect to another component of the preparation including, for example, another form of the compound) amount of the compound or form that is present in a reference preparation of interest (e.g., in a primary sample from a source of interest such as a biological or environmental source) is distinct from the compound as it exists in the reference preparation or source. Thus, in some embodiments, for example, a preparation of a single stereoisomer of a small molecule compound may be considered to be a different form of the compound than a racemic mixture of the compound; a particular salt of a small molecule compound may be considered to be a different form from another salt form of the compound; a preparation that contains only a form of the compound that contains one conformational isomer ((Z) or (E)) of a double bond may be considered to be a different form of the compound from one that contains the other conformational isomer ((E) or (Z)) of the double bond; a preparation in which one or more atoms is a different isotope than is present in a reference preparation may be considered to be a different form; etc.

[0085] Those skilled in the art will further appreciate that, in small molecule structures, the symbol , as used herein, refers to a point of attachment between two atoms. Additionally or alternatively, the symbol refers to a point of attachment ring in a spirocyclic manner.

[0086] Treat: As used herein, the terms “treat,” “treatment,” or “treating” refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and / or condition, for example, for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. Compositions

[0087] The present disclosure provides, among other things, compositions comprising a cationic lipid and an anionically ionizable lipid useful for delivery of a nucleic acid agent. In some embodiments, the present disclosure encompasses an insight that particular combinations of cationic lipids and anionically ionizable lipids are particularly useful, and have particular Page 26 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 characteristics, that make them useful for the delivery of nucleic acid agents. For example, the present disclosure encompasses an insight that “lipid salts”, that is, combinations of cationic lipids and anionically ionizable lipids in a composition, in particular ratios, lead to improved properties of the resulting compositions, such as improved transfection.

[0088] As described herein, compositions described here comprising cationic lipids, anionic lipids and a nucleic acid can form particles, e.g., nanoparticles. Said nanoparticles can be included as part of a suspension useful for administration to a subject.

[0089] Moreover, the present disclosure encompasses an insight that use of cationic and anionically ionizable lipids having particular lipidic groups with prescribed molecular volumes yields particles having desirable shapes useful for improving transfection and delivery of agents, including nucleic acid agents. Accordingly, in some embodiments, the present disclosure provides a composition comprising a cationic lipid, an anionically ionizable lipid, and a nucleic acid, wherein: the cationic lipid comprises a cationic head group, a first bridge group, and a first lipid tail group; the anionically ionizable lipid comprises an anionically ionizable head group, a second bridge group, and a second lipid tail group; the cationic lipid has a lipidic volume from about 700 Å3to about 1500 Å3, and / or the anionically ionizable lipid has a lipidic volume from about 700 Å3to about 1500 Å3; or the cationic lipid and the anionically ionizable lipid have a combined lipidic volume greater than 1400 Å3.

[0090] As described herein, “lipidic volume” refers to the molecular volume of the lipidic groups of a cationic or anionically ionizable lipid compound described. The lipidic volume of the cationic lipid is the combined molecular volume occupied by the first bridge group and first lipid tail group. The lipidic volume of the anionically ionizable lipid is the combined molecular volume occupied by the second bridge group and the second lipid tail group. The molecular volume of a portion of a compound can be determined according to methods known of those of skill in the art. For example, methods for calculating molecular volume is provided in PCT App. Pub. Nos. WO2008 / 043575 and WO2009 / 047006, and Connolly, M. J. Am. Chem. Soc.1985, 107, 1118 – 1124, Zhao, Y.H., Abraham, M.H. and Zissimos, A.M. J. Org. Chem.2003, 68, 7368-7373, each of which is incorporated by reference. Within the context of the present disclosure, reference to a molecular volume for a lipidic group refers to the portion of a lipid that is not part of the “head group.” Generally, lipid compounds are described as comprising a “head group”, which is polar, and can be ionic or ionizable, and a “tail group”, which is generally aliphatic and hydrophobic. In Page 27 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 between the head group and the tail group may exist a “bridge group”. The “bridge group” and the “tail group” together are referred to as a “lipidic group.” For example:Therefore, it is to be understood that a “lipidic volume” refers to the molecular volume of the lipidic group of a lipid compound, as illustrated above.

[0091] Molecular volume is commonly calculated by assigning a value called a van der Waals radius, rivdW, to each atom type in such a way that the sum of these quantities for a given atom pair, i and j, is equal to their closest possible distance (dij): rivdW+ rjvdW≤ dij

[0092] Many different tables of “best” van der Waals radii exist, even though the values for corresponding atoms coming from different authors are similar. In geometric terms, the van der Waals radius may be imagined as a spherical “shield” surrounding the atom, and the closest distance between two non-bonded atoms is when their respective shields touch. However, the shields of covalently bonded atoms intersect since bond lengths are shorter than the sum of the van Page 28 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 der Waals radii partaking atoms. A molecular van der Waals surface, also called a van der Waals envelope, is composed of the spheres for individual atoms with their intersecting sections removed.

[0093] For a single molecule (i.e., molecule for which there is a path between any two atoms along covalent bonds), the van der Waals envelope is a closed surface, and hence, it contains volume. This volume is called the molecular volume, or van der Waals volume, and is usually given in Å3. The straightforward way of calculating molecular volume on a computer is by numerical integration.

[0094] In some embodiments, molecular volume is calculated according to the formula by Zhao, Y.H., Abraham, M.H. and Zissimos, A.M. J. Org. Chem. 2003, 68, 7368-7373, reported here as Method 1. Method 1 Molecular Volume = (sum of all atom contributions) – 5.92NB – 14.7RA, - 3.8RNR where: the sum of all atom contributions is the sum of the total number of atoms multiplied by its contribution value, provided in Table M1; NB is the number of bonds present, determined according to the following equation: NB= N – 1 + RA+ RNARAis the number of aromatic rings; RNA is the number of non-aromatic rings; and N is the total number of atoms. Table M1 Atom ContributionPage 29 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0095] In some embodiments, lipidic volume is determined according to Method 1.

[0096] In some embodiments, the present disclosure provides a composition comprising a cationic lipid, an anionically ionizable lipid, and a nucleic acid, wherein: the cationic lipid comprises a cationic head group, a first bridge group, and a first lipid tail group; the anionically ionizable lipid comprises an anionically ionizable head group, a second bridge group, and a second lipid tail group; the cationic lipid has a lipidic volume from about 700 Å3to about 1500 Å3, and / or the anionically ionizable lipid has a lipidic volume from about 700 Å3to about 1500 Å3; or the cationic lipid and the anionically ionizable lipid have a combined lipidic volume greater than 1400 Å3; and wherein the lipidic volume for each of the cationic lipid, the anionic lipid, or the combined lipidic volume is determined according to Method 1.

[0097] The present disclosure encompasses an insight that particular cationic or anionically ionizable lipids are particularly useful for preparing compositions described herein. Previous cationic lipids, such as those described in WO 97 / 16024, WO 97 / 43363, WO 98 / 05678, WO 01 / 55098, WO 08 / 137758, US 5965434, US 11541010, and Radloff et al. Mol Ther Nucleic Acids, 2023 Oct 29:34:102068, did not include or otherwise appreciate the role that molecular volume plays on LNP shape formation, especially when combined into a lipid salt with an aninonically ionizable lipid. In some embodiments, the present disclosure provides compositions that do not include one or more of palmitoyl-oleoyl-nor-arginine (PONA); guanidino- di[(heptadecyl)methyl]carboxylic acid (GUADACA); 4-methylpyridinium- di(heptadecyl)methylcarboxylic acid (MPDACA); 1,2-dioleoyl-3 trimethylammonium propane (DOTAP); 1,2-dioleoyl-3-dimethylammomium propane (DODAP) and / or dioleoyl- glycerol- hemisuccinate (DOGS) or dioleoyl- phosphatidic acid (DOPA).

[0098] As described herein, a cationic lipid comprises a first lipid tail group and a first bridge group, which are used to determine the lipidic volume. In some embodiments, a cationic lipid has a lipidic volume greater than 650 Å3. In some embodiments, a cationic lipid has a lipidic volume from about 700 Å3to about 1500 Å3. In some embodiments, a cationic lipid has a lipidic volume from about 800 Å3to about 1500 Å3. In some embodiments, a cationic lipid has a lipidic volume that is from about 700 Å3to about 1400 Å3. In some embodiments, a cationic lipid has a lipidic volume that is from about 700 Å3to about 1300 Å3. In some embodiments, a cationic lipid has a lipidic volume that is from about 700 Å3to about 1200 Å3. In some embodiments, a cationic lipid Page 30 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 has a lipidic volume that is from about 700 Å3to about 1100 Å3. In some embodiments, a cationic lipid has a lipidic volume that is from about 700 Å3to about 1000 Å3. In some embodiments, a cationic lipid has a lipidic volume that is from about 700 Å3to about 900 Å3. In some embodiments, a cationic lipid has a lipidic volume that is about 700 Å3, about 750 Å3, about 800 Å3, about 850 Å3, about 900 Å3, about 950 Å3, about 1000 Å3, about 1050 Å3, about 1100 Å3, about 1150 Å3, about 1200 Å3, about 1250 Å3, about 1300 Å3, about 1350 Å3, about 1400 Å3, about 1450 Å3, or about 1500 Å3.

[0099] In some embodiments, a cationic lipid comprising a first lipid tail group is or comprises an optionally substituted C10-C50 aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -Cy-, -NRZ-, -N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, - OC(O)N(RZ)-, -N(RZ)C(O)N(RZ) -, -C(O)N(RZ)SO2(RZ)-, -SO2(RZ)N(RZ)C(O)-,-OC(O)O-, -O-, - C(O)-, -OC(O)-, -C(O)O, SO, or SO2; where each -Cy- is independently an optionally substituted C3-C6 cycloaliphatic, 5- to 12-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, S, 5- to 6-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, S; and each RZis independently H or an optionally substituted group selected from C1-C20 aliphatic, or C3-C12 cycloaliphatic. In some embodiments, a first lipid tail group is or comprises optionally substituted C10-C50aliphatic, wherein one or more carbon atoms are optionally and independently replaced by -C(O)O- or -OC(O)-. In some embodiments, a first lipid tail group is or comprises optionally substituted branched C10-C50 aliphatic, wherein one or more carbon atoms are optionally and independently replaced by -C(O)O- or -OC(O)-. In some embodiments, a first lipid tail group is or comprises optionally substituted C10-C50aliphatic. In some embodiments, a first lipid tail group is or comprises optionally substituted C10-C50 alkyl. In some embodiments, a first lipid tail group is or comprises optionally substituted C10-C50 alkenyl. In some embodiments, the first lipid tail group is non-ionizable.

[0100] As described herein, a cationic lipid comprises a first bridge group. A first bridge group is a multivalent group bonded to the cationic head group and the first lipid tail group. In some embodiments, a first bridge group comprises a chiral carbon atom. In some embodiments, a first bridge group is a C2-C5aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -NRZ-, -N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, - N(RZ)C(O)N(RZ)-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-. In some embodiments, a first bridge group is a C2-C5aliphatic group, wherein one or more carbon atoms are optionally and Page 31 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 independently replaced by -N(RZ)C(O)-, -C(O)N(RZ)-, -OC(O)-, or -C(O)O-. In some embodiments, a first bridge group is a C2-C5 aliphatic group comprising at least one chiral carbon atom, wherein one or more carbon atoms are optionally and independently replaced by - N(RZ)C(O)-, -C(O)N(RZ)-, -OC(O)-, or -C(O)O-. In some embodiments, a first bridge group is a C2-C5 aliphatic group, wherein one carbon atom is replaced by -N(RZ)C(O)- or -C(O)N(RZ)-, and a second carbon atom is replaced by -OC(O)-, or -C(O)O-. In some embodiments, a first bridge group is a C2-C5aliphatic group, wherein two carbon atoms are each independently replaced by - N(RZ)C(O)- or -C(O)N(RZ)-. In some embodiments, a first bridge group is a C2-C5 aliphatic group, wherein two carbon atoms are each independently replaced by or -C(O)N(RZ)-, wherein RZis optionally substituted C1-C20aliphatic. In some embodiments, a first bridge group is non- ionizable.

[0101] As described herein, a cationic lipid comprises a cationic head group. A “cationic head group” as used herein, refers to an ionized moiety that is positively charged between pH 4 and pH 8.. In some embodiments the cationic head group contains a single positive charge that is charged at pH 8 or greater (e.g., pH 9, pH 10, pH 11, pH 12, pH 13, or pH 14). In some embodiments a cationic head group has one or more positive charges, wherein at least one of the charge centers has a pKaof 9 or greater (e.g., pKa10, pKa11, pKa12, pKa13, pKa14. In some embodiments, a cationic head group is or comprises a guanidinium moiety, an amidinium moiety, an amine moiety, an optionally substituted 5- to 6-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 4- to 6-membered heterocycle comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, a cationic head group is or comprises a guanidinium moiety or an amine moiety. In some embodiments, a cationic head group is or comprises a guanidinium moiety. In some embodiments, a cationic head group is or comprises an amidinium moiety. In some embodiments, a cationic head group comprises an amine moiety. In some embodiments, a cationic head group comprises an optionally substituted 5- to 6- membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, a cationic head group is imidazolium or pyridinium. In some embodiments, a cationic head group is an alkylated imidazolium or pyridinium. In some embodiments, a cationic head group comprises an optionally substituted 4- to 6-membered heterocycle comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, a cationic head group is piperidine or pyrrolidine. In some embodiments, a cationic head group is an alkylated piperidinium or Page 32 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 pyrrolidinium. As used herein, reference to an “alkylated” imidazolium, pyridinium, piperidinium or pyrrolidinium refers to a moiety wherein the nitrogen atom is alkylated by one or more methyl groups, thereby providing a positive charge to the nitrogen atom, e.g , or .

[0102] In some embodiments, a cationic lipid does not comprise a steroid moiety, a vitamin D moiety, a vitamin E moiety, or a lipidic group (e.g., a combination of a first bridge group and a first tail group) comprising less than 40 carbon atoms.

[0103] In some embodiments, a cationic lipid is one described in any one of formula ICL, ICL- II, ICL-IIa, ICL-IIb, ICL-IIc, ICL-IId, ICL-III, ICL-IIIa, ICL-IIIb, ICL-IIIc, ICL-IIId, ICL-IV, ICL-IVa, ICL-IVb, ICL-IVc, or ICL-IVd, or a pharmaceutically acceptable salt thereof.

[0104] As described herein, an anionically ionizable lipid comprises a second bridge group and a second lipid tail group, which are used to determine the lipidic volume. In some embodiments, an anionically ionizable lipid has a lipidic volume greater than 400 Å3. In some embodiments, an anionically ionizable lipid has a lipidic volume greater than 450 Å3. In some embodiments, an anionically ionizable lipid has a lipidic volume greater than 500 Å3. In some embodiments, an anionically ionizable lipid has a lipidic volume greater than 550 Å3. In some embodiments, an anionically ionizable lipid has a lipidic volume greater than 600 Å3.In some embodiments, an anionically ionizable lipid has a lipidic volume greater than 650 Å3. In some embodiments, an anionically ionizable lipid has a lipidic volume greater than 700 Å3. In some embodiments, an anionically ionizable lipid has a lipidic volume from about 450 Å3to about 700 Å3. In some embodiments, an anionically ionizable lipid has a lipidic volume from about 700 Å3to about 1500 Å3. In some embodiments, an anionically ionizable lipid has a lipidic volume that is from about 700 Å3to about 1400 Å3. In some embodiments, an anionically ionizable lipid has a lipidic volume that is from about 700 Å3to about 1300 Å3. In some embodiments, an anionically ionizable lipid has a lipidic volume that is from about 700 Å3to about 1200 Å3. In some embodiments, an anionically ionizable lipid has a lipidic volume that is from about 700 Å3to about 1100 Å3. In some embodiments, an anionically ionizable lipid has a lipidic volume that is from about 700 Å3to about 1000 Å3. In some embodiments, an anionically ionizable lipid has a lipidic Page 33 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 volume that is from about 500 Å3to about 700 Å3. In some embodiments, an anionically ionizable lipid has a lipidic volume that is from about 700 Å3to about 900 Å3. In some embodiments, an anionically ionizable lipid has a lipidic volume that is about 400 Å3, about 450 Å3, about 500 Å3, about 550 Å3, about 600 Å3, 650 Å3, about 700 Å3, about 750 Å3, about 800 Å3, about 850 Å3, about 900 Å3, about 950 Å3, about 1000 Å3, about 1050 Å3, about 1100 Å3, about 1150 Å3, about 1200 Å3, about 1250 Å3, about 1300 Å3, about 1350 Å3, about 1400 Å3, about 1450 Å3, or about 1500 Å3.

[0105] In some embodiments, an anionically ionizable lipid comprising a second lipid tail group is or comprises an optionally substituted C10-C50 aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -Cy-, -NRZ-, -N(RZ)C(O)-, - C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ) -, -O-, -C(O)-, -OC(O)-, -C(O)O, SO, or SO2; where each -Cy- is independently an optionally substituted C3-C6 cycloaliphatic, 5- to 12-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, S, 5- to 6-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, S; and each RZis independently H or an optionally substituted group selected from C1-C20 aliphatic, or C3-C12 cycloaliphatic. In some embodiments, a second lipid tail group is or comprises optionally substituted C10-C50 aliphatic, wherein one or more carbon atoms are optionally and independently replaced by -C(O)O- or - OC(O)-. In some embodiments, a second lipid tail group is or comprises optionally substituted branched C10-C50 aliphatic, wherein one or more carbon atoms are optionally and independently replaced by -C(O)O- or -OC(O)-. In some embodiments, a second lipid tail group is or comprises optionally substituted C10-C50aliphatic. In some embodiments, a second lipid tail group is or comprises optionally substituted C10-C50 alkyl. In some embodiments, a second lipid tail group is or comprises optionally substituted C10-C50 alkenyl. In some embodiments, the second lipid tail group is non-ionizable.

[0106] As described herein, an anionically ionizable lipid comprises a second bridge group. A second bridge group is a multivalent group bonded to the anionically ionizable head group and the second lipid tail group. In some embodiments, a second bridge group comprises a chiral carbon atom. In some embodiments, a second bridge group is a C2-C5aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by - NRZ-, -N(RZ)C(O)-, - C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ)-, -O-, -C(O)-, -OC(O)-, - C(O)O-, -SO-, or -SO2-. In some embodiments, a second bridge group is a C2-C5aliphatic group, Page 34 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 wherein one or more carbon atoms are optionally and independently replaced by -OC(O)-, or - C(O)O-. In some embodiments, a second bridge group is non-ionizable.

[0107] As described herein, an anionically ionizable lipid comprises an anionically ionizable head group. As referred to herein, an “anionically ionizable head group” refers to a moiety that that changes its protonation state between pH 4 and pH8. For example, an anionically ionizable head group, in some embodiments, is neutral at acidic pH (e.g., pH of about4) but becomes negatively charged at neutral to alkaline pH (e.g., a pH greater of about 8). In some embodiments, an anionically ionizable head group is or comprises a carboxylic acid moiety or a dihydrogen phosphate moiety. In some embodiments, an anionically ionizable head group is or comprises a carboxylic acid moiety. In some embodiments, an anionically ionizable head group is or comprises or a dihydrogen phosphate moiety.

[0108] In some embodiments, an anionically ionizable lipid comprises an amino acid moiety comprising a carboxylic acid side chain, e.g., glutamic acid or aspartic acid. In some embodiments, an anionically ionizable lipid is one that comprises a hemimalonate, hemiglutarate, hemiadipate, hemipimelate or hemisuberate moiety.

[0109] In some embodiments, an anionically ionizable lipid does not comprise a steroid moiety, a vitamin D moiety, a vitamin E moiety, or a second tail group comprising less than 40 carbon atoms.

[0110] In some embodiments, a composition described herein does not comprise dioleoyl glycerol hemisuccinate (DOGS); dipalmitoyl glycerol hemisuccinate (DPGS); dimyristoyl glycerol hemisuccinate (DMGS); palmitoyl oleoyl glycerol hemisuccinate (POGS); distearoyl glycerol hemisuccinate (DSGS); and cholesteryl hemisuccinate (CHEMS). In some embodiments, a composition described herein does not comprise tetradecanoic acid (myristic acid) and octadecanoic acid (stearic acid).

[0111] In some embodiments, a composition described herein comprises a cationic lipid having a lipidic volume that is from about 700 Å3to about 1500 Å3and an anionically ionizable lipid having a lipidic volume that is from about 700 Å3to about 1500 Å3.

[0112] In some embodiments of a composition described herein, a cationic lipid and an anionically ionizable lipid have a combined lipidic volume that is greater than 1400 Å3. In some embodiments of a composition described herein, a cationic lipid and an anionically ionizable lipid have a combined lipidic volume that is greater than 1450 Å3. In some embodiments of a Page 35 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 composition described herein, a cationic lipid and an anionically ionizable lipid have a combined lipidic volume that is greater than 1500 Å3. In some embodiments of a composition described herein, a cationic lipid and an anionically ionizable lipid have a combined lipidic volume that is from about 1400 Å3to about 3000 Å3. In some embodiments of a composition described herein, a cationic lipid and an anionically ionizable lipid have a combined lipidic volume that is from about 1400 Å3to about 2000 Å3. In some embodiments of a composition described herein, a cationic lipid and an anionically ionizable lipid have a combined lipidic volume that is about 1400 Å3, about 1450 Å3, about 1500 Å3, about 1550 Å3, about 1600 Å3, about 1650 Å3, about 1700 Å3, about 1750 Å3, about 1800 Å3, about 1850 Å3, about 1900 Å3, about 1950 Å3, about 2000 Å3, about 2050 Å3, about 2100 Å3, about 2150 Å3, about 2200 Å3, about 2250 Å3, about 2300 Å3, about 2350 Å3, about 2400 Å3, about 2450 Å3, about 2500 Å3, about 2550 Å3, about 2600 Å3, about 2650 Å3, about 2700 Å3, about 2750 Å3, about 2800 Å3, about 2850 Å3, about 2900 Å3, about 2950 Å3, or about 3000 Å3.

[0113] In some embodiments, a composition described herein comprises a cationic lipid having a lipidic volume that is from about 450 Å3to about 700 Å3and an anionically ionizable lipid having a lipidic volume that is from about 450 Å3to about 700 Å3.

[0114] In some embodiments of a composition described herein, a cationic lipid and an anionically ionizable lipid have a combined lipidic volume that is greater than 900 Å3. In some embodiments of a composition described herein, a cationic lipid and an anionically ionizable lipid have a combined lipidic volume that is greater than 1000 Å3. In some embodiments of a composition described herein, a cationic lipid and an anionically ionizable lipid have a combined lipidic volume that is greater than 2500 Å3. In some embodiments of a composition described herein, a cationic lipid and an anionically ionizable lipid have a combined lipidic volume that is from about 900 Å3to about 1400 Å3. In some embodiments of a composition described herein, a cationic lipid and an anionically ionizable lipid have a combined lipidic volume that is from about 1000 Å3to about 1400 Å3. In some embodiments of a composition described herein, a cationic lipid and an anionically ionizable lipid have a combined lipidic volume that is about 900 Å3, about 950 Å3, about 1000 Å3, about 1500 Å3, about 2000 Å3, about 2500 Å3, about 3000 Å3, about 3500 Å3, or about 4000 Å3.

[0115] In some embodiments, a cationic lipid has a lipidic volume that is about 500 Å3, and an anionically ionizable lipid has a lipidic volume that is about 900 Å3or greater. In some Page 36 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 embodiments, a cationic lipid has a lipidic volume that is about 600 Å3, and an anionically ionizable lipid has a lipidic volume that is about 800 Å3or greater In some embodiments, a cationic lipid has a lipidic volume that is about 650 Å3, and an anionically ionizable lipid has a lipidic volume that is about 750 Å3or greater In some embodiments, a cationic lipid has a lipidic volume that is about 750 Å3or greater and an anionically ionizable lipid has a lipidic volume that is about 650 Å3. In some embodiments, a cationic lipid has a lipidic volume that is about 800 Å3or greater and an anionically ionizable lipid has a lipidic volume that is about 600 Å3. In some embodiments, a cationic lipid has a lipidic volume that is about 900 Å3or greater and an anionically ionizable lipid has a lipidic volume that is about 500 Å3.

[0116] In some embodiments, the present disclosure provides a composition comprising a cationic lipid and an anionically ionizable lipid and a nucleic acid, wherein the nucleic acid is an mRNA. The present disclosure encompasses an insight that previous formulations, such as those reported in WO2008 / 043575 and WO2009 / 047006 describe use of siRNA in combination with a cationic lipid and an anionically ionizable lipid, but do not report use of mRNA. The present disclosure provides compositions useful for delivery of mRNA using cationic lipids and anionically ionizable lipids such as those provided herein. In some embodiments, a cationic lipid is palmitoyl-oleoyl-nor-arginine (PONA) and an anionically ionizable lipid is dioleoylglycerol hemisuccinate (DOGS). In some embodiments, a cationic lipid is palmitoyl-oleoyl-nor-arginine (PONA). In some embodiments, an anionically ionizable lipid is selected from cholesteryl hemisuccinate (CHEMS), dimyristoylglycerolhemisuccinate (DMGS), and dioleoylglycerolhemisuccinate (DOGS). In some embodiments, an anionically ionizable lipid is cholesteryl hemisuccinate (CHEMS). In some embodiments, an anionically ionizable lipid is dimyristoylglycerolhemisuccinate (DMGS). In some embodiments, an anionically ionizable lipid is dioleoylglycerolhemisuccinate (DOGS). In some embodiments, a cationic lipid is palmitoyl- oleoyl-nor-arginine (PONA) and an anionically ionizable lipid is selected from cholesteryl hemisuccinate (CHEMS), dimyristoylglycerolhemisuccinate (DMGS), and dioleoylglycerolhemisuccinate (DOGS). In some embodiments, an anionically ionizable lipid is tetradecanoic acid (myristic acid). In some embodiments, an anionically ionizable lipid is octadecanoic acid (stearic acid). Page 37 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0117] As described herein, the present disclosure encompasses an insight that identification of particular ratios of a cationic lipid and an anionic or anionically ionizable lipid in a lipid nanoparticle (LNP) formulation can improve transfection and delivery of nucleic acid agents to target cells, and further, in some embodiments, avoid the use of polymer conjugated lipids (such as PEG lipids) to achieve substantially equivalent efficacy. For example, as described herein, a molar ratio of a cationic lipid to an anionically ionizable lipid is referred to as a C / A ratio, and, in some embodiments, is between 1.0 and 1.5. Reference to a single digit as part of the ratio is intended to be said digit over 1. For example, a C / A ratio of 1.5 is intended to refer to a molar ratio of cationic lipid to anionically ionizable lipid in a given composition that is 1.5:1 of the cationic lipid to the anionically ionizable lipid.

[0118] In some embodiments, a composition described herein comprises a C / A ratio from about 0.5 to about 2.5. In some embodiments, a composition described herein comprises a C / A ratio from about 0.67 to about 1.50. In some embodiments, a composition described herein comprises a C / A ratio from about 0.75 to about 1.33. In some embodiments, a composition described herein comprises a C / A ratio from about 0.82 to about 1.20. In some embodiments, a composition described herein comprises a C / A ratio from about 0.90 to about 1.10. In some embodiments, a composition described herein comprises a C / A ratio from about 0.60 to about 1.50. In some embodiments, a composition described herein comprises a C / A ratio that is about 0.60, about 0.65, about 0.70, about 0.75, about 0.80, about 0.85, about 0.90, about 0.95, or about 1.00. In some embodiments a composition described herein comprises a C / A ratio that is about 0.90, about 0.95, about 1.00, about 1.05, about 1.10, about 1.15, or about 1.20. In some embodiments, a composition described herein comprises a C / A ratio that is from about 0.80 to about 1.30. In some embodiments, a composition described herein comprises a C / A that is about 0.80, about 0.85, about 0.90, about 0.95, about 1.00, about 1.05, about 1.10, about 1.15, about 1.20, about 1.25, or about 1.3. In some embodiments, a C / A ratio is about 0.90, about 0.95, about 1.0, about 1.05, or about 1.10. Cationic Lipids

[0119] The present disclosure provides compositions comprising cationic lipids described herein, including, new and useful cationic lipids. In some embodiments, a cationic lipid is a compound represented by formula ICL Page 38 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802or a pharmaceutically acceptable salt thereof, wherein: G1is -L1a-L1b-G1a; L1ais a bond, or optionally substituted C1-C6 aliphatic; L1bis optionally substituted C1-C6aliphatic; G1ais -N(Ra)C(N(Ra)2+)N(Ra)2, -N(Ra)3+, -N(Ra)C(N(Ra))N(Ra)2, -N(Ra)(Rb), - C(Ra)2C(N(Ra)2+)N(Ra)2, -N(Ra)C(O)N(Ra)2, -N(Ra)C(S)N(Ra)2, an optionally substituted 5- to 6-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 4- to 6-membered heterocycle comprising 1 to 3 heteroatoms selected from N, O, and S; B1and B2are each independently -NRZ-, -N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, - OC(O)N(RZ)-, -N(RZ)C(O)N(RZ)-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; T1and T2are each independently optionally substituted C5-C35 aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -Cy-, -NRZ-, -N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ) -, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; each Rais independently H or optionally substituted C1-C6 aliphatic; each Rbis independently selected from H, optionally substituted C1-C6 aliphatic, optionally substituted 5- to 6-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S, and optionally substituted 4- to 6-membered heterocycle comprising 1 to 3 heteroatoms selected from N, O, and S; each -Cy- is independently an optionally substituted C3-C6cycloaliphatic, optionally substituted 5- to 12-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, or optionally substituted 5- to 6-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S; and each RZis independently selected from H, optionally substituted C1-C20aliphatic, and optionally substituted C3-C12 cycloaliphatic.

[0120] As described herein G1is -L1a-L1b-G1a. Page 39 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0121] As described herein, L1ais a bond or optionally substituted C1-C6aliphatic. In some embodiments, L1ais a bond. In some embodiments, L1ais optionally substituted C1-C6 aliphatic. In some embodiments, L1ais C1-C6 aliphatic. In some embodiments, L1ais C1-C6 alkylene. In some embodiments, L1ais methylene, ethylene, propylene, butylene, pentylene, or hexylene.

[0122] In some embodiments, L1bis optionally substituted C1-C6 aliphatic. In some embodiments, L1bis C1-C6 aliphatic. In some embodiments, L1bis C1-C6 alkylene. In some embodiments, L1bis methylene, ethylene, propylene, butylene, pentylene, or hexylene. In some embodiments, L1bis methylene.

[0123] In some embodiments, L1ais a bond, and L1bis optionally substituted C1-C6 aliphatic. In some embodiments, L1ais optionally substituted C1-C6aliphatic, and L1bis optionally substituted C1-C6aliphatic. In some embodiments, L1ais methylene, and L1bis optionally substituted C1-C6 aliphatic. In some embodiments, L1ais methylene, and L1bis methylene or ethylene.

[0124] As described herein, G1ais -N(Ra)C(N(Ra)2+)N(Ra)2, -N(Ra)3+, -N(Ra)C(N(Ra))N(Ra)2, -N(Ra)(Rb), -C(Ra)2C(N(Ra)2+)N(Ra)2, -N(Ra)C(O)N(Ra)2, -N(Ra)C(S)N(Ra)2, an optionally substituted 5- to 6-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 4- to 6-membered heterocycle comprising 1 to 3 heteroatoms selected from N, O, and S.

[0125] In some embodiments, G1ais -N(Ra)C(N(Ra)2+)N(Ra)2. In some embodiments, G1ais - N(H)C(N(H)2+)N(H)2. In some embodiments, G1ais -N(CH3)C(N(H)2+)N(H)2. In some embodiments, G1ais -N(CH3)C(N(H)2+)N(H)2. In some embodiments, G1ais - N(H)C(N(H)2+)N(CH3)2. In some embodiments, G1ais -N(H)C(N(H)2+)N(H)(CH3). In some embodiments, G1ais -N(H)C(N(H)(CH3)+)N(H)2. In some embodiments, G1ais - N(H)C(N(H)(CH3)+)N(CH3)(H).

[0126] In some embodiments, G1ais -N(Ra)3+. In some embodiments, G1ais -N(H)3+. In some embodiments, G1ais -N(H)2(CH3)+. In some embodiments, G1ais -N(H)(CH3)2+. In some embodiments, G1ais -N(CH3)3+.

[0127] In some embodiments, G1ais -N(Ra)C(N(Ra))N(Ra)2. In some embodiments, G1ais - N(H)C(N(H))N(H)2. In some embodiments, G1ais -N(CH3)C(N(H))N(H)2. In some embodiments, G1ais -N(H)C(N(CH3))N(H)2. In some embodiments, G1ais - N(H)C(N(H))N(H)(CH3). In some embodiments, G1ais -N(H)C(N(H))N(CH3)2. Page 40 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0128] In some embodiments, G1ais -N(Ra)(Rb). In some embodiments, G1ais -NH2. In some embodiments, G1ais N(H)(CH3). In some embodiments, G1ais N(H)(Rb), wherein Rbis optionally substituted 5- to 6-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, G1ais N(H)(Rb), wherein Rbis imidazole, oxazole, oxadiazole, or triazole. In some embodiments, G1ais N(H)(Rb), wherein Rbis selected from: . Ra)2C(N(Ra)2+)N(Ra)2. In some embodiments, G1ais-C(H)2C(N(H)2+)N(H)2. In some embodiments, G1ais -C(H)2C(N(H)(CH3)+)N(H)2. In some embodiments, G1ais -C(H)2C(N(H)2+)N(H)(CH3). In some embodiments, G1ais - C(H)2C(N(H)2+)N(CH3)2.

[0130] In some embodiments, G1ais -N(Ra)C(O)N(Ra)2. In some embodiments, G1ais - N(H)C(O)N(H)2. In some embodiments, G1ais -N(CH3)C(O)N(H)2. In some embodiments, G1ais -N(H)C(O)N(H)(CH3). In some embodiments, G1ais -N(H)C(O)N(CH3)2.

[0131] In some embodiments, G1ais -N(Ra)C(S)N(Ra)2. In some embodiments, G1ais - N(H)C(S)N(H)2. In some embodiments, G1ais -N(CH3)C(S)N(H)2. In some embodiments, G1ais -N(H)C(S)N(H)(CH3). In some embodiments, G1ais -N(H)C(S)N(CH3)2.

[0132] In some embodiments, G1ais an optionally substituted 5- to 6-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, G1ais imidazole, oxazole, triazole, or oxadiazole.

[0133] In some embodiments, G1ais an optionally substituted 4- to 6-membered heterocycle comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, G1ais selected from: .

[0134] nts, G1ais selected from the group consisting of: Page 41 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 , ,, , , , ,Page 42 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0137] As described herein, B1and B2are each independently -NRZ-, -N(RZ)C(O)-, - C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ)-, -O-, -C(O)-, -OC(O)-, - C(O)O-, -SO-, or -SO2-. In some embodiments, B1is selected from NRZ-, -N(RZ)C(O)-, - C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ)-, -O-, -C(O)-, -OC(O)-, - C(O)O-, -SO-, or -SO2-. In some embodiments, B1is selected from the group consisting of - C(O)O-, -OC(O)-, -C(O)N(RZ)-, and -N(RZ)C(O)-. In some embodiments, B1is selected from the group consisting of -C(O)N(RZ)- and -N(RZ)C(O)-. In some embodiments, B1is -C(O)N(RZ)-. In some embodiments, B1is -N(RZ)C(O)-. In some embodiments, B1is -C(O)N(H)-. In some embodiments, B1is -C(O)N(RZ)- and wherein RZis optionally substituted C1-C20 aliphatic. In some embodiments, B1is -N(H)C(O)-.

[0138] In some embodiments, B2is selected from NRZ-, -N(RZ)C(O)-, -C(O)N(RZ)-, - N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ)-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-. In some embodiments, B2is selected from the group consisting of -C(O)O-, -OC(O)-, - C(O)N(RZ)-, and -N(RZ)C(O)-. In some embodiments, B2is selected from the group consisting of -C(O)N(RZ)- and -N(RZ)C(O)-. In some embodiments, B2is -C(O)N(RZ)-. In some embodiments, B2is -N(RZ)C(O)-. In some embodiments, B2is -C(O)N(H)-. In some embodiments, B2is - C(O)N(RZ)-,wherein RZis optionally substituted C1-C20aliphatic. In some embodiments, B2is - N(H)C(O)-.

[0139] In some embodiments, B1and B2are each independently selected from -C(O)O-, - OC(O)-, -C(O)N(RZ)-, and -N(RZ)C(O)-. In some embodiments, B1and B2are each independently selected from -C(O)N(RZ)-, and -N(RZ)C(O)-. In some embodiments, one of B1and B2is - C(O)N(RZ)-, and the other of B1and B2is -N(RZ)C(O)-. In some embodiments, one of B1and B2is -C(O)N(H)-, and the other of B1and B2is -N(H)C(O)-.

[0140] As described herein, T1and T2are each independently optionally substituted C5-C35aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -Cy-, -NRZ-, -N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ) -, -O- , -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-. In some embodiments, T1is optionally substituted C10-C35aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -Cy-, -NRZ-, -N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, - N(RZ)C(O)N(RZ) -, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-. In some embodiments, T1is optionally substituted C10-C35aliphatic group, wherein one or more carbon atoms are optionally Page 43 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 and independently replaced by -OC(O)- or -C(O)O-. In some embodiments, T1is optionally substituted C10-C35 aliphatic group. In some embodiments, T1is an optionally substituted branched C10-C35 aliphatic group.

[0141] In some embodiments, T1is represented by formula: wherein:X1is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ)-, or -N(RZ)C(O)-; T1ais H or optionally substituted C1-C20aliphatic; T1bis optionally substituted C1-C20 aliphatic; and n1 is 1 to 10.

[0142] As described herein, in some embodiments, X1is a bond, -C(O)O-, -OC(O)-, - C(O)N(RZ)-, or -N(RZ)C(O)-. In some embodiments, X1is a bond. In some embodiments, X1is -C(O)O- or -OC(O)-. In some embodiments, X1is -C(O)O-. In some embodiments, X1is -OC(O)- . In some embodiments, X1is -C(O)N(RZ)-. In some embodiments, X1is -C(O)N(H)-. In some embodiments, X1is -C(O)N(RZ), wherein RZis optionally substituted C1-C20aliphatic. In some embodiments, X1is -C(O)N(RZ), wherein RZis optionally substituted C1-C20 aliphatic, and wherein T1ais H. In some embodiments, X1is -N(RZ)C(O)-. In some embodiments, X1is - N(H)C(O)-.

[0143] As described herein, T1ais H or optionally substituted C1-C20 aliphatic. In some embodiments, T1ais H. In some embodiments, T1ais optionally substituted C1-C10 aliphatic.

[0144] As described herein, T1bis optionally substituted C1-C20aliphatic. In some embodiments, T1bis optionally substituted C1-C10 aliphatic.

[0145] As described herein, n1 is 1 to 10. In some embodiments, n1 is 1 to 7. In some embodiments, n1 is 2 to 5. In some embodiments, n1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0146] In some embodiments, T1is selected from the group consisting of ,Page 44 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 nd orof , nd orPage 45 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0149] In some embodiments, T1is selected from ,and p, wherein oneor more carbon atoms are optionally and independently replaced by -Cy-, -NRZ-, -N(RZ)C(O)-, - C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ) -, -O-, -C(O)-, -OC(O)-, - C(O)O-, -SO-, or -SO2-. In some embodiments, T2is optionally substituted C10-C35 aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -OC(O)- or -C(O)O-. In some embodiments, T2is optionally substituted C10-C35aliphatic group. In some embodiments, T2is an optionally substituted branched C10-C35 aliphatic group.

[0151] In some embodiments, T2is represented by formula: whereinX2is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ), or -N(RZ)C(O)-; T2ais H or optionally substituted C1-C20aliphatic; T2bis optionally substituted C1-C20aliphatic; and n2 is 1 to 10.

[0152] As described herein, in some embodiments, X2is a bond, -C(O)O-, -OC(O)-, - C(O)N(RZ)-, or -N(RZ)C(O)-. In some embodiments, X2is a bond. In some embodiments, X2is -C(O)O- or -OC(O)-. In some embodiments, X2is -C(O)O-. In some embodiments, X2is -OC(O)- . In some embodiments, X2is -C(O)N(RZ)-. In some embodiments, X2is -C(O)N(H)-. In some embodiments, X2is -C(O)N(RZ), wherein RZis optionally substituted C1-C20aliphatic. In some embodiments, X2is -C(O)N(RZ), wherein RZis optionally substituted C1-C20 aliphatic, and wherein T2ais H. In some embodiments, X2is -N(RZ)C(O)-. In some embodiments, X2is - N(H)C(O)-.

[0153] As described herein, T2ais H or optionally substituted C1-C20aliphatic. In some embodiments, T2ais H. In some embodiments, T2ais optionally substituted C1-C10 aliphatic. Page 46 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0154] As described herein, T2bis optionally substituted C1-C20aliphatic. In some embodiments, T2bis optionally substituted C1-C10 aliphatic.

[0155] As described herein, n2 is 1 to 10. In some embodiments, n2 is 1 to 7. In some embodiments, n2 is 2 to 5. In some embodiments, n2 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0156] In some embodiments, T2is selected from the group consisting of , and or, g g of , and12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 or,andbstituted C1-C6aliphatic. In some embodiments, Rais H. In some embodiments, Rais optionally substituted C1- C6 aliphatic. In some embodiments, Rais methyl, ethyl, or propyl. In some embodiments, Rais - CH3.

[0161] As described herein, each Rbis independently selected from H, optionally substituted C1-C6 aliphatic, optionally substituted 5- to 6-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S, and optionally substituted 4- to 6-membered heterocycle comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, Rbis H. In some embodiments, Rbis optionally substituted C1-C6 aliphatic. In some embodiments, Rbis methyl, ethyl, or propyl.

[0162] In some embodiments, Rbis optionally substituted 5- to 6-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, Rbis imidazole, oxazole, oxadiazole, or triazole. In some embodiments, Rbis selected from: ., tionally substituted 4- to 6-membered heterocycle comprising 1 to 3 heteroatoms selected from N, O, and S. Page 48 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0164] As described herein, each -Cy- is independently an optionally substituted C3-C6cycloaliphatic, optionally substituted 5- to 12-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, or optionally substituted 5- to 6-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S.

[0165] As described herein, each RZis independently selected from H, optionally substituted C1-C20 aliphatic, and optionally substituted C3-C12 cycloaliphatic. In some embodiments, RZis H. In some embodiments RZis optionally substituted C1-C20aliphatic. In some embodiments, RZis optionally substituted C3-C12 cycloaliphatic.

[0166] In some embodiments of formula ICL, moiety: is a lipidic group, and is the relevant moidetermining lipidic volume of a cationic lipid, e.g., a compound of formula ICL.

[0167] In some embodiments of formula ICL, moiety: has a lipidic volume from about 700 Å3t30 Å . In some embodiments of formula ICL, moiety: has a lipidic volume from about 700 Å3t0 Å3, or from about 700 Å3to about 1300 Å3, or from about 700 Å3to about 1200 Å3, or from about 700 Å3to about 1100 Å3, or from about 700 Å3to about 1000 Å3, or from about 700 Å3to about 900 Å3, or from about 800 Å3to about 1500 Å3, or from about 900 Å3to about 1400 Å3.

[0168] In some embodiments of formula ICL, moiety:Page 49 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 has a lipidic volume of about 700 Å3, about 750 Å3, about 800 Å3, about 850 Å3, about 900 Å3, about 950 Å3, about 1000 Å3, about 1050 Å3, about 1100 Å3, about 1150 Å3, about 1200 Å3, about 1250 Å3, about 1300 Å3, about 1350 Å3, about 1400 Å3, about 1450 Å3, or about 1500 Å3.

[0169] In some embodiments, a compound described herein is a compound of formula ICL, or a pharmaceutically acceptable salt thereof, wherein: L1ais optionally substituted C1-C6 aliphatic; L1bis optionally substituted C1-C6aliphatic; G1ais -N(Ra)C(N(Ra)2+)N(Ra)2 or -N(Ra)3+; B1and B2are each independently -N(RZ)C(O)-, -C(O)N(RZ)-, -OC(O)-, or -C(O)O-; T1and T2are each independently optionally substituted C10-C35aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -OC(O)- or - C(O)O-; each Rais independently H or optionally substituted C1-C6 aliphatic; and each RZis independently selected from H, optionally substituted C1-C20aliphatic, and optionally substituted C3-C12 cycloaliphatic.

[0170] In some embodiments, a compound of formula ICL is represented by formula ICL-IIor a pharmaceutically acceptable salt thereof, wherein G1, B1, B2, n1, n2, X1, X2, T1a, T1b, T2a, and T2bare as described in classes and subclasses herein, both singly and in combination.

[0171] In some embodiments, a compound of formula ICL-II is represented ICL-IIa or ICL- IIb: Page 50 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802or a pharmaceutically acceptable salt thereof, wherein G1, n1, n2, X1, X2, T1a, T1b, T2a, and T2bare as described in classes and subclasses herein, both singly and in combination.

[0172] In some embodiments, a compound of formula ICL-II is represented ICL-IIc or ICL- IId:or a pharmaceutically acceptable salt thereof, wherein G1, n1, n2, X1, X2, T1a, T1b, T2a, and T2bare as described in classes and subclasses herein, both singly and in combination, and wherein * represents a chiral carbon atom.

[0173] In some embodiments, a compound of formula ICL-II is represented by formula ICL- III:or a pharmaceutically acceptable salt thereof, wherein n1, n2, X1, X2, T1a, T1b, T2a, and T2bare as described in classes and subclasses herein, both singly and in combination. Page 51 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0174] In some embodiments, a compound of formula ICL-III is represented by formula ICL- IIIa or ICL-IIIb:or a pharmaceutically acceptable salt thereof, wherein n1, n2, X1, X2, T1a, T1b, T2a, and T2bare as described in classes and subclasses herein, both singly and in combination.

[0175] In some embodiments, a compound of formula ICL-III is represented by formula ICL- IIIc or ICL-IIId:or a pharmaceutically acceptable salt thereof, wherein n1, n2, X1, X2, T1a, T1b, T2a, and T2bare as described in classes and subclasses herein, both singly and in combination.

[0176] In some embodiments, a compound of formula ICL-II is represented by formula ICL- IV:or a pharmaceutically acceptable salt thereof, wherein n1, n2, X1, X2, T1a, T1b, T2a, and T2bare as described in classes and subclasses herein, both singly and in combination. Page 52 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0177] In some embodiments, a compound of formula ICL-IV is represented by formula ICL- IVa or ICL-IVb:or a pharmaceutically acceptable salt thereof, wherein n1, n2, X1, X2, T1a, T1b, T2a, and T2bare as described in classes and subclasses herein, both singly and in combination.

[0178] In some embodiments, a compound of formula ICL-III is represented by formula ICL- IVc or ICL-IVd:or a pharmaceutically acceptable salt thereof, wherein n1, n2, X1, X2, T1a, T1b, T2a, and T2bare as described in classes and subclasses herein, both singly and in combination.

[0179] With respect to formulas ICL-IIc, ICL-IId, ICL-IIIc, ICL-IIId, ICL-IVc, and ICL-IVd, * indicates a chiral carbon atom. In some embodiments of formulas ICL-IIc, ICL-IId, ICL-IIIc, ICL-IIId, ICL-IVc, and ICL-IVd, * indicates a carbon atom having (S) stereochemical configuration. In some embodiments of formulas ICL-IIc, ICL-IId, ICL-IIIc, ICL-IIId, ICL-IVc, and ICL-IVd, * indicates a carbon atom having (R) stereochemical configuration.

[0180] In some embodiments, a cationic lipid is selected from Table 1 Table 1 Page 53 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound No12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 55 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 56 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 57 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 58 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 59 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound No12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 61 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound Noage o 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound No12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 64 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 65 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 66 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 67 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 68 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 69 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 70 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 71 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 72 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 73 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 74 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 75 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 76 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound No12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound No

[0181] In some embodiments, a cationic lipid is a compound of formula ICL, and is selected from Table 2A: Table 2A Structure Compound NoPage 78 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 79 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 80 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 81 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 82 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound Nog 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound No12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 85 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound Noage o 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 87 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 88 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 89 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 90 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 91 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 92 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 93 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 94 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 95 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 96 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 97 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 98 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 99 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 100 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 101 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound No12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound No dTable 2B Structure Compound NoPage 103 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound No

[0183] In some embodiments, provided compounds are provided and / or utilized in a salt form (e.g., a pharmaceutically acceptable salt form). Reference to a compound provided herein is understood to include reference to salts thereof, unless otherwise indicated. Anionically Ionizable Lipids

[0184] The present disclosure provides compositions comprising anionically ionizable lipids described herein, including, new and useful anionically ionizable lipids. In some embodiments, an anionically ionizable lipid is represented by formula IAL:or a pharmaceutically acceptable salt thereof, wherein: G2is -L2a-L2b-G2a-G2b; L2ais a bond or optionally substituted C1-C6 aliphatic; Page 104 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 L2bis a bond, -C(O)O-, or -OC(O)-; G2ais a optionally substituted C1-C6 aliphatic; G2bis -C(O)OH or -P(O)2(OH)2; B3and B4are each independently selected from –C(O)O-, -OC(O)-, or a C2-C7aliphatic group, wherein one carbon atom is optionally replaced with -OC(O)- or -C(O)O-; T3and T4are each independently optionally substituted C5-C35 aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -Cy-, -NRZ-, -N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ) -, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; each -Cy- is independently an optionally substituted C3-C6cycloaliphatic, optionally substituted 5- to 12-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, S, or optionally substituted 5- to 6-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, S; and each RZis independently selected from H, optionally substituted C1-C20aliphatic, and optionally substituted C3-C12 cycloaliphatic.

[0185] As described generally herein, G2is -L2a-L2b-G2a-G2b.

[0186] As described generally herein, L2ais bond or optionally substituted C1-C6aliphatic. In some embodiments, L2ais a bond. In some embodiments, L2ais optionally substituted C1-C6aliphatic. In some embodiments, L2ais optionally substituted C1-C6 alkylene. In some embodiments, L2ais methylene, ethylene, propylene, butylene, pentylene, or hexylene. In some embodiments, L2ais methylene.

[0187] As described generally herein, L2bis bond, -C(O)O-, or -OC(O)-. In some embodiments, L2bis a bond. In some embodiments, L2bis -C(O)O-. In some embodiments, L2bis -OC(O)-.

[0188] As described generally herein, G2ais optionally substituted C1-C6aliphatic. In some embodiments, G2ais optionally substituted C1-C6 alkylene. In some embodiments, G2ais methylene, ethylene, propylene, butylene, pentylene, or hexylene.

[0189] As described generally herein, G2bis -C(O)OH or -P(O)2(OH). In some embodiments, G2bis -C(O)OH. In some embodiments, G2bis -P(O)2(OH).

[0190] In some embodiments, L2ais methylene, L2bis -C(O)O-, G2ais C1-C6 aliphatic, and G2bis -C(O)OH. Page 105 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 , -, -OC(O)-, and a C2-C7 aliphatic group, wherein one carbon atom of the C2-C7 aliphatic group is optionally replaced with -OC(O)- or -C(O)O-. In some embodiments, B3is selected from –C(O)O- , -OC(O)-, and a C2-C7aliphatic group, wherein one carbon atom of the C2-C7aliphatic group is optionally replaced with -OC(O)- or -C(O)O-. In some embodiments, B3is -C(O)O-. In some embodiments, B3is -OC(O)-. In some embodiments, B3is a C2-C7aliphatic group, wherein one carbon atom of the C2-C7aliphatic group is optionally replaced with -OC(O)- or -C(O)O-. In some embodiments, B3is selected from , wherein ss represents a point of attachment to T3.

[0193] In some embodiments, B4is selected from –C(O)O-, -OC(O)-, and a C2-C7 aliphatic group, wherein one carbon atom of the C2-C7aliphatic group is optionally replaced with -OC(O)- or -C(O)O-. In some embodiments, B4is -C(O)O-. In some embodiments, B4is -OC(O)-. In some embodiments, B4is a C2-C7 aliphatic group, wherein one carbon atom of the C2-C7 aliphatic group is optionally replaced with -OC(O)- or -C(O)O-. In some embodiments, B4is selected from , wherein tt represents a point of attachment to T4.

[0194] In some embodiments, B3is selected from , and B4isselected from , wherein ss represents a point of attachment to T3,and tt represents a point of attachment to T4. Page 106 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0195] As described herein, T3and T4are each independently optionally substituted C5-C35aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -Cy-, -NRZ-, -N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ) -, -O- , -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-. In some embodiments, T3is optionally substituted C10-C35 aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -Cy-, -NRZ-, -N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, - N(RZ)C(O)N(RZ) -, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-. In some embodiments, T3is optionally substituted C10-C35 aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -OC(O)- or -C(O)O-. In some embodiments, T3is optionally substituted C10-C35aliphatic group. In some embodiments, T3is an optionally substituted branched C10-C35aliphatic group.

[0196] In some embodiments, T3is represented by formula: whereinX3is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ)-, or -N(RZ)C(O)-; T3ais H or optionally substituted C1-C20aliphatic; T3bis optionally substituted C1-C20aliphatic; and n3 is 1 to 10.

[0197] As described herein, in some embodiments, X3is a bond, -C(O)O-, -OC(O)-, - C(O)N(RZ)-, or -N(RZ)C(O)-. In some embodiments, X3is a bond. In some embodiments, X3is - C(O)O- or -OC(O)-. In some embodiments, X3is -C(O)O-. In some embodiments, X3is -OC(O)- .

[0198] As described herein, T3ais H or optionally substituted C1-C20aliphatic. In some embodiments, T3ais H. In some embodiments, T3ais optionally substituted C1-C10aliphatic.

[0199] As described herein, T3bis optionally substituted C1-C20 aliphatic. In some embodiments, T3bis optionally substituted C1-C10aliphatic.

[0200] As described herein, n3 is 1 to 10. In some embodiments, n3 is 1 to 7. In some embodiments, n3 is 2 to 5. In some embodiments, n3 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Page 107 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0201] In some embodiments, T3is selected from the group consisting of nd, of ndPage 108 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 ,and .2 I i T4i ed by formula:whereinX4is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ)-, or -N(RZ)C(O)-; T4ais H or optionally substituted C1-C20 aliphatic; T4bis optionally substituted C1-C20 aliphatic; and n4 is 1 to 10.

[0206] As described herein, in some embodiments, X4is a bond, -C(O)O-, -OC(O)-, - C(O)N(RZ)-, or -N(RZ)C(O)-. In some embodiments, X4is a bond. In some embodiments, X4is - C(O)O- or -OC(O)-. In some embodiments, X4is -C(O)O-. In some embodiments, X4is -OC(O)- .

[0207] As described herein, T4ais H or optionally substituted C1-C20 aliphatic. In some embodiments, T4ais H. In some embodiments, T4ais optionally substituted C1C10aliphatic.

[0208] As described herein, T4bis optionally substituted C1-C20aliphatic. In some embodiments, T4bis optionally substituted C1-C10 aliphatic.

[0209] As described herein, n4 is 1 to 10. In some embodiments, n4 is 1 to 7. In some embodiments, n4 is 2 to 5. In some embodiments, n4 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0210] In some embodiments, T4is selected from the group consisting ofPage 109 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 and, of nd ,age o 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0213] In some embodiments, T4is selected from and .

[0214] As described herein each Rais independently H or optionally substituted C1-C6aliphatic. In some embodiments, Rais H. In some embodiments, Rais optionally substituted C1- C6 aliphatic. In some embodiments, Rais methyl, ethyl, or propyl. In some embodiments, Rais - CH3.

[0215] As described herein, each -Cy- is independently an optionally substituted C3-C6cycloaliphatic, optionally substituted 5- to 12-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, or optionally substituted 5- to 6-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S.

[0216] As described herein, each RZis independently selected from H, optionally substituted C1-C20 aliphatic, and optionally substituted C3-C12 cycloaliphatic. In some embodiments, RZis H. In some embodiments RZis optionally substituted C1-C20aliphatic. In some embodiments, RZis optionally substituted C3-C12 cycloaliphatic.

[0217] In some embodiments of formula IAL, moiety: is a lipidic group, and is the relevant moier determining lipidic volume of an anionically ionizable lipid, e.g., a compound of formula IAL.

[0218] In some embodiments of formula IAL, moiety: has a lipidic volume from about 650 Å3to0 Å3. In some embodiments of formula IAL, moiety: has a lipidic volume from about 700 Å3to00 Å3, or from about 700 Å3to about 1200 Å3, or from about 700 Å3to about 900 Å3. Page 111 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0219] In some embodiments, a compound of formula IAL is a compound represented by IAL- II:or a pharmaceutically acceptable salt thereof, wherein G2, T3, and T4, are as described in classes and subclasses herein, both singly and in combination.

[0220] In some embodiments, a compound of formula IAL-II is a compound represented by IAL-III:or a pharmaceutically acceptable salt thereof, wherein G2is as described in classes and subclasses herein, both singly and in combination; X3is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ)-, or -N(RZ)C(O)-; T3ais H or optionally substituted C1-C20aliphatic; T3bis optionally substituted C1-C20 aliphatic; n3 is 1 to 10; X4is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ)-, or -N(RZ)C(O)-; T4ais H or optionally substituted C1-C20 aliphatic; T4bis optionally substituted C1-C20 aliphatic; and n4 is 1 to 10.

[0221] In some embodiments, a compound represented by formula IAL-II is a compound represented by IAL-IV: Page 112 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802or a pharmaceutically acceptable salt thereof, wherein T3and T4are as described in classes and subclasses herein, both singly and in combination.

[0222] In some embodiments, a compound represented by formula IAL-II is a compound represented by IAL-V:or a pharmaceutically acceptable salt thereof, wherein T3and T4are as described in classes and subclasses herein, both singly and in combination, and wherein * indicates a chiral carbon atom. In some embodiments of formula IAL-V, * indicates a carbon atom having (S) stereochemical configuration. In some embodiments of formula IAL-V, * indicates a carbon atom having (R) stereochemical configuration.

[0223] In some embodiments, a compound described herein is a compound of formula IAL, or a pharmaceutically acceptable salt thereof, wherein: G2is -L2a-L2b-G2a-G2b; L2ais optionally substituted C1-C6 aliphatic; L2bis a -C(O)O- or -OC(O)-; G2ais optionally substituted C1-C6 aliphatic; G2bis -C(O)OH; B3and B4are each independently selected from –C(O)O-, -OC(O)-, or a C2-C7aliphatic group, wherein one carbon atom is optionally replaced with -OC(O)- or -C(O)O-; and T3and T4are each independently optionally substituted C5-C35 aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -OC(O)- or - C(O)O-. Page 113 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0224] In some embodiments, an anionically ionizable lipid is selected from Table 3 Table 3 Structure Compound NoPage 114 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 115 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 116 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound No

[0225] In some embodiments, an anionically ionizable lipid is a compound of formula IAL, and is selected from Table 4 Table 4 Structure Compound NoPage 117 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound No12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 119 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Structure Compound NoPage 120 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0226] In some embodiments, provided compounds are provided and / or utilized in a salt form (e.g., a pharmaceutically acceptable salt form). Reference to a compound provided herein is understood to include reference to salts thereof, unless otherwise indicated. Nanoparticles

[0227] In some embodiments, a composition described herein is or comprises particles, e.g., nanoparticles or nucleic acid particles or lipid nanoparticles. That is, as used herein, a composition can, in some embodiments, comprise the components of a nanoparticle, e.g., a lipid nanoparticle, and said components can form nanoparticles when present in the same composition. As used in the present disclosure, “nanoparticle,” refers to a particle having an average diameter suitable for parenteral administration and is less than 1000 nm in diameter. In some embodiments, a composition comprising nanoparticles can have an average nanoparticle size (e.g., mean diameter) of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 70 to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, a composition comprising nanoparticles can have an average nanoparticle size (e.g., mean diameter) of about 50 nm to about 100 nm. In some embodiments, a composition comprising nanoparticles can have an average nanoparticle size (e.g., mean diameter) of about 50 nm to about 150 nm. In some embodiments, a composition comprising nanoparticles can have an average nanoparticle size (e.g., mean diameter) of about 60 nm to about 120 nm. In some embodiments, a composition comprising nanoparticles can have an average nanoparticle size (e.g., mean diameter) of about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. The term “average diameter” or “mean diameter” refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so- called cumulant algorithm, which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PDI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here “average diameter,” “mean diameter,” “diameter,” or “size” for particles is used synonymously with this value of the Z-average.

[0228] A composition comprising nanoparticles can be characterized by its polydispersity index, that is, the relative uniformity of particles within a given composition. For example, Page 121 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 compositions described herein may exhibit a polydispersity index less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less of said nanoparticles. By way of example, a composition comprising nucleic acid particles described herein can exhibit a polydispersity index in a range of about 0.1 to about 0.3 or about 0.2 to about 0.3. The polydispersity index of a given composition that comprises nanoparticles can be calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the “average diameter.”

[0229] Nanoparticles that comprise a nucleic acid (also referred to as nucleic acid particles), as described herein, can be characterized by an “N / P ratio,” which is the molar ratio of cationic (nitrogen) groups (the “N” in N / P) in the cationic polymer to the anionic (phosphate) groups (the “P” in N / P) in RNA. It is understood that a cationic group is one that is either in cationic form (e.g., N+), or one that is ionizable to become cationic. Use of a single number in an N / P ratio (e.g., an N / P ratio of about 5) is intended to refer to that number over 1, e.g., an N / P ratio of about 4 is intended to mean about 4:1. In some embodiments, a nucleic acid particle (e.g., a ribonucleic acid particle) described herein has an N / P ratio greater than or equal to 4. In some embodiments, a nucleic acid particle (e.g., a ribonucleic acid particle) described herein has an N / P ratio that is about 4 to about 12. In some embodiments, a nucleic acid particle (e.g., a ribonucleic acid particle) described herein has an N / P ratio that is about 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, an N / P ratio for a nucleic acid particle (e.g., a ribonucleic acid particle) described herein is from about 6.

[0230] In a nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) composition, it is possible that each nucleic acid species is separately formulated as an individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulation. In that case, each individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulation will comprise one nucleic acid species. The individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulations may be present as separate entities, e.g., in separate containers. Such formulations are obtainable by providing each nucleic acid species separately (typically each in the form of a nucleic acid-containing solution) together with a particle-forming agent, thereby allowing the formation of particles. Respective particles will contain exclusively the specific Page 122 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 nucleic acid species that is being provided when the particles are formed (individual particulate formulations).

[0231] In some embodiments, a composition such as a pharmaceutical composition comprises more than one individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulation. Respective pharmaceutical compositions are referred to as “mixed particulate formulations.” Mixed particulate formulations according to the invention are obtainable by forming, separately, individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulations, as described above, followed by a step of mixing of the individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulations. By the step of mixing, a formulation comprising a mixed population of nucleic acid-containing particles is obtainable. Individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) populations may be together in one container, comprising a mixed population of individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulations.

[0232] Alternatively, it is possible that different nucleic acid species are formulated together as a “combined particulate formulation.” Such formulations are obtainable by providing a combined formulation (typically combined solution) of different nucleic acid species together with a particle-forming agent, thereby allowing the formation of particles. As opposed to a “mixed particulate formulation,” a “combined particulate formulation” will typically comprise particles that comprise more than one nucleic acid species. In a combined particulate composition different nucleic acid species are typically present together in a single particle.

[0233] As described herein, in some embodiments, provided particles are lipid nanoparticles (LNPs). It is understood that a lipid nanoparticle is structurally distinct from other nanoparticles previously used for nucleic acid delivery, such as a liposome, or a lipoplex, in that lipid nanoparticles described herein do not comprise a lipid bilayer or a single core that comprises a nucleic acid agent. See, e.g., ACS Nano 2021, 15, 11, 16982–17015. Moreover, lipid nanoparticles are generally understood to not only lack a lipid bilayer, but instead present a micelle-like structure that encapsulates an agent for delivery (e.g., an RNA) inside a non-aqueous core. See Aldosari, et al., Pharmaceutics, 2021, 13, 206.

[0234] In some embodiments, the present disclosure provides a lipid nanoparticles comprising a cationic lipid, an anionically ionizable lipid, a helper lipid, and a steroid. In some embodiments, Page 123 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 a lipid nanoparticle comprises a polymer-conjugated lipid. In some embodiments, a lipid nanoparticle described herein does not comprise a polymer-conjugated lipid. (i) Helper lipids

[0235] In some embodiments, compositions (e.g. nanoparticles) described herein comprise a helper lipid (also referred to as a neutral lipid). In some embodiments, a helper lipid is a phospholipid. In some embodiments, a helper lipid is or comprises 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl- sn-glycero-3- phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phophatidyl ethanol amines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelins (SM), 1,2‑diacylglyceryl-3-O-4'-(N,N,N-trimethyl)-homoserine (DGTS), ceramides, cholesterol, steroids, such as sterols and their derivatives.

[0236] In some embodiments, a helper lipid is or comprises phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin. In some embodiments, a helper lipid is or comprises diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC) and phosphatidylethanolamines, including, for example diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPG), 1,2-dipalmitoyl-sn-glycero-3- phospho-(1′-rac-glycerol) (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM). In some embodiments, a Page 124 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 helper lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the helper lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the helper lipid is DSPC.

[0237] Helper lipids may be synthetic or naturally derived. Other helper lipids suitable for use in a lipid nanoparticle are described in WO2021 / 026358, WO 2017 / 075531, and WO 2018 / 081480, the entire contents of each of which are incorporated herein by reference.

[0238] In some embodiments, a nanoparticle described herein comprises about 0 to about 50 mol% of a helper lipid. In some embodiments, a nanoparticle described herein comprises about 10 to about 40 mol% of a helper lipid. In some embodiments, a nanoparticle described herein comprises about 5 to about 15 mol% of a helper lipid. In some embodiments, a nanoparticle comprises about 5 to about 15 mol% of a phospholipid. In some embodiments, a nanoparticle comprises about 8 to about 12 mol% of a phospholipid. In some embodiments, a nanoparticle comprises about 10 mol% of a phospholipid. In some embodiments, a nanoparticle comprises about 5 to about 15 mol% of DSPC. In some embodiments, a nanoparticle comprises about 8 to about 12 mol% of DSPC. In some embodiments, a nanoparticle comprises about 10 mol% of DSPC. (ii) Steroids

[0239] In some embodiments, compositions (e.g., nanoparticles) described herein further comprise a steroid. In some embodiments, a steroid is a sterol. In some embodiments, a sterol is β-sitosterol, stigmasterol, cholesterol, cholecalciferol, ergocalciferol, calcipotriol, botulin, lupeol, ursolic acid, oleanolic acid, cycloartenol, lanosterol, or α-tocopherol. In some embodiments, a sterol is β-sitosterol. In some embodiments, a sterol is stigmasterol. In some embodiments, a sterol is cholesterol. In some embodiments, a sterol is cholecalciferol. In some embodiments, a sterol is ergocalciferol. In some embodiments, a sterol is calcipotriol. In some embodiments, a sterol is botulin. In some embodiments, a sterol is lupeol. In some embodiments, a sterol is ursolic acid. In some embodiments, a sterol is oleanolic acid. In some embodiments, a sterol is cycloartenol. In some embodiments, a sterol is lanosterol. In some embodiments, a sterol is α- tocopherol.

[0240] In some embodiments, a nanoparticle comprises about 39 to about 49 mol% of a steroid. In some embodiments, a nanoparticle comprises about 40 to about 46 mol% of a steroid. In some embodiments, a nanoparticle comprises about 40 to about 44 mol% of a steroid. In some Page 125 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 embodiments, a nanoparticle comprises about 20 to about 45 mol% of a steroid. In some embodiments, a nanoparticle comprises about 40, 41, 42, 43, 44, or 45 mol% of a steroid. (iii) Polymer Conjugated Lipids

[0241] In some embodiments, compositions described herein comprise a polymer-conjugated lipid. In some embodiments, a polymer conjugated lipid is a lipid conjugated to polyethylene glycol (a “PEG-lipid”). In some embodiments, a PEG lipid is selected from pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)- 2,3- dimyristoylglycerol, (PEG-DMG) (e.g., 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG)), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000 amine), a pegylated ceramide (PEG-cer), a PEG dialkoxypropylcarbamate such as ω-m ethoxy (polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate, and 2,3-di(tetradecanoxy)propy 1- Ν-(ω methoxy(polyethoxy)ethyl)carbamate, or pegylated dimyristophosphatidylethanolamine (PEG-DMPE) such as 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (DMPE-PEG2k).

[0242] In some embodiments, a PEG-lipid is PEG2000-DMG:

[0243] In some embodi

[0244] In some embodiments, a PEG-lipid is DMPE-PEG2k (PEG2000-DMPE):

[0245] In some embodiments, a PEG-lipid is provided in WO2021 / 026358, WO 2017 / 075531, or WO 2018 / 081480, each of which is incorporated by reference in its entirety.

[0246] In some embodiments, a PEG-lipid is a compound of Formula PCL-I: Page 126 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802or a pharmaceutically acceptable salt thereof, wherein, as applied to formula PCL-I, R8and R9are each independently C10-C30 aliphatic, optionally interrupted by one or more ester bonds, and w is an integer from 30 to 60.

[0247] In some embodiments, a compound of Formula PCL-I is 2-[(polyethylene glycol)-2000]- N,N-ditetradecylacetamide (ALC-0159). In some embodiments, a compound of Formula PCL-I is: or a pharmaceuticallabout 45 to about 50.

[0248] In some embodiments, the PEG-lipid is represented by: wherei0. In one embodiment, the PEG-conjugated lipid (pegylated lipid) is PEG2000-C-DMA which preferably refers to 3-N-[(ω-methoxy poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (MPEG-(2 kDa)-C-DMA) or methoxy-polyethylene glycol-2,3- bis(tetradecyloxy)propylcarbamate (2000).

[0249] In some embodiments, a PEG-lipid is selected from PEG-DAG, PEG-PE, PEG-S-DAG, PEG2000-DMG, PEG-S-DMG, PEG-cer, a PEG dialkyoxypropylcarbamate (e.g., ^- methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3- di(tetradecanoxy)propyl-N-(^ methoxy(polyethoxy)ethyl)carbamate), ALC-0159, and combinations thereof. In some embodiments, a PEG-lipid is ALC-0159 or PEG2000-DMG. In some embodiments, a PEG-lipid is ALC-0159. In some embodiments, a PEG-lipid is PEG2000- Page 127 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 DMG. In some embodiments, a PEG-lipid is PEG-DAG. In some embodiments, a PEG-lipid is PEG-PE. In some embodiments, a PEG-lipid is PEG-S-DAG. In some embodiments, a PEG-lipid is PEG-cer. In some embodiments, a PEG-lipid is a PEG dialkyoxypropylcarbamate.

[0250] In some embodiments, a PEG group that is part of a PEG-lipid has, on average in a composition comprising one or more PEG-lipid molecules, a number average molecular weight (Mn) of about 2000 g / mol.

[0251] In some embodiments, a polymer-conjugated lipid is a polysarcosine-conjugated lipid, also referred to herein as sarcosinylated lipid or pSar-lipid. The term "sarcosinylated lipid" refers to a molecule comprising both a lipid portion and a polysarcosine (poly(N-methylglycine) portion.

[0252] In some embodiments, a polymer-conjugated lipid is a polyoxazoline (POX)-conjugated and / or polyoxazine (POZ)-conjugated lipid, also referred to herein as a conjugate of a POX and / or POZ polymer and one or more hydrophobic chains or as oxazolinylated and / or oxazinylated lipid or POX- and / or POZ-lipid. The term "oxazolinylated lipid" or "POX-lipid" refers to a molecule comprising both a lipid portion and a polyoxazoline portion. The term "oxazinylated lipid" or "POZ-lipid" refers to a molecule comprising both a lipid portion and a polyoxazine portion. The term "oxazolinylated / oxazinylated lipid" or "POX / POZ-lipid" or "POXZ-lipid" refers to a molecule comprising both a lipid portion and a portion of a copolymer of polyoxazoline and polyoxazine.

[0253] In some embodiments, an LNP herein may comprise an oxazolinylated and / or / oxazinylated lipid. In some embodiments, the nucleic acid compositions (such as DNA or RNA compositions, especially mRNA compositions) described herein comprise an oxazolinylated and / or / oxazinylated lipid and are substantially free of a pegylated lipid (or do not contain a pegylated lipid).

[0254] In some embodiments, a polymer-conjugated lipid is one described in WO2024 / 028325, which is incorporated herein by reference in its entirety.

[0255] In some embodiments, a polymer conjugated lipid comprises “n” monomers of the following structure:Page 128 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0256] In some embodiments of the above formulas, n is 5 to 50. In some embodiments, n is 5 to 25. In some embodiments, n is 7 to 14. In some embodiments, n is 10 to 25. In some embodiments, n is 14 to 17. In some embodiments, n is 8 or 14.

[0257] In some embodiments, a polymer conjugated lipid comprises monomers of the following structure: .

[0258] In some embodiments, aelected from the table below: Page 129 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 DSPE- AEEA14- AC

[0259] In some embodiments, a nanoparticle comprises about 0.5 to about 5.0 mol% of a polymer- conjugated lipid. In some embodiments, a nanoparticle comprises about 1.0 to about 2.5 mol% of a polymer-conjugated lipid. In some embodiments, a nanoparticle comprises about 1.5 to about 2.0 mol% of a polymer-conjugated lipid. In some embodiments, a nanoparticle comprises about 1.5 to about 1.8 mol% of a polymer-conjugated lipid. In some embodiments, a nanoparticle comprises about 1.0 mol% of a polymer-conjugated lipid. In some embodiments, a nanoparticle comprises about 1.5 mol% of a polymer-conjugated lipid. Page 130 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0260] The term “average diameter” or “mean diameter” refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PDI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here “average diameter,” “mean diameter,” “diameter,” or “size” for particles is used synonymously with this value of the Z-average.

[0261] The “polydispersity index” is preferably calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the “average diameter.” Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of ribonucleic acid nanoparticles (e.g., ribonucleic acid nanoparticles).

[0262] Different types of nucleic acid particles have been described previously to be suitable for delivery of nucleic acid in particulate form (e.g. Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60). For non-viral nucleic acid delivery vehicles, nanoparticle encapsulation of nucleic acid physically protects nucleic acid from degradation and, depending on the specific chemistry, can aid in cellular uptake and endosomal escape.

[0263] Some embodiments described herein relate to compositions, methods and uses involving more than one, e.g., 2, 3, 4, 5, 6 or even more nucleic acid species. The nucleic acid species may be RNA and / or DNA. For example, the particles described herein may contain one species of RNA (e.g., one species of mRNA) and one species of DNA.

[0264] In a nucleic acid particle composition, it is possible that each nucleic acid species is separately formulated as an individual nucleic acid particle formulation. In that case, each individual nucleic acid particle formulation will comprise one nucleic acid species. The individual nucleic acid particle formulations may be present as separate entities, e.g., in separate containers. Such formulations are obtainable by providing each nucleic acid species separately (typically each in the form of a nucleic acid-containing solution) together with a particle-forming agent, thereby allowing the formation of particles. Respective particles will contain exclusively the specific nucleic acid species that is being provided when the particles are formed (individual particulate formulations). Page 131 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 (iv) Methods of Making Lipid Nanoparticles

[0265] Lipids and lipid nanoparticles comprising nucleic acids and their method of preparation are known in the art, including, e.g., as described in U.S. Patent Nos. 8,569,256, 5,965,542 and U.S. Patent Publication Nos. 2016 / 0199485, 2016 / 0009637, 2015 / 0273068, 2015 / 0265708, 2015 / 0203446, 2015 / 0005363, 2014 / 0308304, 2014 / 0200257, 2013 / 086373, 2013 / 0338210, 2013 / 0323269, 2013 / 0245107, 2013 / 0195920, 2013 / 0123338, 2013 / 0022649, 2013 / 0017223, 2012 / 0295832, 2012 / 0183581, 2012 / 0172411, 2012 / 0027803, 2012 / 0058188, 2011 / 0311583, 2011 / 0311582, 2011 / 0262527, 2011 / 0216622, 2011 / 0117125, 2011 / 0091525, 2011 / 0076335, 2011 / 0060032, 2010 / 0130588, 2007 / 0042031, 2006 / 0240093, 2006 / 0083780, 2006 / 0008910, 2005 / 0175682, 2005 / 017054, 2005 / 0118253, 2005 / 0064595, 2004 / 0142025, 2007 / 0042031, 1999 / 009076 and PCT Pub. Nos. WO 99 / 39741, WO 2018 / 081480, WO 2017 / 004143, WO 2017 / 075531, WO 2015 / 199952, WO 2014 / 008334, WO 2013 / 086373, WO 2013 / 086322, WO 2013 / 016058, WO 2013 / 086373, W02011 / 141705, and WO 2001 / 07548, the full disclosures of which are herein incorporated by reference in their entirety for the purposes described herein.

[0266] For example, in some embodiments, lipids that are useful for delivery of nucleic acids are solubilized in ethanol at a pre- determined weight or molar ratios / percentages (e.g., ones described herein). In some embodiments, lipid nanoparticles (LNP) are prepared at a total lipid to RNA or DNA molar ratio of approximately 6:1 to 30:1. In some embodiments, such RNA or DNA can be diluted to 0.2 mg / mL in acetate buffer.

[0267] In some embodiments, using an ethanol injection technique, a colloidal lipid dispersion comprising nucleic acids (e.g., RNA or DNA) can be formed as follows: an ethanol solution comprising lipids, such as cyclic ionizable lipids described herein, neutral lipids, steroids, and, optionally polymer-conjugated lipids or anionic amphiphiles, is injected into an aqueous solution comprising nucleic acids (e.g., RNA or DNA).

[0268] In some embodiments, lipid and nucleic acid solutions can be mixed at room temperature by pumping each solution (e.g., a lipid solution comprising a cyclic ionizable lipid compound described herein, a neutral lipid, steroids, and the optional polymer conjugated lipids or anionic amphiphile or any other additives) at controlled flow rates into a mixing unit, for example, using piston pumps. In some embodiments, the flow rates of a lipid solution and a nucleic acid solution into a mixing unit are maintained at a ratio of 1:3. Upon mixing, nucleic acid-lipid particles are formed as the ethanolic lipid solution is diluted with aqueous RNAs. The lipid Page 132 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 solubility is decreased, while cationic lipids bearing a positive charge interact with the negatively charged nucleic acid.

[0269] In some embodiments, a solution comprising nucleic acid (e.g., RNA)-encapsulated lipid nanoparticles can be processed by one or more of concentration adjustment, buffer exchange, formulation, and / or filtration.

[0270] In some embodiments, a composition or complex described herein further comprises a pharmaceutically acceptable surfactant. In some embodiments, a pharmaceutically acceptable surfactant is selected from a polysorbate (e.g., polysorbate 20 (Tween20), polysorbate 40 (Tween40), polysorbate 60 (Tween60), and polysorbate 80 (Tween80)), poloxamers, and an amphiphilic group comprising a moiety selected from polyalkylene glycols (e.g., polyethylene glycol), poly(2-oxazoline), poly(2-methyl-2-oxazoline), polysarcosine, polyvinylpyrrolidone, and poly[N-(2-hydroxypropyl)methacrylamide, wherein the moiety is bound to one or more C12-C20 aliphatic groups. RNA

[0271] In some embodiments, a particle described herein comprises one or more oligosaccharide compositions and a nucleic acid. In some embodiments, a nucleic acid is RNA.

[0272] In some embodiments, an RNA amenable to technologies described herein is a single- stranded RNA. In some embodiments, an RNA as disclosed herein is a linear RNA. In some embodiments, a single-stranded RNA is a non-coding RNA in that its nucleotide sequence does not include an open reading frame (or complement thereof). In some embodiments, a single- stranded RNA has a nucleotide sequence that encodes (or is the complement of a sequence that encodes) a polypeptide or a plurality of polypeptides (e.g., epitopes) of the present disclosure.

[0273] In some embodiments, an RNA is or comprises an siRNA, an miRNA, or other non- coding RNA.

[0274] In many embodiments, a relevant RNA includes at least one open reading frame (ORF) (e.g., is an mRNA); in some embodiments, a relevant RNA includes a single ORF; in some embodiments, a relevant RNA includes more than one ORF.

[0275] In some embodiments, an RNA comprises an ORF, e.g., encoding a polypeptide of interest or encoding a plurality of polypeptides of interest. In some embodiments, an RNA produced in accordance with technologies provided herein comprises a plurality of ORFs (e.g., encoding a plurality of polypeptides). In some embodiments, an RNA produced in accordance with Page 133 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 technologies herein comprises a single ORF that encodes a plurality of polypeptides. In some such embodiments, polypeptides are or comprise antigens or epitopes thereof (e.g., relevant antigens).

[0276] In some embodiments, an ORF for use in accordance with the present disclosure encodes a polypeptide that includes a signal sequence, e.g., that is functional in mammalian cells, such as an intrinsic signal sequence or a heterologous signal sequence. In some embodiments, a signal sequence directs secretion of an encoded polypeptide, in some embodiments, a signal sequence directs transport of an encoded polypeptide into a defined cellular compartment, preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal-lysosomal compartment.

[0277] In some embodiments, an ORF encodes a polypeptide that includes a multimerization element (e.g., an intrinsic or heterologous multimerization element). In some embodiments, an ORF that encodes a surface polypeptide (e.g., that includes a signal sequence directing surface localization) includes a multimerization element.

[0278] In some embodiments, an ORF encodes a polypeptide that includes a transmembrane element or domain.

[0279] In some embodiments, an ORF is codon-optimized for expression in a cells of a particular host, e.g., a mammalian host, e.g., a human.

[0280] In some embodiments, an RNA includes unmodified uridine residues; an RNA that includes only unmodified uridine residues may be referred to as a “uRNA”. In some embodiments, an RNA includes one or more modified uridine residues; in some embodiments, such an RNA (e.g., an RNA including entirely modified uridine residues) is referred to as a “modRNA”. In some embodiments, an RNA may be a self-amplifying RNA (saRNA). In some embodiments, an RNA may be a trans-amplifying RNA (taRNA) (see, for example, WO2017 / 162461).

[0281] In some embodiments, a relevant RNA includes a polypeptide-encoding portion or a plurality of polypeptide-encoding portions. In some particular embodiments, such a portion or portions may encode a polypeptide or polypeptides that is or comprises a biologically active polypeptide or portion thereof (e.g., an enzyme or cytokine or therapeutic protein such as a replacement protein or antibody or portion thereof). In some particular embodiments, such a portion or portions may encode a polypeptide or polypeptides that is or comprises an antigen (or an epitope thereof), a cytokine, an enzyme, etc. In some embodiments, an encoded polypeptide or polypeptides may be or include one or more neoantigens or neoepitopes associated with a tumor. Page 134 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 In some embodiments, an encoded polypeptide or polypeptides may be or include one or more antigens (or epitopes thereof) of an infectious agent (e.g., a bacterium, fungus, virus, etc.). In certain embodiments, an encoded polypeptide may be a variant of a wild type polypeptide.

[0282] In some embodiments, a single-stranded RNA (e.g., mRNA) may comprise a secretion signal-encoding region (e.g., a secretion signal-encoding region that allows an encoded target entity or entities to be secreted upon translation by cells). In some embodiments, such a secretion signal-encoding region may be or comprise a non-human secretion signal. In some embodiments, such a secretion signal-encoding region may be or comprise a human secretion signal.

[0283] In some embodiments, a single-stranded RNA (e.g., mRNA) may comprise at least one non-coding element (e.g., to enhance RNA stability and / or translation efficiency). Examples of non-coding elements include but are not limited to a 3’ untranslated region (UTR), a 5’ UTR, a cap structure (e.g., in some embodiments, an enzymatically-added cap; in some embodiments, a co-transcriptional cap), a poly adenine (polyA) tail (e.g., that, in some embodiments, may be or comprise 100 A residues or more, and / or in some embodiments may include one or more “interrupting” [i.e., non-A] sequence elements), and any combinations thereof. Exemplary embodiments of such non-coding elements may be found, for example, in WO2011015347, WO2017053297, US 10519189, US 10494399, WO2007024708, WO2007036366, WO2017060314, WO2016005324, WO2005038030, WO2017036889, WO2017162266, and WO2017162461, each of which is incorporated herein by referenced in its entirety. Formats

[0284] At least four formats useful for RNA pharmaceutical compositions (e.g., immunogenic compositions or vaccines) have been developed, namely non-modified uridine containing mRNA (uRNA), nucleosidemodified mRNA (modRNA), self-amplifying mRNA (saRNA), and trans- amplifying RNAs.

[0285] Features of a non-modified uridine platform may include, for example, one or more of intrinsic adjuvant effect, good tolerability and safety, and strong antibody and T cell responses.

[0286] Features of modified uridine (e.g., pseudouridine) platform may include reduced adjuvant effect, blunted immune innate immune sensor activating capacity and thus augmented antigen expression, good tolerability and safety, and strong antibody and CD4-T cell responses. As noted herein, the present disclosure provides an insight that such strong antibody and CD4 T cell responses may be particularly useful for vaccination. Page 135 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0287] Features of self-amplifying platform may include, for example, long duration of polypeptide (e.g., protein) expression, good tolerability and safety, higher likelihood for efficacy with very low vaccine dose.

[0288] In some embodiments, a self-amplifying platform (e.g., saRNA) comprises a nucleic acid molecule, encoding both a replicase (e.g., a viral replicase) and a gene of interest, wherein the nucleic acid molecule is capable of being replicated by said replicase in cis (cis-replication system).In some embodiments, a trans-amplifying platform (e.g., taRNA) comprises two nucleic acid molecules, wherein one nucleic acid molecule encodes a replicase (e.g., a viral replicase) and the other nucleic acid molecule is capable of being replicated (e.g., a replicon) by said replicase in trans (trans-replication system). In some embodiments, a self / trans-amplifying platform (e.g., RNA) comprises a plurality of nucleic acid molecules, wherein said nucleic acids encode a plurality of replicases and / or replicons.

[0289] In some embodiments, a trans-replication system comprises the presence of both nucleic acid molecules in a single host cell.

[0290] In some such embodiments, a nucleic acid encoding a replicase (e.g., a viral replicase) is not capable of self-replication in a target cell and / or target organism. In some such embodiments, a nucleic acid encoding a replicase (e.g., a viral replicase) lacks at least one conserved sequence element important for (-) strand synthesis based on a (+) strand template and / or for (+) strand synthesis based on a (-) strand template.

[0291] In some embodiments, a self-amplifying RNA comprises a 5’-cap; in some trans- replication systems, at least an RNA encoding a replicase is capped. Without wishing to be bound by any one theory, it has been found that a 5’-cap can be important for high level expression of a gene of interest in trans.

[0292] In some embodiments, a self / trans-amplifying platform does not require propagation of virus particles (e.g., is not associated with undesired virus-particle formation). In some embodiments, a self / trans-amplifying platform is not capable of forming virus particles.

[0293] In some embodiments, an RNA may comprise an Internal Ribosomal Entry Site (IRES) element. In some embodiments, an RNA does not comprise an IRES site; in particular, in some embodiments, an saRNA does not comprise an IRES site. In some such embodiments, translation of a gene of interest and / or replicase is not driven by an IRES element. In some embodiments, an Page 136 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 IRES element is substituted by a 5’-cap. In some such embodiments, substitution by a 5’-cap does not affect the sequence of a polypeptide encoded by an RNA.

[0294] In some embodiments, a complex described herein comprises modRNA, saRNA, taRNA, or uRNA. In some embodiments, a complex comprises modRNA. In some embodiments, a complex comprises saRNA. In some embodiments, a complex comprises taRNA. In some embodiments, a complex comprises uRNA. Methods of Use

[0295] Particles described herein are useful in the treatment and prophylaxis in a subject of diseases, disorders, and conditions described herein. In some embodiments, the present disclosure provides a method of treating a disease, disorder or condition comprising administering to a patient a composition comprising particles described herein. In some embodiments, the present disclosure provides use of a composition comprising particles described herein for the treatment of a disease, disorder, or condition. In some embodiments, a disease, disorder, or condition is an infectious disease, cancer, an autoimmune disease, or a rare disease.

[0296] In some embodiments, an infectious disease is caused by or associated with a viral pathogen. In some embodiments, a viral pathogen is of a family selected from poxviridae, rhabdoviridae, filoviridae, paramyxoviridae, hepadnaviridae, coronaviridae, caliciviridae, picornaviridae, reoviridae, retroviridae, and orthomyxoviridae. In some embodiments, an infectious disease is caused by or associated with a virus selected from SARS-CoV-2, influenza, Crimean-Congo Hemorhhagic Fever (CCHF), Ebola virus, Lassa virus, Marburg virus, HIV, Nipah virus, and MERS-CoV.

[0297] In some embodiments, an infectious disease is caused by or associated with a bacterial pathogen. In some embodiments, a bacterial pathogen is of a species selected from Actinomyces israelii, bacillus antracis, Bacteroides fragilis, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campolobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium idphteriae, Ehrlichia canis, Ehrlichia chaffeensis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Page 137 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Leptospira, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Nocardia asteroids, Rickettsia ricektssii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Shigella dysenteriae, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, Treponema pallidum, Vibrio cholerae, and Yersinia pestis.

[0298] In some embodiments, an infectious disease is caused by or associated with a parasite. In some embodiments, a parasite is of a family selected from Plasmodium, Leishmania, Cryptosporidium, Entamoeba, Trypanosomas, Schistosomes, Ascaris, Echinococcus and Taeniidae.

[0299] In some embodiments, a disease, disorder, or condition is a cancer. In some embodiments, a cancer is selected from bladder cancer, breast cancer, colorectal cancer, kidney cancer, lung cancer, lymphoma, melanoma, oral / oropharyngeal cancer, pancreatic cancer, prostate cancer, thyroid cancer, and uterine cancer.

[0300] In some embodiments, a disease, disorder, or condition is a genetic disorder. In some embodiments, a genetic disorder is associated with a gain-of-function mutation or a loss-of- function mutation.

[0301] In some embodiments, a disease, disorder, or condition is an autoimmune disease. In some embodiments, an autoimmune disease is selected from Addison disease, celiac disease, rheumatoid arthritis, lupus, inflammatory bowel disease, dermatomyositis, multiple sclerosis, diabetes, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, pernicious anemia, graves’ disease, Hashimoto’s thyroiditis, myasthenia gravis, and vasculitis Sjogren syndrome.

[0302] In some embodiments, a disease, disorder, or condition is a rare disease. As described herein, a rare disease refers to a life-threatening or chronically debilitating diseases which are of such low prevalence (e.g., fewer than 1 / 2000 people) that special combined efforts are needed to address them.

[0303] In some embodiments, the present disclosure provides complexes that can selectively target particular systems within a body. As used herein, reference to “targeting” a particular system refers to causing increased expression of RNA derived from cargo in the complex in the Page 138 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 desired system. For example, in some embodiments, complexes described herein can selectively target the lungs, liver, spleen, heart, brain, lymph nodes, bladder, kidneys, and pancreas. As described herein, a complex “selectively targets” an organ when a single target expresses mRNA in an amount that is 65% or greater than expression in other organs post administration (e.g., 65% or more of mRNA throughout the body is expressed from a single organ, with the remaining 35% distributed between one or more different organs). In some embodiments, a complex described herein selectively targets the lungs. In some embodiments, a complex described herein selectively targets the liver. In some embodiments, a complex described herein selectively targets the spleen. In some embodiments, a complex described herein selectively targets the heart. Methods of Delivery

[0304] The present disclosure provides, among other things, a particle that is incorporated into a composition (e.g., a pharmaceutical composition or a pharmaceutical formulation, as referred to herein) to be administered to a subject. For example, in some embodiments, a composition comprising particles described herein is administered as a monotherapy. In some embodiments, a a composition comprising particles described herein is administered as part of a combination therapy. In some embodiments, a concentration of total RNA (e.g., a total concentration of all of the one or more RNA molecules) in a composition described herein is of about 0.01 mg / mL to about 1.0 mg / mL, or about 0.03 mg / mL to about 0.3, or about 0.05 to about 0.15 mg / mL.

[0305] Compositions (also referred to as pharmaceutical compositions) may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure. Page 139 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0306] In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by the United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0307] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and / or perfuming agents can be present in the composition, according to the judgment of the formulator.

[0308] General considerations in the formulation and / or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

[0309] In some embodiments, pharmaceutical compositions provided herein may be formulated with one or more pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

[0310] Pharmaceutical complexes and compositions described herein can be administered by appropriate methods known in the art. As will be appreciated by a skilled artisan, the route and / or mode of administration may depend on a number of factors, including, e.g., but not limited to stability and / or pharmacokinetics and / or pharmacodynamics of pharmaceutical compositions described herein.

[0311] In some embodiments, pharmaceutical compositions described herein are formulated for parenteral administration, which includes modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, Page 140 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0312] In some embodiments, pharmaceutical compositions described herein are formulated for intravenous administration. In some embodiments, pharmaceutically acceptable carriers that may be useful for intravenous administration include sterile aqueous solutions or dispersions and sterile powders for preparation of sterile injectable solutions or dispersions.

[0313] In some particular embodiments, pharmaceutical compositions described herein are formulated for subcutaneous (s.c) administration. In some particular embodiments, pharmaceutical compositions described herein are formulated for intramuscular (i.m) administration.

[0314] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, dispersion, powder (e.g., lyophilized powder), microemulsion, lipid nanoparticles, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. In some embodiments, prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0315] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.

[0316] In some embodiments, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Page 141 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0317] Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0318] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into pharmaceutical compositions described herein. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0319] Formulations of pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing active ingredient(s) into association with a diluent or another excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0320] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of at least one RNA product produced using a system and / or method described herein.

[0321] In some embodiments, an active agent that may be included in a pharmaceutical composition described herein is or comprises a therapeutic agent administered in a combination therapy described herein. Pharmaceutical compositions described herein can be administered in combination therapy, i.e., combined with other agents. In some embodiments, such therapeutic agents may include agents leading to depletion or functional inactivation of regulatory T cells. For example, in some embodiments, a combination therapy can include a provided pharmaceutical composition with at least one immune checkpoint inhibitor. Page 142 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0322] In some embodiments, pharmaceutical composition described herein may be administered in conjunction with radiotherapy and / or autologous peripheral stem cell or bone marrow transplantation.

[0323] In some embodiments, a pharmaceutical composition described herein can be frozen to allow long-term storage.

[0324] The pharmaceutical compositions of the present disclosure may be in in a frozen form or in a "ready-to-use form" (i.e., in a form, in particular a liquid form, which can be immediately administered to a subject, e.g., without any processing such as thawing, reconstituting or diluting). Thus, prior to administration of a storable form of a pharmaceutical composition, this storable form has to be processed or transferred into a ready-to-use or administrable form. E.g., a frozen pharmaceutical composition has to be thawed. Ready to use injectables can be presented in containers such as vials, ampoules or syringes wherein the container may contain one or more doses.

[0325] In one embodiment, the pharmaceutical composition is lyophilized. In one embodiment, the pharmaceutical composition is spray dried. These techniques are well known to those skilled in the art.

[0326] In some embodiments, the pharmaceutical composition is in frozen form and can be stored at a temperature of about -90°C or higher, such as about -90°C to about -10°C. For example, the frozen pharmaceutical compositions described herein can be stored at a temperature ranging from about -90°C to about -10°C, such as from about -90°C to about -40°C or from about -40°C to about -25°C, or from about -25°C to about -10°C, or a temperature of about -20°C.

[0327] In some embodiments of the pharmaceutical compositions in frozen form, the pharmaceutical composition can be stored for at least 1 week, such as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months, preferably at least 4 weeks. For example, the frozen pharmaceutical composition can be stored for at least 4 weeks, preferably at least 1 month, more preferably at least 2 months, more preferably at least 3 months, more preferably at least 6 months at 20°C.

[0328] In some embodiments of the pharmaceutical compositions in frozen form, when the nucleic acid is mRNA, the mRNA integrity after thawing the frozen pharmaceutical composition is at least 90%, at least 95%, at least 97%, at least 98%, or substantially 100% of the initial mRNA Page 143 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 integrity, e.g., after thawing the frozen composition which has been stored (for at least 1 week, such as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months, preferably at least 4 weeks) at -20°C.

[0329] In some embodiments of the pharmaceutical compositions in frozen form, the size (Zaverage) and / or size distribution and / or PDI of the particles after thawing the frozen pharmaceutical composition is essentially equal to the size (Zaverage) and / or size distribution and / or PDI of the particles of the initial pharmaceutical composition before freezing. For example, if a ready-to-use pharmaceutical composition is prepared from a frozen pharmaceutical composition as described herein, it is preferred that the size (Zaverage) and / or size distribution and / or PDI of the particles contained in the ready-to-use pharmaceutical composition is essentially equal to the initial size (Zaverage) and / or size distribution and / or PDI of the particles contained in the frozen pharmaceutical composition before freezing.

[0330] In some embodiments, when the nucleic acid is mRNA, the size of the mRNA particles and the mRNA integrity of the pharmaceutical composition after one freeze / thaw cycle, preferably after two freeze / thaw cycles, more preferably after three freeze / thaw cycles, more preferably after four freeze / thaw cycles, more preferably after five freeze / thaw cycles or more, are essentially equal to the size of the mRNA particles and the mRNA integrity of the initial pharmaceutical composition (i.e., before the pharmaceutical composition has been frozen for the first time).

[0331] In some embodiments, the pharmaceutical composition is in liquid form and can be stored at a temperature ranging from about 0 °C to about 20 °C. For example, the liquid pharmaceutical compositions described herein can be stored at a temperature ranging from about 1 °C to about 15 °C, such as from about 2 °C to about 10 °C, or from about 2 °C to about 8 °C, or at a temperature of about 5 °C.

[0332] In some embodiments, when the nucleic acid is mRNA, the mRNA integrity of the pharmaceutical composition when stored is at least 70%, preferably at least 80%, more preferably at least 90%, of the initial mRNA integrity (i.e., the mRNA integrity of the initial pharmaceutical composition).

[0333] In some embodiments of the pharmaceutical compositions in liquid form, the pharmaceutical composition can be stored for at least 1 week, such as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at Page 144 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 least 12 months, or at least 24 months, preferably at least 4 weeks. For example, the liquid pharmaceutical composition can be stored for at least 4 weeks, preferably at least 1 month, more preferably at least 2 months, more preferably at least 3 months, more preferably at least 6 months at 5°C.

[0334] In some embodiments of the pharmaceutical composition in liquid form, when the nucleic acid is mRNA, the mRNA integrity of the liquid composition, when stored, e.g., at 0 °C or higher for at least one week, is such that the desired effect, e.g., to induce an immune response, can be achieved. For example, the mRNA integrity of the liquid composition, when stored, e.g., at 0 °C or higher for at least one week (such as for at least 2 weeks, at least three weeks, at least four weeks, at least one month, at least two months, at least three months, at least 4 months, or at least 6 months), may be at least 90%, compared to the mRNA integrity of the initial composition, i.e., the mRNA integrity before the composition has been stored. In some embodiments, the mRNA integrity of the composition after storage for at least four weeks (e.g., for at least three months), preferably at a temperature of 0 °C or higher, such as about 2 °C to about 8 °C, is at least 90%, compared to the mRNA integrity before storage.

[0335] In some embodiments, when the nucleic acid is mRNA, the initial mRNA integrity of the pharmaceutical composition (i.e., after its preparation but before storage) is at least 50% and the mRNA integrity of the pharmaceutical composition after storage for at least one week (such as for at least 2 weeks, at least three weeks, at least four weeks, at least one month, at least two months, or at least 3 months), preferably at a temperature of 0 °C or higher, such as about 2 °C to about 8°C, is at least 90% of the initial mRNA integrity.

[0336] In some embodiments of the pharmaceutical composition in liquid form, the size (Zaverage) (and / or size distribution and / or polydispersity index (PDI)) of the particles of the pharmaceutical composition, when stored, e.g., at 0 °C or higher for at least one week, is such that the desired effect, e.g., to induce an immune response, can be achieved. For example, the size (Zaverage) (and / or size distribution and / or polydispersity index (PDI)) of the particles of the pharmaceutical composition, when stored, e.g., at 0 °C or higher for at least one week, is essentially equal to the size (Zaverage) (and / or size distribution and / or PDI) of the particles of the initial pharmaceutical composition, i.e., before storage.

[0337] In some embodiments, the size (Zaverage) of the particles after storage of the pharmaceutical composition, e.g., at 0 °C or higher for at least one week is between about 50 nm Page 145 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 and about 500 nm, preferably between about 40 nm and about 200 nm, more preferably between about 40 nm and about 120 nm. In some embodiments, the PDI of the particles after storage of the pharmaceutical composition, e.g., at 0 °C or higher for at least one week is less than 0.3, preferably less than 0.2, more preferably less than 0.1.

[0338] In some embodiments, the size (Zaverage) of the particles after storage of the pharmaceutical composition, e.g., at 0 °C or higher for at least one week is between about 50 nm and about 500 nm, preferably between about 40 nm and about 200 nm, more preferably between about 40 nm and about 120 nm, and the size (Zaverage) (and / or size distribution and / or PDI) of the particles after storage of the pharmaceutical composition, e.g., at 0 °C or higher for at least one week is essentially equal to the size (Zaverage) (and / or size distribution and / or PDI) of the particles before storage. In some embodiments, the size (Zaverage) of the particles after storage of the pharmaceutical composition, e.g., at 0 °C or higher for at least one week is between about 50 nm and about 500 nm, preferably between about 40 nm and about 200 nm, more preferably between about 40 nm and about 120 nm, and the PDI of the particles after storage of the pharmaceutical composition, e.g., at 0 °C or higher for at least one week is less than 0.3 (preferably less than 0.2, more preferably less than 0.1).

[0339] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation. Exemplary Embodiments

[0340] The following numbered embodiments, while non-limiting, are exemplary of certain aspects of the disclosure: Embodiment 1. A composition comprising a cationic lipid, an anionically ionizable lipid, and a nucleic acid, wherein: the cationic lipid comprises a cationic head group, a first bridge group, and a first lipid tail group; Page 146 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 the anionically ionizable lipid comprises an anionically ionizable head group, a second bridge group, and a second lipid tail group; the cationic lipid has a lipidic volume from about 700 Å3to about 1500 Å3, and / or the anionically ionizable lipid has a lipidic volume from about 700 Å3to about 1500 Å3; or the cationic lipid and the anionically ionizable lipid have a combined lipidic volume greater than 1400 Å3. Embodiment 2. The composition of Embodiment 1, wherein a molar ratio of the cationic lipid to the anionically ionizable lipid, is from about 0.67 to about 1.5, from about 0.75 to about 1.33, from about 0.82 to about 1.2, or from about 0.9 to about 1.1. Embodiment 3. The composition of Embodiment 2, wherein the molar ratio of the cationic lipid to the anionically ionizable lipid is from about 0.9 to about 1.1. Embodiment 4. The composition of any one of Embodiments 1-3, wherein the cationic lipid has a lipidic volume that is from about 700 Å3to about 1500 Å3, and / or the anionically ionizable lipid has a lipidic volume that is from about 700 Å3to about 1500 Å3. Embodiment 5. The composition of any one of Embodiments 1-3, wherein the cationic lipid has a lipidic volume of about 700 Å3to about 1500 Å3and the anionic lipid has a lipidic volume that is greater than 650 Å3. Embodiment 6. The composition of any one of Embodiments 1-3, wherein the anionically ionizable lipid has a lipidic volume of about 700 Å3to about 1500 Å3and the cationic lipid has a lipidic volume that is greater than 650 Å3. Embodiment 7. The composition of any one of Embodiments 1-3, wherein the cationic lipid has a lipidic volume of about 700 Å3to about 1500 Å3and the anionically ionizable lipid has a lipidic volume that is from about 700 Å3to about 1500 Å3. Page 147 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Embodiment 8. The composition of any one of Embodiments 1-3, wherein the cationic lipid and the anionically ionizable lipid have a combined lipidic volume that is greater than 1400 Å3. Embodiment 9. The composition of any one of Embodiments 1-8, wherein the cationic lipid, the anionically ionizable lipid, and the nucleic acid form nanoparticles. Embodiment 10. The composition of any one of Embodiments 1-9, wherein the cationic head group is or comprises a guanidinium moiety, an amidinium moiety, an amine moiety, an optionally substituted 5- to 6-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 4- to 6-membered heterocycle comprising 1 to 3 heteroatoms selected from N, O, and S. Embodiment 11. The composition of any one of Embodiments 1-10, wherein the first lipid tail group is or comprises an optionally substituted C10-C50 aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -Cy-, -NRZ-, -N(RZ)C(O)- , -C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ) -, -C(O)N(RZ)SO2(RZ)- , -SO2(RZ)N(RZ)C(O)-,-OC(O)O-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; where each -Cy- is independently an optionally substituted C3-C6cycloaliphatic, 5- to 12- membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, S, 5- to 6- membered heteroaryl ring having 1-3 heteroatoms selected from N, O, S; and each RZis independently H or an optionally substituted group selected from C1-C20 aliphatic, or C3- C12cycloaliphatic. Embodiment 12. The composition of Embodiment 11, wherein each of the first lipid tail group comprises an optionally substituted branched C10-C50aliphatic group. Embodiment 13. The composition of any one of Embodiments 1-12, wherein the first bridge group comprises a chiral carbon atom. Page 148 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Embodiment 14. The composition of any one of Embodiments 1-13, wherein the first bridge group is a C2-C5 aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by - NRZ-, -N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, - OC(O)N(RZ)-, -N(RZ)C(O)N(RZ)-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-. Embodiment 15. The composition of Embodiment 1, wherein the cationic lipid is represented by formula ICL:or a pharmaceutically acceptable salt thereof, wherein: G1is -L1a-L1b-G1a; L1ais a bond, or optionally substituted C1-C6 aliphatic; L1bis optionally substituted C1-C6aliphatic; G1ais -N(Ra)C(N(Ra)2+)N(Ra)2, -N(Ra)3+, -N(Ra)C(N(Ra))N(Ra)2, -N(Ra)(Rb), - C(Ra)2C(N(Ra)2+)N(Ra)2, -N(Ra)C(O)N(Ra)2, -N(Ra)C(S)N(Ra)2, an optionally substituted 5- to 6-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 4- to 6-membered heterocycle comprising 1 to 3 heteroatoms selected from N, O, and S; B1and B2are each independently -NRZ-, -N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, - OC(O)N(RZ)-, -N(RZ)C(O)N(RZ)-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; T1and T2are each independently optionally substituted C5-C35 aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -Cy-, -NRZ-, - N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ) -, -O-, - C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; each Rais independently H or optionally substituted C1-C6 aliphatic; each Rbis independently selected from H, optionally substituted C1-C6aliphatic, and optionally substituted 5- to 6-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S, and optionally substituted 4- to 6-membered heterocycle comprising 1 to 3 heteroatoms selected from N, O, and S; Page 149 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 each -Cy- is independently an optionally substituted C3-C6cycloaliphatic, optionally substituted 5- to 12-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, or optionally substituted 5- to 6-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S; and each RZis independently selected from H, optionally substituted C1-C20 aliphatic, and optionally substituted C3-C12 cycloaliphatic. Embodiment 16. The composition of Embodiment 15, wherein G1ais - N(Ra)C(N(Ra)2+)N(Ra)2. Embodiment 17. The composition of Embodiments 15 or 16, wherein L1ais optionally substituted C1-C6 aliphatic, and L1bis optionally substituted C1-C6 aliphatic. Embodiment 18. The composition of Embodiment 15, wherein G1ais selected from the group consisting of: , , Embodoup consisting of: , ,Page 150 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Embod areeach independently selected from the group consisting of -C(O)N(RZ) and -N(RZ)C(O)-. Embodiment 21. The composition of Embodiment 20, wherein one of B1and B2is - C(O)N(RZ) and the other of B1and B2is -N(RZ)C(O)-. Embodiment 22. The composition of any one of Embodiments 15-21, wherein T1and T2are each independently an optionally substituted C10-C35 aliphatic group. Embodiment 23. The composition of Embodiment 22, wherein T1and T2are each independently selected from: , ndEmbodiment 24. The composition of Embodiment 22, wherein T1and T2are each independently an optionally substituted branched C10-C35 aliphatic group. Embodiment 25. The composition of Embodiment 15, wherein T1is represented by formula: wherein:Page 151 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 X1is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ)-, or -N(RZ)C(O)-; T1ais H or optionally substituted C1-C20 aliphatic; T1bis optionally substituted C1-C20 aliphatic; and n1 is 1 to 10. Embodiment 26. The composition of Embodiment 25, wherein T1is selected from: , andEmbodiment 27. The composition of Embodiment 25 or 22, wherein moiety: orEmbodiment 28. The composition of Embodiment 15, wherein T2is represented by formula: whereinX2is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ), or -N(RZ)C(O)-; T2ais H or optionally substituted C1-C20 aliphatic; Page 152 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 T2bis optionally substituted C1-C20aliphatic; and n2 is 1 to 10. Embodiment 29. The composition of Embodiment 28, wherein T2is selected from: , ndEmbodiment 30. The composition of Embodiment 28 or 29, wherein moiety: orEmbodiment 31. The composition of Embodiment 15, wherein a compound of formula ICL is represented ICL-II:- Page 153 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 or a pharmaceutically acceptable salt thereof, wherein X1is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ)-, or -N(RZ)C(O) -; T1ais H or optionally substituted C1-C20 aliphatic; T1bis optionally substituted C1-C20aliphatic; n1 is 1 to 10; X2is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ), or -N(RZ)C(O)-; T2ais H or optionally substituted C1-C20aliphatic; T2bis optionally substituted C1-C20 aliphatic; and n2 is 1 to 10. Embodiment 32. The composition of Embodiment 31, wherein a compound of formula ICL- II is represented ICL-IIa or ICL-IIb:or a pharmaceutically acceptable salt thereof. Embodiment 33. The composition of Embodiment 31, wherein a compound of formula ICL- II is represented by formula ICL-III:or a pharmaceutically acceptable salt thereof. Page 154 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Embodiment 34. The composition of Embodiment 33, wherein a compound of formula ICL- III is represented by formula ICL-IIIa or ICL-IIIb:or a pharmaceutically acceptable salt thereof. Embodiment 35. The composition of Embodiment 1, wherein the cationic lipid is selected from Table 1. Embodiment 36. The composition of any one of Embodiments 1-35, wherein the anionically ionizable head group is or comprises a carboxylic acid moiety or a dihydrogen phosphate moiety. Embodiment 37. The composition of any one of Embodiments 1-36, wherein the second lipid tail group is or comprises an optionally substituted C10-C50 aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -Cy-, -NRZ-, -N(RZ)C(O)- , -C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ) -, -O-, -C(O)-, -OC(O)- , -C(O)O-, -SO-, or -SO2-; where each -Cy- is independently an optionally substituted C3- C6 cycloaliphatic, 5- to 12-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, S, 5- to 6-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, S; and each RZis independently H or an optionally substituted group selected from C1- C20 aliphatic, or C3-C12 cycloaliphatic. Embodiment 38. The composition of Embodiment 37, wherein the second lipid tail group comprises is an optionally substituted branched C10-C50 aliphatic group. Page 155 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Embodiment 39. The composition of any one of Embodiments 1-38, wherein the second bridge group comprises a chiral carbon atom. Embodiment 40. The composition of any one of Embodiments 1-39, wherein the second bridge group is a C2-C5 aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -NRZ-, -N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, - OC(O)N(RZ)-, -N(RZ)C(O)N(RZ) -, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-. Embodiment 41. The composition of Embodiment 1, wherein the anionically ionizable lipid is represented by formula IAL:or a pharmaceutically acceptable salt thereof, wherein: G2is -L2a-L2b-G2a-G2b; L2ais a bond or optionally substituted C1-C6 aliphatic L2bis a bond, -C(O)O-, or -OC(O)-; G2ais an optionally substituted C1-C6aliphatic; G2bis -C(O)OH or -P(O)2(OH)2; B3and B4are each independently selected from –C(O)O-, -OC(O)-, or a C2-C6 aliphatic group, wherein one carbon atom is optionally replaced with -OC(O)- or -C(O)O-; T3and T4are each independently optionally substituted C5-C35 aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -Cy-, -NRZ-, - N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ) -, -O-, - C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; each -Cy- is independently an optionally substituted C3-C6 cycloaliphatic, optionally substituted 5- to 12-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, S, or optionally substituted 5- to 6-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, S; and each RZis independently selected from H, optionally substituted C1-C20 aliphatic, and optionally substituted C3-C12cycloaliphatic. Page 156 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Embodiment 42. The composition of Embodiment 41, wherein G2bis -C(O)OH. Embodiment 43. The composition of Embodiment 41 or 42, wherein L2ais optionally substituted C1-C6 aliphatic, and L2bis -C(O)O- or -OC(O)-. Embodiment 44. The composition of any one of Embodiment 41-43, wherein G2is: ,Embodiment 45. The composition of any one of Embodiments 41-44, wherein B3is selected from , wherein ss represents a point of attachment to T3.Embodiment 46. The composition of any one of Embodiments 41-44, wherein B4is selected from , wherein tt represents a point of attachment to T4.Embodiment 47. The composition of any one of Embodiments 41-46, wherein T3and T4are each independently an optionally substituted C10-C35aliphatic group. Embodiment 48. The composition of any one of Embodiments 41-46, wherein T3is represented by formula: whereinPage 157 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 X3is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ)-, or -N(RZ)C(O)-; T3ais H or optionally substituted C1-C20 aliphatic; T3bis optionally substituted C1-C20 aliphatic; and n3 is 1 to 10. Embodiment 49. The composition of Embodiment 48, T3is selected from the group and ,Embodiment 51. The composition of any one of Embodiments 41-50, wherein T4is represented by formula: whereinX4is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ)-, or -N(RZ)C(O)-; T4ais H or optionally substituted C1-C20aliphatic; Page 158 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 T4bis optionally substituted C1-C20aliphatic; and n4 is 1 to 10. Embodiment 52. The composition of Embodiment 51, wherein T4is selected from the group consisting of: , ,Embodiment 54. The composition of Embodiment 41, wherein a compound represented by formula IAL is a compound represented by IAL-II:- or a pharmaceutically acceptable salt thereof. Page 159 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Embodiment 55. The composition of Embodiment 54, wherein a compound represented by formula IAL-II is a compound represented by IAL-III:or a pharmaceutically acceptable salt thereof, wherein X3is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ)-, or -N(RZ)C(O)-; T3ais H or optionally substituted C1-C20 aliphatic; T3bis optionally substituted C1-C20 aliphatic; n3 is 1 to 10; X4is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ)-, or -N(RZ)C(O)-; T4ais H or optionally substituted C1-C20 aliphatic; T4bis optionally substituted C1-C20aliphatic; and n4 is 1 to 10. Embodiment 56. The composition of Embodiment 54, wherein a compound represented by formula IALII is a compound represented by IAL-IV:or a pharmaceutically acceptable salt thereof. Embodiment 57. The composition of Embodiment 1, wherein the anionically ionizable lipid is selected from Table 3. Page 160 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Embodiment 58. The composition of any one of Embodiments 1-57, wherein the composition further comprises a sterol. Embodiment 59. The composition of Embodiment 58, wherein the sterol is cholesterol. Embodiment 60. The composition of any one of Embodiments 1-59, wherein the composition further comprises a phospholipid. Embodiment 61. The composition of Embodiment 60, wherein the phospholipid is a zwitterionic phospholipid. Embodiment 62. The composition of Embodiment 61, wherein the phospholipid is selected from distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), palmitoyloleoyl-phosphatidylcholine (POPC), dioleoylphosphatidylethanolamine (DOPE) and N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM). Embodiment 63. The composition of any one of Embodiments 1-62, wherein composition further comprises a a polymer-conjugated lipid. Embodiment 64. The composition of any one of Embodiments 1-62, wherein the composition does not comprise a polymer-conjugated lipid. Embodiment 65. The composition of any one of Embodiments 1-64, wherein the nucleic acid is RNA. Embodiment 66. The composition of Embodiment 65, wherein the RNA is mRNA, tRNA, rRNA, snRNA, saRNA, taRNA, ssRNA, dsRNA, siRNA, or miRNA. Page 161 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Embodiment 67. The composition of any one of Embodiments 1-66, wherein the nucleic acid is DNA. Embodiment 68. A compound of formula ICL:or a pharmaceutically acceptable salt thereof, wherein: G1is -L1a-L1b-G1a; L1ais a bond optionally substituted C1-C6 aliphatic; L1bis optionally substituted C1-C6aliphatic; G1ais -N(Ra)C(N(Ra)2+)N(Ra)2, -N(Ra)3+, -N(Ra)C(N(Ra))N(Ra)2, -N(Ra)2, - C(Ra)2C(N(Ra)2+)N(Ra)2, -N(Ra)C(O)N(Ra)2, -N(Ra)C(S)N(Ra)2, an optionally substituted 5- to 6-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 4- to 6-membered heterocycle comprising 1 to 3 heteroatoms selected from N, O, and S; B1and B2are each independently -NRZ-, -N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, - OC(O)N(RZ)-, -N(RZ)C(O)N(RZ)-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; T1and T2are each independently optionally substituted C5-C35 aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -Cy-, -NRZ-, - N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ) -, -O-, - C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; each Rais independently H or optionally substituted C1-C6 aliphatic; each -Cy- is independently an optionally substituted C3-C6cycloaliphatic, 5- to 12- membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, S, 5- to 6- membered heteroaryl ring having 1-3 heteroatoms selected from N, O, S; each RZis independently selected from H, optionally substituted C1-C20aliphatic, and optionally substituted C3-C12cycloaliphatic; , and wherein moiety: Page 162 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 has a lipidic volume from about 7 out 1500 Å3.Embodiment 69. The compound of Embodiment 68, wherein the compound is selected from: Table 2A or Table 2B. Embodiment 70. A compound represented by formula IAL:or a pharmaceutically acceptable salt thereof, wherein: G2is -L2a-L2b-G2a-G2b; L2ais a bond or optionally substituted C1-C6aliphatic; L2bis a bond, -C(O)O-, or -OC(O)-; G2ais optionally substituted C1-C6 aliphatic; G2bis -C(O)OH or -P(O)2(OH)2; B3and B4are each independently selected from C1-C6aliphatic, wherein each carbon atom is optionally and independently replaced by -NRZ-, -N(RZ)C(O)-, -C(O)N(RZ)-, - N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ)-, -O-, -C(O)-, -OC(O)-, - C(O)O-, -SO-, and -SO2-; T3and T4are each independently optionally substituted C5-C35 aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -Cy-, -NRZ-, - N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ) -, -O-, - C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; each Rais independently H or optionally substituted C1-C6 aliphatic; each -Cy- is independently an optionally substituted 3-12 membered bivalent C3-C6 cycloaliphatic, 5- to 12-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, 5- to 6-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S; Page 163 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 each RZis independently selected from H, optionally substituted C1-C20aliphatic, and optionally substituted C3-C12 cycloaliphatic; and wherein moiety: has a lipidic volume from about 70 bout 1500 Å3.Embodiment 71. The compound of Embodiment 70, wherein the compound is selected from: Table 4. Embodiment 72. A suspension comprising a dispersed phase and an aqueous phase, and wherein the dispersed phase comprises the composition of any one of Embodiments 1-67 in the form of particles. Embodiment 73. The suspension of Embodiment 72, wherein the aqueous phase is substantially free of the nucleic acid. Embodiment 74. A method of treating a disease, disorder, or condition comprising administering to a subject the suspension of Embodiment 72 or 73. Embodiment 75. The method of Embodiment 74, wherein the disease, disorder, or condition is selected from an infectious disease, cancer, a genetic disorder, an autoimmune disease, or a rare disease. Embodiment 76. A method of increasing or causing increased expression of RNA in a target in a subject comprising administering to the subject the suspension of Embodiment 72 or 73. Embodiment 77. The method of Embodiment 76, wherein the target is selected from the lungs, liver, spleen, heart, brain, lymph nodes, bladder, kidneys, and pancreas. Page 164 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Embodiment 78. The method of any one of Embodiments 74-77, wherein the suspension is administered intramuscularly, intranasally, intravenously, subcutaneously, or intratumoraly. Embodiment 79. Use of the suspension of Embodiment 72 or 73 for use in medicine. Embodiment 80. Use of the suspension of Embodiment 72 or 73 for the treatment of a disease, disorder or condition. EXAMPLES

[0341] As described in the Examples below, in certain exemplary embodiments, compounds are prepared according to the following general procedures. It will be appreciated that, although the general methods depict the synthesis of certain compounds of the present disclosure, the following general methods and other methods known to one of ordinary skill in the art can be applied to all compounds and subclasses and species of each of these compounds, as described herein. Table of Abbreviations Ac acetyl ACN i ilPage 165 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Fmoc-dab Nα-Fmoc-Nγ-Boc-L-2,4-diaminobutyric acid GAC general carboxylic acidGeneral Structures of Fragments Used to Assemble Cationic Lipids

[0342] The present disclosure provides cationic lipids (i.e., lipids of Table 1 and / or Table 2) that can be assembled in general using fragments depicted in Table 5 below. In some embodiments, the general structure of cationic lipids carry a guanidino-headgroup and two optionally branched (with Ak) hydrophobic tails (X and Y). Table 5 ral Structure =Page 166 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0343] A person of skill in the art will appreciate that hydrophobic tails of cationic lipids of the present disclosure can be connected to the guanidino-headgroup either via two amide connections or via one amide and one ester connection. In some embodiments, cationic lipids of the present disclosure bearing an ester / amide connection have a structure similar to the compound depicted in Table 5. X and Y, when optionally combined with Ak, deliver fragments for the synthesis of cationic lipids. In some embodiments such fragments are amines (i.e., X fragments). In some embodiments, such fragments are alcohols (i.e., X fragments). In some embodiments, such fragments are carboxylic acids (i.e., Y fragments). Exemplary amine (GAM-001-009), alcohol (GAL 001-009), and carboxylic acid (GAC-001-009) fragments are depicted in Table 6. Table 6 Page 167 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Amine Fragments =

[0344] One of skill in the art may appreciate that in some embodiments, convergent synthesis employing a guanidino-headgroup (i.e., that depicted in Table 5), an amine fragment (i.e., GAM- 001-009), and a carboxylic acid fragment (i.e., GAC-001-009) delivers a cationic lipid of the present disclosure bearing two amide connections to the headgroup. In some embodiments, Page 168 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 convergent synthesis employing a guanidino-headgroup (i.e., that depicted in Table 5), an alcohol fragment (i.e., GAL-001-009), and a carboxylic acid fragment (i.e., GAC-001-009) delivers a cationic lipid of the present disclosure bearing one amide and one ester connection to the headgroup. Example 1 – General Synthesis of Amine Fragments

[0345] Amine fragments of the present disclosure (i.e., GAM-001-009) can be made by the scheme provided below.Example 2 – General Synthesis of Alcohol Fragments

[0346] Alcohol fragments of the present disclosure (i.e., GAL-001-009) can be made by the scheme provided below.Example 3 – General Synthesis of Carboxylic Acid Fragments

[0347] Carboxylic Acid fragments of the present disclosure (i.e., GAC-001-009) can be made by the scheme provided below. Page 169 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802ide and One Ester Connection to a Guanidino-Headgroup

[0348] Compounds of the present disclosure can be made by the scheme provided below. The present example demonstrates a general convergent synthesis employing a guanidino-headgroup (i.e., analogous to that depicted in Table 5), an alcohol fragment (i.e., GAL-002), and a carboxylic acid fragment (i.e., GAC-001) to deliver a cationic lipid of the present disclosure bearing one amide connection and one alcohol connection to the headgroup. One of skill in the art may appreciate that the below method can be applied to the synthesis of other exemplified cationic lipids of the present disclosure (i.e., those derived from other analogous guanidino-headgroups as well as GAL and GAC fragments). Page 170 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802Example 5 – General Procedure B: General Synthesis of a Cationic Lipid Bearing Two Amide Connections to a Guanidino-Headgroup

[0349] Compounds of the present disclosure can be made by the scheme provided below. The present example demonstrates a general convergent synthesis employing a guanidino-headgroup (i.e., analogous to that depicted in Table 5), an amine fragment (i.e., GAM-002), and a carboxylic acid fragment (i.e., GAC-001) to deliver a cationic lipid of the present disclosure bearing two Page 171 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 amide connections to the headgroup. One of skill in the art may appreciate that the below method can be applied to the synthesis of other exemplified cationic lipids of the present disclosure (i.e., those derived from other analogous guanidino-headgroups as well as GAL and GAC fragments).mide and One Ester Connection to a Guanidino-Headgroup

[0350] Compounds of the present disclosure can be made by the scheme provided below. The present example demonstrates a general convergent synthesis employing a guanidino-headgroup Page 172 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 (i.e., analogous to that depicted in Table 5), an alcohol fragment (i.e., GAL-001), and a carboxylic acid fragment (i.e., GAC-001) to deliver a cationic lipid of the present disclosure bearing one amide connection and one alcohol connection to the headgroup. One of skill in the art may appreciate that the below method can be applied to the synthesis of other exemplified cationic lipids of the present disclosure (i.e., those derived from other analogous guanidino-headgroups as well as GAL and GAC fragments). Page 173 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Example 7g Two Amide Connections to a Guanidino-Headgroup

[0351] Compounds of the present disclosure can be made by the scheme provided below. The present example demonstrates a general convergent synthesis employing a guanidino-headgroup (i.e., analogous to that depicted in Table 5), an amine fragment (i.e., GAM-002), and a carboxylic Page 174 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 acid fragment (i.e., GAC-001) to deliver a cationic lipid of the present disclosure bearing two amide connections to the headgroup. One of skill in the art may appreciate that the below method can be applied to the synthesis of other exemplified cationic lipids of the present disclosure (i.e., those derived from other analogous guanidino-headgroups as well as GAL and GAC fragments).f a Cationic Lipid Bearing One Amide and One Ester Connection to a Guanidino-Headgroup

[0352] Compounds of the present disclosure can be made by the method provided below. The present example demonstrates a representative convergent synthesis employing a guanidino- headgroup (i.e., analogous to that depicted in Table 5), an alcohol fragment (i.e., GAL-009), and a carboxylic acid fragment (i.e., GAC-001) to deliver cationic lipid A-16-i bearing one amide and one ester connection to the headgroup. One of skill in the art may appreciate that the below method Page 175 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 can be applied to the synthesis of other exemplified cationic lipids of the present disclosure (i.e., those derived from other analogous guanidino-headgroups as well as GAL and GAC fragments).

[0353] 5 mL) and cooled down to 0 °C. DMAP (9.2 mg; 0.1 eq.), 8-hydroxyoctyl 2-hexyldecanoate (GAL-009; 300 mg, 1.2 eq.) and DCC (186.3 mg; 1.2 eq.) were added. The reaction was then allowed to warm up slowly to RT and was stirred overnight. The reaction progress was controlled by LC / MS. The reaction was filtered. The organic layer was then extracted with water and washed with NaCl solution. The organic layer was then dried with NaSO4and the solvent was removed under reduced pressure. The crude product (600 mg) was purified by automated flash chromatography with Flash Pure EcoFlex Silica 40 g and a gradient of 40 minutes EA in PE (10%-30%) to yield 8-(((S)-2- ((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4-((tert- butoxycarbonyl)amino)butanoyl)oxy)octyl 2-hexyldecanoate as an oil (465.2 mg, 75.3%).Page 176 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0354] Step 2: 8-(((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4-((tert- butoxycarbonyl)amino)butanoyl)oxy)octyl 2-hexyldecanoate (442.2 mg; 0.548 mmol; 1eq) was dissolved in dry DMF (10 mL) and piperidine (0.553 mL, 10 eq.) was slowly added. The mixture was stirred at room temperature for 2 hours. The reaction was monitored by LC / MS. Water was added to the reaction mixture and the suspension was extracted with ethyl acetate (2x). The combined organic layers were dried over anhydrous Na2SO4, filtered off and the solvent was removed under reduced pressure. The crude product (426.7 mg) was dissolved in DCM and was filtered. The filtrate was purified by automated column chromatography (column: Büchi EcoFlex 40 g, gradient: DCM / MeOH 0-5%) to afford 8-(((S)-2-Amino-4-((tert- butoxycarbonyl)amino)butanoyl)oxy)octyl 2-hexyldecanoate (249.7 mg, 77.4%). [0352- hexyldecanoate (102 mg; 0.174 mmol; 1 eq) and 4-((2-butyloctanoyl)oxy)butanoic acid (GAC001; 49 mg, 1 eq) were dissolved in dry DMF (10 mL) and afterwards treated with DIPEA (60.1 µL) at 0 °C for 15 min. Then, a solution of HATU (72 mg) in 1 mL DMF was added dropwise and the resulting yellow reaction mixture was stirred at room temperature for 2.5 hours. The reaction progress was controlled by TLC and LC / MS. The solvent was removed under reduced pressure. The crude product (279.3 mg) was purified by automated flash chromatography with Flash Pure EcoFlex Silica 25 g and a gradient of 10 minutes pure DCM, 30 minutes of DCM and a premix of DCM an MeOH (8 / 2) from 0% to 25% (0%-5% MeOH) to afford 8-(((2S)-4-((tert- butoxycarbonyl)amino)-2-(4-((2-butyloctanoyl)oxy)butanamido)butanoyl)oxy)octyl 2- hexyldecanoate as an oil (136 mg, 90%). Page 177 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 [035 ((2-butyloctanoyl)oxy)butanamido)butanoyl)oxy)octyl 2-hexyldecanoate (134 mg; 0.158 mmol; 1 eq.) in 10 mL dry DCM was added trifluoroacetic acid (TFA, 2 mL, 20% of solvent volume) dropwise at 0 °C. The colorless solution was stirred at 0 °C for 1h. The reaction mixture was diluted with DCM and washed with aqu. sat. NaHCO3 (3x). The organic phase was dried over Na2SO4and filtered off. The product was directly used (in DCM solution) for the next reaction without purification.

[0357] y)octyl 2-hexyldecanoate (1 eq., 133,127 mg) was dissolved in DMF (15 mL). After addition of N,N- diisopropylethylamine (4 eq., 118 μL), 1H-pyrazole-1-carboxamidine monohydrochloride (2,5 eq., 63,6 mg) was given to the reaction mixture. The reaction was stirred over the weekend. The reaction mixture was concentrated in vacuo. The crude product was purified on RP by automated flash chromatography (XSelect C18 50x150, gradient: buffer A (5mM ammonium acetate buffer) / mobile phase B (ammonium acetate buffer / iPrOH / ACN 5:62:33) 80-100%) to afford the final product A-16-i as a colorless oil (98.8 mg; 69.1%). Purity (CAD): 92.5%;1H NMR (300 MHz, CDCl3) δ = 9.15 (s, 1H, NH), 7.22 (d, J=7.4, 1H, NH), 4.49 (m, 1H, stereocenter CH), 4.19 Page 178 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 – 4.02 (m, 6H, O-CH2), 3.34 – 3.20 (m, 2H, CH), 2.56 (bs, 2H, NH2), 2.37 – 2.25 (m, 4H), 2.00 (s, 4H), 1.97 (s, 1H), 1.70 – 1.51 (m, 8H), 1.44 (q, J=7.3, 4H), 1.32 (s, 10H), 1.26 (m, 30H), 0.92 – 0.83 (m, 12H, CH3). Example 9 – Synthesis of A-56 via General Procedure B: Representative Synthesis of a Cationic Lipid Bearing One Amide and One Ester Connection to a Guanidino-Headgroup

[0358] Compounds of the present disclosure can be made by the method provided below. The present example demonstrates a representative convergent synthesis employing a guanidino- headgroup (i.e., analogous to that depicted in Table 5), an alcohol fragment (i.e., GAL-001), and a carboxylic acid fragment (i.e., GAC-008) to deliver cationic lipid A-56 bearing one amide and one ester connection to the headgroup. One of skill in the art may appreciate that the below method can be applied to the synthesis of other exemplified cationic lipids of the present disclosure (i.e., those derived from other analogous guanidino-headgroups as well as GAL and GAC fragments).

[0359] St0.121 g, 72.6 mmol) in aqueous 5M NaOH (14.5 mL) and 55 mL CH2Cl2 was added benzylchloroformate (38.285 mg, 31.9 mL, 217.7 mmol) via a dropping funnel at 0 °C in 10 minutes. The reaction mixture was then allowed to warm up to room temperature and stirred for 48 h. The reaction was monitored by TLC (CH2Cl2= 100). The 2-methyl-2-thiopseudourea was stained using a previously made sodium amminepentacyanoferrate(II) staining solution and an acidic PtCl2 staining solution. The reaction was diluted with ultrapure water (100 mL) and extracted with CH2Cl2 (2 x 80 mL). The organic layers were combined, dried over anhydrous Na2SO4, filtered, and evaporated to achieve a colorless oil, which was purified via silica column chromatography using a Büchi Silica EcoFlex F0330 column on an automated Büchi C-850 column chromatography system. Solvent A: Petroleum Ether (PE); Solvent B: Ethyl Acetate (EA). The product-containing fractions were combined and concentrated in vacuo to yield 7.217 g of N,N'-di-benzyloxycarbonyl-S- Page 179 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 methylisothiourea as a colorless oil, which quickly solidified to a wax-like white solid at room temperature (27.4%).

[0360] urea (7.2 g,20.1 mmol, 1 eq) in 150 mL anhydrous MeOH at 50 °C was added 4-dimethylaminopyridine (246 mg, 0.1 eq.) and N-alpha-t-butyloxycarbonyl-L-2,4-diaminobutyric acid (8.3 g, 1.80 eq.). The beige suspension was stirred at 50 °C. After 3 days, 4-dimethylaminopyridine (369 mg, 0.15 eq.) was added and the mixture was stirred at 50 °C. After 1 day the temperature was decreased to 45 °C and the mixture was stirred for 3 days. The reaction was monitored by TLC. The solvent was removed under reduced pressure. The crude was suspended in ethyl acetate (200 mL) and the beige suspension was washed with citric acid (2x 80 mL, aqu., 10%) and aqu. sat. NaHCO3(2x 80 mL). The separated aqueous layers were both reextracted with ethyl acetate. The organic phase was dried over Na2SO4 and concentrated in vacuo. The crude product was purified via silica column chromatography using a Büchi Silica EcoFlex F0220 column on an automated Büchi C-850 column chromatography system (gradient: dichlormethane (A) / methanol (B) 0-10% B) to afford 7.1 g of the product, yield: 67%.

[0361] , ar was charged with a solution of the product of Step 2 (350.3 mg, 0.662 mmol, 1 eq.) in CH2Cl2(7 mL). The flask was placed in an ice bath and 4-dimethylaminopyridine (26.5 mg, 0.166 mmol, 0.25 eq.), a solution of 4-hydroxybutyl 2-butyloctanoate (GAL-001; 232.0 mg, 0.795 mmol, 1.2 eq.) in 1 mL CH2Cl2 and 1,3-dicyclohexylcarbodiimide (165.0 mg, 0.795 mmol, 1.2 eq.) were added. The white Page 180 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 suspension was allowed to warm up to rt. Stirring at rt was continued for 3 h. The precipitate was filtered off, CH2Cl2 (15 mL) was added to the flask and the colorless solution was extracted with H2O (2x15 mL) and brine (1x15 mL). The organic layer was dried over anhydrous Na2SO4, filtered off and concentrated in vacuo. The product was redissolved in CH2Cl2and undissolvable solids were filtered off to achieve the crude product as a colorless, murky oil. It was then purified via silica column chromatography using an Interchim Silica 25g column on an automated Büchi C850 column chromatography system (Solvent A: CH2Cl2; Solvent B: premix: CH2Cl2 / MeOH = 95 / 5). The product-containing fractions were concentrated in vacuo to yield 499.3 mg of (S,E)-5- (((benzyloxy)carbonyl)amino)-9-((tert-butoxycarbonyl)amino)-3,10-dioxo-1-phenyl-2,11-dioxa- 4,6-diazapentadec-4-en-15-yl 2-butyloctanoate as a colorless oil (96.3%).

[0362] Sten of (S,E)-5- (((benzyloxy)carbonyl)amino)-9-((tert-butoxycarbonyl)amino)-3,10-dioxo-1-phenyl-2,11-dioxa- 4,6-diazapentadec-4-en-15-yl 2-butyloctanoate (663.6 mg, 0.848 mmol, 1 eq.) in 8.0 mL dry CH2Cl2. TFA (2500 µL, excess) was dropwise added at 0 °C. The colorless solution was stirred at 0 °C for 6 h. The reaction mixture was diluted with 20 mL CH2Cl2and washed with aqu. sat. NaHCO3 (3 x 15 mL). The organic phase was dried over Na2SO4, filtered off and concentrated in vacuo to around 3 mL. 4 mL anhydrous DMF was added and the remaining CH2Cl2 was evaporated off. The dissolved crude product (S,E)-9-amino-5-(((benzyloxy)carbonyl)amino)-3,10- dioxo-1-phenyl-2,11-dioxa-4,6-diazapentadec-4-en-15-yl 2-butyloctanoate was used for the next step without further purification. A 100% conversion for the reaction was assumed. Page 181 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0363] String bar was charged with a solution of (S,E)-9-amino-5-(((benzyloxy)carbonyl)amino)-3,10-dioxo-1-phenyl- 2,11-dioxa-4,6-diazapentadec-4-en-15-yl 2-butyloctanoate (100% conversion of the compound in the previous step was assumed: 435.5 mg, 0.638 mmol, 1 eq.) in 4 mL DMF. The flask was put in an ice bath and a solution of 6-((2-hexyldecanoyl)oxy)hexanoic acid (GAC-008; 250.8 mg, 0.670 mmol, 1.05 eq.) in 1 mL DMF and N,N-diisoproylethylamine (527.0 µL, 3.189 mmol, 5 eq.) was added. After stirring at 0 °C for 15 min a solution of HATU (295.2 mg, 0.765 mmol, 1.2 eq.) in 0.5 mL DMF was added. The colorless solution turned yellow and stirring was continued at rt for 15 min. A reaction control by LC / MS showed complete conversion. Thus, the yellowish solution was concentrated in vacuo. The crude product was dissolved in 5 mL of CH2Cl2, undissolvable crystals were filtered off and the filtrate was again concentrated in vacuo to achieve the crude product as an orange oil. The orange oil was purified via silica column chromatography using a Büchi Silica EcoFlex F0040 column on an automated Büchi C-850 column chromatography system (Solvent A: CH2Cl2; Solvent B: premix: CH2Cl2 / MeOH = 95 / 5) to afford 569.3 mg of the product as a yellowish oil (86.2 %). Page 182 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0364] ring bar was charged with the product of Step 5 (569.3 mg, 549.8 µmol, 1 eq.) and dry EtOH (4.0 mL). Pd(OAc)2(50.1 mg, 69.5 µmol, 0.4 eq.) was added and the atmosphere was replaced with H2. After stirring for 30 min the orange suspension turned black. A reaction control by LC / MS showed complete conversion after 4.5 h. The black precipitate was filtered off using CELITE. The clear brownish-black filtrate was concentrated in vacuo and purified via reversed phase column chromatography using a Waters XSelect CSH Prep C18 5µm OBD 50x150mm column on an automated Büchi C-850 column chromatography system (Solvent A: 5 mM Ammonium acetate; Solvent B: iPrOH / ACN / aqu. sat. NH3= 62:33:5). The product containing fractions were combined and concentrated in vacuo to achieve the product as a clear, colorless oil. Purity control by UPLC showed a purity of only around 80%. To improve purity, the compound was repurified by a Waters autopurification system using a XSelect CSH prep. phenyl-hexyl 5µm OBD 30x150mm column to afford 163.4 mg of final product A-56 as a colorless, oily solid (38.7%). Purity (CAD): 99.1%;1H NMR (300 MHz, CDCl3) δ = 8.29 (s, 3H, NH), 7.02 (d, J=7.4, 1H, NH), 4.69 (bs, 5H), 4.49 – 4.37 (m, 1H, stereocenter CH), 4.29 – 4.06 (m, J=4.8, 2H), 4.05 – 3.84 (m, 4H, O-CH2), 3.35 – 3.13 (m, 2H, NH-CH2), 2.33 – 2.16 (m, 4H), 2.14 – 2.04 (m, 1H), 1.94 – 1.76 (m, 1H), 1.71 – 1.58 (m, 4H), 1.58 – 1.44 (m, 2H), 1.42 – 1.27 (m, 4H), 1.27 – 1.09 (m, 33H, CH2), 0.81 (t, J=1.5, 12H, CH3). Page 183 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Example 10 – Synthesis of A-20 via General Procedure C: Representative Synthesis of a Cationic Lipid Bearing Two Amide Connections to a Guanidino-Headgroup

[0365] Compounds of the present disclosure can be made by the method provided below. The present example demonstrates a representative convergent synthesis employing a guanidino- headgroup (i.e., analogous to that depicted in Table 5), an amine fragment (i.e., GAM-001), and a carboxylic acid fragment (i.e., GAC-001) to deliver cationic lipid A-20 bearing two amide connections to the headgroup. One of skill in the art may appreciate that the below method can be applied to the synthesis of other exemplified cationic lipids of the present disclosure (i.e., those derived from other analogous guanidino-headgroups as well as GAL and GAC fragments). [03Dab(Boc)-OH (350 mg, 795 µmol.) were dissolved in dry DMF (8 mL). DIPEA (139.8 µL, 1 eq.) was added, and the mixture was stirred at 0 °C for 15 min. Then, a solution of HATU (335.7 mg, 1.1 eq.) in 1 mL DMF was added dropwise and the resulting yellow reaction mixture was stirred at room temperature. After 4 hours, DIPEA (139.8 µL, 1 eq.) was added, and the mixture was stirred at room temperature. After 1 hour, DIPEA (139.8 µL, 1 eq.) was added, and the mixture was stirred at room temperature for 20 hours. The reaction progress was controlled by TLC (8% MeOH in CH2Cl2) and LC / MS. The solvent was removed under reduced pressure. The crude product was purified by automated column chromatography (column: Büchi EcoFlex 40g, gradient: CH2Cl2 / MeOH 0-10%). The fractions were not completely clean. Therefore, the product was repurified by automated column chromatography (column: Büchi EcoFlex 40g, CH2Cl2 (A): CH2Cl2 / MeOH 8:2(B), 0 to 25% eluent B) to afford 415.3 mg of 4-((S)-2-((((9H-Fluoren-9- yl)methoxy)carbonyl)amino)-4-((tert-butoxycarbonyl)amino)butanamido)butyl 2-butyloctanoate as an oil (75%).Page 184 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0367] Step 2: 4-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4-((tert- butoxycarbonyl)amino)butanamido)butyl 2-butyloctanoate (478.9 mg) was dissolved in dry DMF (10 mL) and piperidine (1367 µL, 20 eq.) was slowly added. The mixture was stirred at room temperature for 3 hours. The reaction was monitored by TLC (3% MeOH in CH2Cl2, product is not visible at the TLC) and LC / MS. Water was added and the suspension was extracted with ethyl acetate (3x). The combined organic layers were dried over anhydrous Na2SO4, filtered off and the solvent was removed under reduced pressure. The crude product was purified by automated column chromatography (column: Büchi EcoFlex 25g, gradient: CH2Cl2 / 7N NH3 in MeOH 0- 10%) to afford 176.6 mg of 4-((S)-2-Amino-4-((tert-butoxycarbonyl)amino)butanamido)butyl 2- butyloctanoate as an oil (54%).

[0368] utyl 2- butyloctanoate (170.1 mg, 361 µmol) and 4-((2-butyloctanoyl)oxy)butanoic acid (GAC-001, 103.3 mg, 1 eq.) were dissolved in dry DMF (5 mL). DIPEA (127.2 µL, 2 eq.) was added, and the mixture was stirred at 0 °C for 15 min. Then, a solution of HATU (152.4 mg, 1.1 eq.) in 1 mL DMF was added dropwise and the resulting yellow reaction mixture was stirred at room temperature for 2 hours. The reaction progress was controlled by TLC (8% MeOH in CH2Cl2) and LC / MS. The solvent was removed under reduced pressure. The crude product was purified by automated column chromatography (column: Büchi EcoFlex 40 g, gradient: CH2Cl2(A) / premix CH2Cl2 / MeOH (B) 0-40% B). The product was not completely clean. Therefore, the reaction was further purified by automated column chromatography (column: Büchi EcoFlex 25 g, gradient: CH2Cl2(A) / premix CH2Cl2 / MeOH (B) 0-40% B) to afford 190.7 mg of 4-(4-((tert- Butoxycarbonyl)amino)-2-(4-((2-butyloctanoyl)oxy)butanamido)butanamido)butyl 2- butyloctanoate as colorless oil (71%). Page 185 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0369] 2-(4-((2-butyloctanoyl)oxy)butanamido)butanamido)butyl 2-butyloctanoate (190.7 mg, 258 µmol) was dissolved in 5 mL dry CH2Cl2. The mixture was cooled to 0 °C. TFA (0.5 mL, 10% of solvent volume) was added dropwise at 0 °C and the reaction mixture was stirred at 0 °C for 5 hours. CH2Cl2was added and the mixture was washed with aq. sat. NaHCO3. The organic layer was dried over Na2SO4 and filtered off. The solvent was concentrated in vacuo to around 2 mL. Anhydrous DMF (5 mL) were added and the remaining CH2Cl2 was evaporated. The dissolved crude product was used for the next step without further purification (100% yield was assumed).

[0370] oxobutan-2- yl)amino)-4-oxobutyl 2-butyloctanoate (164.9 mg, 258 µmol) was dissolved in dry N,N- dimethylformamide (5 mL). N,N-Diisopropylethylamine (4 eq., 167.2 μL) and 1H-pyrazole-1- carboxamidine monohydrochloride (1.5 eq., 56.7 mg) were added.1 mL chloroform was added to the suspension and all reagents have been completely dissolved. The mixture was stirred at room temperature. The solvent was removed under reduced pressure. The crude product was purified on RP by automated flash chromatography (XSelect C18 50x150, gradient: buffer A (5mM ammonium acetate buffer) / mobile phase B (ammonium acetate Page 186 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 buffer / iPrOH / acetonitrile 5:62:33) 80-100% B) to afford 72.3 mg of final product A-20 (41%). Purity (CAD): 98.4%;1H NMR (300 MHz, CDCl3) δ = 9.19 (s, 1H, NH), 8.62 (s, 1H, NH), 8.55 (t, J=5.76, 1H, NH), 7.17 (d, J=7.7, 1H, NH), 4.65 – 4.47 (m, 1H, CH stereocenter), 4.18 – 3.98 (m, 4H, O-CH2), 3.37 – 3.12 (m, 4H, NH-CH2), 2.41 – 2.22 (m, 4H, CH), 2.15 – 1.76 (m, 5H), 1.75 – 1.36 (m, 12H, CH2), 1.36 – 1.11 (m, 24H, CH2), 0.87 (t, J=3.3, 12H, CH3). Example 11 – Synthesis of A-18 via General Procedure D: Representative Synthesis of a Cationic Lipid Bearing Two Amide Connections to a Guanidino-Headgroup

[0371] Compounds of the present disclosure can be made by the method provided below. The present example demonstrates a representative convergent synthesis employing a guanidino- headgroup (i.e., analogous to that depicted in Table 5), an amine fragment (i.e., GAM-008), and a carboxylic acid fragment (i.e., GAC-009) to deliver cationic lipid A-18 bearing two amide connections to the headgroup. One of skill in the art may appreciate that the below method can be applied to the synthesis of other exemplified cationic lipids of the present disclosure (i.e., those derived from other analogous guanidino-headgroups as well as GAL and GAC fragments).

[0372] Step 1 and 2: Analogous to step 1 and 2 in Example 9.Page 187 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0373] Step 3: A 25-mL, round-bottom flask equipped with a magnetic stirring bar was charged with the product of Step 2 (396.0 mg, 0.749 mmol, 1 eq.) and 6-aminohexyl 2- hexyldecanoate (GAM-008; 352.5 mg, 0.899 mmol, 1 eq.) in dry DMF (4 mL). The flask was put in an ice bath and N,N-diisoproylethylamine (0.371 mL, 2.248 mmol, 1 eq.) was added. After stirring for 15 min a solution of HATU (320.1 mg, 0.824 mmol, 1.1 eq.) in DMF (0.5 mL) was added. The colorless suspension turned yellow and was stirred at 0 °C for 1 h. A reaction control after 1h showed complete conversion. Therefore, the yellowish solution was concentrated in vacuo and purified via silica column chromatography using a Büchi Silica EcoFlex F0040 column on an automated Büchi C-850 column chromatography system (Solvent A: CH2Cl2; Solvent B: CH2Cl2 / MeOH: 0-60% B) The product containing vials were combined and concentrated in vacuo to achieve 600.5 mg of (S,E)-5-(((benzyloxy)carbonyl)amino)-9-((tert-butoxycarbonyl)amino)- 3,10-dioxo-1-phenyl-2-oxa-4,6,11-triazaheptadec-4-en-17-yl 2-hexyldecanoate as a yellowish oil (92.6%).- (((benzyloxy)carbonyl)amino)-9-((tert-butoxycarbonyl)amino)-3,10-dioxo-1-phenyl-2-oxa- 4,6,11-triazaheptadec-4-en-17-yl 2-hexyldecanoate (300.3 mg, 0.347 mmol, 1 eq.) in 6.0 mL dry CH2Cl2. TFA (1200 µL, excess) was dropwise added at 0 °C. The colorless solution was stirred at 0 °C for 4 h. A reaction control by LC / ELSD showed almost complete conversion. The reaction mixture was diluted with 20 mL CH2Cl2 and washed with aqu. sat. NaHCO3 (3 x 20 mL). The organic phase was dried over Na2SO4, filtered off and concentrated in vacuo to around 3 mL.4mL anhydrous DMF was added and the remaining CH2Cl2was evaporated off. The dissolved crude product (S,E)-9-amino-5-(((benzyloxy)carbonyl)amino)-3,10-dioxo-1-phenyl-2-oxa-4,6,11- triazaheptadec-4-en-17-yl 2-hexyldecanoate was used in the next step without further purification (100% conversion assumed). Page 188 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0375] bar was charged with a solution of (S,E)-9-amino-5-(((benzyloxy)carbonyl)amino)-3,10-dioxo-1-phenyl- 2-oxa-4,6,11-triazaheptadec-4-en-17-yl 2-hexyldecanoate (100% conversion was assumed: 265.545 mg, 0.347 mmol, 1 eq.) in 4 mL DMF. The flask was put in an ice bath. A solution of 8- ((2-hexyldecanoyl)oxy)octanoic acid (GAC-009; 153.0 mg, 0.381 mmol, 1.1 eq.) in 1 mL DMF and N,N-diisoproylethylamine (286 µL, 1.733 mmol, 5 eq.) was added. After stirring at 0 °C for 15 min a solution of HATU (160.2 mg, 0.416 mmol, 1.2 eq.) in 0.5 mL DMF was added. The colorless solution turned yellow and the flask was left in the thawing ice bath for 18 h. A reaction control by LC / MS showed complete conversion. Thus, the yellowish solution was concentrated in vacuo and dried at high vacuum over 16 h. The crude product was dissolved in 5 mL of CH2Cl2, undissolved crystals were filtered off and the filtrate was again concentrated in vacuo to achieve the crude product as an orange oil. The orange oil was purified via silica column chromatography using an Interchim Silica 40g column on an automated Büchi C-850 column chromatography system (Solvent A: CH2Cl2; Solvent B: premix: CH2Cl2 / MeOH = 95 / 5: 0-60% B). The product containing fractions were concentrated in vacuo to achieve 328.4 mg of the product as a colorless oil (82.6 %, 2 steps). Page 189 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0376] s charged with the product of step 5 (328.4 mg, 286.4 µmol, 1 eq.) and dry EtOH (4.0 mL). Pd(OAc)2 (27.0 mg, 114.5 µmol, 0.4 eq.) was added and the atmosphere was replaced with H2. After stirring for 30 min the orange suspension turned black. A reaction control by LC / MS showed complete conversion after 2 h. The black precipitate was filtered off using CELITE. The clear brownish- black filtrate was concentrated in vacuo (250.9 mg) and purified via reversed phase column chromatography using a Waters XSelect CSH Prep C18 5µm OBD 50x150mm column on an automated Büchi C-850 column chromatography system (Solvent A: 5 mM Ammonium acetate; Solvent B: iPrOH / ACN / aqu. sat. NH3 = 62:33:5; 80-100% B). The product containing fractions were combined and concentrated in vacuo to achieve 150.8 mg of A-18 as a colorless oil. Purity (CAD): 94.95%;1H NMR (300 MHz, CDCl3) δ = 9.01 (s, 1H, NH), 8.48 (t, J=5.6, 1H, NH), 7.31 (d, J=7.6, 1H, NH), 5.19 (bs, 6H), 4.60 – 4.43 (m, 1H, CH, stereocenter), 4.04 (t, J=6.65, 4H, O- CH2), 3.22 (m, 4H, NH-CH2), 2.36-2.27 (m, 2H, -CH), 2.22 (t, J=7.56, 2H, CH), 1.92 – 1.74 (m, 2H), 1.66 – 1.28 (m, 20H, CH2), 1.25 (s, 41H), 0.94 – 0.77 (t, J=6.2612H, CH3).

[0377] The following examples in Table 7 and Table 8 were prepared using standard chemical manipulations and procedures similar to those used for the preparation of the previous examples. Page 190 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Table 7 Acid09Page 191 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Table 8 Acid09Example 12 – Lipid Compositions Comprising mRNA

[0378] Ethanolic solutions of cationic lipids, anionically ionizable lipids, cholesterol, and DMPE-PEG2k were prepared separately. The different lipid solutions were dispensed in predefined ratios into a 96-well plate in the following order: cationic lipid, cholesterol, DMPE- PEG2k, and anionic lipid. Enough ethanol was then added to complete a final volume of 33 µL. mRNA encoding for luciferase was dissolved in 35mM Tris(hydroxymethyl)aminomethane and 40mM acetic acid pH5.5 at a concentration of 110 ng / µL. The nucleic acid solution completed the final volume of 200 µL. Aliquots were taken and further diluted stepwise using 50mM Tris:Acetate pH7.5 buffer, to a final nucleic acid concentration of 10 ng / µL. Particle size was monitored at this Page 192 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 point using a Wyatt Dynapro III Dynamic Light Scattering Instrument. The obtained particles were used without further purification.

[0379] For transfection studies, lipid formulations comprising mRNA encoding luciferase were prepared as described above. Next, 10 µL of each sample was prediluted in 10 µL of either buffer (denoted -S) or human serum (denoted +S). The mixtures were incubated at room temperature for 30 minutes before transfection. After incubation, HEK293 or HepG2 cells were transfected with a total mRNA content of 50 ng for both the serum-naive and serum-containing nanoparticles and incubated for 24 h. The HEK293 or HepG2 cells were then analyzed for luciferase expression. For analysis, the signals of the top 15% individual formulations per design space were averaged and compared to an established reference. The expression of small aggregates (< 40 nm) and large particles (> 250 nm in diameter) was not considered. Example 13 – Analysis of various polar head groups

[0380] Lipid formulations were generated as described above using cationic guanido lipid, dioleoylglycerol hemisuccinate (DOGS), cholesterol and DMPE-PEG2k lipid. The design space comprised 44 samples having between 20 and 50 mol% cholesterol and 0.15 mol% of DMPE- PEG2k lipid, the remainder being cationic guanido lipid and DOGS in ratios between 0.67 and 1.5. The cationic guanido lipids were selected from A-1 A)Page 193 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 A-2 ^^^-dimethyl-PONAr human serum and tested for expression of luciferase using HepG2 cells. Lipid formulations comprising mRNA encoding luciferase were prepared, incubated in human serum (denoted +S) or buffer (denoted-S) and expression of luciferase was monitored in HepG2 cells. Signals for the best 15% of the individual formulations per formulation design space were averaged.

[0382] As depicted in Figure 1, PONA, POA and PONA methylated at the ^-nitrogen show activity comparable to the reference. Single or double methylations of the ^-nitrogen are less tolerated and cyclization of the terminal nitrogens also results in a loss of activity. Without wishing to be bound to any particular theory, it is concluded that elongation or methylation of the polar linker is tolerated, but modification of the terminal nitrogens is less preferred. Example 14 – Analysis of Lipidic Portions of Cationic Lipids

[0383] Lipid formulations were generated as described above using cationic guanido lipid, anionic lipid, cholesterol and DMPE-PEG2k lipid. The anionic lipid was dioleoyl glycerol hemisuccinate (DOGS). The design space comprised 44 samples in a matrix having between 20 and 50 mol% cholesterol and 0.15 mol% of DMPE-PEG2k lipid, the remainder being cationic guanido lipid and anionic lipid used in ratios between 0.5 and 1.5. The cationic guanido lipids were the materials of example 4 to 11, having a nor-arginine moiety wherein the carboxyl is amidated (X=NH) or esterified (X=O) to an apolar moiety and the N^-amine is amidated to an apolar moiety.

[0384] The transfection of mRNA was observed in different cell lines and with or without pre- incubation in full human serum which resembles a physiological condition.

[0385] As described above with respect to various embodiments, for cationic lipids of formula ICL described herein, lipidic volume is determined by the following moiety: Page 194 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0386] By way of example of a catiowhep g he dashed line.

[0387] The lipidic volume for DOGS is 689Å3. As shown in Figure 2A, formulations wherein the combined lipidic volumes of the cationic and anionic lipid is 1400Å3or more can have an efficacy within an order of magnitude of the reference formulation, in contrast to those having a smaller lipidic volume. It has been surprisingly discovered that the combined lipidic volume is a predictor for the activity of the formulations. This is demonstrated in Figure 2B which analyzes the relative frequency of formulations having 15% or more activity compared to the reference. This frequency is >25% for materials wherein the combined lipidic volumes are 1400 Ų or more.

[0388] An analysis for the cationic lipids shows that cationic lipids having a lipidic volume of 700 Å3or more can have an efficacy within the order of magnitude of the reference formulation, while cationic lipids having a lipidic volume of less than 700Å3have only a low efficacy. See Figure 3A. The improvement of efficacy can be observed for compounds wherein X= NH such that both T1and T2(referring to formula ICL, above) are connected to the nor-arginine scaffold through amide bonds, denoted as “amide-amide” in Figure 3A; it can also be observed for Page 195 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 compounds wherein X= O so that T1is connected through an ester and T2is linked through an amide, denoted as “ester-amide” in Figure 3A.

[0389] Between formulations having the same anionic lipid, which is DOGS in this particular example, the greater cationic lipidic volume shows relative improvement of provided formulations. This is demonstrated in Figure 3B which analyzes the relative frequency of formulations having 15% or more activity compared to the reference. This frequency is >25% for materials wherein the combined lipidic volumes are 700 Å3or more. Example 15 – Calculation example for molecular volumes of cationic lipids

[0390] Molecular volumes can be calculated using the method of Zhao YH, Abraham, MH and Zissimos AM (2003) in J Org Chem 68:7368-7373. No structural information is required and the molecular volume for compounds comprising carbon, hydrogen, nitrogen and oxygen is calculated as volume=20.58*C+7.24*H+15.6*N+14.71*O- 5.92*(C+H+N+O-1), wherein C, H, N and O represents the number of carbon, hydrogen, nitrogen and oxygen atoms in a given molecule or moiety and the volume is calculated in Å3.

[0391] The table below provides molecular volumes for ionized cationic lipids of Figure 4 and portions thereof calculated using the method of Zhao et al. The dotted line in Figure 4 demarcates the cationic head group head group moiety, the remainder is considered the lipidic group and used to determine lipidic volume. molecular volumePage 196 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 molecular volume Compound No. complete lipid lipidic group T1 T2

[0392] Molecular volumes of lipids and lipidic portions within the group of TC1 to TC6 wherein X is O or NH or wherein X1in T1or X2in T2is -C(O)O-, -OC(O)-, -C(O)N(RZ)-, or - N(RZ)C(O)- (as within formula ICL) are similar. The group of lipids TC7 to TC10 are homologues. The calculations illustrate the method of calculating molecular volumes for the cationically ionized lipids and highlights number of carbons within constitutional isomers or related compounds as a main driver of the molecular volumes. Cationic lipids having non-ionizable lipidic segments known in the state of the art have lipidic volumes below 700Å3as demonstrated for PONA, DOTAP or TC14. The Figure 4 also shows representative of inventive cationic lipids having an ammonium or imidazolium group as part of G1 and it is understood that homologs or constitutional isomers are incorporated as well. Example 16 – Analysis of Lipidic Portions of Anionically Ionizable Lipids

[0393] Lipid formulations were generated as described above using cationic guanido lipid, anionically ionizable lipid, cholesterol and DMPE-PEG2k lipid. The cationic lipid was palmitoyl- oleoyl-nor-arginine, PONA. The design space comprised 44 samples in a matrix having between 20 and 50 mol% cholesterol and 0.15 mol% of DMPE-PEG2k lipid, the remainder being cationic guanido lipid and anionically ionizable lipid used in ratios (i.e., the C / A ratio) between 0.5 and 1.5. The anionically ionizable lipids were the materials of Examples 4 to 11, selected from the group of diacylglycerol hemisuccinates. Page 197 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0394] As described above with respect to various embodiments, for anionically ionizable lipids of formula IAL described herein, lipidic volume is determined by the following moiety:

[0395] Anionically ionizable lipids of ple have the following general structure:where lipidic volume is calculated based on the chemical material below the dashed line

[0396] The transfection of mRNA was observed in different cell lines and with or without pre- incubation in full human serum which resembles a physiological condition.

[0397] As shown in Figure 6, formulations wherein the lipidic volume of the anionically ionizable lipid is 700Å3or more can have an efficacy within an order of magnitude of the reference formulation, in contrast to those having a smaller lipidic volume.

[0398] The high efficacy can be observed for compounds wherein X3or X4are -OC(O)- or - C(O)N(RZ)-such that both T3and T4(within the context of formula IAL) comprise ester or amide moieties. Example 17 – Calculation of Molecular Volumes for Anionically Ionizable Lipids

[0399] The table below provides molecular volumes for anionically ionizable lipids of Figure 5 and portions thereof calculated using the method of Zhao et al. . The dotted line in Figure 5 Page 198 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 demarcates the G2 segment, the remainder is considered the lipidic segment having a lipidic volume. molecular volume Compound No complete lipid lipidic portion T3 T4 A4wherein X3 in T3 or X4 in T4 are -C(O)O-, -OC(O)-, -C(O)N(R )-, or -N(R )C(O)- are close to each other. TA5 is a homologue. The calculations illustrate the method of calculating molecular volumes for the anionically ionizable lipids and highlights number of carbons within constitutional isomers or related compounds as a main driver of the molecular volumes. Anionically ionizable lipids in the state of the art have lipidic volumes below 700Å3as demonstrated for DOGS, DMGS or DOPA. Page 199 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Example 18 – General Synthetic scheme for the Preparation of B3, B-6 and B-7

[0401] Synthetic scheme for the preparation of B-3Page 200 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0402] Synthetic scheme for the preparation of intermediate DG-001-13

[0403] Synthesis of (S)-4-(((4-Methoxybenzyl)oxy)methyl)-2,2-dimethyl-1,3-dioxolane

[0404] To a suspension of L-acetone glycerol (1.00 eq., 10.0 g, 9.35 mL, 75.7 mmol) in anhydrous DMF (187 mL) was added NaH (60%) (1.17 eq., 3.55 g, 88.8 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 45 min under argon. After the addition of 4- methoxybenzylchloride (1.05 eq., 10.8 mL, 79.6 mmol), the mixture was allowed to warm to room temperature and then stirred for 3 hours under argon. Water (200 mL) was added to the mixture, and then extracted with EtOAc (2 × 250 mL). The combined organic layers were washed with brine (3 x 200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was solubilized in i-PrOAc (400 mL) and washed with brine (3x150 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressureto afford the crude product (22 g) as a yellow oil. The residue was split in two and purified by flash chromatography over silica gel (irregular SiOH, 50 µm, 330 g, solid loading) using cyclohexane / ethyl acetate as eluent (from 100:0 to 85:15 over 30 min and then 85:15 for 15 min) to afford the desired compound (16.2 g, 64.2 mmol, 85%) as a colorless oil.

[0405] 1H NMR (CDCl3, 400 MHz): δ ppm 7.30 – 7.22 (m, 2H), 6.92 – 6.84 (m, 2H), 4.56 – 4.44 (m, 2H), 4.33 – 4.23 (m, 1H), 4.04 (dd, J = 8.3, 6.4 Hz, 1H), 3.81 (s, 3H), 3.72 (dd, J = 8.3, 6.3 Hz, 1H), 3.53 (dd, J = 9.8, 5.7 Hz, 1H), 3.44 (dd, J = 9.8, 5.6 Hz, 1H), 1.42 (s, 3H), 1.36 (s, 3H). Page 201 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0406] Synthesis of of ((R)-3-((4-Methoxybenzyl)oxy)propane-1,2-diol DG-001-13

[0407] To a solution of (S)-4-(((4-Methoxybenzyl)oxy)methyl)-2,2-dimethyl-1,3-dioxolane (1.00 eq., 16.4 g, 64.2 mmol) in THF (170 mL) was added an aqueous solution of HCl (1N) (2.77 eq., 178 mL, 178 mmol). The reaction mixture was stirred at room temperature for 17 hours. An aqueous saturated solution of NaHCO3 was added to quench the reaction and the reaction mixture was extracted with EtOAc (2x350 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude compound DG-001-13 (12.5 g) as a colorless oil. The aqueous layer was reextracted with EtOAc (2x200 mL), the layers were separated and the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to afford crude compound DG-001-13 (0.6 g) as a colorless oil. The residues were purified by flash chromatography over silica gel (regular SiOH, 15 µm, 330 g, solid loading) using DCM / MeOH as eluent (from 100:0 to 80:20 over 35 min, then 80:20 for 10 min). The product fractions were then combined and concentrated to give compound DG-001-13 (12.25 g, 54.2 mmol, 84%) as a white solid, contaminated by 5.8 wt% of DCM.

[0408] 1H NMR (CDCl3, 400 MHz): δ ppm 7.28 – 7.21 (m, 2H), 6.92 – 6.83 (m, 2H), 4.47 (s, 2H), 3.90 – 3.81 (m, 1H), 3.79 (s, 3H), 3.66 (dd, J = 11.5, 3.8 Hz, 1H), 3.58 (dd, J = 11.4, 5.6 Hz, 1H), 3.56 – 3.44 (m, 2H), 2.72 (s, 2H).

[0409] Synthesis of (S)-3-((4-Methoxybenzyl)oxy)propane-1,2-diyl dioleate DG-015-01

[0410] ( . eq., . g, . mmo ) an ( . eq., . g, . mmol) were added to a solution of compound DG-001-13 (1.00 eq., 1.66 g, 7.82 mmol) and oleic acid (2.20 eq., 4.86 g, 17.2 mmol) in DCM (155 mL) at room temperature. The reaction mixture was stirred at room temperature for 23 hours and was filtered. The filtrate was concentrated under reduced Page 202 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 pressure to afford crude compound DG-015-01 as a white semi-solid. The residue was purified by flash chromatography over silica gel (regular SiOH, 50 µm, 80 g, solid loading) using cyclohexane / ethyl acetate as eluent (from 100:0 to 92:8 over 30 min and 92:8 for 15 min) to give pure compound DG-015-01 (4.22 g, 5.69 mmol, 73%) as a colorless oil.

[0411] 1H NMR (400 MHz, CDCl3): δ ppm 7.23 (d, J = 8.2 Hz, 2H), 6.87 (d, J = 8.2 Hz, 2H), 5.39 – 5.28 (m, 4H), 5.22 (p, J = 5.2 Hz, 1H), 4.52 – 4.40 (m, 2H), 4.33 (dd, J = 11.9, 3.8 Hz, 1H), 4.17 (dd, J = 11.9, 6.4 Hz, 1H), 3.80 (s, 3H), 3.55 (d, J = 5.2 Hz, 2H), 2.29 (dt, J = 15.6, 7.5 Hz, 4H), 2.01 (q, J = 6.4 Hz, 8H), 1.67 – 1.56 (m, 4H), 1.36 – 1.23 (m, 40H), 0.88 (t, J = 6.5 Hz, 6H).

[0412] Synthesis of (S)-3-Hydroxypropane-1,2-diyl dioleate DG-015-02

[0413] DG-015-01 (1.00 eq., 500 mg, 0.67 mmol) in a mixture of DCM (4.80 mL) and water (0.25 mL) at 0 °C. The reaction flask was completely wrapped in aluminum foil and stirred from 0 °C to room temperature for 5.5 hours (the reaction mixture turned from dark green to a dark shade of red). The mixture was diluted with DCM (20 mL) and filtered through a pad of celite. The filtrate was washed with an aqueous saturated NaHCO3solution (3 x 50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford crude compound DG- 015-02 (493 mg, 0.65 mmol, 97%) as a red oil, contaminated by 17.8 wt% of 4- methoxybenzaldehyde. The crude product was used as such in the next step.

[0414] 1H NMR (400 MHz, CDCl3): δ ppm 5.41 – 5.28 (m, 4H), 5.12 – 5.04 (m, 1H), 4.36 – 4.19 (m, 2H), 3.73 (dd, J = 5.0, 1.3 Hz, 2H), 2.39 – 2.28 (m, 4H), 2.06 – 1.96 (m, 8H), 1.61 (s, 5H), 1.35 – 1.24 (m, 40H), 0.92 – 0.84 (m, 6H).

[0415] Synthesis of 5-((R)-2,3-Bis(oleoyloxy)propoxy)-5-oxopentanoic acid B-3 (DG-015) Page 203 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0416] To a solution of crude compound DG-015-02 (1.00 eq., 321 mg, 0.42 mmol) in anhydrous DCM (13 mL) at 0 °C were added glutaric anhydride (1.10 eq., 52.8 mg, 0.46 mmol), DMAP (0.20 eq., 10.3 mg, 0.084 mmol) and anhydrous Et3N (2.50 eq., 0.15 mL, 1.05 mmol). The reaction mixture was stirred from 0 °C to room temperature for 22.5 hours. Glutaric anhydride (1.10 eq., 53 mg, 0.46 mmol) was added at room temperature and the reaction mixture was stirred at room temperature for 21 hours. The reaction mixture was quenched with water (25 mL). An aqueous hydrochloric acid solution (1N, 10 mL) was added until pH~2-3. The layers were separated and the aqueous layer was extracted with DCM (3 x 25 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford crude compound DG-015 (381 mg) as an orange oil. The residue was purified by flash chromatography over silica gel (irregular SiOH, 15 µm, 24 g, solid loading) using dichloromethane / (dichloromethane / methanol 9:1) as eluent (from 100:0 to 60:40 over 30 min) to give impure compound DG-015 (281 mg) as a pale yellow oil. The residue was purified again by flash chromatography over silica gel (irregular SiOH, 15 µm, 12 g, solid loading) using (cyclohexane / acetone 95:5) / (cyclohexane / acetone 6:4) as eluent (from 100:0 for 3 minutes, to 75:25 over 35 min) to give pure compound DG-015 (226 mg, 0.307 mmol, 73%) as a colorless oil.

[0417] LC / MS: Rt = 9.77 min, 100% ELSD, [M+NH4+] = 752.97

[0418] CAD: Rt = 9.34 min, 98.34%

[0419] 1H NMR (CDCl3, 400 MHz): δ ppm 5.42 – 5.29 (m, 4H), 5.29 – 5.21 (m, 1H), 4.30 (ddd, J = 11.6, 8.8, 4.3 Hz, 2H), 4.14 (ddd, J = 11.9, 6.0, 2.3 Hz, 2H), 2.43 (q, J = 7.0 Hz, 4H), 2.31 (td, J = 7.6, 3.1 Hz, 4H), 2.05 – 1.90 (m, 10H), 1.65 – 1.55 (m, 4H), 1.38 – 1.24 (m, 40H), 0.88 (t, J = 6.7 Hz, 6H). Page 204 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 Example 19 – General Synthetic Scheme for the Preparation of B-11, B-13, B-14, B-15, B-18 and B-19

[0001] Synthetic Scheme for Preparation of B-15 Page 205 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0420] Synthesis of (S)-3-((4-Methoxybenzyl)oxy)propane-1,2-diyl bis(6-((tetrahydro-2H- pyran-2-yl)oxy)hexanoate) DG-007-2 Page 206 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0421] To a solution of compound DG-001-013 (1.00 eq., 1.00 g, 4.71 mmol) and compound DG-007-01 (2.50 eq., 3.40 g, 11.8 mmol) in DCM (59 mL) were added DCC (3.00 eq., 2.92 g, 14.1 mmol) and DMAP (0.20 eq., 0.12 g, 0.94 mmol) at room temperature. The reaction mixture was stirred at room temperature for 20 hours. The reaction mixture was filtered and the cake was rinsed with DCM (50 mL). The filtrate was concentrated under vacuum to afford crude product DG-007-02 (5.04 g) as a yellow oil. The residue was purified by reverse phase chromatography over C18 (50 µm, 240 g, solid loading) using water / acetonitrile as eluent (from 60:40 to 0:100 over 55 min then 0:100 for 5 min) to afford compound DG-007-02 (2.74 g, 4.35 mmol, 92%) contaminated by 3.37 wt% of DCM as a colorless oil.

[0422] LC / MS: Rt = 2.27 min, 100%, [M+NH4]+= 626.78 / [M+Na]+= 631.71

[0423] CAD: Rt = 2.03 min, 96.69%

[0424] 1H NMR (CDCl3, 400 MHz): δ ppm 7.25 – 7.20 (m, 2H), 6.91 – 6.84 (m, 2H), 5.21 (dtd, J = 6.4, 5.2, 3.8 Hz, 1H), 4.59 – 4.53 (m, 2H), 4.51 – 4.41 (m, 2H), 4.36 – 4.13 (m, 2H), 3.85 (ddd, J = 11.1, 7.4, 3.4 Hz, 2H), 3.80 (s, 3H), 3.73 (dt, J = 9.7, 6.7 Hz, 2H), 3.55 (dd, J = 5.2, 1.1 Hz, 2H), 3.53 – 3.44 (m, 2H), 3.41 – 3.33 (m, 2H), 2.37 – 2.25 (m, 4H), 1.87 – 1.76 (m, 2H), 1.70 – 1.51 (m, 18H), 1.46 – 1.33 (m, 4H).

[0425] Synthesis of (S)-3-((4-Methoxybenzyl)oxy)propane-1,2-diyl bis(6-hydroxyhexanoate) DG-007-03

[0426] To a solution of compound DG-007-02 (1.00 eq., 2.47 g, 4.06 mmol) in MeOH (24.7 mL) was added activated Amberlyst 15 (H+) (7.50 g). The reaction mixture was stirred on the rotary evaporator for 6 hours and filtered through cotton to remove Amberlyst. The filtrate was concentrated in vacuo to afford crude compound DG-007-03 (1.8 g) as a yellow oil. The residue was purified by flash chromatography over silica gel (irregular SiOH, 50 µm, 80 g, solid loading) using cyclohexane / acetone as eluent (from 100:0 to 60:40 over 40 min) to afford compound DG- Page 207 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 007-03 (1.49 g, 3.31 mmol, 82%) contaminated by 2.29 wt% of DCM and 2.30 wt% of cyclohexane as a colorless oil.

[0427] LC / MS: Rt = 5.61 min, 93.21%, [M+H+] = 441.50 / [M+Na+] = 463.44

[0428] CAD: Rt = 7.01 min, 93.04%

[0429] 1H NMR (CDCl3, 400 MHz): δ ppm 7.26 – 7.21 (m, 2H), 6.91 – 6.83 (m, 2H), 5.23 (dtd, J = 6.5, 5.2, 3.6 Hz, 1H), 4.52 – 4.41 (m, 2H), 4.39 – 4.12 (m, 2H), 3.81 (s, 3H), 3.63 (td, J = 6.4, 2.5 Hz, 4H), 3.55 (dd, J = 5.2, 1.5 Hz, 2H), 2.32 (dt, J = 16.0, 7.4 Hz, 4H), 1.71 – 1.52 (m, 10H), 1.42 – 1.34 (m, 4H).

[0430] Synthesis of (((S)-3-((4-Methoxybenzyl)oxy)propane-1,2-diyl)bis(oxy))bis(6- oxohexane-6,1-diyl) bis(2-butyloctanoate) DG-008-04

[0431] To5 mmol) and 2- butyloctanoic acid (3.00 eq., 1.11 g, 5.56 mmol) in DCM (35.6 mL) were added EDC.HCl (3.00 eq., 1.07 g, 5.56 mmol) and DMAP (4.00 eq., 905 mg, 7.41 mmol) at room temperature. The reaction mixture was stirred at room temperature for 20 hours. The reaction mixture was diluted with DCM (15 mL) and water (15 mL). The layers were separated and the organic layer was washed with water (15 mL) and brine (15 mL), dried over over anhydrous sodium sulfate, filtered and concentrated under vacuum to afford crude compound DG-008-04 (2.6 g) as a colorless oil. The residue was purified by flash chromatography over silica gel (irregular SiOH, 50 µm, 80 g, solid loading) using cyclohexane / acetone as eluent (from 100:0 to 90:10 over 30 min and then 90:10 for 10 min) to afford compound DG-008-04 (1.14 g, 1.38 mmol, 74%) contaminated by 2.2 wt% of cyclohexane as a colorless oil.

[0432] LC / MS: Rt = 6.98 min, 99.24%, [M+Na+] = 828.15

[0433] CAD: Rt = 3.55 min, 99.12%

[0434] 1H NMR (CDCl3, 400 MHz): δ ppm 77.25 – 7.20 (m, 2H), 6.90 – 6.84 (m, 2H), 5.22 (dtd, J = 6.4, 5.2, 3.8 Hz, 1H), 4.51 – 4.42 (m, 2H), 4.33 (dd, J = 11.9, 3.8 Hz, 1H), 4.16 (dd, J = 11.9, 6.4 Hz, 1H), 4.06 (td, J = 6.7, 1.1 Hz, 4H), 3.80 (s, 3H), 3.55 (dd, J = 5.2, 1.1 Hz, 2H), 2.37 Page 208 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 – 2.24 (m, 6H), 1.72 – 1.57 (m, 10H), 1.49 – 1.34 (m, 8H), 1.34 – 1.18 (m, 26H), 0.94 – 0.82 (m, 12H).

[0435] Synthesis of (((S)-3-Hydroxypropane-1,2-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl) bis(2-butyloctanoate) DG-008-05

[0436] Dound DG-008-04 (1.00 eq., 1.11 g, 1.38 mmol) in a mixture of DCM (9.80 mL) and H2O (0.52 mL) at 0 °C. The reaction flask was completely wrapped in aluminum foil and stirred from 0 °C to room temperature for 6 hours (the reaction mixture turned from dark green to a dark shade of red). The mixture was diluted with DCM (30 mL) and filtered through a pad of celite. The collected filtrate was washed with an aqueous saturated solution of NaHCO3(3 x 50 mL). The resulting organic solution was dried over anhydrous anhydrous sodium sulfate, filtered and concentrated under vacuum to afford crude compound DG-008-05 (976 mg, 1.20 mmol, 87%) as a light orange oil contaminated by 15.4 wt% of 4-methoxybenzaldehyde. The crude was used as such in the next step.

[0437] 1H NMR (CDCl3, 400 MHz): δ ppm 5.08 (p, J = 5.0 Hz, 1H), 4.33 (dd, J = 11.9, 4.6 Hz, 1H), 4.23 (dd, J = 11.9, 5.6 Hz, 1H), 4.10 – 4.04 (m, 4H), 3.73 (dd, J = 5.0, 2.1 Hz, 2H), 2.40 – 2.25 (m, 6H), 2.08 (br s, 1H), 1.74 – 1.51 (m, 10H), 1.51 – 1.35 (m, 8H), 1.35 – 1.16 (m, 26H), 0.93 – 0.81 (m, 12H).

[0438] Synthesis of Benzyl ((2R)-2,3-bis((6-((2-butyloctanoyl)oxy)hexanoyl)oxy)propyl) succinate DG-008-06Page 209 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0439] Benzyl (2,5-dioxopyrrolidin-1-yl) succinate (1.20 eq., 441 mg, 1.44 mmol) was added to a solution of compound DG-008-05 (1.00 eq., 824 mg, 1.20 mmol) in DCM (7.50 mL) at room temperature. DMAP (0.20 eq., 29.4 mg, 0.24 mmol) was added and the reaction mixture was stirred at room temperature for 18 hours. DMAP (0.10 eq., 14.7 mg, 0.12 mmol) was added and the reaction mixture was stired at room temperature for 4 hours. The reaction mixture was diluted with DCM (20 mL) and washed with water (20 mL). The aqueous layer was extracted with DCM (20 mL) and the combined organic layers were washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford crude compound DG- 008-06 (1.07 g) as a pale yellow oil. The residue was purified by flash chromatography over silica gel (regular SiOH, 15 µm, 40 g, solid loading) using cyclohexane / acetone as eluent from (100:0 to 90:10 over 30 min then 90:10 for 20 min) to afford compound DG-008-06 (0.25 g, 0.28 mmol, 24%) as a colorless oil.

[0440] LC / MS: Rt = 7.36 min, 100%, [M+H+] = 876.29

[0441] CAD: Rt = 8.24 min, 97.87%

[0442] 1H NMR (CDCl3, 400 MHz): δ ppm 7.40 – 7.29 (m, 5H), 5.24 (tt, J = 5.8, 4.3 Hz, 1H), 5.13 (s, 2H), 4.29 (ddd, J = 11.5, 6.9, 4.3 Hz, 2H), 4.14 (td, J = 11.9, 5.9 Hz, 2H), 4.06 (td, J = 6.6, 1.0 Hz, 4H), 2.72 – 2.61 (m, 4H), 2.37 – 2.25 (m, 6H), 1.70 – 1.53 (m, 12H), 1.48 – 1.34 (m, 8H), 1.34 – 1.18 (m, 24H), 0.93 – 0.82 (m, 12H).

[0443] Synthesis of 4-((2R)-2,3-bis((6-((2-Butyloctanoyl)oxy)hexanoyl)oxy)propoxy)-4- oxobutanoic acid B-15 (DG-008)

[0444] To an argon purged solution of compound DG-008-06 (1.00 eq., 240 mg, 0.27 mmol) in EtOAc (4.69 mL) was added 10% Pd / C (0.20 eq., 117 mg, 0.055 mmol) at room temperature. The reaction mixture was purged with argon (x3) then with hydrogen (x3) and stirred at room temperature for 5 hours. The reaction mixture was filtered through PTFE filter (0.45 µm) and Page 210 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 rinsed with EtOAc (10 mL). The filtrate was concentrated under vacuum to afford crude compound compound DG-008 (203 mg) as a colorless oil. The residue was solubilized in MeOH (2 mL) and loaded onto SPE-thiol cartridge (conditioned with 5 mL of MeOH). SPE cartridge was rinsed with MeOH (3 x 3 mL, by gravity) and the filtrate was concentrated under reduced pressure to afford a colorless oil (186 mg). The residue was purified by flash chromatography over silica gel (regular SiOH, 15 µm, 12 g, solid loading) using cyclohexane / EtOAc as eluent (from 100:0 to 60:40 in 30 min then 60:40 for 10 min) to afford compound DG-008 (151 mg, 0.19 mmol, 70%) as a colorless oil.

[0445] LC / MS: Rt = 5.87 min, 100% ELSD, [M+H+] = 786.10

[0446] CAD: Rt = 6.63 min, 98.66%

[0447] 1H NMR (CDCl3, 400 MHz): δ ppm 5.26 (tt, J = 5.8, 4.3 Hz, 1H), 4.32 (ddd, J = 17.9, 11.9, 4.4 Hz, 2H), 4.17 (ddd, J = 14.3, 11.9, 5.8 Hz, 2H), 4.12 – 4.02 (m, 4H), 2.75 – 2.58 (m, 4H), 2.39 – 2.26 (m, 6H), 1.71 – 1.53 (m, 10H), 1.49 – 1.34 (m, 8H), 1.34 – 1.18 (m, 26H), 0.93 – 0.83 (m, 12H).

[0448] B-11 and B-19 were prepared according to analogous methods described herein.

[0449] 4-((2R)-2,3-bis((8-((2-Propylheptanoyl)oxy)octanoyl)oxy)propoxy)-4-oxobutanoic acid B-11 (DG-010)

[0450] . , , . 8.08

[0451] CAD: Rt = 6.28 min, 97.66%

[0452] 1H NMR (CDCl3, 400 MHz): δ ppm 5.26 (tt, J = 5.9, 4.3 Hz, 1H), 4.31 (ddd, J = 16.1, 11.9, 4.4 Hz, 2H), 4.16 (ddd, J = 15.5, 11.9, 5.9 Hz, 2H), 4.06 (td, J = 6.7, 1.8 Hz, 4H), 2.70 – 2.61 (m, 4H), 2.37 – 2.28 (m, 6H), 1.66 – 1.53 (m, 12H), 1.45 – 1.22 (m, 32H), 0.92 – 0.84 (m, 12H).

[0453] 4-((2R)-2,3-bis((6-((2-Hexyldecanoyl)oxy)hexanoyl)oxy)propoxy)-4-oxobutanoic acid B-19 (DG-009) Page 211 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0454] LC / MS: Rt = 8.60 min, 99.71% ELSD, [M+Na+] = 920.45

[0455] CAD: Rt = 9.36 min, 95.92%

[0456] 1H NMR (CDCl3, 400 MHz): δ ppm 5.26 (p, J = 5.2 Hz, 1H), 4.38 – 4.27 (m, 2H), 4.18 (ddd, J = 13.8, 11.9, 5.8 Hz, 2H), 4.11 – 4.05 (m, 4H), 2.69 – 2.61 (m, 4H), 2.36 – 2.27 (m, 6H), 1.68 – 1.56 (m, 10H), 1.48 – 1.37 (m, 8H), 1.31 – 1.22 (m, 42H), 0.90 – 0.85 (m, 12H). Example 20 – General synthetic scheme for the preparation of B-12, B-16, and B-17 Page 212 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802Page 213 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0457] Synthetic scheme for preparing B-12Page 214 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0458] Synthesis of (S)-O,O'-(3-(Benzyloxy)propane-1,2-diyl) di-tert-butyl diadipate DG- 003-02

[0459] To a solution mg, 3.84 mmol) and 6-(tert-butoxy)-6-oxohexanoic acid (2.20 eq., 1.71 g, 8.45 mmol) in DCM (70 mL) were added DCC (2.50 eq., 1.98 g, 9.60 mmol) and DMAP (0.20 eq., 0.094 g, 0.77 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 days.6-(tert-Butoxy)-6-oxohexanoic acid (0.50 eq., 0.39 g, 1.92 mmol) and DMAP (0.20 eq., 0.094 g, 0.77 mmol) were added at room temperature. The reaction mixture was stirred at room temperature for 4 hours and filtered. The cake was rinsed with diethyl ether (30 mL). The filtrate was concentrated under vacuum to afford crude product DG-003-02 (3.02 g) as a pale-yellow oil. The residue was purified by flash chromatography over silica gel (irregular SiOH, 50 µm, 80 g, solid loading) using cyclohexane / ethyl acetate as eluent (from 100:0 to 85:15 over 30 minutes and then 85:15 for 20 minutes) to afford product DG-003-02 (1.71 g, 3.11 mmol, 81%) as a colorless oil.

[0460] LC / MS: Rt = 2.00 min, 100%, [M+NH4]+= 569.3

[0461] CAD: Rt = 2.36 min, 98.63%

[0462] 1H NMR (CDCl3, 400 MHz): δ ppm 7.39 – 7.27 (m, 5H), 5.23 (dtd, J = 6.3, 5.2, 3.8 Hz, 1H), 4.59 – 4.47 (m, 2H), 4.35 (dd, J = 11.9, 3.9 Hz, 1H), 4.18 (dd, J = 11.9, 6.3 Hz, 1H), 3.58 (dd, J = 5.2, 0.8 Hz, 2H), 2.38 – 2.26 (m, 4H), 2.22 (td, J = 7.0, 1.8 Hz, 4H), 1.69 – 1.58 (m, 8H), 1.46 – 1.40 (m, 18H).

[0463] Synthesis of (S)-6,6'-((3-(Benzyloxy)propane-1,2-diyl)bis(oxy))bis(6-oxohexanoic acid) DG-003-03Page 215 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0464] TFA (12.1 eq., 2.8 mL, 37.7 mmol) was added to solution of compound DG-003-02 (1.00 eq., 1.71 g, 3.11 mmol) in DCM (17 mL) at 0 °C. The reaction mixture was stirred from 0 °C to room temperature for 7 hours. The reaction mixture was quenched with water (25 mL), the product was extracted DCM (2 x 25 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtrated and concentrated under reduced pressure. The residue was dissolved in DCM (17 mL) and TFA (12.1 eq., 2.8 mL, 37.7 mmol) was added at 0 °C. The reaction mixture was stirred from 0 °C to room temperature for 5.5 hours. The reaction mixture was quenched with water (25 mL), the product was extracted DCM (2 x 25 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford crude product DG-003-03 (1.49 g) as a colorless oil. The residue was purified by flash chromatography over silica gel (regular SiOH, 15 µm, 40 g, solid loading) using mixtures (cyclohexane / acetone 95:5) / (cyclohexane / acetone 60:40) as eluent (from 100:0 to 0:100 over 30 minutes and then 0:100 for 10 minutes) to afford compound DG-003-03 (1.29 g, 2.70 mmol, 87%) contaminated by 7.44 wt% of DCM and possible traces of cyclohexane as a colorless oil.

[0465] LC / MS: Rt = 2.44 min, 100%, [M-H+] = 437.5

[0466] CAD: Rt = 7.08 min, 98.78%

[0467] 1H NMR (CDCl3, 400 MHz): δ ppm 7.39 – 7.26 (m, 5H), 5.29 – 5.21 (m, 1H), 4.59 – 4.48 (m, 2H), 4.37 (dd, J = 12.1, 3.0 Hz, 1H), 4.21 (dd, J = 12.1, 6.6 Hz, 1H), 3.64 – 3.52 (m, 2H), 2.41 – 2.27 (m, 8H), 1.74 – 1.61 (m, 8H).

[0468] Synthesis of O,O'-((S)-3-(Benzyloxy)propane-1,2-diyl) bis(2-hexyldecyl) diadipate DG-003-04

[0469] To a solution of compound DG-003-03 (1.00 eq., 1.27 g, 2.67 mmol) and 2- hexyldecan-1-ol (4.00 eq., 3.10 mL, 10.7 mmol) in DCM (51 mL) were added EDC.HCl (4.00 eq., 2.048 g, 10.68 mmol) and DMAP (4.00 eq., 1.31 g, 10.7 mmol) at room temperature. The reaction Page 216 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 mixture was stirred at room temperature for 23.5 hours. The reaction mixture was diluted with DCM (50 mL) and water (50 mL). An aqueous solution of hydrogen chloride was added (1N, 50 mL). The layers were separated and the organic layer was washed with an aqueous solution of hydrogen chloride (1N, 50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by flash chromatography over silica gel (irregular SiOH, 50 µm, 80 g, solid loading) using cyclohexane / ethyl acetate as eluent (from 100:0 to 85:15 in 30 minutes and then 85:15 for 15 minutes) to afford compound DG-003-04 (1.55 g, 1.75 mmol, 65%) as a colorless oil.

[0470] LC / MS: Rt = 3.27 min, 100%, [M+Na+] = 909.7

[0471] CAD: Rt = 4.49 min, 98.12%

[0472] 1H NMR (CDCl3, 400 MHz): δ ppm 7.38 – 7.27 (m, 5H), 5.27 – 5.19 (m, 1H), 4.59 – 4.47 (m, 2H), 4.35 (dd, J = 11.9, 3.9 Hz, 1H), 4.18 (dd, J = 11.9, 6.3 Hz, 1H), 3.96 (d, J = 5.8 Hz, 4H), 3.58 (d, J = 5.2 Hz, 2H), 2.39 – 2.27 (m, 8H), 1.70 – 1.56 (m, 10H), 1.34 – 1.24 (m, 48H), 0.92 – 0.84 (m, 12H).

[0473] Synthesis of bis(2-Hexyldecyl) O,O'-((S)-3-hydroxypropane-1,2-diyl) diadipate DG- 003-05

[0474] To an argon purged solution of compound DG-003-04 (1.00 eq., 1.55 g, 1.75 mmol) in EtOAc (31 mL) was added 10% Pd / C (0.26 eq., 0.48 g, 0.45 mmol) at room temperature. The reaction mixture was purged with argon (x3) then with hydrogen (x3) and stirred at room temperature for 16.5 hours. The reaction mixture was filtered through PTFE filter (0.45 µm) and rinsed with EtOAc (10 mL). The filtrate was concentrated under vacuum to afford crude product DG-003-05 (1.37 g, 1.71 mmol, 98%) as a colorless oil. The crude product was used as such in the next step.

[0475] CAD: Rt = 3.08 min, 94.13% Page 217 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0476] 1H NMR (CDCl3, 400 MHz): δ ppm 5.09 (p, J = 5.0 Hz, 1H), 4.32 (dd, J = 11.9, 4.6 Hz, 1H), 4.23 (dd, J = 11.9, 5.7 Hz, 1H), 3.97 (dd, J = 5.8, 1.5 Hz, 4H), 3.74 (dt, J = 6.9, 4.6 Hz, 2H), 2.43 – 2.29 (m, 9H), 1.72 – 1.60 (m, 10H), 1.34 – 1.24 (m, 48H), 0.88 (t, J = 6.7 Hz, 12H).

[0477] Synthesis of O,O'-((R)-3-((4-(Benzyloxy)-4-oxobutanoyl)oxy)propane-1,2-diyl) bis(2- hexyldecyl) diadipate DG-003-06

[0478] DMAP (0.26 eq., 0.053 g, 0.43 mmol) was added to a solution of compound DG-003- 05 (1.00 eq., 1.35 g, 1.69 mmol) and benzyl (2,5-dioxopyrrolidin-1-yl) succinate (1.20 eq., 0.62 g, 2.03 mmol) in DCM (17.0 mL) at room temperature. The reaction mixture was stirred at room temperature for 18 hours. Then, DMAP (0.12 eq., 0.024 g, 0.20 mmol) was added and the reaction mixture was stirred at room temperature for 4.5 hours. The reaction mixture was quenched with water (25 mL) and the layers were separated. The aqueous layer was extracted with dichloromethane (3 x 25 mL) and the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduce pressure to afford crude product DG-003-06 (1.85 g) as a pale yellow oil. The residue was purified by flash chromatography over silica gel (regular SiOH, 15 µm, 80 g, solid loading) using cyclohexane / ethyl acetate as eluent (from 1000:0 to 85:15 in 35 minutes and 85:15 for 25 minutes) to afford compound DG-003-06 (0.670 g, 0.68 mmol, 40%) as a colorless oil.

[0479] LC / MS: Rt = 9.62 min, 100%, [M+H+] = 988.25

[0480] CAD: Rt = 4.05 min, 98.92%

[0481] 1H NMR (CDCl3, 400 MHz): δ ppm 7.39 – 7.28 (m, 5H), 5.29 – 5.17 (m, 1H), 5.16 – 5.07 (m, 2H), 4.33 – 4.24 (m, 2H), 4.20 – 4.08 (m, 2H), 3.99 – 3.90 (m, 4H), 2.66 (p, J = 3.9 Hz, 4H), 2.42 – 2.23 (m, 8H), 1.71 – 1.56 (m, 10H), 1.32 – 1.20 (m, 48H), 0.92 – 0.80 (m, 12H). Page 218 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0482] Synthesis of 4-((2R)-2,3-bis((6-((2-hexyldecyl)oxy)-6-oxohexanoyl)oxy)propoxy)-4- oxobutanoic acid B-12 (DG-003)

[0483] , 0.67 mmol)in EtOAc (11.5 mL) was added 10% Pd / C (0.20 eq., 144 mg, 0.13 mmol) at room temperature. The reaction mixture was purged with argon (x3) then with hydrogen (x3) and stirred at room temperature for 3.5 hours. The reaction mixture was filtered through PTFE filter (0.45 µm) and the filter rinsed with EtOAc (20 mL). The filtrate was concentrated under vacuum to afford crude compound DG-003 (608 mg) as a colorless oil. The residue was solubilized in MeOH (1.5 mL) and loaded onto SPE-thiol cartridge (conditioned with 5 mL of MeOH). SPE cartridge was rinsed with MeOH (3 x 5 mL, by gravity) and the filtrate was concentrated under reduced pressure to afford crude compound DG-003 (590 mg) of a colorless oil. The residue was purified by flash chromatography over silica gel (regular SiOH, 15 µm, 24 g, solid loading) using cyclohexane / ethyl acetate as eluent (from 100:0 to 60:40 in 30 minutes and 60:40 for 15 minutes) to afford compound DG-003 (493 mg, 0.55 mmol, 81%) contaminated by 0.19 wt% of DCM as a colorless oil.

[0484] LC / MS: Rt = 8.92 min, 100% ELSD, [M+Na]+= 920.21

[0485] CAD: Rt = 9.59 min, 98.88%

[0486] 1H NMR (CDCl3, 400 MHz): δ ppm 5.25 (p, J = 5.1 Hz, 1H), 4.41 – 4.25 (m, 2H), 4.25 – 4.10 (m, 2H), 3.97 (dd, J = 5.9, 2.6 Hz, 4H), 2.66 (tt, J = 5.8, 3.4 Hz, 4H), 2.42 – 2.29 (m, 8H), 1.74 – 1.55 (m, 10H), 1.34 – 1.17 (m, 48H), 0.88 (t, J = 6.8 Hz, 12H). Example 21 – General Synthetic Scheme for the Preparation of B-8, B-9 and B-5 Page 219 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0487] Synthetic scheme of B-9 Page 220 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0488] Synthesis of (R)-Benzyl ((2,2-dimethyl-1,3-dioxolan-4-yl)methyl) succinate DG-001- 05Page 221 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0489] To a solution of compound DG-001-03 (1.00 eq., 10.0 g, 48.0 mmol) and (S)-(2,2- dimethyl-1,3-dioxolan-4-yl)methanol (1.00 eq., 5.93 mL, 48.0 mmol) in DCM (163 mL) were added EDC.HCl (1.50 eq., 13.8 g, 72.0 mol) and DMAP (0.15 eq., 0.88 g, 7.21 mmol) at room temperature. The reaction mixture was stirred for 2 hours at room temperature. The reaction mixture was washed with an aqueous saturated solution of NaHCO3 (150 mL) and brine (150 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to afford crude product DG-001-05 (16 g) as a colorless oil. The residue was purified by flash chromatography over silica gel (irregular SiOH, 50 µm, 330 g, solid loading) using cyclohexane / ethyl acetate as eluent (from 100:0 to 60:40 over 40 min) to afford compound DG- 001-05 (13.5 g, 41.9 mmol, 87%) as a colorless oil.

[0490] 1H NMR (CDCl3, 400 MHz): δ ppm 7.41 – 7.28 (m, 5H), 5.14 (s, 2H), 4.28 (qd, J = 6.1, 4.8 Hz, 1H), 4.20 – 4.07 (m, 2H), 4.05 (dd, J = 8.5, 6.5 Hz, 1H), 3.72 (dd, J = 8.5, 6.1 Hz, 1H), 2.69 (s, 4H), 1.43 (s, 3H), 1.36 (s, 3H).

[0491] Synthesis of (R)-Benzyl (2,3-dihydroxypropyl) succinate DG-001-06

[0492] To a solution of compound DG-001-05 (1.00 eq., 5.75 g, 17.8 mmol) in MeOH (57.5 mL) was added activated Amberlyst 15 (H+) (5 g). The reaction mixture was stirred on the rotary evaporator for 21 hours and filtered. The resin was rinsed with MeOH (100 mL). The filtrate was concentrated under vacuum to afford crude compound DG-001-06 (5.1 g) as a pale-yellow oil. The residue was purified by flash chromatography over silica gel (regular SiOH, 15 µm, 330 g, solid loading) using DCM / (DCM / MeOH 9:1) as eluent from (100:0 to 50:50 over 35 min) to afford compound DG-001-06 (3.5 g, 12.4 mmol, 69%) as a colorless oil.

[0493] LC / MS: Rt = 2.09 min, 100%, [M+Na]+= 305.1 Page 222 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0494] 1H NMR (CDCl3, 400 MHz): δ ppm 7.41 – 7.28 (m, 5H), 5.19 – 5.03 (m, 2H), 4.26 – 4.08 (m, 2H), 3.90 (tt, J = 6.1, 4.3 Hz, 1H), 3.67 (dd, J = 11.5, 4.1 Hz, 1H), 3.58 (dd, J = 11.4, 5.7 Hz, 1H), 2.80 – 2.51 (m, 5H), 2.04 (s, 1H).

[0495] Synthesis of (R)-O,O'-(3-((4-(Benzyloxy)-4-oxobutanoyl)oxy)propane-1,2-diyl) di- tert-butyl diadipate DG-001-08

[0496] To a solutionmol) and 6-(tert-butoxy)-6- oxohexanoic acid (2.05 eq., 4.00 g, 198 mmol) in DCM (185 mL) were added DCC (2.50 eq., 4.98 g, 24.1 mmol) and DMAP (0.20 eq., 0.24 g, 1.93 mmol) at room temperature. The reaction mixture was stirred at room temperature for 18 hours. DCC (0.50 eq., 1.00 g, 4.82 mmol) and DMAP (0.05 eq., 0.059 g, 0.48 mmol) were added at room temperature and the reaction mixture was stirred for 2 hours. The reaction mixture was filtered and rinsed with DCM (200 mL). The filtrate was concentrated under vacuum to afford crude compound DG-001-08 (10.5 g) as a semi-solid. The residue was purified by flash chromatography over silica gel (regular SiOH, 15 µm, 330 g, solid loading) using cyclohexane / ethyl acetate as eluent (from 100:0 to 70:30 over 45 minutes and 70:30 for 10 minutes) to afford compound DG-001-08 (3.75 g, 5.76 mmol, 60%) contaminated by 0.7 wt% of ethyl acetate as a white semi-solid.

[0497] LC / MS: Rt = 8.53 min, 84.44%, [M+NH4+] = 668.62

[0498] CAD: Rt = 9.92 min, 89.33%

[0499] 1H NMR (CDCl3, 400 MHz): δ ppm 7.41 – 7.28 (m, 5H), 5.28 – 5.20 (m, 1H), 5.13 (s, 2H), 4.34 – 4.24 (m, 2H), 4.20 – 4.07 (m, 2H), 2.72 – 2.61 (m, 4H), 2.34 (td, J = 7.1, 1.9 Hz, 4H), 2.28 – 2.19 (m, 4H), 1.67 – 1.59 (m, 8H), 1.45 – 1.42 (m, 18H).

[0500] Synthesis of (R)-6,6'-((3-((4-(Benzyloxy)-4-oxobutanoyl)oxy)propane-1,2- diyl)bis(oxy))bis(6-oxohexanoic acid) DG-001-10 Page 223 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0501] To a solution of 0 g, 6.30 mmol) in DCM (47.7mL) was added TFA (20.0 eq., 9.36 mL, 126 mmol) at 0 °C. The reaction mixture was stirred at room temprature for 1 hour. TFA (15.0 eq., 7.02 mL, 94.5 mmol) was added at 0 °C and the reaction mixture was stirred for 2 hours at room temprature. The reaction mixture was quenched with water (150 mL) and the layers were separated. The organic layer was washed with water (3x150 mL), dried anhydrous sodium sulfate, filtered and concentrated under vacuum to afford crude product DG-001-10 (3.3 g) as a yellow oil. The residue was purified by flash chromoatography over silica gel (regular SiOH, 15 µm, 120 g, solid loading) using DCM / MeOH as eluent (from 100:0 to 9:1 over 42 minutes) to afford pure product DG-001-10 (3.00 g, 5.16 mmol, 82%) contaminated by 7.4 wt% of EtOAc as a yellow oil.

[0502] CAD: Rt = 7.27 min, 96.65%

[0503] 1H NMR (CDCl3, 400 MHz): δ ppm 7.40 – 7.28 (m, 5H), 5.30 – 5.23 (m, 1H), 5.14 (s, 2H), 4.34 – 4.26 (m, 2H), 4.21 – 4.14 (m, 2H), 2.72 – 2.62 (m, 4H), 2.41 – 2.32 (m, 8H), 1.72 – 1.65 (m, 8H).

[0504] Synthesis of O,O'-((R)-3-((4-(Benzyloxy)-4-oxobutanoyl)oxy)propane-1,2-diyl) bis(2- butyloctyl) diadipate DG-002-02

[0505] To, , 1 mmol) and 2- butyloctan-1-ol (4.00 eq., 830 mg, 4.46 mmol) in DCM (21.4 mL) were added EDC.HCl (4.00 eq., 854 mg, 4.46 mmol) and DMAP (4.00 eq., 544 mg, 4.46 mmol) at room temprature. The reaction Page 224 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 mixture was stirred at room temprature for 47 hours. The reaction mixture was diluted with DCM (50 mL) and water (50 mL). The layers were separated and the organic layer was washed with water (2x50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to afford crude product DG-002-02 (1.83 g) as a pale yellow semi- solid. The residue was purified by flash chromatography over silica gel (irregular SiOH, 50 µm, 80 g, solid loading) using cyclohexane / EtOAc as eluent (from 100:0 to 85:15 in 30 minutes, 85:15 for 30 minutes and to 75:25 in 15 minutes) to afford impure product DG-002-02 (0.804 g) as pale- yellow oil. The residue was purified by flash chromatography over silica gel (regular SiOH, 15 µm, 40 g, solid loading) using cyclohexane / (cyclohexane / acetone 95:5) as eluent (from 100:0 to 0:100 in 30 minutes and 0:100 for 25 minutes) to afford pure product DG-002-02 (611 mg, 0.69 mmol, 62%) contaminated by 1.07 wt% of cyclohexane as a colorless oil.

[0506] CAD: Rt = 8.37 min, 96.50%

[0507] 1H NMR (CDCl3, 400 MHz): δ ppm 7.41 – 7.28 (m, 5H), 5.29 – 5.19 (m, 1H), 5.13 (s, 2H), 4.34 – 4.24 (m, 2H), 4.14 (td, J = 11.8, 5.9 Hz, 2H), 3.97 (d, J = 5.8 Hz, 4H), 2.72 – 2.62 (m, 4H), 2.38 – 2.27 (m, 8H), 1.70 – 1.58 (m, 10H), 1.34 – 1.24 (m, 32H), 0.93 – 0.84 (m, 12H).

[0508] Synthesis of 4-((2R)-2,3-bis((6-((2-butyloctyl)oxy)-6-oxohexanoyl)oxy)propoxy)-4- oxobutanoic acid B-9 (DG-002)

[0509] To an argon-purged solution of compound DG-002-02 (1.00 eq., 543 mg, 0.62 mmol) in EtOAc (10.8 mL) was added 10% Pd / C (0.20 eq., 132 mg, 0.12 mmol). The reaction mixture was purged with argon (x3) and then with hydrogen (3x), and stirred at room temperature for 24 hours. The reaction mixture was purged with argon, filtered on a PTFE filter and the filter was rinsed with EtOAc (20 mL). The filtrate was concentrated under vacuum to afford crude product DG-002 (536 mg) as a colorless oil. The residue was solubilized in MeOH (1.5 mL) and loaded onto SPE-thiol cartridge (conditioned with 5 mL of MeOH). The SPE cartridge was rinsed with Page 225 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 MeOH (3 x 5 mL, by gravity) and the filtrate was concentrated under reduced pressure to afford the crude product DG-002 (520 mg) as a colorless oil. The residue was purified by flash chromatography over silica gel (regular SiOH, 15 µm, 24 g, solid loading) using mixtures (cyclohexane / acetone 95:5) / (cyclohexane / acetone 6:4) as eluent (from 100:0 to 40:60 in 30 minutes) to afford impure product DG-002 (457 mg) as a colorless oil. The resdiue was purified by flash chromatography over silica gel (regular SiOH, 15 µm, 24 g, solid loading) using cyclohexane / EtOAc as eluent (from 100:0 to 60:40 in 30 minutes and 60:40 for 15 minutes) to afford compound DG-002 (355 mg, 0.45 mmol, 73%) as a colorless oil.

[0510] LC / MS: Rt = 6.07 min, 99.90% ELSD, [M+H]+= 786.10

[0511] CAD: Rt = 6.70 min, 96.48%

[0512] 1H NMR (CDCl3, 400 MHz): δ ppm 5.25 (tt, J = 5.7, 4.3 Hz, 1H), 4.39 – 4.25 (m, 2H), 4.25 – 4.11 (m, 2H), 3.98 (dd, J = 5.9, 2.6 Hz, 4H), 2.72 – 2.59 (m, 4H), 2.40 – 2.29 (m, 8H), 1.74 – 1.55 (m, 10H), 1.37 – 1.18 (m, 32H), 0.95 – 0.81 (m, 12H). Example 22 – General Synthesis of B-10 Page 226 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802

[0513] Synthesis of (R)-benzyl (2,3-bis((8-(dihexylamino)-8-oxooctanoyl)oxy)propyl) succinate DG-013-02

[0514] To a solution of compound DG-004-04 (obtained using an analogous procedure to compound DG-001-10, 1.00 eq., 450 mg, 0.76 mmol) and dihexylamine (4.00 eq., 561 mg, 3.03 mmol) in DCM (14.6 mL) were added HATU (4.00 eq., 1.15 g, 3.03 mmol) and DIEA (4.00 eq., Page 227 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 0.527 mL, 3.03 mmol) at room temprature. The reaction mixture was stirred at room temprature for 20 hours. The reaction mixture was diluted with DCM (15 mL) and water (15 mL). The layers were separated and the organic layer was washed with water (2x15 mL) and brine (15 mL), dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to afford crude product DG-013-02 (1.50 g) as a colorless oil. The residue was purified by flash chromatography over silica gel (irregular SiOH, 15 µm, 40 g, solid loading) using mixtures (cyclohexane / acetone 95:5) / (cyclohexane / acetone 6:4) as eluents (from 100:0 to 0:100 in 47 minutes) to afford compound DG-013-02 (460 mg, 0.49 mmol, 65%) as a pale yellow oil.

[0515] CAD: Rt = 6.92 min, 100%

[0516] 1H NMR (CDCl3, 400 MHz): δ ppm 7.43 – 7.30 (m, 5H), 5.30 – 5.21 (m, 1H), 5.16 (s, 2H), 4.30 (ddd, J = 11.9, 10.5, 4.3 Hz, 2H), 4.16 (td, J = 11.9, 5.9 Hz, 2H), 3.34 – 3.26 (m, 4H), 3.25 – 3.17 (m, 4H), 2.74 – 2.65 (m, 4H), 2.38 – 2.25 (m, 8H), 1.71 – 1.61 (m, 8H), 1.56 – 1.47 (m, 7H), 1.39 – 1.24 (m, 33H), 0.96 – 0.86 (m, 12H).

[0517] Synthesis of (R)-4-(2,3-bis((8-(dihexylamino)-8-oxooctanoyl)oxy)propoxy)-4- oxobutanoic acid B-10 (DG-013)

[0518] To an argon-purged solution of compound DG-013-02 (1.00 eq., 409 mg, 0.44 mmol) in EtOAc (8 mL) was added 10% Pd / C (0.20 eq., 94.0 mg, 0.088 mmol). The reaction mixture was purged with argon (x3) and then with hydrogen (3x), and stirred at room temperature for 24 hours. The reaction mixture was purged with argon, filtered on a PTFE filter and the filter was rinsed with EtOAc (20 mL). The filtrate was concentrated under vacuum to afford crude product DG- 013 (372 mg) as a colorless oil. The residue was solubilized in MeOH (1.5 mL) and loaded onto SPE-thiol cartridge (conditioned with 5 mL of MeOH). The SPE cartridge was rinsed with MeOH (3 x 5 mL, by gravity) and the filtrate was concentrated under reduced pressure to afford crude product DG-013 (362 mg) as a colorless oil. ...

Claims

Attorney Docket No.2013237-1441 Client Ref. No. P1802 CLAIMS 1. A composition comprising a cationic lipid, an anionically ionizable lipid, and a nucleic acid, wherein: the cationic lipid comprises a cationic head group, a first bridge group, and a first lipid tail group; the anionically ionizable lipid comprises an anionically ionizable head group, a second bridge group, and a second lipid tail group; the cationic lipid has a lipidic volume from about 700 Å3to about 1500 Å3, and / or the anionically ionizable lipid has a lipidic volume from about 700 Å3to about 1500 Å3; or the cationic lipid and the anionically ionizable lipid have a combined lipidic volume greater than 1400 Å3.

2. The composition of claim 1, wherein a molar ratio of the cationic lipid to the anionically ionizable lipid, is from about 0.67 to about 1.5, from about 0.75 to about 1.33, from about 0.82 to about 1.2, or from about 0.9 to about 1.

1.

3. The composition of claim 2, wherein the molar ratio of the cationic lipid to the anionically ionizable lipid is from about 0.9 to about 1.

1.

4. The composition of any one of claims 1-3, wherein the cationic lipid has a lipidic volume that is from about 700 Å3to about 1500 Å3, and / or the anionically ionizable lipid has a lipidic volume that is from about 700 Å3to about 1500 Å3.

5. The composition of any one of claims 1-3, wherein the cationic lipid has a lipidic volume of about 700 Å3to about 1500 Å3and the anionic lipid has a lipidic volume that is greater than 650 Å3. Page 232 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 6. The composition of any one of claims 1-3, wherein the anionically ionizable lipid has a lipidic volume of about 700 Å3to about 1500 Å3and the cationic lipid has a lipidic volume that is greater than 650 Å3.

7. The composition of any one of claims 1-3, wherein the cationic lipid has a lipidic volume of about 700 Å3to about 1500 Å3and the anionically ionizable lipid has a lipidic volume that is from about 700 Å3to about 1500 Å3.

8. The composition of any one of claims 1-3, wherein the cationic lipid and the anionically ionizable lipid have a combined lipidic volume that is greater than 1400 Å3.

9. The composition of any one of claims 1-8, wherein the cationic lipid, the anionically ionizable lipid, and the nucleic acid form nanoparticles.

10. The composition of any one of claims 1-9, wherein the cationic head group is or comprises a guanidinium moiety, an amidinium moiety, an amine moiety, an optionally substituted 5- to 6-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 4- to 6-membered heterocycle comprising 1 to 3 heteroatoms selected from N, O, and S.

11. The composition of any one of claims 1-10, wherein the first lipid tail group is or comprises an optionally substituted C10-C50 aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -Cy-, -NRZ-, -N(RZ)C(O)-, -C(O)N(RZ)-, - N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ) -, -C(O)N(RZ)SO2(RZ)-, - SO2(RZ)N(RZ)C(O)-,-OC(O)O-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; where each -Cy- is independently an optionally substituted C3-C6cycloaliphatic, 5- to 12- membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, S, 5- to 6- membered heteroaryl ring having 1-3 heteroatoms selected from N, O, S; and each RZis independently H or an optionally substituted group selected from C1-C20 aliphatic, or C3- C12cycloaliphatic. Page 233 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 12. The composition of claim 11, wherein each of the first lipid tail group comprises an optionally substituted branched C10-C50 aliphatic group.

13. The composition of any one of claims 1-12, wherein the first bridge group comprises a chiral carbon atom.

14. The composition of any one of claims 1-13, wherein the first bridge group is a C2-C5 aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by - NRZ-, -N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, - N(RZ)C(O)N(RZ)-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-.

15. The composition of claim 1, wherein the cationic lipid is represented by formula ICL:or a pharmaceutically acceptable salt thereof, wherein: G1is -L1a-L1b-G1a; L1ais a bond, or optionally substituted C1-C6 aliphatic; L1bis optionally substituted C1-C6 aliphatic; G1ais -N(Ra)C(N(Ra)2+)N(Ra)2, -N(Ra)3+, -N(Ra)C(N(Ra))N(Ra)2, -N(Ra)(Rb), - C(Ra)2C(N(Ra)2+)N(Ra)2, -N(Ra)C(O)N(Ra)2, -N(Ra)C(S)N(Ra)2, an optionally substituted 5- to 6-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 4- to 6-membered heterocycle comprising 1 to 3 heteroatoms selected from N, O, and S; B1and B2are each independently -NRZ-, -N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, - OC(O)N(RZ)-, -N(RZ)C(O)N(RZ)-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; T1and T2are each independently optionally substituted C5-C35aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -Cy-, -NRZ-, - N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ) -, -O-, - C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; Page 234 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 each Rais independently H or optionally substituted C1-C6aliphatic; each Rbis independently selected from H, optionally substituted C1-C6 aliphatic, and optionally substituted 5- to 6-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S, and optionally substituted 4- to 6-membered heterocycle comprising 1 to 3 heteroatoms selected from N, O, and S; each -Cy- is independently an optionally substituted C3-C6 cycloaliphatic, optionally substituted 5- to 12-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, or optionally substituted 5- to 6-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S; and each RZis independently selected from H, optionally substituted C1-C20aliphatic, and optionally substituted C3-C12cycloaliphatic.

16. The composition of claim 15, wherein G1ais -N(Ra)C(N(Ra)2+)N(Ra)2.

17. The composition of claims 15 or 16, wherein L1ais optionally substituted C1-C6 aliphatic, and L1bis optionally substituted C1-C6 aliphatic.

18. The composition of claim 15, wherein G1ais selected from the group consisting of: , ,19. The composition of claim 15, wherein G1is selected from the group consisting of: Page 235 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 , ,20. The composition of any one of claims 15-19, wherein B1and B2are each independently selected from the group consisting of -C(O)N(RZ) and -N(RZ)C(O)-.

21. The composition of claim 20, wherein one of B1and B2is -C(O)N(RZ) and the other of B1and B2is -N(RZ)C(O)-.

22. The composition of any one of claims 15-21, wherein T1and T2are each independently an optionally substituted C10-C35 aliphatic group.

23. The composition of claim 22, wherein T1and T2are each independently selected from: , nd24. The composition of claim 22, wherein T1and T2are each independently an optionally substituted branched C10-C35 aliphatic group. Page 236 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 25. The composition of claim 15, wherein T1is represented by formula: wherein:X1is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ)-, or -N(RZ)C(O)-; T1ais H or optionally substituted C1-C20 aliphatic; T1bis optionally substituted C1-C20aliphatic; and n1 is 1 to 10.

26. The composition of claim 25, wherein T1is selected from: , nd27. The composition of claim 25 or 22, wherein moiety: or28. The composition of claim 15, wherein T2is represented by formula: Page 237 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 wherein 2X is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ), or -N(RZ)C(O)-; T2ais H or optionally substituted C1-C20aliphatic; T2bis optionally substituted C1-C20aliphatic; and n2 is 1 to 10.

29. The composition of claim 28, wherein T2is selected from: , nd30. The composition of claim 28 or 29, wherein moiety: or31. The composition of claim 15, wherein a compound of formula ICL is represented ICL-II: Page 238 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802or a pharmaceutically acceptable salt thereof, wherein X1is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ)-, or -N(RZ)C(O) -; T1ais H or optionally substituted C1-C20 aliphatic; T1bis optionally substituted C1-C20 aliphatic; n1 is 1 to 10; X2is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ), or -N(RZ)C(O)-; T2ais H or optionally substituted C1-C20 aliphatic; T2bis optionally substituted C1-C20aliphatic; and n2 is 1 to 10.

32. The composition of claim 31, wherein a compound of formula ICL-II is represented ICL- IIa or ICL-IIb:or a pharmaceutically acceptable salt thereof.

33. The composition of claim 31, wherein a compound of formula ICL-II is represented by formula ICL-III: Page 239 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 or a pharmaceutically34. The composition of claim 33, wherein a compound of formula ICL-III is represented by formula ICL-IIIa or ICL-IIIb:or a pharmaceutically acceptable salt thereof.

35. The composition of claim 1, wherein the cationic lipid is selected from Table 1.

36. The composition of any one of claims 1-35, wherein the anionically ionizable head group is or comprises a carboxylic acid moiety or a dihydrogen phosphate moiety.

37. The composition of any one of claims 1-36, wherein the second lipid tail group is or comprises an optionally substituted C10-C50 aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -Cy-, -NRZ-, -N(RZ)C(O)-, - C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ) -, -O-, -C(O)-, -OC(O)-, - C(O)O-, -SO-, or -SO2-; where each -Cy- is independently an optionally substituted C3-C6 cycloaliphatic, 5- to 12-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, S, 5- to 6-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, S; Page 240 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 and each RZis independently H or an optionally substituted group selected from C1-C20aliphatic, or C3-C12 cycloaliphatic.

38. The composition of claim 37, wherein the second lipid tail group comprises is an optionally substituted branched C10-C50 aliphatic group.

39. The composition of any one of claims 1-38, wherein the second bridge group comprises a chiral carbon atom.

40. The composition of any one of claims 1-39, wherein the second bridge group is a C2-C5aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -NRZ-, -N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, - N(RZ)C(O)N(RZ) -, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-.

41. The composition of claim 1, wherein the anionically ionizable lipid is represented by formula IAL:or a pharmaceutically acceptable salt thereof, wherein: G2is -L2a-L2b-G2a-G2b; L2ais a bond or optionally substituted C1-C6 aliphatic L2bis a bond, -C(O)O-, or -OC(O)-; G2ais an optionally substituted C1-C6aliphatic; G2bis -C(O)OH or -P(O)2(OH)2; B3and B4are each independently selected from –C(O)O-, -OC(O)-, or a C2-C6 aliphatic group, wherein one carbon atom is optionally replaced with -OC(O)- or -C(O)O-; T3and T4are each independently optionally substituted C5-C35aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -Cy-, -NRZ-, - N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ) -, -O-, - C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; Page 241 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 each -Cy- is independently an optionally substituted C3-C6cycloaliphatic, optionally substituted 5- to 12-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, S, or optionally substituted 5- to 6-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, S; and each RZis independently selected from H, optionally substituted C1-C20 aliphatic, and optionally substituted C3-C12 cycloaliphatic.

42. The composition of claim 41, wherein G2bis -C(O)OH.

43. The composition of claim 41 or 42, wherein L2ais optionally substituted C1-C6aliphatic, and L2bis -C(O)O- or -OC(O)-.

44. The composition of any one of claim 41-43, wherein G2is: ,45. The composition of any one of claims 41-44, wherein B3is selected fromand , wherein ss represents a point of attachment to T3.

46. The composition of any one of claims 41-44, wherein B4is selected froand , wherein tt r4epresents a point of attachment to T . Page 242 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 47. The composition of any one of claims 41-46, wherein T3and T4are each independently an optionally substituted C10-C35 aliphatic group.

48. The composition of any one of claims 41-46, wherein T3is represented by formula: whereinX3is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ)-, or -N(RZ)C(O)-; T3ais H or optionally substituted C1-C20 aliphatic; T3bis optionally substituted C1-C20 aliphatic; and n3 is 1 to 10.

49. The composition of claim 48, T3is selected from the group consisting of , ,Page 243 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 51. The composition of any one of claims 41-50, wherein T4is represented by formula: whereinX4is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ)-, or -N(RZ)C(O)-; T4ais H or optionally substituted C1-C20aliphatic; T4bis optionally substituted C1-C20 aliphatic; and n4 is 1 to 10.

52. The composition of claim 51, wherein T4is selected from the group consisting of: , 53.,54. The composition of claim 41, wherein a compound represented by formula IAL is a compound represented by IAL-II: Page 244 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802or a pharmaceutically acceptable salt thereof.

55. The composition of claim 54, wherein a compound represented by formula IAL-II is a compound represented by IAL-III:or a pharmaceutically acceptable salt thereof, wherein X3is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ)-, or -N(RZ)C(O)-; T3ais H or optionally substituted C1-C20aliphatic; T3bis optionally substituted C1-C20 aliphatic; n3 is 1 to 10; X4is a bond, -C(O)O-, -OC(O)-, -C(O)N(RZ)-, or -N(RZ)C(O)-; T4ais H or optionally substituted C1-C20 aliphatic; T4bis optionally substituted C1-C20 aliphatic; and n4 is 1 to 10.

56. The composition of claim 54, wherein a compound represented by formula IALII is a compound represented by IAL-IV:- Page 245 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 or a pharmaceutically acceptable salt thereof.

57. The composition of claim 1, wherein the anionically ionizable lipid is selected from Table 3.

58. The composition of any one of claims 1-57, wherein the composition further comprises a sterol.

59. The composition of claim 58, wherein the sterol is cholesterol.

60. The composition of any one of claims 1-59, wherein the composition further comprises a phospholipid.

61. The composition of claim 60, wherein the phospholipid is a zwitterionic phospholipid.

62. The composition of claim 61, wherein the phospholipid is selected from distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), palmitoyloleoyl-phosphatidylcholine (POPC), dioleoylphosphatidylethanolamine (DOPE) and N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM).

63. The composition of any one of claims 1-62, wherein composition further comprises a a polymer-conjugated lipid.

64. The composition of any one of claims 1-62, wherein the composition does not comprise a polymer-conjugated lipid.

65. The composition of any one of claims 1-64, wherein the nucleic acid is RNA. Page 246 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 66. The composition of claim 65, wherein the RNA is mRNA, tRNA, rRNA, snRNA, saRNA, taRNA, ssRNA, dsRNA, siRNA, or miRNA.

67. The composition of any one of claims 1-66, wherein the nucleic acid is DNA.

68. A compound of formula ICL:or a pharmaceutically acceptable salt thereof, wherein: G1is -L1a-L1b-G1a; L1ais a bond optionally substituted C1-C6 aliphatic; L1bis optionally substituted C1-C6 aliphatic; G1ais -N(Ra)C(N(Ra)2+)N(Ra)2, -N(Ra)3+, -N(Ra)C(N(Ra))N(Ra)2, -N(Ra)2, - C(Ra)2C(N(Ra)2+)N(Ra)2, -N(Ra)C(O)N(Ra)2, -N(Ra)C(S)N(Ra)2, an optionally substituted 5- to 6-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S, or an optionally substituted 4- to 6-membered heterocycle comprising 1 to 3 heteroatoms selected from N, O, and S; B1and B2are each independently -NRZ-, -N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, - OC(O)N(RZ)-, -N(RZ)C(O)N(RZ)-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; T1and T2are each independently optionally substituted C5-C35aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -Cy-, -NRZ-, - N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ) -, -O-, - C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; each Rais independently H or optionally substituted C1-C6aliphatic; each -Cy- is independently an optionally substituted C3-C6 cycloaliphatic, 5- to 12- membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, S, 5- to 6- membered heteroaryl ring having 1-3 heteroatoms selected from N, O, S; each RZis independently selected from H, optionally substituted C1-C20 aliphatic, and optionally substituted C3-C12 cycloaliphatic; , and wherein moiety: Page 247 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 has a lipidic volume from about 7 out 1500 Å3.

69. The compound of claim 68, wherein the compound is selected from: Table 2A or Table 2B.

70. A compound represented by formula IAL:or a pharmaceutically acceptable salt thereof, wherein: G2is -L2a-L2b-G2a-G2b; L2ais a bond or optionally substituted C1-C6aliphatic; L2bis a bond, -C(O)O-, or -OC(O)-; G2ais optionally substituted C1-C6 aliphatic; G2bis -C(O)OH or -P(O)2(OH)2; B3and B4are each independently selected from C1-C6aliphatic, wherein each carbon atom is optionally and independently replaced by -NRZ-, -N(RZ)C(O)-, -C(O)N(RZ)-, - N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ)-, -O-, -C(O)-, -OC(O)-, - C(O)O-, -SO-, and -SO2-; T3and T4are each independently optionally substituted C5-C35 aliphatic group, wherein one or more carbon atoms are optionally and independently replaced by -Cy-, -NRZ-, - N(RZ)C(O)-, -C(O)N(RZ)-, -N(RZ)C(O)O-, -OC(O)N(RZ)-, -N(RZ)C(O)N(RZ) -, -O-, - C(O)-, -OC(O)-, -C(O)O-, -SO-, or -SO2-; each Rais independently H or optionally substituted C1-C6 aliphatic; each -Cy- is independently an optionally substituted 3-12 membered bivalent C3-C6 cycloaliphatic, 5- to 12-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S, 5- to 6-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S; Page 248 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 each RZis independently selected from H, optionally substituted C1-C20aliphatic, and optionally substituted C3-C12 cycloaliphatic; and wherein moiety: has a lipidic volume from about 70 bout 1500 Å3.

71. The compound of claim 70, wherein the compound is selected from: Table 4.

72. A suspension comprising a dispersed phase and an aqueous phase, and wherein the dispersed phase comprises the composition of any one of claims 1-67 in the form of particles.

73. The suspension of claim 72, wherein the aqueous phase is substantially free of the nucleic acid.

74. A method of treating a disease, disorder, or condition comprising administering to a subject the suspension of claim 72 or 73.

75. The method of claim 74, wherein the disease, disorder, or condition is selected from an infectious disease, cancer, a genetic disorder, an autoimmune disease, or a rare disease.

76. A method of increasing or causing increased expression of RNA in a target in a subject comprising administering to the subject the suspension of claim 72 or 73.

77. The method of claim 76, wherein the target is selected from the lungs, liver, spleen, heart, brain, lymph nodes, bladder, kidneys, and pancreas. Page 249 of 251 12832328v1Attorney Docket No.2013237-1441 Client Ref. No. P1802 78. The method of any one of claims 74-77, wherein the suspension is administered intramuscularly, intranasally, intravenously, subcutaneously, or intratumoraly.

79. Use of the suspension of claim 72 or 73 for use in medicine.

80. Use of the suspension of claim 72 or 73 for the treatment of a disease, disorder or condition. Page 250 of 251 12832328v1

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