Conformationally tunable lipid compositions and therapeutic uses thereof

Abstract

Disclosed are lipid nanoparticle formulations comprising a lipidoid as defined in the application. Also disclosed are nanoparticle compositions comprising a lipidoid of the invention that are capable of delivering a therapeutic agent. The application also discloses a pharmaceutical composition comprising a lipidoid composition of the invention.

Description

Attorney Docket No.: FAZ-00925CONEOEMATIONALLY TUNABLE LIPID COMPOSITIONS AND THERAPEUTIC USES_ THEREOFRELATED APPLICATIONS

[0001] This application claims the benefit of priority to U. S. Provisional Patent Application serial number 63 / 650,499, filed May 22, 2024, the contents of which are incorporated herein by reference.BACKGROUND

[0002] The effective targeted delivery of biologically active substances such as small molecule drugs, proteins, and nucleic acids represents a continuing medical challenge.

[0003] Lipid-containing nanoparticles can be effective transport vehicles into cells and / or intracellular compartments for biologically active substances. For a more efficient and targeted payload delivery, designs of such nanoparticles should consider certain cell environmental stimuli, including but not limited to pH.

[0004] There is a need to develop pH-sensitive amphiphilic molecules for use in lipid nanoparticles that can deliver therapeutic agents, such as nucleic acid molecules, proteins, and small molecule drugs with safety, efficacy, and specificity.SUMMARY

[0005] In certain aspects, provided here is a lipid nanoparticle, comprising a lipidoid or a salt thereof and a second lipidoid or a salt thereof; wherein:the lipidoid is selected from:-1- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-2- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-3- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925the second lipidoid has the structure of formula (K):° (K); or a salt thereof;-4- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925wherein:RA1and RA2are each independently methyl or ethyl;RBand RB1are each independently H or methyl; andR1and R2are each independently branched (C8-C30)alkyl; wherein a branched (C8-C30)alkyl may comprise more than one branch point.

[0006] In certain aspects, provided here is a lipid nanoparticle, comprising a lipidoid or a salt thereof, wherein the lipidoid is selected from:-5- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-6- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-7- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0007] In certain embodiments, the invention provides nanoparticle compositions comprising a lipidoid of the invention.

[0008] In certain such embodiments, the invention provides nanoparticle compositions comprising a pay load, such as a therapeutic agent (e.g., a nucleic acid molecule, a protein, or a small molecule drug) or an antigen (e.g., a protein o a nucleic acid).

[0009] The invention also provides a method of delivering a therapeutic agent, comprising administering to a subject in need thereof an effective amount of a nanoparticle composition of the invention, wherein the nanoparticle composition comprises a therapeutic agent such as a protein or small molecule drug.

[0010] Also provided herein is a pharmaceutical composition, comprising a lipid nanoparticle composition of the invention, and a pharmaceutically acceptable excipient.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 shows a series of bar graphs demonstrating size, mRNA encapsulation efficiency and pKa of lipid nanoparticles (LNPs) comprising the tested lipidoids.

[0012] Figure 2 shows the in vivo hEPO expression levels of LNPs comprising four tested lipidoids compared to a control nanoparticle formulation comprising SM-102.

[0013] Figure 3 shows the in vivo hEPO expression levels of LNP formulations in which two lipidoids (a lipidoid and a second lipidoid) are coformulated, and a series of bar graphs evaluating their size, encapsulation efficiency, and pKa.

[0014] Figure 4 shows the in vivo hEPO expression levels of LNP formulations having a lipidoid as a helper lipid replacement, and a series of bar graphs evaluating their size, encapsulation efficiency, and pKa.-8- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0015] Figure 5 shows the in vivo hEPO expression levels of LNP formulations having variable concentrations of lipidoid 1 as a helper lipid replacement.

[0016] Figure 6 shows the in vivo hEPO expression levels of LNP formulations having a lipidoid as a helper lipid replacement at a 2 mg / kg dose.DETAILED DESCRIPTION

[0017] The present invention is based on the surprising discovery that lipid nanoparticles comprising a lipidoid described herein effectively encapsulate and deliver therapeutic agents. The lipidoids described herein can undergo a conformational change upon protonation when exposed to a pH change.Definitions

[0018] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well-known and commonly used in the art.

[0019] The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g. “Principles of Neural Science”, McGraw-Hill Medical, New York, N. Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed.”, W. H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.”, W. H. Freeman & Co., N. Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.”, Sinauer Associates, Inc., Sunderland, MA (2000).

[0020] Chemistry terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, C. A. (1985).

[0021] All of the above, and any other publications, patents and published patent applications referred to in this application are specifically incorporated by reference-9- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925herein. In case of conflict, the present specification, including its specific definitions, will control.

[0022] The term “lipid nanoparticle” or “LNP” refers to, without limiting the meaning, a particle that comprises a plurality of lipid molecules (e.g., lipidoids) physically associated with each other by intermolecular forces. For example, the LNP may comprise an ionizable lipid that allows its entry into cells via endocytosis, and allows subsequent endosomal escape. The LNP typically also contains other lipids, a sterol such as cholesterol, and polyethylene glycol (PEG), for example. The LNPs may be, e.g., microspheres and can comprise a core. In some embodiments the core comprises a payload such as an RNA molecule or a fragment thereof.

[0023] The term “agent” is used herein to denote a chemical compound (such as an organic or inorganic compound, a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof as well as humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portion thereof, e.g., a peptide, a lipid, a carbohydrate), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents include, for example, agents whose structure is known, and those whose structure is not known.

[0024] A “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats).

[0025] “Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0026] The term “preventing” is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and-10- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0027] “Administering” or “administration of’ a substance (e.g., a composition such as a composition comprising a compound or an agent), a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0028] Appropriate methods of administering a substance, a compound or an agent to a subject will also depend, for example, on the age and / or the physical condition of the subject and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability and toxicity). In some embodiments, a compound or an agent is administered orally, e.g., to a subject by ingestion. In some embodiments, the orally administered compound or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.

[0029] As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the patient, which may include synergistic effects of the two agents). For example, the different therapeutic-11- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925compounds can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic agents.

[0030] A “therapeutically effective amount” or a “therapeutically effective dose” of a drug or agent is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect. The full therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject’s size, health and age, and the nature and extent of the condition being treated, such as cancer or a viral infection. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.

[0031] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which it does not. For example, “optionally substituted alkyl” refers to the alkyl may be substituted as well as where the alkyl is not substituted.

[0032] It is understood that substituents and substitution patterns on the compounds of the present invention can be selected by one of ordinary skilled person in the art to result chemically stable compounds which can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.

[0033] As used herein, the term “optionally substituted” refers to the replacement of one to six hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, amino, alkylamino, dialkylamino, amido (-C(O)NH2), carboxyl (-C(O)OH), cyano, haloalkyl, haloalkoxy, -OCO-CH2-O-alkyl, -OP(O)(O-alkyl)2 or -CH2-OP(O)(O-alkyl)2. Preferably, “optionally substituted” refers to the replacement of one to four hydrogen radicals in a given structure with the substituents mentioned above. More preferably, one to three-12- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925hydrogen radicals are replaced by the substituents as mentioned above. It is understood that the substituent can be further substituted.

[0034] As used herein, the term “alkyl” refers to saturated aliphatic groups, including but not limited to C1-C10 straight-chain alkyl groups or C1-C10 branched-chain alkyl groups. Preferably, the “alkyl” group refers to Ci-Ce straight-chain alkyl groups or Ci- C<> branched-chain alkyl groups. Most preferably, the “alkyl” group refers to C1-C4 straight-chain alkyl groups or C1-C4 branched-chain alkyl groups. Examples of “alkyl” include, but are not limited to, methyl, ethyl, 1 -propyl, 2-propyl, n-butyl, secbutyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neo-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1- heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1 -octyl, 2-octyl, 3-octyl or 4-octyl and the like. The “alkyl” group may be optionally substituted. A “linear” alkyl group refers to a straight-chain alkyl group without a branching point. A “branched” alkyl group refers to an alkyl group having at least one branch point. Examples of branched alkyl groups include, e.g., isopropyl, sec-butyl, and tert-butyl.

[0035] Moreover, the term “alkyl” as used throughout the specification, examples, and claims is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc.

[0036] The term “Cx.y” or “Cx-Cy”, when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups that contain from x to y carbons in the chain. Coalkyl indicates a hydrogen where the group is in a terminal position, a bond if internal. A Chalky I group, for example, contains from one to six carbon atoms in the chain.

[0037] The term "alkenyl" as used herein means a straight or branched chain hydrocarbon radical containing from 2 to 10 carbons and containing at least one carbon-carbon double bond (i.e., an olefin) formed by the removal of two hydrogens. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2- propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-l -heptenyl, and 3- decenyl. The unsaturated bond(s) of the alkenyl group can be located anywhere in the moiety and can have either the (Z) or the (E) configuration about the double bond(s).

[0038] The term "alkynyl" as used herein means a straight or branched chain hydrocarbon radical containing from 2 to 10 carbon atoms and containing at least one carbon--13- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925carbon triple bond. Representative examples of alkynyl include, but are not limited, to acetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, and 1-butynyl.

[0039] The term “acyl” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0040] The term “acylamino” is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-, preferably alkylC(O)NH-.

[0041] The term “acyloxy” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

[0042] The term “alkoxy” refers to an alkyl group appended to the parent molecular moiety through an oxygen atom. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like.

[0043] The term “alkoxyalkyl” refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula -alkyl-O-alkyl.

[0044] The term “alkylamino”, as used herein, refers to an amino group substituted with at least one alkyl group. A “dialkylamino” refers to an amino group substituted with two alkyl groups.

[0045] The term “alkylthio”, as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.

[0046] The term “amide”, as used herein, refers to a groupO VXN-R9R10wherein R9and R10each independently represent a hydrogen or hydrocarbyl group, or R9and R10taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure. In some embodiments, “amido” refers to the group -C(O)NH2.

[0047] The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented by R9R9N or N-R10R10RW’wherein R9, R10, and R10’ each independently represent a hydrogen or a hydrocarbyl group, or R9and R10taken together with the N atom to which they are-14- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925attached complete a heterocycle having from 4 to 8 atoms in the ring structure. In some embodiments, “amino” refers to the group -NH2.

[0048] The term “aminoalkyl”, as used herein, refers to an alkyl group substituted with an amino group.

[0049] The term “arylalkyl”, as used herein, refers to an alkyl group substituted with an aryl group.

[0050] The term “aryl” as used herein include substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0051] The term “carbamate” is art-recognized and refers to a groupO OAoA^ or A^AO, R’"R9R9wherein R9and R10independently represent hydrogen or a hydrocarbyl group.

[0052] The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group.

[0053] The term “carbocycle” includes 5-7 membered monocyclic and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated and aromatic rings. Carbocycle includes bicyclic molecules in which one, two or three or more atoms are shared between the two rings. The term “fused carbocycle” refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated and aromatic bicyclic rings, as valence permits, is included in the definition of carbocyclic. Exemplary “carbocycles” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2, 3, 4--15- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene and adamantane.Exemplary fused carbocycles include decalin, naphthalene, 1, 2,3,4- tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-lH-indene and bicyclo[4.1.0]hept-3-ene. “Carbocycles” may be substituted at any one or more positions capable of bearing a hydrogen atom.

[0054] The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group.

[0055] The term “carbonate” is art-recognized and refers to a group -OCO2-.

[0056] The term “carboxy” or “carboxyl”, as used herein, refers to a group represented by the formula -CO2H.

[0057] The term “cycloalkyl” includes substituted or unsubstituted non-aromatic single ring structures, preferably 4- to 8-membered rings, more preferably 4- to 6-membered rings. The term “cycloalkyl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is cycloalkyl and the substituent (e.g., R100) is attached to the cycloalkyl ring, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Cycloalkyl groups include, e.g., cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0058] The term “ester”, as used herein, refers to a group -C(O)OR9wherein R9represents a hydrocarbyl group.

[0059] The term “ether”, as used herein, refers to a hydrocarbyl group linked through an oxygen to another hydrocarbyl group. Accordingly, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. Ethers may be either symmetrical or unsymmetrical. Examples of ethers include, but are not limited to, heterocycle-O- heterocycle and aryl-O-heterocycle. Ethers include “alkoxyalkyl” groups, which may be represented by the general formula alkyl-O-alkyl.

[0060] The terms “halo” and “halogen” as used herein means halogen and includes chloro, fluoro, bromo, and iodo.

[0061] The terms “heteroaralkyl” and “heteroarylalkyl”, as used herein, refers to an alkyl group substituted with a heteroaryl group.

[0062] The term “heteroaryl” includes substituted or unsubstituted aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four-16- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925heteroatoms, more preferably one or two heteroatoms. The term “heteroaryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.

[0063] The term “heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0064] The terms “heterocycloalkylalkyl” and “heterocyclylalkyl”, as used herein, refers to an alkyl group substituted with a heterocycle group.

[0065] The terms “heterocycloalkyl,” “heterocyclyl”, “heterocycle”, and “heterocyclic” refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10- membered rings, more preferably 3- to 7-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heterocycloalkyl,” “heterocyclyl”, “heterocycle”, and “heterocyclic” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls.Heterocycloalkyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0066] The term “hydrocarbyl”, as used herein, refers to a group that is bonded through a carbon atom that does not have a =0 or =S substituent, and typically has at least one carbon-hydrogen bond and a primarily carbon backbone, but may optionally include heteroatoms. Thus, groups like methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered to be hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has a =0 substituent on the linking carbon) and ethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

[0067] The term “hydroxy alkyl”, as used herein, refers to an alkyl group substituted with a hydroxy group.-17- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0068] The term “lower” when used in conjunction with a chemical moiety, such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups where there are ten or fewer atoms in the substituent, preferably six or fewer. A “lower alkyl”, for example, refers to an alkyl group that contains ten or fewer carbon atoms, preferably six or fewer. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents, such as in the recitations hydroxyalkyl and aralkyl (in which case, for example, the atoms within the aryl group are not counted when counting the carbon atoms in the alkyl substituent).

[0069] The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls) in which two or more atoms are common to two adjoining rings, e.g., the rings are “fused rings”. Each of the rings of the polycycle can be substituted or unsubstituted. In certain embodiments, each ring of the polycycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7.

[0070] The term “sulfate” is art-recognized and refers to the group -OSO3H, or a pharmaceutically acceptable salt thereof.

[0071] The term “sulfonamide” is art-recognized and refers to the group represented by the general formulae_ o10o Rw5— S-N or6H8*-n-r9wherein R9and R10independently represents hydrogen or hydrocarbyl.

[0072] The term “sulfoxide” is art-recognized and refers to the group-S(O)-.

[0073] The term “sulfonate” is art-recognized and refers to the group SO3H, or a pharmaceutically acceptable salt thereof.

[0074] The term “sulfone” is art-recognized and refers to the group -S(O)2-.

[0075] The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo-18- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.

[0076] The term “thioalkyl”, as used herein, refers to an alkyl group substituted with a thiol group.

[0077] The term “thioester”, as used herein, refers to a group -C(O)SR9or -SC(O)R9

[0078] wherein R9represents a hydrocarbyl.

[0079] The term “thioether”, as used herein, is equivalent to an ether, wherein the oxygen is replaced with a sulfur.

[0080] The term “urea” is art-recognized and may be represented by the general formulaOwherein R9and R10independently represent hydrogen or a hydrocarbyl.

[0081] The term “modulate” as used herein includes the inhibition or suppression of a function or activity (such as cell proliferation) as well as the enhancement of a function or activity.

[0082] The phrase “pharmaceutically acceptable” is art-recognized. In certain embodiments, the term includes compositions, excipients, adjuvants, polymers and-19- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925other materials 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.

[0083] “Pharmaceutically acceptable salt” or “salt” is used herein to refer to an acid addition salt or a basic addition salt which is suitable for or compatible with the treatment of patients.

[0084] The term “pharmaceutically acceptable acid addition salt” as used herein means any non-toxic organic or inorganic salt of any base compounds disclosed herein. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of compounds of Formula I are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts, e.g., oxalates, may be used, for example, in the isolation of compounds of Formula I for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

[0085] The term “pharmaceutically acceptable basic addition salt” as used herein means any non-toxic organic or inorganic base addition salt of any acid compounds disclosed herein. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide. Illustrative organic bases which form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The selection of the appropriate salt will be known to a person skilled in the art.

[0086] In certain embodiments, the lipidoids useful in the methods and compositions of this disclosure have at least one stereogenic center in their structure. This stereogenic-20- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925center may be present in a R or a S configuration, said R and S notation is used in correspondence with the rules described in Pure Appl. Chem. (1976), 45, 11-30. The disclosure contemplates all stereoisomeric forms such as enantiomeric and diastereoisomeric forms of the compounds, salts, prodrugs or mixtures thereof (including all possible mixtures of stereoisomers). See, e.g., WO 01 / 062726.

[0087] Some of the lipidoids may also comprise chemical compounds which exist in tautomeric forms. Such forms, although not explicitly indicated in the formulae described herein, are intended to be included within the scope of the present disclosure.

[0088] The phrase “pharmaceutically acceptable excipient” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material useful for formulating a drug for medicinal or therapeutic use.Nanoparticle Compositions / Lipid Nanoformulations / Lipid-Based Carriers

[0089] In certain embodiments, provided herein is a lipid nanoparticle, comprising a lipidoid or a salt thereof and a second lipidoid or a salt thereof; wherein:the lipidoid is selected from:-21- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-22- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-23- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925the second lipidoid has the structure of formula (K):° (K);or a salt thereof;wherein:RA1and RA2are each independently methyl or ethyl;RBand RB1are each independently H or methyl; andR1and R2are each independently branched (C8-C30)alkyl; wherein a branched (C8-C30)alkyl may comprise more than one branch point.

[0090] In certain embodiments, provided herein is a lipid nanoparticle, comprising a lipidoid or a salt thereof, wherein the lipidoid is selected from:-24- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-25- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-26- FoleyHoagUS 12883713.3Attorney Docket No.: FAZ-00925

[0091] In certain embodiments, the lipidoid is selected from:-27- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925(lipidoid 5);(lipidoid 7);-28- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925(lipidoid 11);(lipidoid 8); and

[0092] In certain preferred embodiments, the lipidoid is selected from:

[0093] In certain preferred embodiments, the lipidoid is selected from:-29- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925; and

[0094] The lipidoids described herein exhibit the effect of undergoing a conformational change (i.e., a cyclohexane chair flip) upon protonation under acidic conditions. This conformational change surprisingly facilitates delivery of a payload housed in a lipid nanoparticle composition comprising the lipidoid. Furthermore, use of a lipidoid described herein as a replacement for a “helper lipid” (e.g., a phospholipid) often used in LNP formulations surprisingly led to excellent payload delivery even at molar concentrations as low as 5-10 mol % of the LNP formulation.

[0095] The lipidoids described herein have been characterized in the following references: Pharmaceutics 2011, 3, 379-405; Journal of Liposome Research, 2012; 22(4): 319-328; BioMol Concepts 2014; 5(2): 131-141; Biochimica et Biophysica Acta 1848 (2015) 3113-3125; Chemistry and Physics of Lipids 210 (2018) 129-141; International Journal of Nanomedicine 2019:14 1039-1049, “New insights on the release and self-healing model of stimuli- sensitive liposomes” Journal of Colloid and Interface Science, January 2023; the contents of which are herein incorporated by reference in their entireties.-30- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0096] In certain embodiments, provided herein is a nanoparticle composition comprising a lipidoid described herein, or a pharmaceutically acceptable salt thereof. In certain embodiments, the nanoparticle composition comprises a plurality of lipidoids described herein, or a pharmaceutically acceptable salt thereof.

[0097] As used herein, “nanoparticle composition” is used interchangeably with the terms “lipid-based carrier,” “lipid nanoformulation,” and “lipid nanoparticle.”

[0098] In some embodiments, compounds described herein are formulated into a lipid- based carrier (or lipid nanoformulation). In some embodiments, the lipid-based carrier (or lipid nanoformulation) is a liposome or a lipid nanoparticle (LNP). In certain embodiments, the lipid-based carrier is an LNP.

[0099] In some embodiments, the lipid nanoparticle further comprises a non-cationic lipid (e.g., a phospholipid), a structural lipid (e.g., a sterol such as cholesterol), and / or a PEGylated lipid.

[0100] In some embodiments, the lipid nanoparticle further comprises a phospholipid, a sterol, and / or a PEGylated lipid.

[0101] In some embodiments, the lipid nanoparticle further comprises a second lipidoid.

[0102] In some embodiments, the lipid nanoparticle further comprises a second lipidoid (e.g., an ionizable lipid), a non-cationic lipid (e.g., phospholipid), a structural lipid (e.g., a sterol such as cholesterol), and / or a PEGylated lipid.

[0103] In certain embodiments, the second lipidoid is a cationic, anionic, ionizable, or zwitterionic lipidoid.

[0104] All descriptions and all embodiments discussed herein relating to the aspects of the lipidoids described herein are all applicable to these aspects of the invention relating to the lipid-based carriers (or a lipid nanoformulation).

[0105] As described herein, suitable compounds to be used in the lipid-based carrier (or lipid nanoformulation) include all the isomers and isotopes of the compounds described above, as well as all the pharmaceutically acceptable salts, solvates, or hydrates thereof, and all crystal forms, crystal form mixtures, and anhydrides or hydrates.

[0106] One or more naturally occurring and / or synthetic lipid compounds may be used in the preparation of the lipid-based carrier (or lipid nanoformulation).-31- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0107] The lipid-based carrier (or lipid nanoformulation) may contain positively charged (cationic) lipids, neutral lipids, negatively charged (anionic) lipids, or a combination thereof.

[0108] In some embodiments, the lipid nanoparticle of the invention may be conjugated to a targeting moiety (e.g., an antibody or antigen-binding fragment thereof) through a linking group. Various linking groups known in the art may be used in the lipid nanoparticles of the invention, and can comprise one or more of optionally substituted alkylene, optionally substituted heteroalkylene, optionally substituted alkenylene, optionally substituted heteroalkenylene, optionally substituted alkynylene, optionally substituted heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, a peptide moiety, a dipeptide moiety, -(C=O)-, a disulfide, a hydrazone, thioester, sulfone, sulfoxide, thiosulfinate, thiosulfonate, sulfate, sulfonate, sulfonylurea, ether, thioether, ester, amide, carbonate, carbamate, urea, sulfamide, succinimide, maleimide, phosphate, diphosphate, triazole, or a saccharide, or a combination thereof. Suitable linking groups are described, e.g., in WO 2024 / 015229, WO 2024 / 006272, and WO 2023 / 225359.Cationic Lipids (Positively Charged) and / or Ionizable Lipids

[0109] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises one or more cationic lipids, e.g., a cationic lipid that can exist in a positively charged or neutral form depending on pH, or an amine-containing lipid that can be readily protonated. In some embodiments, the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions.

[0110] Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. Examples of positively charged (cationic) lipids include, but are not limited to, N, N'-dimethyl-N, N'-dioctacyl ammonium bromide (DDAB) and chloride DDAC), N-(l-(2,3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTMA), 3P-[N-(N', N'-dimethylaminoethyl)carbamoyl) cholesterol (DC-chol), 1,2- dioleoyloxy-3-[trimethylammonio] -propane (DOTAP), l,2-dioctadecyloxy-3- [trimethylammonio] -propane (DSTAP), and l,2-dioleoyloxypropyl-3-dimethyl- hydroxy ethyl ammonium chloride (DORI), N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2- Dioleoyl-3-Dimethylammonium-propane (DODAP), 1,2-Dioleoylcarbamyl-3--32- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925Dimethylammonium-propane (DOCDAP), 1,2-Dilmeoyl-3-Dimethylammomum- propane (DLINDAP), 3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)- 1- (cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)- 3'-oxapentoxy)-3-dimethyl-l-(cis, cis-9',12'-octadecadienoxy)propane (CpLin DMA), N, N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA), and the cationic lipids described in e.g. Martin et al., Current Pharmaceutical Design, pages 1-394, which is herein incorporated by reference in its entirety. In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises more than one cationic lipid.

[0111] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises a cationic lipid having an effective pKa over 6.0. In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises a second cationic lipid having a different effective pKa (e.g., greater than the first effective pKa) than the first cationic lipid.

[0112] In some embodiments, cationic lipids that can be used in the lipid-based carrier (or lipid nanoformulation) include, for example those described in Table 4 of WO 2019 / 217941, which is incorporated by reference.

[0113] In some embodiments, the cationic lipid is an ionizable lipid (e.g., a lipid that is protonated at low pH, but that remains neutral at physiological pH). In some embodiments, the lipid-based carrier (or lipid nanoformulation) may comprise one or more additional ionizable lipids, different than the ionizable lipids (e.g., the lipidoids and second lipidoids) described herein. Exemplary ionizable lipids include, but are not limited to,(LP01),(SM-086),FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925(SM-102),(ALC-0315),(Lipid 10),(Lipid A9), and(DLin-MC3-DMA), (see WO 2017 / 004143A1, which is incorporated herein by reference in its entirety).

[0114] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises one or more compounds described by WO 2021 / 113777 (e.g., a lipid of Formula (3) such as a lipid of Table 3 of WO 2021 / 113777), which is incorporated herein by reference in its entirety.

[0115] In some embodiments, the ionizable lipid is a lipid disclosed in Hou, X., et al. Nat Rev Mater 6, 1078-1094 (2021). (e.g., L319, C12-200, and DLin-MC3-DMA), (which is incorporated by reference herein in its entirety).

[0116] Examples of other ionizable lipids that can be used in lipid-based carrier (or lipid nanoformulation) include, without limitation, one or more of the following formulas: X of US 2016 / 0311759; I of US 20150376115 or in US 2016 / 0376224; Compound 5 or Compound 6 in US 2016 / 0376224; I, IA, or II of US 9,867,888; I, II or III of US 2016 / 0151284; I, IA, II, or IIA of US 2017 / 0210967; I-c of US 2015 / 0140070; A of US 2013 / 0178541; I of US 2013 / 0303587 or US 2013 / 0123338; I of US 2015 / 0141678; II, III, IV, or V of US 2015 / 0239926; I of US 2017 / 0119904; I or II of -34- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925WO 2017 / 117528; A of US 2012 / 0149894; A of US 2015 / 0057373; A of WO 2013 / 116126; A of US 2013 / 0090372; A of US 2013 / 0274523; A of US 2013 / 0274504; A of US 2013 / 0053572; A of WO 2013 / 016058; A of WO 2012 / 162210; I of US 2008 / 042973; I, II, III, or IV of US 2012 / 01287670; I or II of US 2014 / 0200257; I, II, or III of US 2015 / 0203446; I or III of US 2015 / 0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of US 2014 / 0308304; of US 2013 / 0338210; I, II, III, or IV of WO 2009 / 132131; A of US 2012 / 01011478; I or XXXV of US 2012 / 0027796; XIV or XVII of US 2012 / 0058144; of US 2013 / 0323269; I of US 2011 / 0117125; I, II, or III of US 2011 / 0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US 2012 / 0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of US 2011 / 0076335; I or II of US 2006 / 008378; I of W02015 / 074085 (e.g., ATX-002); I of US 2013 / 0123338; I or X-A-Y-Z of US 2015 / 0064242; XVI, XVII, or XVIII of US 2013 / 0022649; I, II, or III of US 2013 / 0116307; I, II, or III of US 2013 / 0116307; I or II of US 2010 / 0062967; I-X of US 2013 / 0189351; I of US 2014 / 0039032; V of US 2018 / 0028664; I of US 2016 / 0317458; I of US 2013 / 0195920; 5, 6, or 10 of US 10,221,127; III-3 of WO 2018 / 081480; 1-5 or 1-8 of WO 2020 / 081938; I of WO 2015 / 199952 e.g., compound 6 or 22) and Table 1 therein; 18 or 25 of US 9,867,888; A of US 2019 / 0136231; II of WO 2020 / 219876; 1 of US 2012 / 0027803; OF-02 of US 2019 / 0240349; 23 of US 10,086,013; cKK-E12 / A6 of Miao et al (2020); C12-200 of WO 2010 / 053572; 7C1 of Dahlman et al (2017); 304-013 or 503-013 of Whitehead et al; TS-P4C2 of U S9, 708, 628; I of WO 2020 / 106946; I of WO 2020 / 106946; (1), (2), (3), or (4) of WO 2021 / 113777; and any one of Tables 1-16 of WO 2021 / 113777, all of which are incorporated herein by reference in their entirety.

[0117] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further includes biodegradable ionizable lipids, for instance, (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate, also called 3- ((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9, 12- dienoate). See, e.g., lipids of WO 2019 / 067992, WO 2017 / 173054, WO 2015 / 095340, and WO 2014 / 136086, which are incorporated herein by reference in their entirety.-35- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0118] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises an ionizable lipid. In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises a second lipidoid. As used herein, “lipidoid compounds” refers to the lipidoids (as described herein) and / or the second lipidoids (as described below).

[0119] In some embodiments, the second lipidoid has the structure of formula (I):pA2 _ _ _I RB\ RB1OX A / L1-Y1-R1RAI W ^X^NI2'Y2-R2(I);or a salt thereof;wherein:RA1and RA2are each independently H, alkyl, or hydroxyalkyl;or wherein RA1and RA2, taken together with the intervening nitrogen, form an optionally substituted heterocyclic ring;RBand RB1are each independently H, optionally substituted alkyl, alkenyl, cycloalkyl, arylalkyl, heteroarylalkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, or thioalkyl;or wherein RBand RB1, taken together with the intervening carbon, form an optionally substituted cycloalkyl or heterocyclic ring;or RA1and RBtaken together with the intervening N-CH atoms to which they are attached form an N-containing heterocyclic ring;L1and L2are each independently -CH2CH2-, -(CH2)3-, -(CH2)4-, -CH(CH3)CH2-, or -CH2CH(CH3)-;X is a bond, -CH2-, or -CH2CH2-;W is a bond, -CH2-, or -CH2CH2-;Y1and Y2are each independently -O(C=O)-, -S(C=O)-, or -O(C=O)O-; and R1and R2are each independently linear (Cs-C3o)alkyl, branched (Cs-C3o)alkyl, linear (Cs-C3o)alkenyl, branched (Cs-C3o)alkenyl, linear (Cs-C3o)alkynyl, or branched (Cs-C3o)alkynyl; wherein a branched (Cs-C3o)alkyl, a branched (Cs-C3o)alkenyl, or a branched (Cs-C3o)alkynyl may comprise more than one branch point, and a linear or branched (Cs-C3o)alkenyl may comprise more than one olefin, and a linear or branched (Cs-C3o)alkynyl may comprise more than one carbon-carbon triple bond.-36- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0120] In certain embodiments, if X and W are both a bond, and RBis H, then RA1and RA2are not both methyl; andthe compound is not:L-Lysine-bis(O, O'-oleoyl-P-hydroxyethyl)amide;L-Lysine-bis(O, O'-palmitoyl-P-hydroxyethyl)amide;L-Lysine-bis(O, O'-myristoyl-P-hydroxyethyl)amide;L-Ornithine-bis(O, O'-palmitoyl-P-hydroxyethyl)amide;L-Ornithine-bis(O, O'-oleoyl-P-hydroxyethyl)amide;L-Ornithine-bis(O, O'-myristoyl-P-hydroxyethyl)amide;L-Arginine-bis(O, O'-palmitoyl-P-hydroxyethyl)amide;L-Arginine-bis(O, O'-oleoyl-P-hydroxyethyl)amide;L-Serine-bis(O, O'-oleoyl-P-hydroxyethyl)amide;Glycine-bis(O, O'-palmitoyl-P-hydroxyethyl)amide;Sarcosine-bis(O, O'-palmitoyl-P-hydroxyethyl)amide;L-Histidine-bis(O, O'-palmitoyl-P-hydroxyethyl)amide;L-Glutamine-bis(O, O'-palmitoyl-P-hydroxyethyl)amide;L-Glutamic acid-y-N'-bis(O, O'-palmitoyl-P-hydroxyethyl)amide;L-aspartic acid-P-N'-bis(O, O'-palmitoyl-P-hydroxyethyl)amide; orL-aspartic acid-a-N'-bis(O, O'-palmitoyl-P-hydroxyethyl)amide.

[0121] In certain embodiments, RA1and RA2are the same. In exemplary embodiments, RA1and RA2are each H.

[0122] In alternative embodiments, RA1and RA2are different. In certain such embodiments, RA1is hydroxyalkyl, such as -CH2CH2OH; and RA2is H.

[0123] In certain embodiments, RBis H, optionally substituted alkyl, arylalkyl, heteroarylalkyl, hydroxyalkyl, or aminoalkyl. In more specific embodiments, RBis H, methyl, -CH2PI1, -CH2OH, -(CEh^NEh, or -CH2(imidazolyl). In certain preferred embodiments, RBis methyl.

[0124] In certain embodiments, RB1is hydrogen.

[0125] In certain embodiments, at least one of X and W is a bond.

[0126] In certain embodiments, X and W are both a bond.

[0127] In certain such embodiments, the second lipidoid has the structure of formula (la):-37- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925RA2ON A / L1-Y1-R1R Y NRLl „QY -R (la) or a salt thereof;wherein the second lipidoid has the absolute stereochemistry shown.

[0128] In alternative such embodiments, the second lipidoid has the structure of formula (lb):RA2ON JL / L1-Y1-R1rA< Lb"RL2,,,Y2-R2(lb) or a salt thereof;wherein the second lipidoid has the absolute stereochemistry shown.

[0129] In certain embodiments, X is -CH2-; and W is a bond.

[0130] In certain such embodiments, the second lipidoid has the structure of formula (Ic):RBOrA1JL Jk / L1-Y1-R1I I RA2L22 2Y -R (Ic) or a salt thereof;wherein the second lipidoid has the absolute stereochemistry shown.

[0131] In alternative such embodiments, the second lipidoid has the structure of formula (Id):RBO RA1Jk / L1-Y1-R1N N I I RA2L22 2Y -R (Id) or a salt thereof;wherein the second lipidoid has the absolute stereochemistry shown.

[0132] In certain embodiments, X is -CH2CH2-; and W is a bond.

[0133] In certain such embodiments, the second lipidoid has the structure of formula (le):RA2ON / L1-Y1-R1RA<V NRL2,,Y -R (le) or a salt thereof;wherein the second lipidoid has the absolute stereochemistry shown.

[0134] In alternative such embodiments, the second lipidoid has the structure of formula (If):-38- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925RA2Ohk AXL1-Y1-R1rA< 2rYRLl,,Y -R (If) or a salt thereof;wherein the second lipidoid has the absolute stereochemistry shown.

[0135] In certain embodiments, W is a bond.

[0136] In certain embodiments, Y1and Y2are each -O(C=O)-.

[0137] In certain embodiments, L1and L2each represent -CH2CH2-.

[0138] In certain embodiments, R1and R2are each independently linear (Cs-C24)alkyl, branched (Cs-C24)alkyl, linear (Cs-C24)alkenyl, or branched (Cs-C24)alkenyl.

[0139] In certain embodiments, the second lipidoid tails are linear. In certain such embodiments, R1and R2are each independently linear (Cs-C24)alkyl or linear (Cs- C24)alkenyl, preferably linear (Cs-C24)alkyl, more preferably linear (C9-Ci?)alkyl. Alternatively, R1and R2can each independently be linear (Cs-C24)alkenyl, preferably linear (Ci7)alkenyl.

[0140] In certain embodiments, R1and R2are each independently branched (Cs-C24)alkyl, preferably branched (Cis-Ci9)alkyl.

[0141] In certain embodiments, the second lipidoid has the structure of formula (Ig):1 1RA2L*OO R2(Ig) or a salt thereof.

[0142] In certain preferred embodiments, RBis aminoalkyl, X and W are both a bond, RA1and RA2are each H, L1and L2are each -CH2CH2-; and R1and R2are each independently branched (Cs-C3o)alkyl.

[0143] In other preferred embodiments, RBis hydroxyalkyl, X and W are both a bond, RA1and RA2are each H, L1and L2are each -CH2CH2-; and R1and R2are each independently linear or branched (Cs-C3o)alkyl.

[0144] In other preferred embodiments, RBis H, X and W are both a bond, RA1is H, RA2is H or methyl, L1and L2are each -CH2CH2-; and R1and R2are each independently branched (Cs-C3o)alkyl, linear (Cs-C3o)alkenyl, or branched (Cs-C3o)alkenyl.

[0145] In still other preferred embodiments, RBis heteroarylalkyl, X and W are both a bond, RA1and RA2are each H, L1and L2are each -CH2CH2-; and R1and R2are each-39- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925independently branched (Cs-C3o)alkyl, linear (Cs-C3o)alkenyl, or branched (Cs- C30) alkenyl.

[0146] In any of the preceding embodiments, Y1and Y2can each be -O(C=O)-.

[0147] Representative second lipidoids include those in Table A below.-40- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-41- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-42- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-43- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-44- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-45- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-46- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-47- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-48- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-49- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-50- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-51- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-52- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-53- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-54- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-55- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-56- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-57- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-58- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-59- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-60- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-61- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-62- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-63- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-64- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-65- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-66- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-67- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-68- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-69- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-70- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-71- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-72- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-73- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-74- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-75- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-76- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-77- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-78- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0148] In certain embodiments, the second lipidoid has the structure of formula (II):R3ixY3RA3RB2RB2A O L3N JL xY-1RA4X" IT N X4R4RH(II);or a salt thereof;wherein:RA3and RA4are each independently H, alkyl, or hydroxyalkyl;or wherein RA3and RA4, taken together with the intervening nitrogen, form an optionally substituted heterocycloalkyl ring;RB2and RB2Aare each independently H, optionally substituted alkyl, alkenyl, cycloalkyl, arylalkyl, heteroarylalkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, or thioalkyl;or wherein RB2and RB2A, taken together with the intervening carbon, form an optionally substituted cycloalkyl or heterocycloalkyl ring;-79- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925or RA3and RB2taken together with the intervening N-CH atoms to which they are attached form an N-containing heterocyclic ring;L3and L4are each independently -CH2-, -CH2CH2-, -(CH2)3-, -(CH2)4-, -CH(CH3)CH2- or -CH2CH(CH3)-;V is a bond, -CH2- or -CH2CH2-;U is a bond, -CH2- or -CH2CH2-;Y3and Y4are each independently -O(C=O)-, -S(C=O)-, or -O(C=O)O-; and R3and R4are each independently linear (Cs-C3o)alkyl, branched (Cs-C3o)alkyl, linear (Cs-C3o)alkenyl, branched (Cs-C3o)alkenyl, linear (Cs-C3o)alkynyl, or branched (Cs-C3o)alkynyl; wherein a branched (Cs-C3o)alkyl, a branched (Cs-C3o)alkenyl, or a branched (Cs-C3o)alkynyl may comprise more than one branch point, and a linear or branched (Cs-C3o)alkenyl may comprise more than one olefin, and a linear or branched (Cs-C3o)alkynyl may comprise more than one carbon-carbon triple bond.

[0149] In certain such embodiments, representative second lipidoids include those in Table B below:Table B: Further representative second lipidoids-80- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0150] In any of the preceding embodiments, the salt of the second lipidoid may be a pharmaceutically acceptable salt.

[0151] In certain embodiments, the second lipidoid is:or a pharmaceutically acceptable salt thereof.Non-Cationic Lipids (e.g.. Phospholipids)

[0152] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises one or more non-cationic lipids. In some embodiments, the non-cationic lipid is a phospholipid. In some embodiments, the non-cationic lipid is a phospholipid-81- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925substitute or replacement. In some embodiments, the non-cationic lipid is a negatively charged (anionic) lipid.

[0153] Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn- glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoy 1-pho sphatidy lethanolamine (DOPE), p almitoy loleoy Ipho sphatidy Icholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidy lethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl- phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, 1- stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoy Ipho sphatidy Icholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl- sn-glycero-3-phosphocholine (DLPC), Sodium 1,2- ditetradecanoyl-sn-glycero-3-phosphate (DMPA), phosphatidylcholine (lecithin), phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), phosphatidylethanolamine (cephalin), cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, paimitoyl, stearoyl, or oleoyl. Additional exemplary lipids, in certain embodiments, include, without limitation, those described in Kim et al. Nano Lett. 2020, 20 (6) 4543-4549, which is incorporated herein by reference. Such lipids include, in some embodiments, plant lipids found to improve liver transfection with mRNA e.g., DGTS).-82- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0154] In some embodiments, the lipid-based carrier (or lipid nanoformulation) may comprise a combination of distearoylphosphatidylcholine / cholesterol, dipalmitoylphosphatidylcholine / cholesterol, dimyrystoylphosphatidylcholine / cholesterol, l,2-Dioleoyl-sn-glycero-3- phosphocholine (DOPC) / cholesterol, or egg sphingomyelin / cholesterol.

[0155] Other examples of suitable non-cationic lipids include, without limitation, nonphosphorous lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, and the like. Other non-cationic lipids are described in WO 2017 / 099823 or US 2018 / 0028664, which are incorporated herein by reference in their entirety.

[0156] In one embodiment, the lipid-based carrier (or lipid nanoformulation) further comprises one or more non-cationic lipid that is oleic acid or a compound of Formula I, II, or IV of US 2018 / 0028664, which is incorporated herein by reference in its entirety.

[0157] The non-cationic lipid content can be, for example, 0-30% (mol) of the total lipid components present. In some embodiments, the non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid components present.

[0158] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises a neutral lipid, and the molar ratio of an ionizable lipid to a neutral lipid ranges from about 2:1 to about 8:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1).

[0159] In some embodiments, the lipid-based carrier (or lipid nanoformulation) does not include any phospholipids.

[0160] In some embodiments, the lipid-based carrier (or lipid nanoformulation) can further include one or more phospholipids, and optionally one or more additional molecules of similar molecular shape and dimensions having both a hydrophobic moiety and a hydrophilic moiety (e.g., cholesterol).

[0161] Exemplary anionic lipids include dimyrystoyl-, dipalmitoyl-, and distearoyl- phasphatidylglycerol; dimyrystoyl-, dipalmitoyl-, and dipalmitoyl-phosphatidic acid; dimyrystoyl-, dipalmitoyl-, and dipalmitoyl-phosphatidylethanolamine; and their unsaturated diacyl and mixed acyl chain counterparts as well as cardiolipin.-83- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0162] Exemplary neutral lipids include DLPC (1,2-dilauroyl- sn-glycero-3- phosphocholine), DMPC (l,2-dimyristoyl-sn-glycero-3-phosphocholine), DPPC (1,2- dipalmitoyl-sn-glycero-3-phosphocholine), DSPC (l,2-distearoyl-sn-glycero-3- phosphocholine), DOPC (l,2-dioleoyl-sn-glycero-3-phosphocholine), DMPA (Sodium 1,2- ditetradecanoyl-sn-glycero-3-phosphate), DPPE (1,2-Dipalmitoyl-sn- glycero-3- phosphoethanolamine), and DOPE (l,2-dioleoyl-sn-glycero-3- phosphoethanolamine).

[0163] Exemplary phospholipids include, but are not limited to, phosphatidylcholine (lecithin), lysolecithin, lysophosphatidylethanol-amine, phosphatidylserine, phosphatidylinositol, sphingomyelin, phosphatidylethanolamine (cephalin), cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, phosphatidylcholine, and dipalmitoy 1-pho sphatidy Igly cerol.Structural Lipids

[0164] The lipid-based carrier (or lipid nanoformulation) described herein may further comprise one or more structural lipids. As used herein, the term “structural lipid” refers to sterols (e.g., cholesterol and derivatives thereof) and to lipids containing sterol moieties.

[0165] Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can be selected from the group including but not limited to, cholesterol or cholesterol derivative, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. In certain embodiments, the structural lipid is a steroid. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In certain embodiments, the structural lipid is alpha-tocopherol.

[0166] In some embodiments, structural lipids may be incorporated into the lipid-based carrier at molar ratios ranging from about 0.1 to 1.0 (cholesterol phospholipid).

[0167] In some embodiments, sterols, when present, can include one or more of cholesterol or cholesterol derivatives, such as those described in WO 2009 / 127060 or US 2010 / 0130588, which are incorporated herein by reference in their entirety.Additional exemplary sterols include phytosterols, including those described in-84- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925Eygeris et al. (2020), Nano Lett. 2020;20(6):4543-4549, incorporated herein by reference.

[0168] In some embodiments, the structural lipid is a cholesterol derivative. Nonlimiting examples of cholesterol derivatives include polar analogues such as 5a- cholestanol, 53-coprostanol, cholesteryl-(2’-hydroxy)-ethyl ether, cholesteryl-(4'- hydroxy) -butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a- cholestane, cholestenone, 5a-cholestanone, 5p-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analogue, e.g., cholesteryl-(4'-hydroxy)-butyl ether. Exemplary cholesterol derivatives are described in WO 2009 / 127060 and US 2010 / 0130588, each of which is incorporated herein by reference in its entirety.

[0169] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises sterol in an amount of 0-50 mol% e.g., 0-10 mol %, 10-20 mol %, 20-50 mol%, 20-30 mol %, 30-40 mol %, or 40-50 mol %) of the total lipid components. Polymers and Polyethylene Glycol (PEG) Lipids

[0170] In some embodiments, the lipid-based carrier (or lipid nanoformulation) may include one or more polymers or co-polymers, e.g., poly(lactic-co-glycolic acid) (PFAG) nanoparticles.

[0171] In some embodiments, the lipid-based carrier (or lipid nanoformulation) may include one or more polyethylene glycol (PEG) lipid (also referred to as a “PEGylated lipid”). Examples of useful PEG-lipids include, but are not limited to, 1,2-Diacyl-sn- Glycero-3- Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-350] (mPEG 350 PE); 1,2-Diacyl-sn- Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-550] (mPEG 550 PE); 1,2- Diacyl-sn-Glycero-3-Phosphoethanolamine-N- [Methoxy(Polyethylene glycol)-750] (mPEG 750 PE); l,2-Diacyl-sn-Glycero-3- Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)- 1000] (mPEG 1000 PE); l,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)- 2000] (mPEG 2000 PE); l,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N- [Methoxy(Polyethylene glycol)-3000] (mPEG 3000 PE); l,2-Diacyl-sn-Glycero-3- Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-5000] (mPEG 5000 PE); N- Acyl- Sphingosine- l-[Succinyl(Methoxy Polyethylene Glycol) 750] (mPEG 750 Ceramide); N-Acyl- Sphingosine- l-[Succinyl(Methoxy Polyethylene Glycol) 2000] (mPEG 2000 Ceramide); and N- Acyl-Sphingosine-l-[Succinyl(Methoxy-85- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925Polyethylene Glycol) 5000] (mPEG 5000 Ceramide). In some embodiments, the PEG lipid is a polyethyleneglycol-diacylglycerol (i.e., polyethyleneglycol diacylglycerol (PEG-DAG), PEG-cholesterol, or PEG-DMB) conjugate.

[0172] In some embodiments, the lipid-based carrier (or nanoformulation) includes one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers described in Table 5 of WO 2019 / 217941, which is incorporated herein by reference in its entirety). In some embodiments, the one or more conjugated lipids is formulated with one or more ionic lipids (e.g., non-cationic lipid such as a neutral or anionic, or zwitterionic lipid); and one or more sterols (e.g., cholesterol).

[0173] The PEG conjugate can comprise a PEG-dilaurylglycerol (C12), a PEG- dimyristylglycerol (C14), a PEG-dipalmitoylglycerol (C16), a PEG-disterylglycerol (C18), PEG-dilaurylglycamide (C12), PEG-dimyristylglycamide (C14), PEG- dipalmitoylglycamide (C16), and PEG-disterylglycamide (C18).

[0174] In some embodiments, conjugated lipids, when present, can include one or more of PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG- ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl- l-0-(w- methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N- (carbonyl-methoxypolyethylene glycol 2000)- 1,2- distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and those described in Table 2 of WO 2019 / 051289 (which is herein incorporated by reference in its entirety), and combinations of the foregoing.

[0175] Additional exemplary PEG-lipid conjugates are described, for example, in US 5,885,613, US 6,287,591, US 2003 / 0077829, US 2003 / 0077829, US 2005 / 0175682, US 2008 / 0020058, US 2011 / 0117125, US 2010 / 0130588, US 2016 / 0376224, US 2017 / 0119904, US 2018 / 0028664, and WO 2017 / 099823, all of which are incorporated herein by reference in their entirety.

[0176] In some embodiments, the PEG-lipid is a compound of Formula III, III-a-I, Ill-a- 2, III-b-1, III-b-2, or V of US 2018 / 0028664, which is incorporated herein by reference in its entirety. In some embodiments, the PEG-lipid is of Formula II of US 2015 / 0376115 or US 2016 / 0376224, both of which are incorporated herein by reference in their entirety. In some embodiments, the PEG-DAA conjugate can be,-86- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925for example, PEG-dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG- dipalmityloxypropyl, or PEG-distearyloxypropyl. In some embodiments, the PEG- lipid includes one of the following:

[0177] In some embodiments, lipids conjugated with a molecule other than a PEG can also be used in place of PEG-lipid. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (GPL) conjugates can be used in place of or in addition to the PEG-lipid.

[0178] Exemplary conjugated lipids, e.g., PEG-lipids, (POZ)-lipid conjugates, ATTA- lipid conjugates and cationic polymer-lipids, include those described in Table 2 of WO 2019 / 051289A9, which is incorporated herein by reference in its entirety.

[0179] In some embodiments, the conjugated lipid (e.g., the PEGylated lipid) can be present in an amount of 0-20 mol% of the total lipid components present in the lipid- based carrier (or lipid nanoformulation). In some embodiments, the conjugated lipid e.g., the PEGylated lipid) content is 0.5-10 mol% or 2-5 mol% of the total lipid components.

[0180] When needed, the lipid-based carrier (or lipid nanoformulation) described herein may be coated with a polymer layer to enhance stability in vivo (e.g., sterically stabilized LNPs).-87- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0181] Examples of suitable polymers include, but are not limited to, poly(ethylene glycol), which may form a hydrophilic surface layer that improves the circulation half-life of liposomes and enhances the amount of lipid nanoformulations (e.g., liposomes or LNPs) that reach therapeutic targets. See, e.g., Working et al. J Pharmacol Exp Ther, 289: 1128-1133 (1999); Gabizon et al., J Controlled. Release 53: 275-279 (1998); Adlakha Hutcheon et al., Nat Biotechnol 17: 775-779 (1999); and Koning et al., Biochim Biophys Acta 1420: 153-167 (1999), which are incorporated herein by reference in their entirety.

[0182] In certain embodiments, the nanoparticle composition further comprises a PEGylated lipid, a sterol, a phospholipid, and / or a neutral lipid.Percentages of Lipid Nanoformulation Components

[0183] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises one or more of the compounds described herein, optionally a non-cationic lipid (e.g., a phospholipid), a sterol, a neutral lipid, and / or optionally conjugated lipid (e.g., a PEGylated lipid) that inhibits aggregation of particles. The relative amounts of these components can be varied independently and to achieve desired properties. For example, in some embodiments, the ionizable lipid, including, for example, the lipidoid compounds (e.g., the lipidoid and / or the second lipidoid), of the lipid-based carrier (or lipid nanoformulation) described herein is present in an amount from about 20 mol% to about 100 mol% (e.g., about 25-100 mol %, 20-90 mol%, 20-80 mol%, 20-70 mol%, 25-100 mol%, 30-70 mol%, 30-60 mol%, 30-50 mol%, 40-60 mol%, 45-60 mol%, 40-80 mol%, or 50-90 mol%) of the total lipid component; a noncationic lipid (e.g., phospholipid) is present in an amount from about 0 mol% to about 50 mol% (e.g., about 0-40 mol%, 0-30 mol%, 0-20 mol%, 0-10 mol%, 0-5 mol%, 5- 50 mol%, 5-40 mol%, 5-30 mol%, or 5-10 mol%) of the total lipid component, a conjugated lipid (e.g., a PEGylated lipid) is present in an amount from about 0 mol% to about 20 mol% (e.g., about 0-10 mol%, 0.5 to 20 mol %, 1-10 mol%, 1-5 mol%, or 5-10 mol%) of the total lipid component, and a sterol is present in an amount from about 0 mol % to about 70 mol% (e.g., about 0-50 mol%, 10-60 mol%, 10-50 mol%, 15-60 mol%, 15-50 mol%, 20-50 mol%, 20-40 mol%, or 30-40 mol%) of the total lipid component, provided that the total mol% of the total lipid component does not exceed 100%, and wherein the lipidoid is present at more than 0 mol%. As an example, the total lipid component of the lipid nanoparticle may comprise 50 mol% of-88- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925ionizable lipids comprising 25 mol% of SM-102 and 25 mol% of lipidoid 1 (as described herein). Alternatively, the total lipid component of the lipid nanoparticle may comprise 50 mol% of ionizable lipids comprising 50 mol% of lipidoid compounds comprising 25 mol% of lipidoid 1 (a lipidoid as described herein) and 25 mol% of Compound 903 (a second lipidoid as described herein).

[0184] As used herein, the term “total lipid component” of the lipid nanoparticle refers to the aggregated amounts of all lipid and lipidoid components present in the composition. For example, if a composition consisted of 25 mol % lipid A, 25 mol % lipid B, 25 mol % non-lipid C, and 25 mol % non-lipid D, then the total lipid component of the composition is 50 mol %, and each of lipid A and lipid B is 50 mol % of the total lipid component of the composition.

[0185] In some embodiments, the lipid nanoparticle comprises a lipidoid described herein, a PEGylated lipid, a sterol, and / or a phospholipid.

[0186] In certain such embodiments, the total lipid component of the lipid nanoparticle comprises:about 2-100 mol% of the lipidoid,about 0-50 mol% phospholipid,about 0-70 mol% sterol, andabout 0-10 mol% PEGylated lipid.

[0187] In alternative embodiments, the total lipid component of the lipid nanoparticle comprises:about 5-60 mol% of the lipidoid,about 0-30 mol% phospholipid,about 15-60 mol% sterol, andabout 0-10 mol% PEGylated lipid.

[0188] In alternative embodiments, the total lipid component of the lipid nanoparticle comprises:about 5-30 mol% of the lipidoid,about 0-20 mol% phospholipid,about 25-50 mol% sterol, andabout 0.5-5 mol% PEGylated lipid.

[0189] In certain embodiments, the lipid nanoparticle comprises the lipidoid, the second lipidoid as described herein, a PEGylated lipid, and a sterol.-89- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0190] In certain embodiments, the lipid nanoparticle is substantially free of a phospholipid.

[0191] In certain such embodiments, the lipid nanoparticle is substantially free of distearolyphosphatidycholine (DSPC).

[0192] In certain such embodiments, the total lipid component of the lipid nanoparticle comprises:about 1-50 mol% of the lipidoid,about 10-60 mol% second lipidoid,about 5-50 mol% sterol, andabout 0-10 mol% PEGylated lipid.

[0193] In further embodiments, the total lipid component of the lipid nanoparticle comprises:about 1-40 mol% of the lipidoid,about 30-60 mol% second lipidoid,about 15-45 mol% sterol, andabout 0-10 mol% PEGylated lipid.

[0194] In still further embodiments, the total lipid component of the lipid nanoparticle comprises:about 1-30 mol% of the lipidoid,about 40-60 mol% second lipidoid,about 25-45 mol% sterol, andabout 0.5-5 mol% PEGylated lipid.

[0195] In still further embodiments, the total lipid component of the lipid nanoparticle comprises:about 5-20 mol% of the lipidoid,about 40-60 mol% second lipidoid,about 25-45 mol% sterol, andabout 0.5-5 mol% PEGylated lipid.

[0196] In still further embodiments, the total lipid component of the lipid nanoparticle comprises:about 5-15 mol% of the lipidoid,about 40-60 mol% second lipidoid,about 25-45 mol% sterol, and-90- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925about 0.5-5 mol% PEGylated lipid.

[0197] In certain embodiments, the lipid nanoparticle comprises the lipidoid, a second lipidoid as described herein, a PEGylated lipid, a sterol, and a phospholipid.

[0198] In certain embodiments, the total lipid component of the lipid nanoparticle comprises:about 1-50 mol% of the lipidoid,about 10-60 mol% second lipidoid,about 1-25 mol% of the phospholipid;about 5-50 mol% sterol, andabout 0-10 mol% PEGylated lipid.

[0199] In further embodiments, the total lipid component of the lipid nanoparticle comprises:about 10-40 mol% of the lipidoid,about 10-40 mol% second lipidoid,about 5-20 mol% of the phospholipid;about 15-45 mol% sterol, andabout 0-10 mol% PEGylated lipid.

[0200] In still further embodiments, the total lipid component of the lipid nanoparticle comprises:about 20-30 mol% of the lipidoid,about 20-30 mol% second lipidoid,about 5-20 mol% of the phospholipid;about 25-45 mol% sterol, andabout 0.5-5 mol% PEGylated lipid.

[0201] In certain embodiments, the lipidoid and the second lipidoid are in a ratio of about 1:10 to about 10:1.

[0202] In further embodiments, the lipidoid and the second lipidoid are in a ratio of about 1:5 to about 5:1.

[0203] In further embodiments, the lipidoid and the second lipidoid are in a ratio of about 1:5 to about 1:1.5.

[0204] In still further embodiments, the lipidoid and the second lipidoid are in a ratio of about 1:1.-91- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0205] In certain embodiments, the total lipid component of the lipid nanoparticle comprises about 40-70 mol% of the lipidoid compounds (lipidoid and second lipidoid), e.g., about 45-65 mol%, 50-65 mol %, 50-60 mol%, 45-55 mol%, 50 mol%, 55 mol %, or 60 mol%. In certain such embodiments, the lipidoid and second lipidoid are in a ratio of about 1:10 to about 10:1, about 1:5 to about 5:1, about 1:5 to about 1:1.5, or about 1:1.

[0206] In some embodiments, molar ratios of lipidoid compound / sterol / phospholipid (or another structural lipid) / PEG-lipid / additional components is varied in the following ranges: lipidoid compound (i.e., lipidoid and second lipidoid) (25-100%); phospholipid (DSPC) (0-40%); sterol (0-50%); and PEG lipid (0-5%).

[0207] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises, by mol% or wt% of the total lipid component, 50-75% ionizable lipid (including the lipidoid and, if present, second lipidoid compounds as described herein), 20-40% sterol (e.g., cholesterol or derivative), 0 to 10% non-cationic-lipid, and 1-10% conjugated lipid (e.g., the PEGylated lipid).

[0208] In some embodiments, the lipidoid described herein is a component of the lipid- based carrier (or lipid nanoformulation, or nanoparticle composition) and comprises from 1 mol% to 95 mol%, from 1 mol% to 90 mol%, from 1 mol% to 80 mol%, from 5 mol% to 70 mol%, from 5 mol% to 60 mol%, from 20 mol% to 55 mol%, from 20 mol% to 45 mol%, 20 mol% to 40 mol%, from 25 mol% to 50 mol%, from 25 mol% to 45 mol%, from 30 mol% to 50 mol%, from 30 mol% to 45 mol%, from 30 mol% to 40 mol%, from 35 mol% to 45 mol%, or from 37 mol% to 42 mol% (or any fraction of these ranges) of the total lipid component.

[0209] In some embodiments, the lipidoid compounds (i.e., lipidoid and second lipidoid) are components of the lipid-based carrier (or lipid nanoformulation, or nanoparticle composition) and together comprise from 10 mol% to 95 mol%, from 10 mol% to 90 mol%, from 20 mol% to 90 mol%, from 20 mol% to 80 mol%, from 20 mol% to 70 mol%, from 25 mol% to 70 mol%, from 30 mol% to 70 mol%, 35 mol% to 70 mol%, from 40 mol% to 70 mol%, from 45 mol% to 70 mol%, from 50 mol% to 70 mol%, from 52 mol% to 68 mol%, from 55 mol% to 68 mol%, or from 55 mol% to 65 mol%, (or any fraction of these ranges) of the total lipid component.

[0210] In some embodiments, where the lipid-based carrier (or lipid nanoformulation) contains a mixture of phospholipid and sterol (e.g. cholesterol or derivative), the-92- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925mixture may be present up to 40 mol%, 45 mol%, 50 mol%, 55 mol%, or 60 mol% of the total lipid component.

[0211] In some embodiments, the phospholipid component in the mixture may be present from 2 mol% to 20 mol%, from 2 mol% to 15 mol%, from 2 mol% to 12 mol%, from 4 mol% to 15 mol%, from 4 mol% to 10 mol%, from 5 mol% to 10 mol%, (or any fraction of these ranges) of the total lipid component. In some embodiments, the lipid-based carrier (or lipid nanoformulation or nanoparticle composition) is substantially free of a phospholipid. In certain embodiments, the lipid-based carrier (or lipid nanoformulation or nanoparticle composition) is substantially free of distearolyphosphatidycholine (DSPC).

[0212] In some embodiments, the sterol component (e.g. cholesterol or derivative) in the mixture may comprise from 25 mol% to 70 mol%, from 25 mol% to 65 mol%, from 25 mol% to 60 mol%, from 30 mol% to 60 mol%, from 35 mol% to 60 mol%, from 35 mol% to 55 mol%, from 40 mol% to 55 mol%, from 45 mol% to 55 mol%, or from 45 mol% to 50 mol% (or any fraction of these ranges) of the total lipid component.

[0213] In some embodiments, where the lipid-based carrier (or lipid nanoformulation) is phospholipid-free, the sterol component (e.g. cholesterol or derivative) may be present up to 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, 60 mol%, 65 mol%, or 70 mol% of the total lipid component. For instance, the sterol component (e.g. cholesterol or derivative) may be present from 25 mol% to 65 mol%, from 25 mol% to 60 mol%, from 25 mol% to 55 mol%, from 25 mol% to 50 mol%, from 25 mol% to 45 mol%, from 25 mol% to 40 mol%, from 30 mol% to 45 mol%, from 30 mol% to 40 mol%, from 35 mol% to 45 mol%, from 30 mol% to 35 mol%, or from 35 mol% to 40 mol% (or any fraction thereof or range therein) of the total lipid component.

[0214] In some embodiments, the non-ionizable lipid components in the lipid-based carrier (or lipid nano formulation) may be present from 5 mol% to 90 mol%, from 10 mol% to 85 mol%, or from 20 mol% to 80 mol% (or any fraction of these ranges) of the total lipid component.

[0215] The ratio of total lipid components to the cargo (e.g., an encapsulated therapeutic agent such as a nucleic acid) can be varied as desired. For example, the total lipid components to the cargo (mass or weight) ratio can be from about 10:1 to about 30:1. In some embodiments, the total lipid component to the cargo ratio (mass / mass ratio;-93- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925w / w ratio) can be in the range of from about 1:1 to about 25:1, from about 10:1 to about 14:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. The amounts of total lipid components and the cargo can be adjusted to provide a desired N / P ratio, for example, N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or higher. Generally, the lipid-based carrier (or lipid nanoformulation) ’s overall lipid content can range from about 5 mg / mL to about 30 mg / mL.Nitrogen / phosphate ratios (N: P ratio) is evaluated at values between 0.1 and 100.

[0216] In some embodiments, the lipid-based carrier (or lipid nanoformulation) includes the ionizable lipid compound as described herein, phospholipid, cholesterol, and a PEGylated lipid in a molar ratio of 50:10:38.5:1.5. In some embodiments, the lipid- based carrier (or lipid nanoformulation) includes the ionizable lipid compound as described herein, cholesterol and a PEGylated lipid in a molar ratio of 60:38.5:1.5.

[0217] In some embodiments of any of the aspects or embodiments herein, the lipid- based carrier (or lipid nanoformulation) further comprises a tissue targeting moiety. The tissue targeting moiety can be a peptide, oligosaccharide or the like, which can be used for the delivery of the lipid-based carrier (or lipid nanoformulation) to one or more specific tissues such as the liver. In some embodiments, the tissue targeting moiety is a ligand for liver specific receptors. In one embodiment, the ligand of liver specific receptors used for liver targeting is an oligosaccharide such as N- Acetylgalactos amine (GalNAc) which is covalently attached to a component of a lipid-based carrier (or lipid nanoformulation), e.g., PEG-lipid conjugates or the like. In some embodiments, the GalNAc is covalently attached to, for example, PEG-lipid conjugate. In some embodiments, the GalNAc is conjugated to DSPE-PEG2000. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid- based carrier (or lipid nanoformulation) at a molar percentage of 0.2% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid- based carrier (or lipid nanoformulation) at a molar percentage of 0.3% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid- based carrier (or lipid nanoformulation) at a molar percentage of 0.4% of the total-94- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid- based carrier (or lipid nanoformulation) at a molar percentage of 0.5% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid- based carrier (or lipid nanoformulation) at a molar percentage of 0.6% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid- based carrier (or lipid nanoformulation) at a molar percentage of 0.7% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid- based carrier (or lipid nanoformulation) at a molar percentage of 0.8% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid- based carrier (or lipid nanoformulation) at a molar percentage of 0.9% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid- based carrier (or lipid nanoformulation) at a molar percentage of 1.0% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid- based carrier (or lipid nanoformulation) at a molar percentage of about 1.5% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 2.0% of the total lipid.Properties of Lipid Nano formulations

[0218] In some embodiments, the average particle diameter of the lipid-based carrier (or lipid nanoformulation) may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS). In some embodiments, the average particle diameter of the lipid-based carrier (or lipid nanoformulation) ranges from about 1 mm to about 500 mm, from about 5 mm to about 200 mm, from about 10 mm to about 100 mm, from about 20 mm to about 80 mm, from about 25 mm to about 60 mm, from about 30 mm to about 55 mm, from about 35 mm to about 50 mm, from about 38 mm to about 42 mm, from about 40 nm to about 150 nm (such as about 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), from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70-95- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm.

[0219] The lipid-based carrier or lipid nanoformulation (e.g., liposome or LNP) may, in some instances, be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a lipid nanoformulation (e.g., liposome or LNP), e.g., the particle size distribution of the liposome or LNP. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A lipid- based carrier or lipid nanoformulation (e.g., liposome or LNP) may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the lipid-based carrier or lipid nanoformulation (e.g., liposome or LNP) may be from about 0.10 to about 0.20.

[0220] The zeta potential of a lipid-based carrier or a lipid nanoformulation (e.g., liposome or LNP) may be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential may describe the surface charge of a liposome or LNP. Lipid nanoformulations (e.g., liposomes or LNP) with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a liposome or LNP may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0221] The efficiency of encapsulation of a cargo such as a protein and / or nucleic acid, describes the amount of protein and / or nucleic acid that is encapsulated or otherwise associated with a lipid nanoformulation (e.g., liposome or LNP) after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high-96- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925(e.g., at least 70%. 80%. 90%. 95%, close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing the liposome or LNP before and after breaking up the liposome or LNP with one or more organic solvents or detergents. An anion exchange resin may be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence may be used to measure the amount of free protein and / or nucleic acid (e.g., RNA) in a solution. For the liposome or LNP described herein, the encapsulation efficiency of a protein and / or nucleic acid may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.

[0222] The lipid carrier or lipid nanoformulation may optionally include one or more coatings. In some embodiments, the lipid carrier or lipid nanoformulation (e.g., liposome or LNP) may be formulated in a capsule, film, or tablet having a coating. A capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness, or density.

[0223] Additional exemplary lipids, formulations, methods, and characterization of a lipid carrier or lipid nanoformulation (e.g., liposome or LNP) are taught by WO 2020 / 061457 and WO 2021 / 113777, which are incorporated herein by reference in their entirety. Further exemplary lipids, formulations, methods, and characterization of LNPs are taught by Hou et al. Lipid nanoparticles for mRNA delivery. Hou, Nat Rev Mater, 6; 1078-1094 (2021)., which is incorporated herein by reference in its entirety (see, for example, exemplary lipids and lipid derivatives of Figure 2 of Hou et al.).

[0224] In some embodiments, in vitro or ex vivo cell lipofections are performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Mirus Bio). In certain embodiments, LNPs are formulated using the GenVoy_ILM ionizable lipid mix (Precision NanoSystems). In certain embodiments, LNPs are formulated using 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA) or dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which are taught in Jayaraman et al.-97- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925Angew Chem Int Ed Engl 51(34):8529-8533 (2012), incorporated herein by reference in its entirety.

[0225] Lipid nanoformulations (e.g., liposome or LNP) optimized for the delivery of CRISPR-Cas systems, e.g., Cas9-gRNA RNP, gRNA, Cas9 mRNA, are described in WO 2019067992 and WO 2019067910, which are incorporated by reference in their entirety.

[0226] Additional specific lipid nanoformulations (e.g., liposome or LNP) useful for delivery of nucleic acid effector molecules are described in US 8158601 and US 8168775, which are incorporated by reference in their entirety.

[0227] A variety of methods can be used for preparing the lipid carrier or lipid nanoformulation (e.g., liposomes or LNPs) described herein. Such methods are known in the art or disclosed herein, for example, the methods described in Lichtenberg and Barenholz in Methods of Biochemical Analysis, 33:337-462 (1988), which is incorporated herein by reference in its entirety. See also Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980); U. S. Patent Nos. 4,235,871; 4,501,728; and 4,837,028; Liposomes, Marc J. Ostro, ed., Marcel Dekker, Inc., New York, 1983, Chapter 1; and Hope, et al., Chem. Phys. Lip. 40:89 (1986), which are incorporated herein by reference in their entirety. Small unilamellar vesicles (SUV, size < 100 nm) can be prepared by a combination of standard methods of thin-film hydration and repeated extrusion.

[0228] Techniques for sizing the lipid carrier or lipid nanoformulations (e.g., liposomes or LNPs) to a desired size are well-known to one skilled in the art. See, e.g., U. S. Patent No. 4,737,323, and Hope et al., Biochim. Biophys. Acta, 812: 55-65, which are incorporated by reference in their entirety. Sonicating a lipid nanoformulation (e.g., liposome or LNP) suspension either by bath or probe sonication produces a progressive size reduction down to small unilamellar vesicles less than about 50 nm in size. Homogenization or microfluidization are other methods which rely on shearing energy to fragment large lipid nanoformulations (e.g., liposomes or LNPs) into smaller ones. In a typical homogenization procedure, multilamellar vesicles are recirculated through a standard emulsion homogenizer until selected lipid nanoformulation (e.g., liposome or LNP) sizes, typically between about 100 and 500 nm, are observed. In both methods, the particle size distribution can be monitored by conventional laser-beam particle size discrimination.-98- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0229] Extrusion of lipid nanoformulations e.g., liposomes or LNPs) through a smallpore polycarbonate membrane or an asymmetric ceramic membrane is a very effective method for reducing liposome or LNP sizes to a relatively well- defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired liposome or LNP size distribution is achieved. The lipid-based carrier or lipid nanoformulations may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in liposome or LNP size.

[0230] Any of the lipid-based carrier or lipid nanoformulations described herein can be analyzed by methods well-known to one skilled in the art to determine its physical and / or chemical features. For example, a phosphate assay can be used to determine the concentration of the lipid nanoformulations. One phosphate assay is based on the interaction between molybdate and malachite green dye. The main principle involves the reaction of inorganic phosphate with molybdate to form a colorless unreduced phosphomolybdate complex which is converted to a blue colored complex when reduced under acidic conditions. Phosphomolybdate gives 20 or 30 times more color when complexed with malachite green. The final product, reduced green soluble complex is measured by its absorbance at 620 nm and is a direct measure of inorganic phosphate in solution.

[0231] In some embodiments, the lipid-based carrier or lipid nanoformulations disclosed herein are tested for particle size, lipid concentration, and active agent encapsulation.Payloads

[0232] In some embodiments, the lipid nanoparticle further comprises a payload. In certain embodiments, the term “effector” is used interchangeably with “payload.”

[0233] In some embodiments, the present disclosure provides a method of delivering a payload, the method comprising administering, e.g., to a patient, a lipid nanoparticle of the present disclosure, wherein the lipid nanoparticle comprises a payload. In certain embodiments, the pay load comprises a therapeutic agent.Nucleic acid molecule

[0234] In some embodiments, the therapeutic agent is a nucleic acid molecule. The nucleic acid molecule may be any nucleic acid molecule that can function as a therapeutic or diagnostic agent. For instance, the nucleic acid molecule may be a DNA or RNA.-99- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0235] In some embodiments, the nucleic acid molecule is a nucleic acid selected from the group consisting of a plasmid, an immuno stimulatory oligonucleotide, an antisense oligonucleotide, an antagomir, an aptamer, a deoxyribozyme (DNAzyme), and a ribozyme.

[0236] In certain embodiments, the therapeutic agent is a nucleic acid molecule, such as a plasmid, an immuno stimulatory oligonucleotide, an antisense oligonucleotide, an antagomir, an aptamer, a deoxyribozyme (DNAzyme), or a ribozyme. In certain embodiments, the nucleic acid molecule is DNA or RNA. In certain embodiments, the nucleic acid molecule is DNA; and the DNA is a linear DNA, circular DNA, single stranded DNA, or double stranded DNA. In alternative embodiments, the nucleic acid molecule is RNA; and the RNA is messenger RNA (mRNA), miRNA, siRNA or siRNA precursor, RNA aptamer, linear RNA, circular RNA, single stranded RNA, double stranded RNA, tRNA, microRNA (miRNA) or miRNA precursor, Dicer substrate small interfering RNA (dsiRNA), Dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), guide RNA (gRNA), IncRNA, ncRNA, sncRNA, rRNA, snRNA, piRNA, snoRNA, snRNA, scaRNA, exRNA, scaRNA, Y RNA, or hnRNA, preferably mRNA. In yet further embodiments, the nucleic acid molecule comprises a phosphoramide, a phosphorothioate, a phosphorodithioate, an O-methylphosphoroamidate, a morpholino, a locked nucleic acid (LNA), a glycerol nucleic acid (GNA), a threose nucleic acid (TNA), or a peptide nucleic acid (PNA).

[0237] In some embodiments, the therapeutic agent is DNA. The DNA may be selected by one skilled in the art. In some embodiments, the DNA is linear DNA, circular DNA, single stranded DNA, or double stranded DNA.

[0238] In some embodiments, the therapeutic agent is linear DNA.

[0239] In some embodiments, the therapeutic agent is circular DNA.

[0240] In some embodiments, the therapeutic agent is single stranded DNA.

[0241] In some embodiments, the therapeutic agent is double stranded DNA.

[0242] In some embodiments, the therapeutic agent is RNA. The RNA may be selected by one skilled in the art. In certain embodiments, the RNA is mRNA, miRNA, siRNA or siRNA precursor, RNA aptamer, linear RNA, circular RNA, single stranded RNA, double stranded RNA, tRNA, microRNA (miRNA) or miRNA precursor, a Dicer substrate small interfering RNA (dsiRNA), a short hairpin RNA (shRNA), an-100- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925asymmetric interfering RNA (aiRNA), a guide RNA (gRNA), IncRNA, ncRNA, sncRNA, rRNA, snRNA, piRNA, snoRNA, snRNA, scaRNA, exRNA, Y RNA, or hnRNA.

[0243] In certain embodiments, the DNA or RNA encodes a polypeptide.

[0244] In some embodiments, the nucleic acid molecule comprises one or more nucleic acid analogs selected from the group consisting of a phosphoramide, a phosphorothioate, a phosphorodithioate, an O-methylphosphoroamidate, a morpholino, a locked nucleic acid (LNA), a glycerol nucleic acid (GNA), a threose nucleic acid (TNA), and a peptide nucleic acid (PNA).

[0245] In some embodiments, the therapeutic agent is an mRNA (messenger RNA).

[0246] In some embodiments, the therapeutic agent is a miRNA (microRNA) or miRNA precursor.

[0247] In some embodiments, the therapeutic agent is a siRNA (small interfering RNA) or siRNA precursor.

[0248] In some embodiments, the therapeutic agent is a Dicer substrate small interfering RNA (dsiRNA).

[0249] In some embodiments, the therapeutic agent is a short hairpin RNA (shRNA).

[0250] In some embodiments, the therapeutic agent is an asymmetric interfering RNA (aiRNA).

[0251] In some embodiments, the therapeutic agent is a guide RNA (gRNA).

[0252] In some embodiments, the therapeutic agent is an RNA aptamer.

[0253] In some embodiments, the therapeutic agent is a circular RNA, e.g., a circular RNA encoding a therapeutic polypeptide, or a non-coding circular RNA.

[0254] In some embodiments, the therapeutic agent is a tRNA (transfer RNA).

[0255] In some embodiments, the therapeutic agent is a rRNA (ribosomal RNA).

[0256] In some embodiments, the therapeutic agent is a IncRNA (long non-coding RNA).

[0257] In some embodiments, the therapeutic agent is a snRNA (small nuclear RNA).

[0258] In some embodiments, the therapeutic agent is a ncRNA (non-coding RNA).

[0259] In some embodiments, the therapeutic agent is a sncRNA (small noncoding RNA).

[0260] In some embodiments, the therapeutic agent is a snoRNA (small nucleolar RNA).

[0261] In some embodiments, the therapeutic agent is a piRNA (piwi-interacting RNA).-101- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0262] In some embodiments, the therapeutic agent is a scaRNA (small cajal bodyspecific RNA).

[0263] In some embodiments, the therapeutic agent is an exRNA (extracellular RNA).

[0264] In some embodiments, the therapeutic agent is a Y RNA (small non-coding RNAs that are components of the Ro60 ribonucleoprotein particle).

[0265] In some embodiments, the therapeutic agent is a hnRNA (heterogeneous nuclear RNA).

[0266] In some embodiments, the therapeutic agent is a shRNA (small hairpin RNA).

[0267] In some embodiments, the therapeutic agent is an enzymatic nucleic acid molecule. The term “enzymatic nucleic acid molecule” refers to a nucleic acid molecule which has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity which is active to specifically cleave target RNA. That is, the enzymatic nucleic acid molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule. These complementary regions allow sufficient hybridization of the enzymatic nucleic acid molecule to the target RNA and thus permit cleavage. One hundred percent complementarity is preferred, but complementarity as low as 50-75% can also be useful in this invention (see for example Werner et al., Nucleic Acids Research 23:2092-2096 (1995); Hammann et al., Antisense and Nucleic Acid Drug Dev. 9:25-31 (1999), which are incorporated herein by reference in their entirety).

[0268] The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamerbinding ribozyme, regulatable ribozyme, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity.

[0269] In some embodiments, the therapeutic agent is an antisense nucleic acid. The term “antisense nucleic acid” refers to a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid) interactions and alters the activity of the target RNA. Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and / or-102- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925an antisense molecule can bind such that the antisense molecule forms a loop. Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both. In addition, antisense DNA can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex.

[0270] In some embodiments, the nucleic acid molecule may be a 2-5A antisense chimera. The term “2-5A antisense chimera” refers to an antisense oligonucleotide containing a 5 '-phosphorylated 2'-5 '-linked adenylate residue. These chimeras bind to target RNA in a sequence- specific manner and activate a cellular 2-5A-dependent ribonuclease which, in turn, cleaves the target RNA.

[0271] In some embodiments, the nucleic acid molecule may be a triplex forming oligonucleotide. The term “triplex forming oligonucleotide” refers to an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix.

[0272] In some embodiments, the nucleic acid molecule may be a decoy RNA. The term “decoy RNA” refers to a RNA molecule or aptamer that is designed to preferentially bind to a predetermined ligand. Such binding can result in the inhibition or activation of a target molecule.

[0273] In some embodiments, the nucleic acid molecule (e.g., RNA or DNA) encodes a therapeutic peptide or polypeptide. In the case of a DNA, the nucleic acid comprises a promoter operably linked to the sequence encoding the therapeutic peptide or polypeptide. The therapeutic peptide or polypeptide may be, e.g., a transcription factor; a chromatin remodeling factor; an antigen; a hormone; an enzyme (such as a nuclease, e.g., an endonuclease, e.g., a nuclease element of a CRISPR system, e.g., a Cas9, dCas9, aCas9-nickase, Cpf / Casl2a); a Crispr-linked enzyme, e.g., a base editor or prime editor; a mobile genetic element protein (e.g., a transposase, a retrotransposase, a recombinase, an integrase); a Gene Writer; a polymerase; a methylase; a demethylase; an acetylase; a deacetylase; a kinase; a phosphatase; a ligase; a deubiquitinase; an integrase; a recombinase; a topoisomerase; a gyrase; a helicase; a lysosomal acid hydrolase); an antibody; a receptor ligand; a receptor; a clotting factor; a membrane protein; a mitochondrial protein; a nuclear protein; an antibody or other protein scaffold binder such as a centyrin, darpin, or adnectin.-103- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0274] In some embodiments, the nucleic acid molecule (e.g., a DNA or RNA) encodes (if DNA) or is (if RNA) a non-coding RNA, e.g., one or more of a siRNA, a miRNA, long non-coding RNA, a piRNA, a snoRNA, a scaRNA, a tRNA, a rRNA, a therapeutic RNA aptamer, and a snRNA.

[0275] In some embodiments, the therapeutic nucleic acid molecule targets a host gene, e.g., the nucleic acid effector hybridizes to an endogenous gene.

[0276] In some embodiments, the nucleic acid molecule is an antisense RNA; a guide RNA; a nucleic acid that hybridizes to an exogenous nucleic acid such as a viral DNA or RNA, nucleic acid that hybridizes to an RNA; a nucleic acid that interferes with gene transcription; a nucleic acid that interferes with RNA translation; a nucleic acid that stabilizes RNA or destabilizes RNA such as through targeting for degradation; or a nucleic acid that modulates a DNA or RNA binding factor.

[0277] In some embodiments, the nucleic acid molecule targets a sense strand of a host gene. In some embodiments, the nucleic acid molecule targets an antisense strand of a host gene.

[0278] In some embodiments, the nucleic acid molecule is or encodes a guide RNA.Guide RNA sequences are generally designed to have a length of between 15-30 nucleotides (e.g., 17, 19, 20, 21, 24 nucleotides) and complementary to the targeted nucleic acid sequence. Custom gRNA generators and algorithms are available commercially for use in the design of effective guide RNAs. Gene editing has also been achieved using a chimeric "single guide RNA" ("sgRNA"), an engineered (synthetic) single RNA molecule that mimics a naturally occurring crRNA-tracrRNA complex and contains both a tracrRNA (for binding the nuclease) and at least one crRNA (to guide the nuclease to the sequence targeted for editing). Chemically modified sgRNAs have also been demonstrated to be effective in genome editing; see, for example, Hendel et al. (2015) Nature Biotechnol., 985-991. The gRNA may recognize specific DNA sequences (e.g., sequences adjacent to or within a promoter, enhancer, silencer, or repressor of a gene). In some embodiments, the gRNA is used as part of a CRISPR system for gene editing. For the purposes of gene editing, the ssDNA construct or sequence disclosed herein may be designed to include one or multiple sequences encoding guide RNA sequences corresponding to a desired target DNA sequence; see, for example, Cong et al. (2013) Science, 339:819-823; Ran et al. (2013) Nature Protocols, 8:2281-2308.-104- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0279] In some embodiments, the nucleic acid molecule can include a plurality of sequences. The plurality may be the same or different types. The plurality of sequences may be the same or different sequences of the same type.

[0280] All the nucleic acid molecules described herein can be chemically modified. The various modification strategy to the nucleic acid molecules are well known to one skilled in the art. In some embodiments, the nucleic acid molecule comprises one or more modifications selected from the group consisting of pseudouridine, 5- bromouracil, 5-methylcytosine, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, florophores (e.g., rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine. In some embodiments, the antisense oligonucleotide may be a locked nucleic acid oligonucleotide (LNA). The term “locked nucleic acid (LNA)” refers to oligonucleotides that contain one or more nucleotide building blocks in which an extra methylene bridge fixes the ribose moiety either in the C3'-endo (beta-D-LNA) or C2'-endo (alpha-L-LNA) conformation (Grunweller A, Hartmann R K, BioDrugs, 21(4): 235-243 (2007)).

[0281] Additional examples of the nucleic acid molecules (including tumor suppressor genes, antisense oligonucleotides, siRNA, miRNA, or shRNA) may be found in U. S. Published Patent Application No. 2007 / 0065499 and U. S. Patent No. 7,780,882, which are incorporated by reference herein in their entireties.

[0282] In some embodiments, the therapeutic agent can include a plurality of nucleic acid molecules, which may be the same or different types.

[0283] In some embodiments, the N: P ratio of the nucleic acid molecule-encapsulated lipid-based carrier or lipid nanoformulation ranges from 1:1 to 30:1, for instance from 3:1 to 20:1, from 3:1 to 15:1, from 3:1 to 10:1, or from 3:1 to 6:1. An N: P ratio refers to the molar ratio of the amines present in the lipid-based carrier or lipid nanoformulation (e.g., the amines in the ionizable lipids) to the phosphates present in the nucleic acid molecule. It is a factor for efficient packaging and potency. In some embodiments, the N: P ratio of the nucleic acid molecule - encapsulated lipid-based carrier or lipid nanoformulation ranges from 3:1 to 15:1.-105- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925Other Therapeutic Agents

[0284] In alternative embodiments, the therapeutic agent is a peptide, a protein, or small molecule drug.

[0285] In some embodiments, the protein effector may be any peptide or protein molecule that can function as a therapeutic or diagnostic agent.

[0286] In certain embodiments, the protein may be a peptide or polypeptide, e.g., a transcription factor; a chromatin remodeling factor; an antigen; a hormone; an enzyme (such as a nuclease, e.g., an endonuclease, e.g., a nuclease element of a CRISPR system, e.g., a Cas9, dCas9, aCas9-nickase, Cpf / Casl2a); a Crispr-linked enzyme, e.g., a base editor or prime editor; a mobile genetic element protein (e.g., a transposase, a retrotransposase, a recombinase, an integrase); a gene writer; a polymerase; a methylase; a demethylase; an acetylase; a deacetylase; a kinase; a phosphatase; a ligase; a deubiquitinase; an integrase; a recombinase; a topoisomerase; a gyrase; a helicase; a lysosomal acid hydrolase); an antibody; a receptor ligand; a receptor; a clotting factor; a membrane protein; a mitochondrial protein; a nuclear protein; an antibody or other protein scaffold binder, such as a centyrin, darpin, or adnectin.

[0287] In certain embodiments, the protein is a ribonucleoprotein (RNP) that a complex of ribonucleic acid and RNA-binding protein.

[0288] In certain embodiments, the protein is a recombinant cytokine such as IL- 12.

[0289] In certain embodiments, the nanoparticle composition can include a plurality of protein molecules, which may be the same or different types.

[0290] In some embodiments, the therapeutic agent is a small molecule drug, for instance, a small molecule drug approved for use in humans by an appropriate regulatory authority.

[0291] In certain embodiments, the small molecule drug comprises an immune suppressive agent, an endosomal escape agent, or a combination of both.

[0292] In certain embodiments, the small molecule drug is an HD AC inhibitor, a kinase inhibitor, a cytotoxic molecule, a chromatin modulator, an RNAi modulator, transcription factor, an adjuvant, or a combination of two or more.

[0293] In some embodiments, the small molecule drug may be a compound with limited cell permeability properties. For instance, the small molecule drug may be a STING (simulator of interferon genes) modulator.-106- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0294] In some embodiments, the nanoparticle composition can include a plurality of small molecule drugs, which may be the same or different types.

[0295] In some embodiments, the therapeutic agent may be encapsulated in the LNP. For example, the therapeutic agent may be completely or partially located in the interior space of the LNPs, within the lipid layer / membrane, or associated with the exterior surface of the lipid layer / membrane. In some embodiments, incorporation of the therapeutic agent into the LNP protects the therapeutic agents from environments which may contain enzymes or chemicals or conditions that degrade the therapeutic agents and / or systems or receptors that cause the rapid excretion of the therapeutic agents. Moreover, incorporating the therapeutic agent into the LNP may promote uptake of the therapeutic agent, and hence, may enhance the therapeutic effect.

[0296] The ratio of total lipid component to the therapeutic agent can be varied as desired. For example, the total lipid component to the therapeutic agent (mass or weight) ratio can be from about 10: 1 to about 30: 1. In some embodiments, the total lipid component to the therapeutic agent ratio (mass / mass ratio; w / w ratio) can be in the range of from about 1:1 to about 25:1, from about 10:1 to about 14:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. The amounts of total lipid component and the therapeutic agent can be adjusted to provide a desired N: P ratio, for example, N: P ratio of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or higher. Generally, the overall lipid and lipidoid content can range from about 5 mg / mL to about 30 mg / mL in the nanoparticle composition.

[0297] The nanoparticle composition may contain about 5 to about 95% by weight of the therapeutic agent. In some embodiments, the nanoparticle composition contains about 5%, about 10%, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 95% by weight of the therapeutic agent. In some embodiments, the nanoparticle composition contains the therapeutic agent in an amount about 5-95%, about 5-90%, about 5-80 %, about 5-70 %, about 5-60%, about 5-50%, about 5-40%, about 5-30%, about 5-20%, about 5-10%, about 10-95%, about 10-90%, about 10- 80%, about 10-70%, about 10-60%, about 10-50%, about 10-40%, about 10-30%, about 10-20%, about 20-95%, about 20-90%, about 20-80%, about 20-70%, about 20- 60%, about 20-50%, about 20-40%, about 20-30%, about 30-95%, about 30-90%, about 30-80%, about 30-70%, about 30-60%, about 30-50%, about 30-40%, about 40--107- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-0092595%, about 40-90%, about 40-80%, about 40-70%, about 40-60%, about 40-50%, about 50-95%, about 50-90%, about 50-80%, about 50-70%, about 50-60%, about 60- 95%, about 60-90%, about 60-80%, about 60-70%, about 70-95%, about 70-90%, about 70-80%, about 80-95%, about 80-90%, or about 90-95%, based on the weight of the nanoparticle composition.

[0298] In certain embodiments, the nanoparticle composition further comprises an antigen. In some embodiments, the antigen is a protein or a nucleic acid. In certain embodiments, the antigen is a protein. In other embodiments, the antigen is a nucleic acid.

[0299] In further embodiments, the nanoparticle composition further comprises an mRNA molecule comprising a nucleotide sequence that encodes an antigen.

[0300] The nanoparticle compositions described herein are useful for delivering a therapeutic agent.Pharmaceutical Compositions

[0301] In certain embodiments, provided herein is a pharmaceutical composition comprising a lipid nanoparticle of the invention, and a pharmaceutically acceptable excipient. As used throughout this section, a “lipidoid composition” can refer to a nanoparticle composition comprising a lipidoid disclosed herein, optionally with a second lipidoid.

[0302] The compositions and methods of the present invention may be utilized to treat an individual in need thereof. The pharmaceutical composition described herein may comprise a therapeutic or prophylactic composition, or any combination thereof. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the nanoparticle composition or the lipidoid composition is preferably administered as a pharmaceutical composition comprising, for example, a lipidoid composition and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution-108- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0303] A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a lipidoid composition. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a lipidoid composition. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[0304] The phrase "pharmaceutically acceptable" is employed herein to refer to those lipidoid compositions, 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.

[0305] The phrase "pharmaceutically acceptable excipient" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose,-109- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0306] A pharmaceutical composition described herein can be administered parenterally to a subject by any of a number of routes of administration including, for example, intravenously, intramuscularly, or subcutaneously. The pharmaceutical composition may also be formulated for inhalation. In certain embodiments, a pharmaceutical composition may be simply dissolved or suspended in sterile water.

[0307] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.

[0308] In certain embodiments, compositions of the present disclosure may be used alone or conjointly administered with another type of therapeutic agent.Methods of Use

[0309] In certain embodiments, provided herein is a method of delivering a therapeutic agent, comprising administering to a subject in need thereof an effective amount of the lipid nanoparticle of the invention that comprises a therapeutic agent.

[0310] Also provided is a method of vaccination, comprising administering to a subject in need thereof an effective amount of the lipid nanoparticle that comprises an antigen.

[0311] Actual dosage levels of the pharmaceutical compositions may be varied so as to obtain an amount of the payload that is effective to achieve the desired therapeutic-110- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0312] The selected dosage level will depend upon a variety of factors including the activity of the particular payload, composition or combination of compositions employed; the age, sex, weight, condition, general health and prior medical history of the patient being treated; and like factors well known in the medical arts.

[0313] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0314] Desired response or desired results include effects at the cellular level, tissue level, or clinical results. As such, “a therapeutically effective amount” or synonym thereto depends upon the context in which it is being applied. For example, in some embodiments it is an amount of the composition sufficient to achieve a treatment response as compared to the response obtained without administration of the composition. In other embodiments, it is an amount that results in a beneficial or desired result in a subject as compared to a control. As defined herein, a therapeutically effective amount of a composition (e.g., a pharmaceutical composition) disclosed herein may be readily determined by one of ordinary skill by routine methods known in the art. Dosage regimen and route of administration may be adjusted to provide the optimum therapeutic response.

[0315] The patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines, cattle, swine, sheep, cats, and dogs; poultry; and pets in general.

[0316] In general, a suitable daily dose of an active lipidoid composition used in the compositions and methods of the invention will be that amount of the lipidoid composition that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0317] If desired, the effective daily dose of the active lipidoid composition may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage-Ill- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925forms. In certain embodiments of the present invention, the active lipidoid composition may be administered two or three times daily. In preferred embodiments, the active lipidoid composition will be administered once daily.EXAMPLES

[0318] The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention.Example 1: Synthesis and characterization ofLipidoids 1-12Synthesis of intermediates: Intermediates A, B, and C were used to generate the corresponding Lipidoids 1-12Synthetic route to Intermediate AToluene 115 C, 2.5 hrsSynthesis of Compound 3Toluene 115 C, 2.5 hrs-112- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925To a solution of Compound 1 (12.18 g, 65.38 mmol, 2 eq.) in Tol. (50 mL) was added Compound 2 (5 g, 32.69 mmol, 3.55 mL, 1 eq.) dropwise at 20°C. The mixture was heated to 115°C for 2.5 hours. LCMS showed desired MS was detected. TLC (Petroleum ether: Ethyl acetate=5:l, Rf= 0.66) indicated trace of Compound 1 was remained and new spots formed. 4 reactions were combined for workup. The reaction mixture was diluted with DCM (600 mL) and neutralized with solid NaHCO3(~30 g). The organic phase was washed with water (240 mL*2) and brine (240 mL), dried over Na2SO4, filtered and concentrated in vacuo to give crude product. The crude product was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate=l: O to 300:1) to give Compound 3 (57.05 g, 126.02 mmol, 94.14% yield, 100% purity) as white solid.LCMS [M+23]+= 475.4'H NMR (400 MHz, CHLOROFORM-d) 6 = 6.85 (s, 2H), 4.20 (t, J= 6.8 Hz, 4H), 1.74 - 1.62 (m, 4H), 1.43 - 1.17 (m, 36H), 1.01 - 0.76 (m, 6H)Synthesis of Compound 6Compound 3 (25 g, 55.22 mmol, 1 eq.), Compound 4 (9.79 g, 82.83 mmol, 1.5 eq.), and Compound 5 (608.07 mg, 5.52 mmol, 821.71 pL, 0.1 eq.) were mixed and diluted with Isopropyl alcohol (15 mL). The mixture was heated to 120°C for 20 hours in a sealed reactor. LCMS showed Compound 3 was consumed and desired MS was detected. 2 reactions were combined for workup. After cooling, H2O (400 mL) and DCM (700 mL) were added and the mixture was neutralized with solid NaHCO3(~80 g). The organic layer was separated, washed with brine (300 mL), dried over Na2SO4, filtered, and concentrated in vacuo to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate=200: 1 to 30:1) to give Compound 6 (49.55 g, 97.78 mmol, 88.54% yield) as colorless oil. (1 Batch: 25 g, 2 Batches: 110.44 mmol)LCMS [M+23]+= 529.4 452.72, 506.81-113- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925'H NMR (400 MHz, CHLOROFORM-d) 6 = 5.70 (d, J = 2.4 Hz, 2H), 4.17 - 4.00 (m, 4H), 2.91 - 2.80 (m, 2H), 2.43 (br d, J = 17.6 Hz, 2H), 2.25 - 2.12 (m, 2H), 1.69 - 1.59 (m, 4H), 1.36 - 1.21 (m, 36H), 0.89 (t, J= 6.8 Hz, 6H)Synthesis of Intermediate Ao oIntermediate ATo a solution of Compound 6 (15 g, 29.60 mmol, 1 eq.) in DCM (270 mL) was added m- CPBA (7.66 g, 35.52 mmol, 80% purity, 1.2 eq.) in small portions at 0°C. The reaction mixture was stirred at 25 °C for 16 hours. LCMS showed Compound 6 consumed and desired MS was detected. After the consumption of the starting material, DCM (100 mL) was added followed by sat. Na2S2O3 (200 mL). The mixture was stirred for 30 min (PH<=9, the test paper of KI did not change blue), and then the organic phase was washed with sat. NaHCO3(100 mL*2), brine (100 mL) and dried by Na2SO4. Then the organic phase was evaporated to give crude product. The crude product was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate=250: 1 to 100:1) to give Intermediate A (10.5 g, 10.5 g, 20.08 mmol, 67.87% yield, 100% purity) as colorless oil.LCMS [M+l]+= 523.4'H NMR (400 MHz, CHLOROFORM-d) 6 = 4.06 (dq, J= 3.2, 6.8 Hz, 4H), 3.32 - 3.13 (m, 2H), 2.85 (dt, J= 4.8, 10.8 Hz, 1H), 2.62 (dt, J= 6.4, 10.8 Hz, 1H), 2.47 (ddd, J= 1.6, 4.8, 14.8 Hz, 1H), 2.32 (ddd, J = 4.8, 6.4, 15.6 Hz, 1H), 2.07 (dd, J= 10.8, 15.6 Hz, 1H), 1.90 (ddd, J = 2.0, 10.8, 14.8 Hz, 1H), 1.65 - 1.59 (m, 4H), 1.35 - 1.24 (m, 36H), 0.89 (t, J = 6.8 Hz, 6H)Synthetic route to Intermediate B-114- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925Synthesis of Compound 3:To a solution of Compound 1 (18.27 g, 98.06 mmol, 2 eq.) in Tol. (70 mL) was added Compound 2 (7.5 g, 49.03 mmol, 5.32 mL, 1 eq.) dropwise at 25°C. The mixture was stirred at 115°C for 2.5 hours. TLC (Petroleum ether: Ethyl acetate=20:l, Rf= 0.92) indicated Compound 2 was remained and new spots formed. 2 reactions were combined for workup. After cooling, H2O (120 mL) and DCM (125 mL) were added and themixture was neutralized with solid NaHCO3(~60 g). The organic layer was separated, washed with brine (120 mL), dried over Na2SO4, and concentrated in vacuo to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether:Ethyl acetate=100:0 to 99:1) to give Compound 3 (42 g„ 92.7 mmol, 95% yield) as colorless oil (2 batches: 98.06 mmol).-115- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925LCMS [M+23]+= 475.3'H NMR (400 MHz, CHLOROFORM-d) 6 = 6.85 (s, 2H), 4.12 (d, J= 5.8 Hz, 4H), 1.77 - 1.64 (m, 2H), 1.42 - 1.19 (m, 32H), LOO - 0.80 (m, 12H).Synthesis of Compound 6To a solution of Compound 3 (32 g, 70.69 mmol, 1 eq.), Compound 4 (12.53 g, 106.03 mmol, 1.5 eq.) and Compound 5 (778.33 mg, 7.07 mmol, 1.05 mL, 0.1 eq.) were mixed and diluted with IPA (19.2 mL). The mixture was heated to 120°C for 16 hours in a sealed reactor. LCMS showed Compound 3 was consumed completely and desired MS was detected. After cooling, H2O (200 mL) and DCM (250 mL) were added and the mixture was neutralized with solid NaHCO3(~40 g). The organic layer was separated, washed with brine (200 mL), dried over Na2SO4, filtered and concentrated in vacuo to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate=100:0 to 94:6) to give Compound 6 (47 g, crude) as white solid. LCMS [M+23]+= 529.4'H NMR (400 MHz, CHLOROFORM-d) 6 = 5.70 (d, J= 2.4 Hz, 2H), 4.08 - 4.00 (m, 2H), 3.95 (m, 2H), 2.97 - 2.83 (m, 2H), 2.51 - 2.37 (m, 2H), 2.25 - 2.12 (m, 2H), 1.62 (br s, 2H), 1.28 (br s, 32H), 0.94 - 0.83 (m, 12H)Synthesis of Intermediate B-116- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925m-CPBA DCM, 16 hrsTo a solution of Compound 6 (19 g, 37.49 mmol, 1 eq.) in DCM (350 mL) was added m-CPBA (9.70 g, 44.99 mmol, 80% purity, 1.2 eq.) in small portions at 0°C. The reaction mixture was stirred at 25 °C for 16 hours. TLC (Petroleum ether: Ethyl acetate= 5:1, Rf = 0.7) indicated Compound 6 was consumed completely and many new spots formed. 2 reactions were combined for workup. After the consumption of the starting material (TLC), DCM (120 mL) was added followed by sat. Na2S2O3 (500 mL). The mixture was stirred for 30 min (PH<=9, the test paper of KI did not change blue), and then the organic phase was washed with sat. NaHCCh (200 mL*2), and dried by Na2SO4. Then the organic phase was evaporated to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate=97:3 to 95:5) to give Intermediate B (36.5 g, 69.82 mmol, 93.00% yield) as white solid. (2 Batches: 74.98 mmol)LCMS [M+23]+= 545.4'H NMR (400 MHz, CHLOROFORM-d) 6 = 4.07 - 3.98 (m, 2H), 3.97 - 3.89 (m, 2H), 3.27 - 3.23 (m, 1H), 3.20 (t, J = 4.4 Hz, 1H), 2.86 (td, J= 5.2, 10.4 Hz, 1H), 2.64 (dt, J = 6.8, 10.4 Hz, 1H), 2.48 (ddd, 7= 1.6, 4.8, 14.8 Hz, 1H), 2.32 (ddd, 7= 4.8, 6.8, 15.2 Hz, 1H), 2.13 - 2.04 (m, 1H), 1.91 (ddd, 7= 2.0, 10.4, 14.8 Hz, 1H), 1.61 (br s, 2H), 1.27 (br s, 32H), 0.90 (dt, 7= 3.6, 6.4 Hz, 12H)Synthetic route to Intermediate CFoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925Toluene 115 C, 2.5 hrsIntermediate C-118- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925Synthesis of Compound 3COCI CIOC 2 OHToluene 1 115 C, 2.5 hrsTo a solution of Compound 1 (33.24 g, 117.68 mmol, 2 eq.) in Tol. (100 mL) was added Compound 2 (9 g, 58.84 mmol, 6.38 mL, 1 eq.) dropwise at 0°C. The reaction mixture was stirred at 118°C for 16 hours. TLC (Petroleum ether: Ethyl acetate =20:1, Rf= 0.56) indicated Compound 1 was consumed completely and new spots formed. The reaction mixture diluted with ethyl acetate (150 mL) and washed with sat. NaHCO3(100 mL) and brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate=300:l) to give Compound 3 (36.8 g, 57.05 mmol, 96.96% yield) as colorless oil.'H NMR (400 MHz, CHLOROFORM-d) 5 = 6.86 (s, 2H), 4.20 (t, J = 6.8 Hz, 4H), 1.75 - 1.62 (m, 4H), 1.48 - 1.22 (m, 48H), 1.19 - 1.06 (m, 4H), 0.89 (t, J= 6.8 Hz, 6H), 0.65 (s, 4H), 0.58 (dd, J = 4.0, 8.0 Hz, 2H), -0.32 (q, J = 5.2 Hz, 2H)Synthesis of Compound 6-119- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925To a solution of Compound 3 (18.4 g, 28.53 mmol, 1 eq.) in Tol. (50 mL) was added Compound 5 (314.09 mg, 2.85 mmol, 424.45 pL, 0.1 eq.) and Compound 4 (5.39 g, 45.64 mmol, 1.6 eq.) at 25°C. The reaction mixture was stirred at 120°C for 48 hours. TLC (Petroleum ether: Ethyl acetate =20:1, Rf= 0.43) indicated Compound 3 was consumed and one major new spot formed. 2 reactions were combined for workup. The reaction was diluted with ethyl acetate (200 mL) and washed with sat. NaHCO3(100 mL) and brine (100 mL). The organic phase was dried over Na2SO4, filtered and concentrated to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate=50:l) to give Compound 6 (37 g, 52.92 mmol, 92.76% yield) as a colorless oil. (2 Batches: 57.06 mmol)'H NMR (400 MHz, CHLOROFORM-d) 6 = 5.70 (d, J = 2.4 Hz, 2H), 4.18 - 3.98 (m, 4H), 2.86 (dt, J= 2.4, 4.8 Hz, 2H), 2.53 - 2.35 (m, 2H), 2.25 - 2.09 (m, 2H), 1.86 (td, J = 3.2, 6.8 Hz, 3H), 1.71 - 1.59 (m, 4H), 1.41 - 1.26 (m, 45H), 1.19 - 1.08 (m, 4H), 0.94 -0.84 (m, 6H), 0.73 - 0.61 (m, 4H), 0.60 - 0.51 (m, 2H), -0.26 - -0.41 (m, 2H)Synthesis of Intermediate C-120- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925To a solution of Compound 6 (9.73 g, 13.92 mmol, 1 eq. in DCM (100 mL) was added m-CPBA (3.81 g, 18.79 mmol, 85% purity, 1.35 eq.) at 0°C. The reaction mixture was stirred at 25°C for 16 hours. TLC (Petroleum ether: Ethyl acetate =8:1, Rf= 0.3) indicated Compound 6 was consumed completely and new spots formed. 3 reactions were combined for workup. After the consumption of the starting material (TLC), DCM (150 mL) was added followed by sat. Na2S2O3 (150 mL). The mixture was stirred for 30 min (PH<=8, the test paper of KI did not change blue), and then the organic phase was washed with brine (100 mL) and dried by Na2SO4, filtered and evaporated to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate=20:l) to give Intermediate C (29 g, 30.41 mmol, 72.84% yield, 75% purity) as colorless oil. (1 Batch: 9.73 g, 3 Batches)'H NMR (400 MHz, CHLOROFORM-d) 5 = 4.06 (ttd, J= 3.6, 7.2, 10.8 Hz, 4H), 3.31 - 3.24 (m, 1H), 3.19 (t, J= 4.4 Hz, 1H), 2.91 - 2.80 (m, 1H), 2.68 - 2.57 (m, 1H), 2.48 (ddd, J= 1.6, 4.8, 14.8 Hz, 1H), 2.38 - 2.27 (m, 1H), 2.07 (dd, J= 10.8, 15.6 Hz, 1H), 1.90 (ddd, J= 2.0, 10.8, 14.8 Hz, 1H), 1.68 - 1.58 (m, 4H), 1.38 (br s, 10H), 1.36 - 1.22 (m, 38H), 1.21 - 1.06 (m, 4H), 0.93 - 0.84 (m, 6H), 0.66 (br d, J= 5.2 Hz, 4H), 0.61 -0.50 (m, 2H), -0.28 - -0.40 (m, 2H)Specific synthesis of Lipidoids 1-12: Lipidoids 1-12 were synthesized using the intermediates described aboveSynthesis of Lipidoid 1-121- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925OIntermediate A Lipidoid 1 A mixture of Intermediate A (4 g, 7.65 mmol, 1 eq.) and Compound 1 (3.96 g, 45.45 mmol, 4 mL, 5.94 eq.) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 85 °C for 36 hours under N2 atmosphere. LCMS showed trace of Intermediate A was remained and main peak with desired MS was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate=80:l to 15:1) to give Lipidoid 1 (2.33 g, 3.82 mmol, 49.88% yield, 99.91% purity) as light-yellow oil. LCMS [M+l]+= 610.5'H NMR (400 MHz, METHANOL-d4) 5 = 4.18 - 4.03 (m, 4H), 4.03 - 3.97 (m, 1H), 3.70 (t, J= 4.8 Hz, 4H), 3.10 (dt, J= 4.4, 9.2 Hz, 1H), 2.99 (dt, J= 3.6, 9.2 Hz, 1H), 2.65 - 2.54 (m, 2H), 2.53 - 2.43 (m, 2H), 2.30 - 2.18 (m, 1H), 2.03 (dtd, J= 3.2, 10.4, 13.6 Hz, 2H), 1.94 - 1.84 (m, 1H), 1.80 (td, J= 5.2, 13.2 Hz, 1H), 1.69 - 1.58 (m, 4H), 1.41 - 1.25 (m, 36H), 0.94 - 0.88 (m, 6H)Synthesis of Lipidoid 2o H2N CF31Intermediate A Lipidoid 2 A solution of Intermediate A (4.3 g, 8.22 mmol, 1 eq.) in IPA (2 mL) was added Compound 1 (32.56 g, 328.70 mmol, 25.80 mL, 39.96 eq.), and the mixture was stirred at 85°C for 14 days in a sealed tube. LCMS indicated Intermediate A was consumed completely and one main peak with desired MS was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate=95:5 to 85:15) to give Lipidoid 2 (2.02 g, 3.25 mmol, 39.49% yield) as colorless oil.LCMS [M+l]+=622.5-122- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925'H NMR (400 MHz, DMSO-de) 6 = 4.90 (d, J = 3.6 Hz, 1H), 4.06 - 3.88 (m, 4H), 3.67 (br d, J= 2.8 Hz, 1H), 3.30 - 3.13 (m, 2H), 2.84 (br dd, J= 2.8, 6.8 Hz, 2H), 2.68 (br d, J = 3.2 Hz, 1H), 2.57 - 2.53 (m, 1H), 1.70 (br s, 4H), 1.57 - 1.48 (m, 4H), 1.24 (s, 36H), 0.96 - 0.76 (m, 6H)Synthesis of Lipidoid 3Lipidoid 3A mixture of Intermediate A (5 g, 9.56 mmol, 1 eq.), Compound 1 (4.36 g, 47.82 mmol, 3.71 mL, 5 eq.) in IPA (5 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 80°C for 16 hours under N2 atmosphere. LCMS showed Intermediate A was consumed and desired MS was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether: Ethyl acetate=50:l to 20:1) to give Lipidoid 3 (2.00 g, 3.18 mmol, 33.26% yield, 97.55% purity) as white solid.LCMS [M+l]+= 614.6'H NMR (400 MHz, METHANOL-d4) 6 = 4.18 - 4.02 (m, 4H), 3.80 - 3.66 (m, 2H), 3.57 - 3.49 (m, 2H), 3.17 - 2.96 (m, 2H), 2.93 - 2.67 (m, 2H), 2.66 - 2.43 (m, 1H), 2.15 - 1.96 (m, 2H), 1.87 - 1.68 (m, 2H), 1.68 - 1.58 (m, 4H), 1.30 (br s, 36H), 1.00 - 0.74 (m, 6H)Synthesis of Lipidoid 4oIntermediate A Lipidoid 4 A solution of Intermediate A (2 g, 3.83 mmol, 1 eq.) and Compound 1 (2 mL, 1 eq.) in IPA (2 mL) was stirred at 80°C for 72 hours. LCMS showed trace of Intermediate A was remained and main peak with desired MS was detected. 2 reactions were combined-123- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925for workup. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate=40:l to 8:1) to give Lipidoid 4 (2.09 g, 3.60 mmol, 47.00% yield) as white solid. (2 Batches: 7.66 mmol)LCMS [M+l]+= 579.5'H NMR (400 MHz, DMSO-de) 5 = 4.98 (d, J = 3.6 Hz, 1H), 4.04 - 3.88 (m, 4H), 3.72 - 3.65 (m, 2H), 3.65 - 3.54 (m, 1H), 2.89 - 2.66 (m, 4H), 1.83 - 1.64 (m, 4H), 1.51 (br s, 4H), 1.24 (s, 36H), 0.94 - 0.77 (m, 6H)Synthesis of Lipidoid 5Intermediate BA mixture of Intermediate B (2 g, 3.83 mmol, 1 eq.), Compound 1 (1.98 g, 22.73 mmol, 2.00 mL, 5.94 eq.) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 85°C for 16 hours under N2 atmosphere. TLC (Petroleum ether: Ethyl acetate=5:l, Rf= 0.4) indicated Compound 7 was consumed completely and one new spot formed. The reaction mixture was quenched by addition sat. NaHCO3(10 mL) at 20°C, and extracted with ethyl acetate (10 mL * 3). The combined organic layers were washed with brine (10 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate=93:7 to 90:10) to give Lipidoid 5 (2.04 g, 3.34 mmol, 43.72% yield) as white solid.LCMS [M+l]+= 610.5'H NMR (400 MHz, METHANOL-d4) 6 = 4.06 - 3.96 (m, 5H), 3.70 (t, J = 4.4 Hz, 4H), 3.14 - 3.06 (m, 1H), 3.02 (dt, J= 3.6, 9.2 Hz, 1H), 2.63 - 2.54 (m, 2H), 2.54 - 2.45 (m,-124- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-009252H), 2.28 - 2.22 (m, 1H), 2.10 - 1.97 (m, 2H), 1.94 - 1.85 (m, 1H), 1.81 (td, J= 5.2, 13.2 Hz, 1H), 1.64 (br s, 2H), 1.32 (br s, 32H), 1.04 - 0.81 (m, 12H)Synthesis of Lipidoid 6H2N CF31A mixture of Intermediate B (4 g, 7.65 mmol, 1 eq.), Compound 1 (15.16 g, 153.02 mmol, 12.01 mL, 20 eq.) in IPA (15 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 85 °C for 96 hours in a sealed tube. TLC (Petroleum ether: Ethyl acetate= 5:1, Rf= 0.4) indicated Intermediate B was consumed completely and one new spot formed. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCE, Petroleum ether: Ethyl acetate=95:5 to 94:6) to give Lipidoid 6 (2.03 g, 3.26 mmol, 42.67% yield) as white solid.LCMS [M+l]+= 622.5'H NMR (400 MHz, METHANOL-d4) 5 = 4.06 - 3.90 (m, 4H), 3.76 (br d, J= 3.2 Hz, 1H), 3.24 (q, J= 9.6 Hz, 2H), 3.09 - 2.97 (m, 2H), 2.81 (br d, J= 3.6 Hz, 1H), 2.03 - 1.91 (m, 2H), 1.90 - 1.73 (m, 2H), 1.63 (br s, 2H), 1.31 (br s, 32H), 1.04 - 0.81 (m, 12H)Synthesis of Lipidoid 7FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925NH2 y OH OH <|Intermediate BA mixture of Intermediate B (3 g, 5.74 mmol, 1 eq.), Compound 1 (2.61 g, 28.69 mmol, 2.22 mL, 5 eq.) in IPA (7 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 85 °C for 16 hours under N2 atmosphere. LCMS indicated Intermediate B was consumed completely and one main peak with desired MS was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Dichloromethane: Methanol= 100:0 to 1:99) to give Lipidoid 7 (1.91 g, 3.11 mmol, 54.22% yield) as colorless oil.LCMS [M+l]+=614.6'H NMR (400 MHz, DMSO-de) 6 = 4.87 (br s, 1H), 4.68 - 4.39 (m, 2H), 4.00 - 3.78 (m, 4H), 3.65 (br s, 1H), 3.57 - 3.37 (m, 3H), 2.87 (m, 2H), 2.70 - 2.53 (m, 3H), 2.45 - 2.25 (m, 1H), 1.76 (br s, 2H), 1.70 - 1.63 (m, 2H), 1.56 (br s, 2H), 1.24 (br s, 32H), 0.99 - 0.68 (m, 12H)Synthesis of Lipidoid 8To a solution of Intermediate B (2 g, 3.83 mmol, 1 eq.) in IPA (2 mL) was added Compound 1 (214.49 mg, 3.83 mmol, 2 mL, 1 eq.). The mixture was stirred at 85°C for-126- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-0092572 hours. LCMS indicated Intermediate B was consumed completely and one main peak with desired MS was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCE, Petroleum ether: Ethyl acetate= 94:6 to 83:17) to give Lipidoid 8 (2.06 g, 3.55 mmol, 46.44% yield) as yellow oil.LCMS [M+l]+=579.5'H NMR (400 MHz, METHANOL-d4) 6 = 4.58 (s, 1H), 4.05 - 3.94 (m, 4H), 3.79 (q, J = 4.0 Hz, 1H), 3.76 - 3.61 (m, 2H), 3.12 - 3.03 (m, 1H), 3.03 - 2.94 (m, 1H), 2.92 (q, J = 4.0 Hz, 1H), 2.07 - 1.76 (m, 4H), 1.68 - 1.59 (m, 3H), 1.31 (br s, 32H), 1.01 - 0.84 (m, 12H)Synthesis of Lipidoid 9Lipidoid 9A mixture of Intermediate C (1 g, 1.40 mmol, 1 eq.) and Compound 1 (990.00 mg, 11.36 mmol, 1000.00 pL, 8.13 eq.) was stirred at 85°C for 36 hours. TLC (PE: EA=5:1, Rf=0.5) indicated Intermediate C was consumed completely and one new spot formed.3 reactions were combined for workup, which was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCE, DCM: MeOH=50:l to 30:1) to give Lipidoid 9 (2.4 g, 2.99 mmol, 71.21% yield) as colorless oil. (3 Batches: 4.2 mmol)LCMS [M+l]+=802.7'H NMR (400 MHz, METHANOL-d4) 5 = 4.08 (t, J = 6.4 Hz, 4H), 4.00 (d, J = 2.8 Hz, 1H), 3.70 (t, J= 4.4 Hz, 4H), 3.09 (dd, J= 4.4, 9.6 Hz, 1H), 3.03 - 2.94 (m, 1H), 2.66 -2.54 (m, 2H), 2.52 - 2.44 (m, 2H), 2.24 (d, J= 2.8 Hz, 1H), 2.10 - 1.97 (m, 2H), 1.96 - -127- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-009251.73 (m, 2H), 1.70 - 1.57 (m, 4H), 1.50 - 1.26 (m, 48H), 1.20 (br s, 4H), 0.91 (t, J = 6.8 Hz, 6H), 0.69 (d, J= 5.2 Hz, 4H), 0.63 - 0.55 (m, 2H), -0.32 (q, J= 5.2 Hz, 2H)Synthesis of Lipidoid 10Lipidoid 10To a solution of Intermediate C (2 g, 2.80 mmol, 1 eq.) in i-PrOH (15 mL) was added Compound 1 (6.65 g, 67.12 mmol, 5.27 mL, 24 eq.). The mixture was stirred at 85°C for 6 days in a sealed tube. TLC (Petroleum ether: Ethyl acetate =3:1, Rf= 0.43) indicated Intermediate C was remained and new spots formed. 3 reactions were combined for workup. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCE, Petroleum ether: Ethyl acetate=5:l) to give Lipidoid 10 (4.4 g, 5.40 mmol, 64.2% yield) as colorless oil. (3 Batches: 8.4 mmol)LCMS [M+l]+=814.6'H NMR (400 MHz, METHANOL-d4) 6 = 4.07 (t, J = 6.4 Hz, 4H), 3.76 (br d, J = 3.2 Hz, 1H), 3.25 (br dd, J= 4.0, 9.6 Hz, 2H), 3.09 - 2.94 (m, 2H), 2.81 (br d, J= 4.0 Hz, 1H), 2.04 - 1.91 (m, 2H), 1.87 - 1.75 (m, 2H), 1.67 - 1.59 (m, 4H), 1.40 (br s, 12H), 1.35 (br s, 20H), 1.31 (br s, 16H), 1.19 (br d, J = 7.2 Hz, 4H), 0.96 - 0.84 (m, 6H), 0.75 - 0.64 (m, 4H), 0.63 - 0.53 (m, 2H), -0.31 (q, J= 5.2 Hz, 2H)Synthesis of Lipidoid 11-128- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925Lipidoid 11To a solution of Intermediate C (1 g, 1.40 mmol, 1 eq.~) in IPA (1 mL) was added Compound 1 (2.55 g, 27.97 mmol, 20 eq.). The mixture was stirred at 85°C for 72 hours and TLC indicated Intermediate C had been consumed. 5 reactions were combined for workup. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCT, Petroleum ether: Ethyl acetate=40:l to 8:1) to give Lipidoid 11 (1.3 g, 1.61 mmol, 23.06% yield) as yellow oil. (5 Batches, 7.0 mmol)LCMS [M+l]+=806.7'H NMR (400 MHz, METHANOL-d4) 5 = 4.13 - 4.05 (m, 4H), 3.79 - 3.69 (m, 2H), 3.54 (d, J= 5.2 Hz, 2H), 3.16 - 3.03 (m, 2H), 2.92 - 2.56 (m, 3H), 2.16 - 2.02 (m, 2H), 1.84 - 1.71 (m, 2H), 1.64 (br t, J= 6.4 Hz, 4H), 1.44 - 1.28 (m, 48H), 1.23 - 1.12 (m, 4H), 0.91 (t, J= 6.8 Hz, 6H), 0.74 - 0.64 (m, 4H), 0.57 - 0.57 (m, 1H), 0.63 - 0.56 (m, 1H), -0.28 - -0.36 (m, 2H)Synthesis of Lipidoid 12-129- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925A mixture of Intermediate C (4 g, 5.59 mmol, 1 eq.), Compound 1 (3.81 g, 67.96 mmol, 2 mL, 12.15 eq.) in IPA (4 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 85°C for 16 hours under N2 atmosphere. LCMS showed 20% of Intermediate C remained and one main peak with desired MS was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate=50:l to 10:1) to give Lipidoid 12 (1.2 g, 1.56 mmol, 27.82% yield) as colorless oil.LCMS [M+l]+=771.6'H NMR (400 MHz, METHANOL-ch) 5 = 4.07 (q, J = 6.4 Hz, 4H), 3.78 (br d, J = 3.2 Hz, 1H), 3.74 - 3.59 (m, 2H), 3.11 - 2.94 (m, 2H), 2.90 (q, J = 4.0 Hz, 1H), 2.07 - 1.74 (m, 4H), 1.69 - 1.58 (m, 4H), 1.47 - 1.27 (m, 48H), 1.19 (br d, J= 7.2 Hz, 4H), 0.96 - 0.85 (m, 6H), 0.73 - 0.64 (m, 4H), 0.64 - 0.54 (m, 2H), -0.26 - 0.40 (m, 2H)Example 2, Formulations and physical properties of 12 lipidoid candidates for mRNA encapsulation

[0319] In this example, the physical properties of lipid nanoparticle (LNP) formulations were assessed. 12 lipid nanoparticle formulations were synthesized, comprising ionizable lipid: helper lipid: cholesterol: PEGylated lipid in a molar ratio of 50:10:38.5:1.5. Lipidoids 1-12 were used as the sole ionizable lipid in these formulations. The LNPs were characterized by dynamic light scattering (DLS) measurement for measuring its hydrodynamic radius and poly dispersity index (PDI). Encapsulation efficiency (EE%) was quantitated using a fluorescence based Ribogreen assay. Apparent pKa was determined using the 6-(p-Toluidino)-2- naphthalenesulfonic acid (TNS) binding assay. 4 of 12 tested lipidoids (Lipidoids 1, 3, 7, and 11) demonstrated >50% mRNA encapsulation (in vivo study cutoff is -90%). All but 1 tested lipid (Lipidoid 5) demonstrated favorable physical properties (i.e. size) to be formulated into an LNP (in vivo study cutoff is -lOOnrn). The formulations resulting in favorable capsulation of mRNA are shown in Table 1 below, and corresponding bar graphs are shown in Figure 1.-130- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925Example 3. Tested lipids screened b in vivo hEPO expression levels in livid nanoparticle formulations

[0320] In this example, the hEPO expression levels for LNPs comprising lipidoids 1, 3, 7 and 11 in Example 2 were compared to a control LNP comprising SM-102. None of the evaluated LNPs demonstrated in vivo expression, as shown in Figure 2.

[0321] In this example, messenger RNA molecules encoding hEPO proteins were formulated in lipid nanoparticles for delivery in vivo. The lipid nanoparticle formulations comprised of a lipid composition of ionizable lipid: helper lipid: cholesterol: DMG-PEG2k at 50:10:38.5:1.5 mol%. The lipid mixture in ethanol was mixed with hEPO mRNA in RNA acidifying buffer (10 mM citrate, pH 4) at an ionizable-lipid-nitrogen-to-RNA-phosphate ratio (N: P) of 6 using a microfluidic device (Precision NanoSystems, Inc.) at a combined flow rate of 10 mL / min (7.5 mL / min for aqueous buffer, RNA and 2.5 mL / min for ethanol, lipid mix). The resulting particles were neutralized by buffer exchange into a HEPES buffer solution (20mM HEPES pH 7.8, 50mM sodium chloride, 15% sucrose) via PD-10 desalting column. The neutralized particles were concentrated using 100 kDa AMICON® Ultra centrifugal filters and sterile filtered using 0.2 um syringe filters. Samples were characterized, then frozen at -30 °C for storage. Prior to dosing, samples were thawed at room temperature and diluted as needed.

[0322] LNPs were characterized by dynamic light scattering (DLS) measurement for measuring its hydrodynamic radius and poly dispersity index (PDI). Encapsulation efficiency (EE%) and total RNA concentration were quantitated using a fluorescence based Ribogreen assay. The measurements for each of the LNPs are shown in Table 1 below.

[0323] The in vivo studies were performed in C57BL / 6 female mice at 6 to 8 weeks weighing in at approximately 20 g. The LNPs formulated with different ionizable lipids at 0.2 mg / kg of hEPO mRNA were administered by tail vein injection and animals were euthanized at 6 h or 24 h post-administration for blood serum sample collection and liver tissue extraction. The hEPO levels from the samples were analyzed and cross -compared by enzyme-linked immunoassay (ELISA) according to manufacturer’s protocol and shown in Table 1 below.-131- FoleyHoagUS12883713.3Table 1: LNP formulations for in vivo studiesIonizable Lipid:Ionizable Encapsulation Apparent Dose hEPO DSPC: Cholesterol: N: P ratio Size (nm) PDILipid Efficiency % pKa (mg / kg) (ng / mL)DMG-PEG2k (mol%)SM-102 50: 10: 38.5: 1.5 6 79.9 0.07 98.5 6.807 0.2 1900 + / - 188 Lipidoid 1 50: 10: 38.5: 1.5 6 82.6 0.113 61.1 4.217 0.2Lipidoid 3 50: 10: 38.5: 1.5 6 87.0 0.093 96.0 5.099 0.2Lipidoid 7 50: 10: 38.5: 1.5 6 69.7 0.224 98.5 4.561 0.2Lipidoid 11 50: 10: 38.5: 1.5 6 90.5 0.148 95.0 5.619 0.2Attorney Docket No.: FAZ-00925Example 4. Synthesis and characterization of series 900 lipidoids (second lipidoids) General procedure for preparation of Boc-protected diethanolamine lipids

[0324] To a solution of Compound 1 (13.74 g, 53.59 mmol, 2.2 eq.) in DCM (50 mL) was added DMAP (595.21 mg, 4.87 mmol, 0.2 eq.), EDCI (11.67 g, 60.90 mmol, 2.5 eq.), DIEA (9.45 g, 73.08 mmol, 12.73 mL, 3 eq.) and Compound 2 (5 g, 24.36 mmol, 4.61 mL, 1 eq.). The mixture was stirred at 20°C for 16 hours. TLC (Petroleum ether: Ethyl acetate=3:l, Pl: Rf=0.64) indicated Compound 1 and Compound 2 was consumed completely and new spots formed. The reaction mixture was quenched by addition sat. NaHCO3(10 mL) at 20°C, and then diluted with H2O (40 mL) and extracted with DCM (80 mL). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate=200: 1 to 70:1 to 30:1) to give Compound 3 (11.48 g, 16.81 mmol, 52.13% yield, 99.9% purity) as colorless oil. Compound 3: LCMS [M- 100+1]+= 582.5.1H NMR (400 MHz, chloroform-d) 5 = 4.33 - 4.07 (m, 4H), 3.66 - 3.31 (m, 4H), 2.54 - 2.18 (m, 2H), 1.64 - 1.55 (m, 4H), 1.50 - 1.38 (m, 13H), 1.34 - 1.19 (m, 40H), 0.88 (t, J= 6.8 Hz, 12H).General procedure for preparation of deprotected diethanolamine lipids-133- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0325] A solution of Compound 3 (9.48 g, 13.90 mmol, 1 eq.) in HCl / EtOAc (4 M,47.40 mL, 13.64 eq.) was stirred at 20°C for 2 hours. LC-MS showed Compound 3 was consumed and desired MS was detected. The reaction mixture was concentrated under reduced pressure to give Compound 4 (8.6 g, crude, HC1) as colorless oil.Compound 4: LCMS [M+l]+= 582.5.1H NMR (400 MHz, chloroform-d) 5 = 9.87 (br s, 2H), 4.51 (br s, 4H), 3.36 (br s, 4H), 2.52 - 2.31 (m, 2H), 1.71 - 1.56 (m, 4H), 1.53 -1.41 (m, 4H), 1.38 - 1.18 (m, 40H), 0.88 (t, J= 6.8 Hz, 12H).General procedure for preparation of acrylate precursor

[0326] To a solution of Compound 4 (8.6 g, 13.91 mmol, 1 eq., HC1) in DCM (90 mL) was added TEA (42.22 g, 417.20 mmol, 58.07 mL, 30 eq.) and Compound 5 (1.76 g, 19.47 mmol, 1.58 mL, 1.4 eq.) at 0°C. The mixture was stirred at 25 °C for 16 hours. TLC (Petroleum ether: Ethyl acetate=5:l, Pl: Rf=0.50) indicated Compound 4 was-134- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925consumed completely and new spots formed. The reaction mixture was quenched by addition MeOH (15 mL) at 20°C, and then washed with NH4CI (50 mL). The organic layer was washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate=50:l to 26:1) to give Compound 6 (3 g, 4.69 mmol, 33.75% yield, 99.5% purity) as colorless oil.Compound 6: LCMS [M+23]+= 658.5.1H NMR (400 MHz, chloroform-d) 5 = 6.65 (dd, J= 10.4, 16.8 Hz, 1H), 6.40 (dd, J= 2.0, 16.8 Hz, 1H), 5.74 (dd, J = 2.0, 10.4 Hz, 1H), 4.34 - 4.13 (m, 4H), 3.69 (t, J= 5.6 Hz, 4H), 2.43 - 2.24 (m, 2H), 1.62 - 1.51 (m, 4H), 1.49 - 1.38 (m, 4H), 1.25 (br s, 40H), 0.88 (t, J= 6.8 Hz, 12H)General procedure for preparation of Series 900-1Compound 901

[0327] To a solution of Compound 6 (50 mg, 78.62 pmol, 1 eq.) in MeOH (1.5 mL) and THF (1.5 mL) was added N-methylmethanamine (2 M, 78.62 pL, 2 eq.). The mixture was stirred at 20°C for 16 hours. LC-MS showed Compound 6 was consumed completely and one main peak with desired mass was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, DCM: MeOH= 1:0 to 50:1) to give Compound 901 (40 mg, 58.73 pmol, 74.70% yield, 100% purity) as colorless oil.Compound 901 LCMS [M+1]+= 681.6.1H NMR (400 MHz, chloroform-d) 5 = 4.21 (q, J = 5.6 Hz, 4H), 3.75 - 3.53 (m, 4H), 3.03 (br d, J = 2.8 Hz, 2H), 2.85 (br s, 2H), 2.54 (br s, 6H), 2.33 (br dd, J= 5.6, 8.0 Hz, 2H), 1.65 - 1.52 (m, 4H), 1.49 - 1.40 (m, 4H), 1.26 (br s, 40H), 0.88 (br t, J = 6.8 Hz, 12H)General procedure for preparation of HCl salt

[0328] To a solution of Compound 901 (40 mg, 58.73 pmol, 1 eq.) in dioxane (1 mL) was added HCl / dioxane (0.5 M, 234.92 pL, 2 eq.). The mixture was stirred at 20°C-135- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925for 2 hours. The mixture was concentrated and lyophilized to give Compound 901 (27.8 mg, 38.74 pmol, 65.97% yield, HC1) as colorless oil.N N©I OCompound 901: LCMS [M+l]+= 681.6.1H NMR (400 MHz, chloroform-d) 5 = 12.32 (br s, 1H), 4.31 - 4.13 (m, 4H), 3.75 - 3.56 (m, 4H), 3.43 (br s, 2H), 3.14 (br s, 2H), 2.84 (br s, 6H), 2.46 - 2.22 (m, 2H), 1.63 - 1.52 (m, 4H), 1.45 (m, 4H), 1.26 (br s, 40H), 0.88 (t, J= 6.8 Hz, 12H)

[0329] Compound 902 and 903 have varied lipid tails than the other structures. They were prepared with the same method as Compound 901, but using different lipid starting materials.ciNoo.Compound 902: LCMS [M+l]+= 681.6. ’H NMR (400 MHz, chloroform-d) 5 = 12.67 - 12.40 (m, 1H), 4.34 - 4.08 (m, 4H), 3.75 - 3.55 (m, 4H), 3.47 - 3.35 (m, 2H), 3.27 - 3.06 (m, 2H), 2.94 - 2.73 (m, 6H), 2.45 - 2.26 (m, 2H), 1.62 - 1.53 (m, 4H), 1.49 - 1.40 (m, 4H), 1.34 - 1.20 (m, 40H), 0.95 - 0.78 (m, 12H)N© I1Oo.o Compound 903: LCMS [M+l]+= 793.7.1H NMR (400 MHz, chloroform-d) 5 = 12.75 - 12.46 (m, 1H), 4.22 (td, J= 5.6, 15.0 Hz, 4H), 3.72 - 3.56 (m, 4H), 3.42 (br d, J= 5.4 Hz, 2H), 3.15 (br t, J= 6.4 Hz, 2H), 2.82 (d,-136- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925J= 4.8 Hz, 6H), 2.40 - 2.26 (m, 2H), 1.61 - 1.52 (m, 4H), 1.45 (br dd, J= 5.2, 8.4 Hz, 4H), 1.26 (s, 56H), 0.88 (t, J= 6.8 Hz, 12H).ci o Bl N OO.Compound 904: LCMS [M+l]+= 667.6.1H NMR (400 MHz, chloroform-d) 5 = 9.63 - 9.38 (m, 2H), 4.22 (br s, 4H), 3.61 (br s, 4H), 3.27 (br s, 2H), 3.12 (br s, 2H), 2.79 (br s, 3H), 2.40 - 2.28 (m, 2H), 1.65 - 1.52 (m, 4H), 1.49 - 1.40 (m, 4H), 1.35 - 1.19 (m, 40H), 0.89 (t, J = 6.8 Hz, 12H).ci o NBl O O. O Compound 905: LCMS [M+l]+= 681.6. ‘H NMR (400 MHz, chloroform-d) 5 = 9.38 (br s, 2H), 4.22 (br t, J = 5.6 Hz, 4H), 3.68 -3.54 (m, 4H), 3.25 (br s, 2H), 3.19 - 3.04 (m, 4H), 2.41 - 2.29 (m, 2H), 1.62 - 1.52 (m, 4H), 1.51 - 1.41 (m, 7H), 1.36 - 1.16 (m, 40H), 0.88 (t, J = 6.8 Hz, 12H).Cl o H © JL N OO.Compound 906: LCMS [M+l]+= 695.6.1H NMR (400 MHz, chloroform-d) 5 = 11.98 (br s, 1H), 4.22 (td, J= 5.6, 19.6 Hz, 4H), 3.74 - 3.56 (m, 4H), 3.48 (m, 1H), 3.38 - 3.02 (m, 5H), 2.77 (d, J= 4.6 Hz, 3H), 2.38 - 2.27 (m, 2H), 1.65 - 1.52 (m, 4H), 1.51 - 1.41 (m, 7H), 1.35 - 1.17 (m, 40H), 0.88 (t, J= 6.8 Hz, 12H).FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925ci H NCompound 907: LCMS [M+l]+= 709.6. NMR (400 MHz, chloroform-d) 5 = 12.15 (br s, 1H), 4.22 (td, J = 5.6, 18.8 Hz, 4H), 3.70 (br t, J = 5.6 Hz, 2H), 3.61 (t, J = 5.6 Hz, 2H), 3.37 (br d, J = 5.2 Hz, 2H), 3.27 - 3.14 (m, 4H), 3.14 - 3.02 (m, 2H), 2.33 (ddd, J = 2.9, 5.6, 8.4 Hz, 2H), 1.63 - 1.51 (m, 4H), 1.50 -1.38 (m, 10H), 1.35 - 1.15 (m, 40H), 0.98 - 0.81 (m, 12H).ci o Nfl? o o.o Compound 908: LCMS [M+l]+= 695.6.1H NMR (400 MHz, chloroform-d) 5 = 9.38 - 9.18 (m, 2H), 4.28 - 4.16 (m, 4H), 3.67 - 3.56 (m, 4H), 3.26 (br s, 2H), 3.19 - 3.09 (m, 2H), 3.06 - 2.95 (m, 2H), 2.38 - 2.29 (m, 2H), 1.94-1.84 (m, 2H), 1.64 - 1.52 (m, 4H), 1.46 (br d, J= 4.8 Hz, 4H), 1.34 - 1.18 (m, 40H), 1.06 (t, J= 7.2 Hz, 3H), 0.97 - 0.77 (m, 12H).Cl oH ® 1N OO.Compound 909: LCMS [M+l]+= 709.6.1H NMR (400 MHz, chloroform-d) 5 = 11.97 (br s, 1H), 4.32 - 4.14 (m, 4H), 3.74 - 3.56 (m, 4H), 3.49 (m, 1H), 3.34 (m, 1H), 3.18 (m, 2H), 3.14 - 3.01 (m, 1H), 2.93 (br dd, J= 5.6, 12.0 Hz, 1H), 2.79 (d, J = 4.8 Hz, 3H), 2.40 - 2.28 (m, 2H), 2.03 - 1.82 (m, 2H), 1.57 (br dd, 7 = 7.6, 13.6 Hz, 4H), 1.46 (br dd, J = 6.0, 8.0 Hz, 4H), 1.36 - 1.16 (m, 40H), 1.04 (t, J = 7.2 Hz, 3H), 0.88 (t, J = 6.8 Hz, 12H).-138- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925NMR (400 MHz, chloroform-d) 5 = 12.31 - 12.17 (m, 1H), 4.28 - 4.16 (m, 4H), 3.70 (br t, J = 6.0 Hz, 2H), 3.64 - 3.57 (m, 2H), 3.43 - 3.35 (m, 2H), 3.25 - 3.16 (m, 3H), 3.14 - 2.89 (m, 3H), 2.39 - 2.29 (m, 2H), 1.94 - 1.81 (m, 2H), 1.56 - 1.51 (m, 3H), 1.50 - 1.38 (m, 8H), 1.33 - 1.16 (m, 40H), 1.03 (t, J= 7.6 Hz, 3H), 0.89 (t, J= 6.8 Hz, 12H).Compound 911: LCMS [M+l]+= 737.7. 1H NMR (400 MHz, chloroform-d) 5 = 12.31 - 12.11 (m, 1H), 4.31 - 4.11 (m, 4H), 3.69 (br t, J = 5.6 Hz, 2H), 3.63 - 3.55 (m, 2H), 3.43 - 3.33 (m, 2H), 3.27 - 3.16 (m, 2H), 3.06 - 2.88 (m, 4H), 2.40 - 2.27 (m, 2H), 1.95 - 1.80 (m, 4H), 1.58 - 1.49 (m, 4H), 1.49 - 1.37 (m, 4H), 1.33 - 1.18 (m, 40H), 1.07 - 0.97 (m, 6H), 0.89 (t, J= 6.8 Hz, 12H)Compound 912: LCMS [M+l]+=707.6. 1H NMR (400 MHz, chloroform-d) 5 = 12.58 (br s, 1H), 4.21 (td, J= 5.6, 15.1 Hz, 4H), 3.80 - 3.65 (m, 4H), 3.61 (t, J = 5.6 Hz, 2H), 3.45 (br d, J = 5.6 Hz, 2H), 3.16 (br t, J = 6.4 Hz, 2H), 2.87 (br d, J= 6.4 Hz, 2H), 2.33 (dt, J= 2.8, 5.6 Hz, 2H), 2.28 - 2.16 (m, 2H), 2.09 (br s, 2H), 1.60 - 1.52 (m, 4H), 1.46 (br dd, J= 5.6, 8.4 Hz, 4H), 1.36 - 1.20 (m, 40H), 0.88 (t, J = 6.8 Hz, 12H).-139- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925o Compound 913: LCMS [M+l]+=721.6. 1H NMR (400 MHz, chloroform-d) 5 = 12.24 - 12.00 (m, 1H), 4.22 (td, 7= 5.6, 17.6 Hz, 4H), 3.69 (br t, J= 5.6 Hz, 2H), 3.61 (t, J= 5.6 Hz, 2H), 3.50 (br d, J= 11.6 Hz, 2H), 3.34 (q, J = 5.6 Hz, 2H), 3.27 - 3.16 (m, 2H), 2.75 - 2.60 (m, 2H), 2.41 - 2.18 (m, 4H), 1.99 - 1.81 (m, 3H), 1.57 (brdd, 7 = 7.2, 14.4 Hz, 4H), 1.50 - 1.38 (m, 5H), 1.35 - 1.16 (m, 40H), 0.88 (t, 7 = 6.8 Hz, 12H).Compound 914: LCMS [M+l]+= 723.6. 1H NMR (400 MHz, chloroform-d) 5 = 13.10 (br s, 1H), 4.31 - 4.16 (m, 6H), 3.98 (dd, J = 2.8, 12.8 Hz, 2H), 3.67 (br t, 7= 5.6 Hz, 2H), 3.60 (t, 7= 5.6 Hz, 2H), 3.45 - 3.36 (m, 4H), 3.26 - 3.19 (m, 2H), 3.03 - 2.86 (m, 2H), 2.38 - 2.27 (m, 2H), 1.62 - 1.52 (m, 4H), 1.50 - 1.39 (m, 4H), 1.33 - 1.18 (m, 40H), 0.88 (t, J= 6.8 Hz, 12H).o Compound 915: LCMS [M+l]+= 765.7. 1H NMR (400 MHz, chloroform-d) 5 = 12.12 (br s, 1H), 4.28 - 4.15 (m, 4H), 3.73 - 3.66 (m, 2H), 3.60 (br t, J = 5.6 Hz, 2H), 3.37 (br d, J = 3.6 Hz, 2H), 3.21 (br s, 2H), 3.01 (dt, J = 5.6, 12.0 Hz, 4H), 2.38 - 2.28 (m, 2H), 1.79 (brdd, 7= 5.6, 11.2 Hz, 4H), 1.57 (td, 7 = 7.2, 14.0 Hz, 4H), 1.49 - 1.36 (m, 8H), 1.35 - 1.17 (m, 40H), 0.99 (t, 7 = 7.2 Hz, 6H), 0.88 (t, 7= 6.8 Hz, 12H).-140- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-009251H NMR (400 MHz, chloroform-d) 5 = 11.64 (br s, 1H), 4.38 - 4.13 (m, 4H), 4.02 (br s, 2H), 3.76 - 3.56 (m, 5H), 3.41 (br s, 2H), 3.18 (br s, 3H), 2.90 (br d, J= 2.8 Hz, 3H), 2.42 - 2.28 (m, 3H), 1.64 - 1.51 (m, 4H), 1.49 - 1.40 (m, 4H), 1.35 - 1.18 (m, 40H), 0.88 (t, J = 6.8 Hz, 12H)Compound 917e: LCMS [M-100+1] = 807.8. ’H NMR (400 MHz, chloroform-d) 5 = 4.20 (q, J= 5.8 Hz, 4H), 3.67-3.57 (m, 4H), 2.73 (t, J= 7.5 Hz, 2H), 2.56 (t, J= 7.6 Hz, 2H), 2.45 (q, J= 7.2 Hz, 2H), 2.31 (ttd, J= 8.6, 5.7, 2.8 Hz, 2H), 2.25 (s, 3H), 1.64-1.52 (m, 6H), 1.48 - 1.39 (m, 4H), 1.35-1.15 (m, 54H), 1.07 (t, J= 7.2 Hz, 3H), 0.92-0.83 (m, 12H).Compound 917f: LCMS [M-100+1]+= 821.7.1H NMR (400 MHz, chloroform-d) 5 = 4.20 (q, J = 5.8 Hz, 4H), 3.62 (dt, J = 11.4, 6.0 Hz, 4H), 2.81 (t, J = 7.5 Hz, 2H), 2.54 (q, J= 7.1 Hz, 6H), 2.31 (ttd, J= 8.2, 5.5, 2.0 Hz, 2H), 1.64- 1.50 (m, 6H), 1.44 (tt, J= 13.9, 5.0 Hz, 4H), 1.35 - 1.13 (m, 54H), 1.03 (t, J= 7.1 Hz, 6H), 0.96 - 0.80 (m, 12H).-141- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-009256...,.--xO Compound 943: LCMS [M+l]+= 807.8. 'H NMR (400 MHz, chloroform-d) 6 = 12.40 (br s, 1H), 4.27 - 4.15 (m, 4H), 3.89 (d, J = 2.0 Hz, 1H), 3.80 - 3.70 (m, 1H), 3.66 - 3.40 (m, 4H), 2.80 (d, J= 4.8 Hz, 3H), 2.74 (d, J = 4.8 Hz, 3H), 2.71 - 2.62 (m, 1H), 2.37 - 2.25 (m, 2H), 1.61 - 1.51 (m, 4H), 1.44 (q, J = 6.8 Hz, 7H), 1.25 (s, 56H), 0.88 (t, J= 6.8 Hz, 12H)Synthesis and Characterization of Compound 1003A THF, -78C, 2hrs-142- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925Pyr, DMAP DCM, 25C, 12 hrs 4General procedure for preparation of Compound 3:8To a solution of Compound 3A (92 g, 499.14 mmol, 1 eq.~) in THF (1000 mL) was added Compound 3B (94.52 g, 499.14 mmol, 97.45 mL, 1 eq.~) at -78°C. The mixture was stirred at -78°C for 2 hours. TLC (PE: EA = 10:1, Pl: Rf= 0.4) indicated -30% of-143- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925Compound 3A was remained, and one major new spot with larger polarity was detected. The reaction mixture was quenched by addition NH4CI (500 mL) at 0°C, and then diluted with H2O (1000 mL) and extracted with ethyl acetate (1000 mL * 3). The combined organic layers were washed with brine (500 mL * 3), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate=100:l to 30:1) to give Compound 3 (72 g, 266.18 mmol, 53.33% yield) as white solid.'H NMR (CHLOROFORM-d, 400 MHz) 6 = 3.59 (br dd, 1H, 7=4.4, 6.8 Hz), 1.2- 1.5 (m, 30H), 0.8-1.0 (m, 6H)General procedure for preparation of compound 5CDMAP (02 eq ), DIEA (3 eq ), EDCI(1.5 eq ) DCM 25 °C, 1 h5B 5CA mixture of Compound 5A (35 g, 221.19 mmol, 1 eq.), Compound 5B (55.51 g, 265.42 mmol, 45.50 mL, 1.2 eq.), EDCI (63.60 g, 331.78 mmol, 1.5 eq.), DIEA (85.76 g, 663.56 mmol, 115.58 mL, 3 eq.) and DMAP (5.40 g, 44.24 mmol, 0.2 eq.) in DCM (350 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 25°C for 1 hour under N2 atmosphere. TLC (Petroleum ether: Ethyl acetate=10:l, Pl:Rf= 0.66) indicated Compound 5A was consumed completely and one new spot formed. The reaction mixture was diluted with sat. NPLCl (400 mL) and extracted with DCM (400 mL*3). The combined organic layers were dried over Na2SO4 filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCE, Petroleum ether: Ethyl acetate=100:0 to 80:1) to give Compound 5C (50 g, 136.64 mmol, 61.78% yield, 95.47% purity) as yellow oil.LCMS [M+l]+= 349.2'H NMR (400 MHz, CHLOROFORM-d) 5 = 4.05 (t, J = 6.8 Hz, 2H), 3.40 (t, J = 6.8 Hz, 2H), 2.28 (t, J= 7.6 Hz, 2H), 1.90 - 1.71 (m, 2H), 1.61 (quin, J= 6.8 Hz, 4H), 1.49 -1.38 (m, 2H), 1.37 - 1.14 (m, 16H), 0.92 - 0.80 (m, 3H)-144- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925General procedure for preparation of compound 5:5CA mixture of Compound 5C (15 g, 42.94 mmol, 1 eq.), Compound 5D (13.11 g, 214.69 mmol, 12.96 mL, 5 eq.), KI (712.76 mg, 4.29 mmol, 0.1 eq.), K2CO3 (11.87 g, 85.87 mmol, 2 eq.) in ACN (150 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 90°C for 16 hours under N2 atmosphere. TLC (DCM: ME=5:1, Pl: Rf= 0.44) indicated Compound 5C was consumed, and one major new spot with larger polarity was detected. The residue was diluted with ethyl acetate (100 mL) and extracted with H2O (800 mL). The combined organic layers were dried over Na2SO4 filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Dichloromethane: Methanol=50:l to 5:1) to give Compound 5 (6.3 g, 19.12 mmol, 44.53% yield, 100% purity) as white solid.LCMS [M+l]+= 330.3'H NMR (400 MHz, CHLOROFORM-d) 6 = 4.06 (t, J= 6.8 Hz, 2H), 3.71 - 3.61 (m, 2H), 2.89 - 2.74 (m, 2H), 2.64 (t, J = 7.2 Hz, 2H), 2.38 (br s, 2H), 2.29 (t, J = 7.6 Hz, 2H), 1.67 - 1.56 (m, 4H), 1.55 - 1.46 (m, 2H), 1.39 - 1.24 (m, 18H), 0.96 - 0.82 (m, 3H)General procedure for preparation of compound 2:Py (1.5 eq), DCM, 25 °C, 2 hTo a solution of Compound lx (24 g, 114.76 mmol, 19.67 mL, 1 eq.) in DCM (300 mL) was added Py (13.62 g, 172.15 mmol, 13.89 mL, 1.5 eq.) and Compound 1A (27.76 g, 137.72 mmol, 1.2 eq.) in DCM (300 mL) was added dropwise at 0°C. The mixture was-145- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925stirred at 25°C for 2 hours. TLC (Petroleum ether: Ethyl acetate=5:l, Pl: Rf= 0.5) indicated Compound lx was consumed completely and one new spot formed. The reaction mixture was diluted with H2O (250 mL) and extracted with DCM (200 mL *3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate=l: O to 100:1) to give Compound 2 (32 g, 85.51 mmol, 74.51% yield was obtained as a colorless oil.'H NMR (CHLOROFORM-d, 400 MHz) 6 = 8.2-8 3 (m, 2H), 7.3-7.4 (m, 2H), 4.2-43 (m, 2H), 3.4-3.5 (m, 2H), 1.8-1.9 (m, 2H), 1.7-1.8 (m, 2H), 1.3-1.5 (m, 8H)General procedure for preparation of compound 4:4To a solution of Compound 2 (32 g, 85.51 mmol, 1 eq.) in DCM (300 mL) was added Py (13.53 g, 171.02 mmol, 13.80 mL, 2 eq.) and DMAP (20.89 g, 171.02 mmol, 2 eq.). Then Compound 3 (23.13 g, 85.51 mmol, 1 eq.) was added at 0°C. The mixture was stirred at 25°C for 12 hours. TLC (Petroleum ether: Ethyl acetate=20:l, Pl: Rf= 0.5) indicated -30% of Compound 3 was remained, and one major new spot with lower polarity was detected. The reaction was messy according to TLC. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified’©! y column chromatography (SiO2, Petroleum ether: Ethyl acetate=O:l to 160:1) to give Compound 4 (10.4 g, 20.57 mmol, 24.05% yield) was obtained as a white solid.'H NMR (CHLOROFORM-d, 400 MHz) 6 = 4.6-47 (m, 1H), 4.12 (t, 2H, 7=6.4 Hz), 3.40 (t, 2H, 7=6.8 Hz), 1.8-1.9 (m, 2H), 1.5-1.7 (m, 6H), 1.2- 1.5 (m, 34H), 0.88 (t, 6H, 7=6.0 Hz)-146- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925General procedure for preparation of compound 6:A mixture of Compound 5 (2.87 g, 8.71 mmol, 1 eq.), Compound 4 (4.40 g, 8.71 mmol, 1 eq.), K2CO3 (2.41 g, 17.42 mmol, 2 eq.), Nal (130.55 mg, 870.97 pmol, 0.1 eq.) in ACN (28 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 80°C for 16 hours under N2 atmosphere. TLC (Dichloromethane: Methanol=8:l, Rl: Rf= 0.3, R2: Rf= 0.9, Pl: Rf= 0.6) indicated Compound 5 was consumed completely and one new spot formed. The residue was diluted with EA (30 mL). The combined organic layers were washed with NH4CI (30mL *3), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Dichloromethane: Methanol=100:l to 50:1) to give Compound 6 (5.9 g, 7.82 mmol, 29.94% yield, 100% purity) was obtained as a white solid.LCMS [M+l]+= 754.7'H NMR (400 MHz, CHLOROFORM-d) 5 = 4.69 (br t, J = 6.4 Hz, 1H), 4.27 - 4.01 (m, 4H), 3.91 - 3.61 (m, 2H), 3.20 - 2.40 (m, 6H), 2.30 (t, J= 7.6 Hz, 2H), 1.70 - 1.52 (m, 18H), 1.39 - 1.16 (m, 52H), 0.89 (t, J= 6.8 Hz, 9H)General procedure for the Compound 100 HCl-salt:-147- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925HCl / dioxane- >DOM, 25C;2hrsci-3 Compound 100To a solution of Compound 6 (5.9 g, 7.82 mmol, 1 eq.) in DCM (59 mL) was added HCl / dioxane (0.5 M, 46.94 mL, 3 eq.). The mixture was stirred at 20°C for 2 hours. The reaction mixture was concentrated under reduced pressure to give a residue. Then the residue was diluted with acetonitrile (5 mL) and H2O (80 mL). The mixture was lyophilized to give Compound 100 (5.01 g, 6.34 mmol, 81.00% yield, 100% purity, HC1) as yellow gum.LCMS [M+l]+= 754.7'H NMR (400 MHz, DMSO-de) 5 = 9.51 (br s, 1H), 5.30 (br s, 1H), 4.66 - 4.53 (m, 1H), 4.11 - 3.93 (m, 4H), 3.72 (br d, J = 3.0 Hz, 2H), 3.19 - 2.99 (m, 6H), 2.26 (br t, J=72 Hz, 2H), 1.68 - 1.47 (m, 14H), 1.34 - 1.18 (m, 52H), 0.94 - 0.76 (m, 9H)Example 5: In vivo hEPO expression levels in lipid nanoparticles with lipidoid compounds coformulations

[0330] The lipidoids described herein were also explored in LNP formulations in combination with a second lipidoid, Compound 903 (shown below). As shown, the lipidoids described herein modulate protein hEPO expression levels in LNPs comprising the lipidoid and Compound 903. Formulations were tested in which Compound 903 and the lipidoid were formulated in equivalent molar amounts.-148- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925

[0331] In this example, messenger RNA molecules encoding hEPO proteins were formulated in lipid nanoparticles for delivery in vivo. The lipid nanoparticle formulations were synthesized, comprising Compound 903: Lipidoid X: DSPC: cholesterol: DMG-PEG2k at 25:25:10:38.5:1.5 mol%. The lipid mixture in ethanol was mixed with hEPO mRNA in RNA acidifying buffer (10 mM citrate, pH 4) at an ionizable-lipid-nitrogen-to-RNA-phosphate ratio (N: P) of 6 using a microfluidic device (Precision NanoSystems, Inc.) at a combined flow rate of 10 mL / min (7.5 mL / min for aqueous buffer, RNA and 2.5 mL / min for ethanol, lipid mix). The resulting particles were neutralized by buffer exchange into a HEPES buffer solution (20mM HEPES pH 7.8, 50mM sodium chloride, 15% sucrose) via PD-10 desalting column. The neutralized particles were concentrated using 100 kDa AMICON® Ultra centrifugal filters and sterile filtered using 0.2 um syringe filters. Samples were characterized, then frozen at -30 °C for storage. Prior to dosing, samples were thawed at room temperature and diluted as needed. LNPs were characterized as in Example 3, and the measurements for each of the LNPs are shown in Table 2 below.

[0332] The in vivo studies were performed in C57BL / 6 female mice at 6 to 8 weeks weighing in at approximately 20 g. The LNPs formulated with different ionizable lipids (Compound 903 and Lipidoid X) at 0.2 mg / kg of hEPO mRNA were administered by tail vein injection and animals were euthanized at 6 h or 24 h postadministration for blood serum sample collection and liver tissue extraction. The hEPO levels from the samples were analyzed and cross-compared by enzyme-linked immunoassay (ELISA) according to manufacturer’s protocol and shown in Table 2 below.

[0333] Coformulations of the lipidoid with Compound 903 (lipidoid and second lipidoid) lead to sub-60 nm particle sizes for coformulations comprising lipidoids 1, 2, and 6, which were fully able to encapsulate LNPs. All coformulations demonstrated LNP size below 100 nm, and high encapsulation efficiency. Coformulations resulting sub- 60 nm particle size and formulations retaining favorable capsulation of mRNA, are shown in Table 2 below, and corresponding bar graphs are shown in Figure 3. The -149- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925lipidoid: Compound 903 coformulations were also screened by in vivo hEPO expression levels. Three of seven coformulations resulted in high hEPO expression levels.-150- FoleyHoagUS12883713.3Table 2. Lipidoid coformulations for in vivo studiesCompound 903: Lipidoid:Encapsulation Dose hEPO Lipidoid DSPC: Cholesterol: N: P ratio Size (nm) PDIEfficiency % (mg / kg) (ng / ml) DMG-PEG2k (mol%)(none) [control] 50: 0: 10: 38.5: 1.5 6 88.8 0.044 98.3 0.2 1182 + / - 76 Lipidoid 1 25: 25: 10: 38.5: 1.5 6 59.3 0.097 98.8 0.2 360 + / - 136 Lipidoid 2 25: 25: 10: 38.5: 1.5 6 59.0 0.083 99.0 0.2 217 + / - 154 Lipidoid 3 25: 25: 10: 38.5: 1.5 6 63.4 0.12 98.1 0.2 891 + / - 93 Lipidoid 6 25: 25: 10: 38.5: 1.5 6 55.9 0.083 98.6 0.2 880 + / - 152 Lipidoid 7 25: 25: 10: 38.5: 1.5 6 69.1 0.117 97.7 0.2 219 + / - 27 Lipidoid 10 25: 25: 10: 38.5: 1.5 6 61.9 0.063 99.2 0.2 223 + / - 11 Lipidoid 11 25: 25: 10: 38.5: 1.5 6 77.7 0.036 99.5 0.2 2058 + / - 349Attorney Docket No.: FAZ-00925Example 6: In vivo hEPO expression levels in livid nanoparticle formulations where a lipidoid replaces a helper lipid

[0334] The lipidoids described herein were explored in LNP formulations in combination with a second lipidoid, Compound 903 (shown below). As shown, the lipidoids described herein modulate protein hEPO expression levels in LNPs comprising the lipidoid, and a second lipioid, Compound 903. Formulations were tested in which the lipidoid serves as a replacement for the helper lipid (i.e., phospholipid) often used in LNP formulations (e.g., 10 mol% DSPC in LNP formulations was replaced with 10 mol% of one of lipidoids 1-12).O N OO.(Compound 903)

[0335] In this example, messenger RNA molecules encoding hEPO proteins were formulated in lipid nanoparticles for delivery in vivo. The lipid nanoparticle formulations were synthesized, comprising Compound 903: Lipidoid X: cholesterol: DMG-PEG2k at 50:10:38.5:1.5 mol%. The lipid mixture in ethanol was mixed with hEPO mRNA in RNA acidifying buffer (10 mM citrate, pH 4) at an ionizable- nitrogen-to-RNA-phosphate ratio (N: P) of 6 using a microfluidic device (Precision NanoSystems, Inc.) at a combined flow rate of 10 mL / min (7.5 mL / min for aqueous buffer, RNA and 2.5 mL / min for ethanol, lipid mix). The resulting particles were neutralized by buffer exchange into a HEPES buffer solution (20mM HEPES pH 7.8, 50mM sodium chloride, 15% sucrose) via PD-10 desalting column. The neutralized particles were concentrated using 100 kDa AMICON® Ultra centrifugal filters and sterile filtered using 0.2 um syringe filters. Samples were characterized, then frozen at -30 °C for storage. Prior to dosing, samples were thawed at room temperature and diluted as needed. LNPs were characterized as in Example 3, and the measurements for each of the LNPs are shown in Table 3 below.

[0336] The in vivo studies were performed in C57BL / 6 female mice at 6 to 8 weeks weighing in at approximately 20 g. The LNPs formulated with different ionizable lipids (Compound 903 and lipidoid X) at 0.2 mg / kg of hEPO mRNA were administered by tail vein injection and animals were euthanized at 6 h or 24 h post--152- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925administration for blood serum sample collection and liver tissue extraction. The hEPO levels from the samples were analyzed and cross-compared by enzyme-linked immunoassay (ELISA) according to manufacturer’s protocol and shown in Table 3 below.

[0337] Five of seven lipidoids tested as a helper lipid replacement (lipidoids 2, 6, 7, 10, and 11) demonstrated LNP size increase above 100 nm, while 2 lipidoids (lipidoids 1 and 3) retain LNP size below 100 nm. All LNPs showed high encapsulation efficiency. The formulations, resulting particle size and formulations retaining favorable encapsulation of mRNA are shown in Table 3 below, and corresponding bar graphs are shown in Figure 4. Tested LNPs were also screened for in vivo hEPO expression levels. LNPs comprising five of seven tested lipidoids resulted in high hEPO expression.-153- FoleyHoagUS12883713.3Table 3: LNP formulations where lipidoid replaces a helper lipid (10 mol %) for in vivo studiesCompound 903: Helper lipidHelper lipid or Encapsulation hEPO or replacement: Cholesterol: N: P ratio Size (nm) PDI Dose (mg / kg) replacement Efficiency % (ng / mL)DMG-PEG2k (mol%)DSPC (control) 50: 10: 38.5: 1.5 6 73.3 0.005 98.4 0.2 1182 + / - 76 Lipidoid 1 50: 10: 38.5: 1.5 6 97.7 0.035 99.1 0.2 3634 + / - 934 Lipidoid 2 50: 10: 38.5: 1.5 6 102.9 0.038 99.2 0.2 1559 + / - 374 Lipidoid 3 50: 10: 38.5: 1.5 6 96.8 0.055 99.0 0.2 699 + / - 116 Lipidoid 6 50: 10: 38.5: 1.5 6 102.2 0.012 99.1 0.2 1809 + / - 528 Lipidoid 7 50: 10: 38.5: 1.5 6 136.7 0.040 98.9 0.2 1971 + / - 304 Lipidoid 10 50: 10: 38.5: 1.5 6 106.7 0.019 99.2 0.2 4122 + / - 727 Lipidoid 11 50: 10: 38.5: 1.5 6 122.4 0.013 99.4 0.2 812 + / - 144Attorney Docket No.: FAZ-00925Example 7: In vivo hEPO expression levels in livid nanoparticle formulations where a lipidoid replaces a helper lipid at varied molar ratios

[0338] In this example, messenger RNA molecules encoding hEPO proteins were formulated in lipid nanoparticles for delivery in vivo. The lipid nanoparticle formulations were synthesized, comprising Compound 903: Lipidoid 1: cholesterol: DMG-PEG2k indicated in Table 4 (below). The lipid mixture in ethanol was mixed with hEPO mRNA in RNA acidifying buffer (10 mM citrate, pH 4) at the ionizable- lipid-nitrogen-to-RNA-phosphate ratio (N: P) of 6, using a microfluidic device (Precision NanoSystems, Inc.) at a combined flow rate of 10 mL / min (7.5 mL / min for aqueous buffer, hEPO mRNA and 2.5 mL / min for ethanol, lipid mix). The resulting particles were neutralized by buffer exchange into a HEPES buffer solution (20mM HEPES pH 7.8, 50mM sodium chloride, 15% sucrose) via PD-10 desalting column. The neutralized particles were concentrated using 100 kDa AMICON® Ultra centrifugal filters and sterile filtered using 0.2 um syringe filters. Samples were characterized, then frozen at -30 °C for storage. Prior to dosing, samples were thawed at room temperature and diluted as needed. LNPs were characterized as in example 3, and the measurements for each of the LNPs are shown in Table 4 below.

[0339] The in vivo studies were performed in C57BL / 6 female mice at 6 to 8 weeks weighing in at approximately 20 g. The LNPs formulated with different ionizable lipids (Table 4 Ionizable lipids (SM-102 or Compound 903) and Lipidoid 1) at 0.2 mg / kg of hEPO mRNA were administered by tail vein injection and animals were euthanized at 6 h or 24 h post-administration for blood serum sample collection and liver tissue extraction. The hEPO levels from the samples were analyzed and crosscompared by enzyme-linked immunoassay (ELISA) according to manufacturer’s protocol and shown in Table 4 below and in Figure 5.

[0340] Using lipidoid 1 as a replacement for helper lipid DSPC in an LNP composition increases protein expression by more than 5x relative to an LNP composition comprising SM-102 (control). Particularly high protein expression is observed for LNP formulations comprising 5% lipidoid 1 as a helper lipid replacement.-155- FoleyHoagUS12883713.3Table 4. LNP formulations using lipidoid as helper lipid replacement at varied molar ratiosIonizable lipid:Helper lipid or Helper lipid or ZetaN: P Encapsulation Apparent Dose hEPO Ionizable lipid ionizable ionizable replacement Size (nm) PDI potentialratio Efficiency % pKa (mg / kg) (ng / mL) replacement: Cholesterol: DMG- (mV)PEG2k (mol%)SM-102 DSPC 50: 10: 38.5: 1.5 6 73.7 0.084 -4.368 83.6 6.223 0.2 2431 + / -489 Compound 903 DSPC 50: 10: 38.5: 1.5 6 74.5 0.052 -3.711 88.1 6.226 0.2 981 + / - 23913815 + / - Compound 903 Lipidoid 1 (5%) 52.82: 5: 40.68: 1.5 6 99.7 0.025 -7.747 86.3 6.27 0.21359 Compound 903 Lipidoid 1 (10%) 50: 10: 38.5: 1.5 6 100.4 0.054 -0.9694 83.7 6.112 0.2 6777 + / - 682 Compound 903 Lipidoid 1 (15%) 47.18: 15: 36.32: 1.5 6 97.2 0.077 -5.348 83.5 6.171 0.2 3880 + / - 253 Compound 903 Lipidoid 1 (25%) 41.53: 25: 31.97: 1.5 6 96.4 0.013 -5.352 85.9 6.099 0.2 2121 + / - 320Attorney Docket No.: FAZ-00925Example 8: Dose escalation with cytokine profiling

[0341] In this example, messenger RNA molecules encoding hEPO proteins were formulated in lipid nanoparticles for delivery in vivo. The lipid nanoparticle formulations were synthesized, comprising Compound 903: Lipidoid X: cholesterol: DMG-PEG2k at 50:10:38.5:1.5 mol%. The lipid mixture in ethanol was mixed with hEPO mRNA in RNA acidifying buffer (10 mM citrate, pH 4) at an ionizable-lipid- nitrogen-to-RNA-phosphate ratio (N: P) of 6 using a microfluidic device (Precision NanoSystems, Inc.) at a combined flow rate of 10 mL / min (7.5 mL / min for aqueous buffer, RNA and 2.5 mL / min for ethanol, lipid mix). The resulting particles were neutralized by buffer exchange into a HEPES buffer solution (20mM HEPES pH 7.8, 50mM sodium chloride, 15% sucrose) via PD-10 desalting column. The neutralized particles were concentrated using 100 kDa AMICON® Ultra centrifugal filters and sterile filtered using 0.2 um syringe filters. Samples were characterized, then frozen at -30 °C for storage. Prior to dosing, samples were thawed at room temperature and diluted as needed. LNPs were characterized as in Example 3 and the measurements for each of the LNPs are shown in Table 5 below.

[0342] The in vivo studies were performed in C57BL / 6 female mice at 6 to 8 weeks weighing in at approximately 20 g. The LNPs formulated with different ionizable lipids (Compound 903 and Lipidoid X) at 2 mg / kg of hEPO mRNA were administered by tail vein injection and animals are euthanized at 6 h or 24 h postadministration for blood serum sample collection and liver tissue extraction. The hEPO levels from the samples were analyzed and cross-compared by enzyme-linked immunoassay (ELISA) according to manufacturer’s protocol and shown in Table 5 below.

[0343] For cytokine profiling of compounds used in Example 8, the tissue and blood samples were processed and analyzed using a custom ProcartaPlex kit in a Luminex 200. The kit is customized to detect and quantitate IFN-alpha, IFN-beta, IFN-gamma, IL-1 beta, IL-6, IL- 18, IL-22, IP- 10 (CXCL10), MCP-1 (CCL2), and TNF alpha in mouse serum. Cytokine levels for LNPs comprising Compound 903 and one of lipidoids 1, 2, 6, 7, and 10 as the helper lipid replacement were below the lower limit of quantification (LLOQ) for IFN-gamma, IL-1 beta, and TNF-alpha. Cytokine levels for LNPs comprising Compound 903 and one of lipidoids 1, 2, 6, 7, and 10 as the helper lipid replacement were lower than LNPs comprising SM-102 (control) for IFN--157- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925alpha, IL- 18, IP- 10, and MCP-10. Cytokine levels for LNPs comprising Compound 903 and one of lipidoids 2, 6, and 7 as the helper lipid replacement were lower than LNPs comprising SM-102 (control) for IFN-beta, IL-6, and IL-22.

[0344] Using a lipidoid as a replacement for the helper lipid in LNPs comprising Compound 903 (second lipidoid) improves tolerability of the LNP. LNPs comprising lipidoid 6 as helper lipid replacement demonstrated the lower cytokine response across the panel of lipidoids surveyed, and such LNPs with lipidoid 6 also high hEPO expression as shown in the Table 5 below and in Figure 6.-158- FoleyHoagUS12883713.3Table 5. LNP formulations using lipidoid as helper lipid replacement (10 mol%) in 2.0 mg / kg doseIonizable lipid:Helper lipid Helper lipid or ZetaIonizable N: P Size Encapsulation Apparent Dose hEPO or ionizable ionizable replacement: PDI potentiallipid ratio (nm) Efficiency % pKa (mg / kg) (ng / mL) replacement Cholesterol: (mV)DMG-PEG2k (mol%)28369 + / - SM-102 DSPC 50: 10: 38.5: 1.5 6 71.8 0.085 -1.665 86.2 6.146 2.03809 Compound 21220 + / - DSPC 50: 10: 38.5: 1.5 6 72.5 0.066 -4.668 91.3 6.102 2.0903 3045 Compound 64244 + / - Lipidoid 1 50: 10: 38.5: 1.5 6 100.1 0.105 -1.834 92.5 6.22 2.0 903 3485 Compound 64963 + / - Lipidoid 2 50: 10: 38.5: 1.5 6 101.3 0.053 -0.526 88.2 6.265 2.0 903 5753 Compound 86948 + / - Lipidoid 6 50: 10: 38.5: 1.5 6 103.6 0.057 -0.367 97.3 6.031 2.0 903 7390 Compound 61548 + / - Lipidoid 7 50: 10: 38.5: 1.5 6 117.7 0.026 -6.335 94.0 6.325 2.0 903 5556 Compound 14930 + / - Lipidoid 10 50: 10: 38.5: 1.5 6 105.5 0.095 -6.837 89.2 — 2.0 903 2181Attorney Docket No.: FAZ-00925Examyle 8: In vivo hEPO expression levels in livid nanoparticle formulations comyrisins varying combinations of ionizable liyids and ionizable helyer li id replacementsIn this example, messenger RNA molecules encoding hEPO proteins were formulated in LNPs for delivery in vivo. The lipid nanoparticle formulations comprise (i) ionizable lipid: lipidoid: cholesterol: DMG-PEG2k as indicated in Table 6 and 7, or (ii) ionizable lipid: lipidoid A: lipidoid B: cholesterol: DMG-PEG2k as indicated in Table 8 and 9. The lipid mixture in ethanol is mixed with hEPO mRNA in RNA acidifying buffer (10 mM citrate, pH 4) at a N-to-P ratio of 6 using a microfluidic device (Precision NanoSystems, Inc.) at a combined flow rate of 10 mL / min (7.5 mL / min for aqueous buffer, RNA and 2.5 mL / min for ethanol, lipid mix). The resulting particles are neutralized by buffer exchange into 20 mM HEPES (pH 7.8), 50 mM sodium chloride, 15% (w / v) sucrose solution via PD-10 desalting column. The neutralized particles are concentrated using 100 kDa AMICON® Ultra centrifugal filters and sterile-filtered using 0.2-um syringe filters. LNPs are then stored at -80C until use. Prior to in vivo dosing, LNPs are thawed at room temperature and diluted 2-fold with 20 mM HEPES (pH 7.8), resulting in a final dosing buffer of 20 mM HEPES (pH 7.8), 25 mM sodium chloride, 7.5% (w / v) sucrose. Resulting LNPs are characterized by dynamic light scattering (DLS) measurement to measure hydrodynamic diameter and polydispersity index (PDI) and the Zeta potential is measured using a Zetasizer (Malvern). Encapsulation efficiency (EE%) and total RNA concentration are quantitated using a fluorescence-based Ribogreen assay, and the apparent pKa of the LNPs is measured by a fluorescence-based TNS assay. The formulation and measurements for several studies are captured in the tables below.The in vivo studies described in Tables 6-9 are performed in C57BL / 6 female mice at 6 to 8 weeks weighing approximately 20 g. The LNPs formulated with different ionizable lipids at 0.2 mg / kg of hEPO mRNA and 0.2 mg / kg of fLuc mRNA are administered by tail vein injection. Animals are euthanized 6 h post- administration for blood serum sample collection. The hEPO levels in samples are analyzed and crosscompared by enzyme-linked immunoassay (ELISA) according to manufacturer’s protocol. The hEPO expression levels for the LNPs shown in Tables 6-9 include control LNPs comprising either SM-102 or ALC-0315 or (DC Chemicals Cat. No.: DC52025 and DC Chemicals Cat. No.: DC42537, respectively)-160- FoleyHoagUS12883713.3Table 6: LNP formulations using lipidoid as helper lipid replacement (10 mol%) in 0.2 mg / kg doseIonizable Lipid: Helperlipid orHeiner linid Encapsulation hEPO Ionizable Lipid1 1ionizable replacement: Size (nm) PDI pKaor ionizable,,, „ Efficiency % Expression Cholesterol: DMG- replacement PEG2k (mol % )SM-102 DSPC 50:10: 38.5: 1.5 63.2 0.142 88.4 6.332 1892±196 SM-102 Lipidoid 1 50: 10: 38.5: 1.5 74.2 0.089 89.9 6.33 1539±237 Compound 903 Lipidoid 1 50:10: 38.5: 1.5 78.4 0.121 92.5 6.33 3594±407 Compound 907 DSPC 50: 10: 38.5: 1.5 80.8 0.13 88.9 6.341 9672±909 Compound 943 DSPC 50: 10: 38.5: 1.5 64.6 0.102 88.7 6.261 1020±187 Compound 907 Lipidoid 1 50: 10: 38.5: 1.5 90.7 0.083 89.1 6.327 4402±834 Compound 907 Lipidoid 2 50: 10: 38.5: 1.5 86.9 0.103 87.2 6.252 5647 445 Compound 907 Lipidoid 5 50: 10: 38.5: 1.5 89.3 0.118 90.5 6.308 749111183 Compound 907 Lipidoid 6 50: 10: 38.5: 1.5 90.2 0.092 89.4 6.275 45901636 Compound 907 Lipidoid 7 50: 10: 38.5: 1.5 96.8 0.079 88.2 6.326 31341572Compound 907 Lipidoid 9 50: 10: 38.5: 1.5 86.4 0.117 90.1 6.316 8858 1424 Compound 907 Lipidoid 10 50: 10: 38.5: 1.5 91.2 0.049 92 6.286 902011150 Compound 943 Lipidoid 1 50: 10: 38.5: 1.5 83 0.122 95.5 6.318 613711063 Compound 943 Lipidoid 2 50: 10: 38.5: 1.5 79.7 0.101 93.1 6.309 22981324 Compound 943 Lipidoid 5 50: 10: 38.5: 1.5 84.7 0.054 85.6 6.242 896411431 Compound 943 Lipidoid 6 50: 10: 38.5: 1.5 83.5 0.096 87.8 6.243 598611309 Compound 943 Lipidoid 7 50: 10: 38.5: 1.5 91 0.0956.294 59121469 Compound 943 Lipidoid 9 50: 10: 38.5: 1.5 80.7 0.12 91.5 6.262 888511169 Compound 943 Lipidoid 10 50: 10: 38.5: 1.5 89.4 0.0616.241 77001783In this study, formulations using Lipidoid 5, 9 and 10 as a replacement for DSPC showed comparable expression when combined with Compound 907, as did formulations using Lipidoid 5, 9 and 10 as a replacement for DSPC when combined with Compound 943.Table 7: LNP formulations using lipidoid as helper lipid replacement in 0.2 mg / kg doseIonizable Lipid: HelperHelper lipid lipidEncapsulationIonizable Lipid or ionizable or ionizable replacement Size (nm) PDI hEPO Expression Efficiency %replacement: Cholesterol: DMG- PEG2k (mol%)SM-102 DSPC 50: 10: 38.5: 1.5 65.9 0.049 99 4007 ± 745 Compound 903 Lipidoid 1 52.82: 5: 40.68: 1.4 92.9 0.042 99.3 9150 ± 629 Compound 907 Lipidoid 1 52.82: 5: 40.68: 1.4 91.4 0.078 96.5 6923 ± 603 Compound 907 DSPC 50: 10: 38.5: 1.5 83.8 0.097 93.1 7762 ± 1097 Compound 903 DSPC 50: 10: 38.5: 1.5 72.5 0.027 99.1 4228 ± 346 Compound 903 Lipidoid 1 55.37: 0.5: 42.63: 1.5 105.6 0.075 98.4 11524 ± 1393 Compound 903 Lipidoid 1 54.8: 1.5: 42.2: 1.5 99.2 0.013 98.8 7412 ± 741 Compound 903 Lipidoid 1 53.95: 3: 41.55: 1.5 111.5 0.085 99.4 2503 ± 314 Compound 903 Lipidoid 1 50: 10: 38.5: 1.5 90.6 0.099 98.8 6568 ± 768 Compound 907 Lipidoid 1 55.37: 0.5: 42.63: 1.5 99.4 0.114 93.8 8063 ± 187Compound 907 Lipidoid 1 54.8: 1.5: 42.2: 1.5 97.7 0.053 95.8 8714 ± 1148 Compound 907 Lipidoid 1 53.95: 3: 41.55: 1.5 93.3 0.093 95.3 4977 ± 527Compound 907 Lipidoid 1 50: 10: 38.5: 1.5 92.1 0.041 94.2 5926 ± 312In this study, formulations using Lipidoid 1 at 0.5%, 1.5%, 5 % and 10% in combination with Compound 903 showed greater expression compared to formulations with Compound 903 and DSPC. Lipidoid 1 at 0.5%, 1.5%, 5 % and 10% in combination with Compound 903 showed comparable expression compared to formulations with Compound 907 and DSPC.Table 8: LNP formulations using lipidoid (i) as helper lipid replacement or (ii) in addition to phospholipid helper lipid in 0.2 mg / kg doseIonizable Lipid:Helper lipidor ionizableHelper lipid Helper lipidreplacement A:or ionizable or ionizable Size EncapsulationIonizable Lipid Helper lipid PDI pKa hEPO Expression replacement replacement (nm) Efficiency %or ionizableA Breplacement B:Cholesterol: DMG- PEG2k (mol%)SM-102 DSPC - 50: 10: 0: 38.5: 1.5 81.3 0.097 98.7 6.314 1809 ± 469 Compound 907 DSPC - 50: 10: 0: 38.5: 1.5 102.3 0.116 94.8 6.318 7967 ± 1900 ALC-0315 DSPC - 50: 10: 0: 38.5: 1.5 79.3 0.06 90.1 6.101 2592 ± 666 Compound 903 - - 55.65: 0: 0: 42.85: 1.5 108.7 0.036 99 6.295 11492 ± 1921 Compound 903 - - 65.2: 0: 0: 33.3: 1.5 156.1 0.045 97 6.332 323 ± 60 Compound 903 Lipidoid 1 - 50: 10: 0: 38.5: 1.5 96.8 0.083 99.2 6.314 6515 ± 859Compound 903 Lipidoid 1 52.82: 5: 0: 40.68: 1.5 98.9 0.074 99.2 6.316 11496 ± 2312 Compound 903 Lipidoid 1 54.8: 1.5: 0: 42.2: 1.5 105.1 0.125 99.2 6.347 7509 ± 82155.37: 0.5: 0: 42.63:Compound 903 Lipidoid 1 99.4 0.093 99.1 6.297 10815 ± 9181.5Compound 903 DSPC - 50: 10: 0: 38.5: 1.5 71.7 0.042 98.9 6.181 4288 ± 939 Compound 903 DSPC - 52.82: 5: 0: 40.68: 1.5 81.7 0.038 99.1 6.262 13628 ± 1912 Compound 903 DSPC 54.8: 1.5: 0: 42.2: 1.5 105.5 0.051 98.7 6.29 3893 ± 51755.37: 0.5: 0: 42.63:Compound 903 DSPC 99.7 0.11 99 6.339 10375 ± 7881.5Compound 903 Lipidoid 1 DSPC 50: 0.5: 9.5: 38.5: 1.6 69.9 0.053 98.7 6.194 5071 ± 1023 Compound 903 Lipidoid 1 DSPC 50: 5: 5: 38.5: 1.5 83.3 0.047 99.1 6.303 8273 ± 1896 Compound 903 Lipidoid 1 DSPC 50: 9.5: 0.5: 38.5: 1.5 93.4 0.038 98.7 6.339 8273 ± 1051 In this study, formulations with Compound 903 and Lipidoid 1 at 5% and 0.5% showed comparable expression to Compound 903 formulations with no lipidoid as helper and with Compound 903 and DSPC at 5% and 0.5%.Table 9: LNP formulations using lipidoid as a helper lipid in addition to phospholipid helper lipid in 0.2 mg / kg dose Ionizable Lipid: DSPC:Ionizable ionizable replacementSize EncapsulationIonizable Lipid DSPC replacement helper lipid: PDI hEPO (nm) Efficiency %helper lipid Cholesterol: DMG- Expression PEG2k (mol%)SM-102 DSPC - 50: 10: 0: 38.5: 1.5 76 76 76.0 2885 ± 414 ALC-0315 DSPC - 50: 10: 0: 38.5: 1.5 71.5 71.5 71.5 4888 ± 915 Compound 917f DSPC - 50: 10: 0: 38.5: 1.5 73.8 73.8 73.8 2245 ± 213 Compound 917f DSPC Lipidoid 1 50: 5: 5: 38.5: 1.5 80.9 80.9 80.9 4120 ± 576 Compound 917f DSPC Lipidoid 1 45: 10: 5: 38.5: 1.5 66.3 66.3 66.3 1810 ± 428 Compound 917e DSPC - 50: 10: 0: 38.5: 1.5 71.1 71.1 71.1 5268 ± 479 Compound 917e DSPC Lipidoid 1 50: 5: 5: 38.5: 1.5 76.6 76.6 76.6 4237 ± 584 Compound 917e DSPC Lipidoid 1 45: 10: 5: 38.5: 1.5 62.7 62.7 62.7 1634 ± 243 Compound 903 DSPC - 50: 10: 0: 38.5: 1.5 72.7 72.7 72.7 1163 ± 151 Compound 903 DSPC Lipidoid 1 50: 5: 5: 38.5: 1.5 81.8 81.8 81.8 916 ± 143 Compound 903 DSPC Lipidoid 1 45: 10: 5: 38.5: 1.5 64.9 64.9 64.9 1467 ± 207Compound 902 DSPC 50:10:0:38.5:1.5 77.1 77.1 77.1 269 ± 61 Compound 902 DSPC Lipidoid 1 50:5:5:38.5:1.5 77.4 77.4 77.4 1385 ± 214 Compound 902 DSPC Lipidoid l 45:10:5:38.5:1.5 71.6 71.6 71.6 1068 ± 221 Compound 907 DSPC 50:10:0:38.5:1.5 89.7 89.7 89.7 2502 ± 209 Compound 907 DSPC Lipidoid 1 50:5:5:38.5:1.5 91.3 91.3 91.3 4597 ± 931 Compound 907 DSPC Lipidoid l 45:10:5:38.5:1.5 80.2 80.2 80.2 2095 ± 258 Compound 906 DSPC 50:10:0:38.5:1.5 98.7 98.7 98.7 7786 ± 1598 Compound 906 DSPC Lipidoid 1 50:5:5:38.5:1.5 96.8 96.8 96.8 1181 ± 354 Compound 906 DSPC Lipidoid l 45:10:5:38.5:1.5 93 93 93 3295 ± 687 Compound 901 DSPC 50:10:0:38.5:1.5 93.2 93.2 93.2 904 ± 152 Compound 901 DSPC Lipidoid 1 50:5:5:38.5:1.5 81.6 81.6 81.6 1444 ± 173 Compound 901 DSPC Lipidoid l 45:10:5:38.5:1.5 94.6 94.6 94.6 656 ± 132 Compound 100 DSPC 50:10:0:38.5:1.5 78.5 78.5 78.5 1157 ± 132 Compound 100 DSPC Lipidoid 1 50:5:5:38.5:1.5 73.2 73.2 73.2 801 ± 1105 Compound 100 DSPC Lipidoid l 45:10:5:38.5:1.5 73.6 73.6 73.6 282 ± 84Attorney Docket No.: FAZ-00925In this study, formulations with Compound 917f and both DSPC and Lipidoid 1 as helper lipids showed comparable expression to formulations with Compound 917f and DSPC only. The same trend was true for Compound 902 and 903.INCORPORATION BY REFERENCE

[0345] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.EQUIVALENTS

[0346] While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.-169- FoleyHoagUS12883713.3

Claims

Attorney Docket No.: FAZ-00925CLAIMSWe claim:

1. A lipid nanoparticle, comprising a lipidoid or a salt thereof and a second lipidoid or a salt thereof; wherein:the lipidoid is selected from:-170- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-171- FoleyHoagUS 12883713.3Attorney Docket No.: FAZ-00925-172- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925the second lipidoid has the structure of formula (K):° (K);or a salt thereof;wherein:RA1and RA2are each independently methyl or ethyl;RBand RB1are each independently H or methyl; andR1and R2are each independently branched (C8-C30)alkyl; wherein a branched (C8-C30)alkyl may comprise more than one branch point.

2. A lipid nanoparticle, comprising a lipidoid or a salt thereof, wherein the lipidoid is selected from:-173- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-174- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-175- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-009253. The lipid nanoparticle of claim 2, wherein the lipidoid is selected from:-176- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925-177- FoleyHoagUS 12883713.3Attorney Docket No.: FAZ-009254. The lipid nanoparticle of claim 2 or 3, wherein the lipidoid is selected from:

5. The lipid nanoparticle of claim 2 or 3, wherein the lipidoid is selected from:-178- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-009256. The lipid nanoparticle of any one of claims 1-5, further comprising a PEGylated lipid, a sterol, and / or a phospholipid.

7. The lipid nanoparticle of claim 6, wherein the total lipid component of the lipid nanoparticle comprises:about 2-100 mol% of the lipidoid,about 0-50 mol% phospholipid,about 0-70 mol% sterol, andabout 0-10 mol% PEGylated lipid.

8. The lipid nanoparticle of claim 7, wherein the total lipid component of the lipid nanoparticle comprises:about 5-60 mol% of the lipidoid,about 0-30 mol% phospholipid,about 15-60 mol% sterol, andabout 0-10 mol% PEGylated lipid.

9. The lipid nanoparticle of claim 8, wherein the total lipid component of the lipid-179- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925nanoparticle comprises:about 5-30 mol% of the lipidoid,about 0-20 mol% phospholipid,about 25-50 mol% sterol, andabout 0.5-5 mol% PEGylated lipid.

10. The lipid nanoparticle of any one of claims 2-9, further comprising an ionizable lipid.

11. The lipid nanoparticle of claim 10, wherein the ionizable lipid is a second lipidoid.

12. The lipid nanoparticle of claim 11, wherein the second lipidoid has the structure of formula (I):nA2 _ _AZ*NXR\ / RH 1X A / L11 1-Y1-R1RAI W NL2'Y2-R2(I);or a salt thereof;wherein:RA1and RA2are each independently H, alkyl, or hydroxyalkyl;or wherein RA1and RA2, taken together with the intervening nitrogen, form an optionally substituted heterocyclic ring;RBand RB1are each independently H, optionally substituted alkyl, alkenyl, cycloalkyl, arylalkyl, heteroarylalkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, or thioalkyl;or wherein RBand RB1, taken together with the intervening carbon, form an optionally substituted cycloalkyl or heterocyclic ring;or RA1and RBtaken together with the intervening N-CH atoms to which they are attached form an N-containing heterocyclic ring;L1and L2are each independently -CH2CH2-, -(CH2)3-, -(CH2)4-, -CH(CH3)CH2-, or -CH2CH(CH3)-;X is a bond, -CH2-, or -CH2CH2-;W is a bond, -CH2-, or -CH2CH2-;-180- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925Y1and Y2are each independently -O(C=O)-, -S(C=O)-, or -O(C=O)O-; and R1and R2are each independently linear (Cs-C3o)alkyl, branched (Cs-C3o)alkyl, linear (Cs-C3o)alkenyl, branched (Cs-C3o)alkenyl, linear (Cs-C3o)alkynyl, or branched (Cs-C3o)alkynyl; wherein a branched (Cs-C3o)alkyl, a branched (Cs-C3o)alkenyl, or a branched (Cs-C3o)alkynyl may comprise more than one branch point, and a linear or branched (Cs-C3o)alkenyl may comprise more than one olefin, and a linear or branched (Cs-C3o)alkynyl may comprise more than one carbon-carbon triple bond.

13. The lipid nanoparticle of claim 12, wherein the second lipidoid is selected Table A.

14. The lipid nanoparticle of claim 10 or 11, wherein the second lipidoid has the structure of formula (II):R3ixY3RA3RB2 RB2AOL3N / -LXY^rA4' LT V"^ N X4R4RH(ii);or a salt thereof;wherein:RA3and RA4are each independently H, alkyl, or hydroxyalkyl;or wherein RA3and RA4, taken together with the intervening nitrogen, form an optionally substituted heterocycloalkyl ring;RB2and RB2Aare each independently H, optionally substituted alkyl, alkenyl, cycloalkyl, arylalkyl, heteroarylalkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, or thioalkyl;or wherein RB2and RB2A, taken together with the intervening carbon, form an optionally substituted cycloalkyl or heterocycloalkyl ring; or RA3and RB2taken together with the intervening N-CH atoms to which they are attached form an N-containing heterocyclic ring;L3and L4are each independently -CH2-, -CH2CH2-, -(CH2)3-, -(CH2)4-, -CH(CH3)CH2-, or -CH2CH(CH3)-;V is a bond, -CH2- or -CH2CH2-;-181- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925U is a bond, -CH2- or -CH2CH2-;Y3and Y4are each independently -O(C=O)-, -S(C=O)-, or -O(C=O)O-; and R3and R4are each independently linear (Cs-C3o)alkyl, branched (Cs-C3o)alkyl, linear (Cs-C3o)alkenyl, branched (Cs-C3o)alkenyl, linear (Cs-C3o)alkynyl, or branched (Cs-C3o)alkynyl; wherein a branched (Cs-C3o)alkyl, a branched (Cs-C3o)alkenyl, or a branched (Cs-C3o)alkynyl may comprise more than one branch point, and a linear or branched (Cs-C3o)alkenyl may comprise more than one olefin, and a linear or branched (Cs-C3o)alkynyl may comprise more than one carbon-carbon triple bond.

15. The lipid nanoparticle of claim 14, wherein the second lipidoid is selected from the Table B.

16. The lipid nanoparticle of any one of claims 1 and 10-15, wherein the lipid nanoparticle comprises the lipidoid, the second lipidoid, a PEGylated lipid, and a sterol.

17. The lipid nanoparticle of any one of claims 1 and 10-16, wherein the lipid nanoparticle is substantially free of a phospholipid.

18. The lipid nanoparticle of claim 17, wherein the lipid nanoparticle is substantially free of distearolyphosphatidy choline (DSPC).

19. The lipid nanoparticle of any one of claims 1 and 10-18, wherein the total lipid component of the lipid nanoparticle comprises:about 1-50 mol% of the lipidoid,about 10-60 mol% second lipidoid,about 5-50 mol% sterol, andabout 0-10 mol% PEGylated lipid.

20. The lipid nanoparticle of any one of claims 1 and 10-19, wherein the total lipid component of the lipid nanoparticle comprises:about 1-40 mol% of the lipidoid,-182- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925about 30-60 mol% second lipidoid,about 15-45 mol% sterol, andabout 0-10 mol% PEGylated lipid.

21. The lipid nanoparticle of any one of claims 1 and 10-20, wherein the total lipid component of the lipid nanoparticle comprises:about 1-30 mol% of the lipidoid,about 40-60 mol% second lipidoid,about 25-45 mol% sterol, andabout 0.5-5 mol% PEGylated lipid.

22. The lipid nanoparticle of any one of claims 1 and 10-21, wherein the total lipid component of the lipid nanoparticle comprises:about 5-20 mol% of the lipidoid,about 40-60 mol% second lipidoid,about 25-45 mol% sterol, andabout 0.5-5 mol% PEGylated lipid.

23. The lipid nanoparticle of any one of claims 1 and 10-22, wherein the total lipid component of the lipid nanoparticle comprises:about 5-15 mol% of the lipidoid,about 40-60 mol% second lipidoid,about 25-45 mol% sterol, andabout 0.5-5 mol% PEGylated lipid.

24. The lipid nanoparticle of any one of claims 1 and 10-15, wherein the lipid nanoparticle comprises the lipidoid, the second lipidoid, a PEGylated lipid, a sterol, and a phospholipid.

25. The lipid nanoparticle of any one of claims 1 and 10-15 and 24, wherein the total lipid component of the lipid nanoparticle comprises:about 1-50 mol% of the lipidoid,about 1-50 mol% second lipidoid,-183- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925about 1-25 mol% of the phospholipid;about 5-50 mol% sterol, andabout 0-10 mol% PEGylated lipid.

26. The lipid nanoparticle of any one of claims 1 and 10-15 and 24-25, wherein the total lipid component of the lipid nanoparticle comprises:about 10-40 mol% of the lipidoid,about 10-40 mol% second lipidoid,about 5-20 mol% of the phospholipid;about 15-45 mol% sterol, andabout 0-10 mol% PEGylated lipid.

27. The lipid nanoparticle of any one of claims 1 and 10-15 and 24-26, wherein the total lipid component of the lipid nanoparticle comprises:about 20-30 mol% of the lipidoid,about 20-30 mol% second lipidoid,about 5-20 mol% of the phospholipid;about 25-45 mol% sterol, andabout 0.5-5 mol% PEGylated lipid.

28. The lipid nanoparticle of any one of claims 1 and 10-18, wherein the lipidoid and the second lipidoid are in a ratio of about 1:10 to about 10:1.

29. The lipid nanoparticle of any one of claims 1 and 10-18, wherein the lipidoid and the second lipidoid are in a ratio of about 1:5 to about 5:1.

30. The lipid nanoparticle of any one of claims 1 and 10-18, wherein the lipidoid and the second lipidoid are in a ratio of about 1:5 to about 1:1.5.

31. The lipid nanoparticle of any one of claims 1 and 10-18, wherein the lipidoid and the second lipidoid are in a ratio of about 1:1.

32. The lipid nanoparticle of any one of claims 1-31, further comprising a pay load.-184- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-0092533. The lipid nanoparticle of claim 32, wherein the pay load is a therapeutic agent.

34. The lipid nanoparticle of claim 33, wherein the therapeutic agent is a nucleic acid molecule.

35. The lipid nanoparticle of claim 34, wherein the nucleic acid molecule is a plasmid, an immunostimulatory oligonucleotide, an antisense oligonucleotide, an antagomir, an aptamer, a deoxyribozyme (DNAzyme), or a ribozyme.

36. The lipid nanoparticle of claim 34, wherein the nucleic acid molecule is DNA or RNA.

37. The lipid nanoparticle of claim 36, wherein the nucleic acid molecule is DNA; and the DNA is a linear DNA, circular DNA, single stranded DNA, or double stranded DNA.

38. The lipid nanoparticle of claim 36, wherein the nucleic acid molecule is RNA; and the RNA is messenger RNA (mRNA), miRNA, siRNA or siRNA precursor, RNA aptamer, linear RNA, circular RNA, single stranded RNA, double stranded RNA, tRNA, microRNA (miRNA) or miRNA precursor, Dicer substrate small interfering RNA (dsiRNA), Dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), guide RNA (gRNA), IncRNA, ncRNA, sncRNA, rRNA, snRNA, piRNA, snoRNA, snRNA, scaRNA, exRNA, scaRNA, Y RNA, or hnRNA.

39. The lipid nanoparticle of claim 38, wherein the RNA is mRNA.

40. The lipid nanoparticle of claim 36, wherein the DNA or RNA encodes a polypeptide.

41. The lipid nanoparticle of any one of claims 34-40, wherein the nucleic acid molecule comprises one or more nucleic acid analogs selected from the group-185- FoleyHoagUS12883713.3Attorney Docket No.: FAZ-00925consisting of a phosphoramide, a phosphorothioate, a phosphorodithioate, an O- methylphosphoroamidate, a morpholino, a locked nucleic acid (LNA), a glycerol nucleic acid (GNA), a threose nucleic acid (TNA), and a peptide nucleic acid (PNA).

42. The lipid nanoparticle of claim 33, wherein the therapeutic agent is a protein or small molecule drug.

43. The lipid nanoparticle of any one of claims 1-31, further comprising an antigen.

44. The lipid nanoparticle of claim 43, wherein the antigen is a protein or a nucleic acid.

45. The lipid nanoparticle of claim 44, wherein the antigen is a protein.

46. The lipid nanoparticle of claim 44, wherein the antigen is a nucleic acid.

47. The lipid nanoparticle of any one of claims 1-31, wherein the lipid nanoparticle comprises an mRNA molecule comprising a nucleotide sequence encoding an antigen.

48. A pharmaceutical composition, comprising a lipid nanoparticle of any one of claims 1-45, and a pharmaceutically acceptable excipient.

49. A method of delivering a therapeutic agent, comprising administering to a subject in need thereof an effective amount of the lipid nanoparticle of any one of claims 33-42.-186- FoleyHoagUS12883713.3

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