Unsaturated ionizable lipids and nanoparticle compositions thereof

Abstract

Novel unsaturated ionizable lipids are provided. Also provided are novel lipid nanoparticle compositions comprising novel unsaturated ionizable lipids for the delivery of nucleic acid material to cells in vitro and in vivo with different and improved pharmacokinetic profiles as compared to what is typically observed in the art. Also provided are methods for using the compositions in research and as therapeutics.

Description

UNSATURATED IONIZABLE LIPIDS AND NANOPARTICLE COMPOSITIONS THEREOF 1. BACKGROUND

[0001] There are many instances in which delivery of a nucleic acid is desired, where such instances include research, diagnostic and therapeutic applications. An example of such a therapeutic application is gene therapy, which can be used to treat genetic disorders and other conditions. Genetic disorders, although individually rare, collectively represent a significant disease burden, particularly for children, resulting in substantial disability and mortality.

[0002] In the field of gene therapy, viral vectors, such as vectors based on AAV, are commonly employed to deliver genes into cells. However, AAV vectors are limited in the size of genetic cargo that can be packaged. Accordingly, any genetic cargo greater than 4.7kB is not suitable for delivery with AAV vectors, which limits the utility of such vectors for many indications. In addition, viral vectors, such as AAV, induce an antibody response, limiting redosing, which is not suitable for some indications. Moreover, in indications where the target cells are dividing, such as the liver, expression from successfully transduced cells can be reduced or lost with cell division and turnover, requiring redosing – which may not be possible or effective due to immune memory. In addition, many subjects have pre-existing immunity to commonly used viral vectors such as AAV, which can limit even initial treatment with an AAV gene therapy. Furthermore, viral vectors such as AAV can be toxic at the doses that would be required to achieve therapeutic benefit in some indications.

[0003] Lipid nanoparticles (LNPs) provide an alternative to viral gene therapy. While lipid nanoparticles have been developed and employed for delivery of many RNA therapeutics, lipid-nanoparticle-delivered RNA has limited therapeutic longevity. DNA delivered by lipid nanoparticles designed for RNA delivery suffers from poor efficiency and significant innate immune response activation in treated subjects. New delivery vehicles for delivering nucleic acids, such as DNA, to cells, stand to significantly advance numerous scientific pursuits, particularly in vivo for patients in need of therapy but also for research applications. 2. SUMMARY

[0004] Novel unsaturated ionizable lipids are provided. Also provided are novel lipid nanoparticle compositions comprising novel unsaturated ionizable lipids for the delivery ofnucleic acid material to cells in vitro and in vivo with different and improved pharmacokinetic profiles as compared to what is typically observed in the art. Also provided are methods for using the compositions in research and as therapeutics.

[0005] In some embodiments, the present disclosure provides preparations and uses of a compound of Formula I:Formula I or a pharmaceutically acceptable salt thereof; wherein: Zaand Zbis each independently H or an optionally substituted ionizable group; Laand Lbis each independently a bond or an optionally substituted, saturated or partially unsaturated, straight or branched divalent aliphatic C1-12hydrocarbon chain, wherein one or more carbon atoms of the chain are optionally replaced by a divalent group independently selected from -O-, -NRe-, -S-, -S(O)-, -S(NRe)-, - S(O)2-, -S(O)(NRe)-, -S(NRe)2-, and -C(O)-; each Reis independently hydrogen or an optionally substituted C1-6aliphatic group; Wnis an optionally substituted linear alkylene of n carbon atoms, wherein n is 2 to 6; Wmis an optionally substituted linear alkylene of m carbon atoms, wherein m is 2 to 6; p is an integer from 1 to 3; q is an integer from 1 to 3; each Xaand Xbis independently a bond or an optionally substituted, saturated or partially unsaturated, straight or branched divalent aliphatic C1-20hydrocarbon chain, wherein one or more carbon atoms of the chain are optionally replaced by a divalent group independently selected from -O-, -NRe-, -S-, -S(O)-, -S(NRe)-, - S(O)2-, -S(O)(NRe)-, -S(NRe)2-, and -C(O)-; each Raand Rbis independently H or an optionally substituted, saturated or partially unsaturated, straight or branched aliphatic C1-50 hydrocarbon group, wherein one or more carbon atoms of the chain are optionally replaced by a divalent groupindependently selected from -O-, -S-, -S(O)-, -S(NRe)-, -S(O)2-, -S(O)(NRe)-, -S(NRe)2-, -C(O)- and -CyA-; and each CyAindependently comprises an optionally substituted ring system selected from a 3- to 12-membered saturated or partially unsaturated carbocyclene; a phenylene; a 3- to 12-membered saturated or partially unsaturated heterocyclene having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; a 5- to 6- membered heteroarylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a combination of any of these ring systems including fused variants thereof. 3. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description, and accompanying drawings, where:

[0007] FIGURES 1A-1D provide the results of an analysis of the safety and potency of LNPs formulated with ionizable lipid U-1 of the disclosure and a DNA encoding EPO, following intravenous administration to adult wild type BALB / c mice at a dose of 0.3 mg / kg DNA or 1.0 mg / kg DNA. FIG.1A provides the physical characteristics of the LNP comprising U-1 and of LNPs comprising select ionizable lipids from the art (L-17, L-21, ALC0315, or ARCT) for comparison. FIG.1B provides the concentration of IL-6 detected in serum 4 hours post-dosing. FIG.1C and 1D provide the concentration of erythropoietin (EPO) detected in serum 3 days (FIG.1C) and 7 days (FIG.1D) post-dosing.

[0008] FIGURES 2A-2D provide the results of an analysis of the potency and safety of LNPs formulated with the ionizable lipid U-3 of the disclosure and a DNA encoding EPO, following intravenous administration to adult wild type BALB / c mice at a dose of 0.3 mg / kg DNA or 1.0 mg / kg DNA. FIG.2A provides the physical characteristics of the LNP comprising U-3 and of LNPs comprising select ionizable lipids from the art (L-19, L-20, ALC-0315, or CL1) for comparison. FIG.2B and FIG.2C provide the concentration of EPO detected in serum 3 days (FIG.2B) and 7 days (FIG.2C) post-dosing. FIG.2D provides the concentration of IL-6 detected in serum 4 hours post-dosing.

[0009] FIGURES 3A-3L provide the results of an analysis of the potency and safety of LNPs formulated with ionizable lipid U-2 of the disclosure and a DNA encoding human Factor IX (FIX), following intravenous administration to adult wild type BALB / c mice at adose of 0.5 mg / kg DNA. FIG.3A provides the physical characteristics of the LNP comprising U-2 and of LNPs comprising select ionizable lipids from the art (L-15, L-17, L- 18, or CL1). FIG.3B and FIG.3C provide the concentration of FIX detected in serum 7 days (FIG.3B) and 14 days (FIG.3C) post-dosing, reported as a percentage of what would normally be detected in normal humans (100% of normal human FIX levels being approximately 5 µg / ml of FIX protein). FIGs.3D-3L provide the concentrations of IFNα (FIG.3D), IFNβ (FIG.3E), MIP-1α (FIG.3F), MCP-1 (FIG.3G), IP-10 (FIG.3H), TNFα (FIG.3I), IL-6 (FIG.3J), IL-1β (FIG.3K), and IFNγ (FIG.3L) in serum 4 hours post-dosing.

[0010] FIGURES 4A-4D provide the results of an analysis of the potency and safety of LNPs formulated with ionizable lipid U-4 of the disclosure and a DNA encoding human factor IX (FIX), following intravenous administration to adult wild type BALB / c mice at a dose of 0.5 mg / kg DNA. FIG 4A provides the physical characteristics of the LNP comprising U-4 and of LNPs comprising select ionizable lipids from the art (L-15, L18, CL1, A6, A9, or ARCT). FIGs.4B and 4C provide the concentration of FIX detected in serum 7 days (FIG.4B) and 14 days (FIG.4C) post-dosing. FIG.4D provides the concentration of IL- 6 detected in serum 4 hours post-dosing.

[0011] FIGURES 5A-5B provide the results of an analysis of the potency of LNPs formulated with ionizable lipids U-2, U-3, and U-4. FIG 5A provides the physical characteristics of the LNPs comprising U-2, U-3, and U-4. FIG 5B provides the concentration of human factor IX (FIX) detected in serum 7 days after intravenous administration to adult wild type BALB / c mice at a dose of 0.3 mg / kg DNA of LNPs formulated with ionizable lipid U-2, U-3 or U-4, where U-4 is formulated with various helper lipids. 4. DETAILED DESCRIPTION 4.1. Definitions

[0012] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of suchrecitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0013] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0014] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken downinto at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0015] The terms "individual," "subject" and "host" are used interchangeably herein and refer to any subject for whom diagnosis, treatment or therapy is desired. In some aspects, the subject is a mammal. In some aspects, the subject is a human being. In some aspects, the subject is a patient. In some aspects, the subject is a human patient. In some aspects, the subject can have or is suspected of having a disorder or health condition associated with a gene-of-interest (GOI). In some aspects, the subject is a human who is diagnosed with a risk of disorder or health condition associated with a GOI at the time of diagnosis or later. In some cases, the diagnosis with a risk of disorder or health condition associated with a GOI can be determined based on the presence of one or more mutations in the endogenous GOI or genomic sequence near the GOI in the genome that may affect the expression of GOI.

[0016] As used herein, the phrase “therapeutic agent” refers to any agent that has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect, when administered to a subject.

[0017] The term "treatment", when used referring to a disease or condition, means an attenuation of the symptoms associated with the condition afflicting an individual is achieved, where attenuation is used in a broad sense to refer to the stabilization or reduction in the magnitude of a parameter, e.g., a symptom, associated with the condition (e.g., hemophilia A) being treated. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or eliminated entirely such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition. Thus, treatment includes: (i) prevention, that is, reducing the risk of development of clinical symptoms, including causing the clinical symptoms not to develop, e.g., preventing disease progression; (ii) inhibition, that is, arresting the development or further development of clinical symptoms,e.g., reducing the rate of progression of disease; (iii) mitigation, that is, reducing the symptoms of, or completely inhibiting disease.

[0018] The terms "effective amount," "pharmaceutically effective amount," or "therapeutically effective amount" as used herein mean a sufficient amount of the composition to provide the desired utility when administered to a subject having a particular condition. The term "therapeutically effective amount" therefore refers to an amount of therapeutic cells or a composition having therapeutic cells that is sufficient to promote a particular effect when administered to a subject in need of treatment. An effective amount would also include an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using routine experimentation.

[0019] The term "pharmaceutically acceptable excipient" as used herein refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive or diluent for administration of a compound(s) of interest to a subject. "Pharmaceutically acceptable excipient" can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers.

[0020] As used herein, a "pharmaceutical composition" is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human. In general a “pharmaceutical composition” is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade). Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intracheal, intramuscular, subcutaneous, and the like.

[0021] As used herein, the phrase "having the formula" or "having the structure" is not intended to be limiting and is used in the same way that the term "comprising" is commonly used. The term "independently selected from" is used herein to indicate that the recited elements, e.g., R groups or the like, can be identical or different.

[0022] As used herein, the terms “may,” “optional," "optionally," or “may optionally” mean that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not. For example, the phrase "optionally substituted" means that a non-hydrogen substituent mayor may not be present on a given atom, and, thus, the description includes structures wherein a non-hydrogen substituent is present and structures wherein a non-hydrogen substituent is not present.

[0023] “Acyl” refers to the groups H-C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. For example, acyl includes the “acetyl” group CH3C(O)-.

[0024] The term "alkyl" refers to a branched or unbranched saturated hydrocarbon group (i.e., a mono-radical) typically although not necessarily containing 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, as well as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like. Generally, although not necessarily, alkyl groups herein may contain 1 to about 18 carbon atoms, and such groups may contain 1 to about 12 carbon atoms. The term "lower alkyl" intends an alkyl group of 1 to 6 carbon atoms. "Substituted alkyl" refers to alkyl substituted with one or more substituent groups, and this includes instances wherein two hydrogen atoms from the same carbon atom in an alkyl substituent are replaced, such as in a carbonyl group (i.e., a substituted alkyl group may include a -C(=O)- moiety). The terms "heteroatom-containing alkyl" and "heteroalkyl" refer to an alkyl substituent in which at least one carbon atom is replaced with a heteroatom, as described in further detail infra. If not otherwise indicated, the terms "alkyl" and "lower alkyl" include linear, branched, cyclic, unsubstituted, substituted, and / or heteroatom-containing alkyl or lower alkyl, respectively.

[0025] The term “substituted alkyl” is meant to include an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain have been optionally replaced with a heteroatom such as -O-, -N-, -S-, -S(O)n- (where n is 0 to 2), -NR- (where R is hydrogen or alkyl) and having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, - SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NRaRb, wherein R’ and R” may be the same or different and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.

[0026] The term "alkenyl" refers to a linear, branched or cyclic hydrocarbon group of 2 to about 24 carbon atoms containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, and the like. Generally, although again not necessarily, alkenyl groups herein may contain 2 to about 18 carbon atoms, and for example may contain 2 to 12 carbon atoms. The term "lower alkenyl" intends an alkenyl group of 2 to 6 carbon atoms. The term "substituted alkenyl" refers to alkenyl substituted with one or more substituent groups, and the terms "heteroatom-containing alkenyl" and "heteroalkenyl" refer to alkenyl in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the terms "alkenyl" and "lower alkenyl" include linear, branched, cyclic, unsubstituted, substituted, and / or heteroatom-containing alkenyl and lower alkenyl, respectively.

[0027] The term "alkynyl" refers to a linear or branched hydrocarbon group of 2 to 24 carbon atoms containing at least one triple bond, such as ethynyl, n-propynyl, and the like. Generally, although again not necessarily, alkynyl groups herein may contain 2 to about 18 carbon atoms, and such groups may further contain 2 to 12 carbon atoms. The term "lower alkynyl" intends an alkynyl group of 2 to 6 carbon atoms. The term "substituted alkynyl" refers to alkynyl substituted with one or more substituent groups, and the terms "heteroatom- containing alkynyl" and "heteroalkynyl" refer to alkynyl in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the terms "alkynyl" and "lower alkynyl" include linear, branched, unsubstituted, substituted, and / or heteroatom-containing alkynyl and lower alkynyl, respectively.

[0028] The term "aryl", unless otherwise specified, refers to an aromatic substituent generally, although not necessarily, containing 5 to 30 carbon atoms and containing a single aromatic ring or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such that the different aromatic rings are bound to a common group such as a methylene or ethylene moiety). Aryl groups may, for example, contain 5 to 20 carbon atoms, and as a further example, aryl groups may contain 5 to 12 carbon atoms. For example, aryl groups may contain one aromatic ring or two or more fused or linked aromatic rings (i.e., biaryl, aryl-substituted aryl, etc.). Examples include phenyl, naphthyl, biphenyl, diphenylether, diphenylamine, benzophenone, and the like. "Substituted aryl" refers to an arylmoiety substituted with one or more substituent groups, and the terms "heteroatom- containing aryl" and "heteroaryl" refer to aryl substituent, in which at least one carbon atom is replaced with a heteroatom, as will be described in further detail infra. Aryl is intended to include stable cyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated C3-C14moieties, exemplified but not limited to phenyl, biphenyl, naphthyl, pyridyl, furyl, thiophenyl, imidazoyl, pyrimidinyl, and oxazoyl; which may further be substituted with one to five members selected from the group consisting of hydroxy, C1-C8 alkoxy, C1-C8 branched or straight-chain alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro, halogen, trifluoromethyl, cyano, and carboxyl (see e.g. Katritzky, Handbook of Heterocyclic Chemistry). If not otherwise indicated, the term "aryl" includes unsubstituted, substituted, and / or heteroatom-containing aromatic substituents.

[0029] The term "alkylene" refers to a di-radical alkyl group. Unless otherwise indicated, such groups include saturated hydrocarbon chains containing from 1 to 24 carbon atoms, which may be substituted or unsubstituted, may contain one or more alicyclic groups, and may be heteroatom-containing. "Lower alkylene" refers to alkylene linkages containing from 1 to 6 carbon atoms. Examples include, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), 2-methylpropylene (-CH2-CH(CH3)-CH2-), hexylene (-(CH2)6-), and the like.

[0030] Similarly, the terms “alkenylene”, “alkynylene”, “arylene”, “aralkylene”, and “alkarylene” refer to di-radical alkenyl, alkynyl, aryl, aralkyl, and alkaryl groups, respectively.

[0031] The term "amino" refers to the group -NRR’ wherein R and R’ are independently hydrogen or nonhydrogen substituents, with nonhydrogen substituents including, for example, alkyl, aryl, alkenyl, aralkyl, and substituted and / or heteroatom-containing variants thereof.

[0032] “Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems. Examples of suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.

[0033] The term “substituted cycloalkyl” refers to cycloalkyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, - SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0034] “Heteroaryl” refers to an aromatic group of from 1 to 15 carbon atoms, such as from 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (such as, pyridinyl, imidazolyl or furyl) or multiple condensed rings in a ring system (for example as in groups such as, indolizinyl, quinolinyl, benzofuran, benzimidazolyl or benzothienyl), wherein at least one ring within the ring system is aromatic, provided that the point of attachment is through an atom of an aromatic ring. In certain embodiments, the nitrogen and / or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N-oxide (N→O), sulfinyl, or sulfonyl moieties. This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise constrained by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO- heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl, and trihalomethyl.

[0035] The terms “heterocycle,” “heterocyclic” and “heterocyclyl” refer to a saturated or unsaturated group having a single ring or multiple condensed rings, including fused bridged and spiro ring systems, and having from 3 to 15 ring atoms, including 1 to 4 hetero atoms. These ring heteroatoms are selected from nitrogen, sulfur and oxygen, wherein, in fused ring systems, one or more of the rings can be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through the non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, -S(O)-, or –SO2- moieties. 11 of 94

[0036] Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to as thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[0037] Unless otherwise constrained by the definition for the heterocyclic substituent, such heterocyclic groups can be optionally substituted with 1 to 5, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO- heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and fused heterocycle.

[0038] By "substituted" as in "substituted alkyl," "substituted aryl," and the like, as alluded to in some of the aforementioned definitions, is meant that in the alkyl, aryl, or other moiety, at least one hydrogen atom bound to a carbon (or other) atom is replaced with one or more non-hydrogen substituents. Examples of such substituents include, without limitation, functional groups, and the hydrocarbyl moieties C1-C24alkyl (including C1-C18alkyl, further including C1-C12 alkyl, and further including C1-C6 alkyl), C2-C24 alkenyl (including C2-C18 alkenyl, further including C2-C12alkenyl, and further including C2-C6alkenyl), C2-C24alkynyl (including C2-C18 alkynyl, further including C2-C12 alkynyl, and further including C2- C6alkynyl), C5-C30aryl (including C5-C20aryl, and further including C5-C12aryl), and C6-C30aralkyl (including C6-C20 aralkyl, and further including C6-C12 aralkyl). The above-mentioned hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated. Unless otherwise indicated, any of the groups described herein are to be interpreted as including substituted and / or heteroatom-containing moieties, in addition to unsubstituted groups.

[0039] The term “heteroaliphatic” refers to an aliphatic moiety that contains at least one heteroatom in the chain, for example, an amine, carbonyl, carboxy, oxo, thio, phosphate, phosphonate, nitrogen, phosphorus, silicon, or boron atoms in place of a carbon atom. In some embodiments, the only heteroatom is nitrogen. In some embodiments, the only heteroatom is oxygen. In some embodiments, the only heteroatom is sulfur. “Heteroaliphatic” is intended herein to include, but is not limited to, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, and heterocycloalkynyl moieties. In some embodiments, “heteroaliphatic” is used to indicate a heteroaliphatic group (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) having 1-40 carbon atoms. In some embodiments, the heteroaliphatic group is optionally substituted in a manner that results in the formation of a stable moiety. Nonlimiting examples of heteroaliphatic moieties are polyethylene glycol, polyalkylene glycol, amide, polyamide, polylactide, polyglycolide, thioether, ether, alkyl-heterocycle-alkyl, —O-alkyl-O-alkyl, alkyl-O-haloalkyl, etc.

[0040] By "linking" or "linker" as in "linking group," "linker moiety," etc., is meant a linking moiety that connects two groups via covalent bonds. The linker may be linear, branched, cyclic or a single atom. Examples of such linking groups include alkyl, alkenylene, alkynylene, arylene, alkarylene, aralkylene, and linking moieties containing functional groups including, without limitation: amido (-NH-CO-), ureylene (-NH-CO-NH-), imide (-CO-NH- CO-) , epoxy (-O-), epithio (-S-), epidioxy (-O-O-), carbonyldioxy (-O-CO-O-), alkyldioxy (- O-(CH2)n-O-), epoxyimino (-O-NH-), epimino (-NH-), carbonyl (-CO-), etc. In certain cases, one, two, three, four or five or more carbon atoms of a linker backbone may be optionally substituted with a sulfur, nitrogen or oxygen heteroatom. The bonds between backbone atoms may be saturated or unsaturated, usually not more than one, two, or three unsaturated bonds will be present in a linker backbone. The linker may include one or more substituent groups, for example with an alkyl, aryl or alkenyl group. A linker may include, without limitations, poly(ethylene glycol) unit(s) (e.g., -(CH2-CH2-O)-); ethers, thioethers, amines, alkyls (e.g., (C1-C12)alkyl) , which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1- methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like. The linker backbone may include a cyclic group, for example, an aryl, a heterocycle or a cycloalkyl group, where 2 or more atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included in the backbone. A linker may be cleavable or non-cleavable. Any convenient orientation and / or connections of the linkers to the linked groups may be used.

[0041] When the term "substituted" appears prior or after a list of possible substituted groups, it is intended that the term apply to every member of that group. For example, the phrase "substituted alkyl and aryl" is to be interpreted as "substituted alkyl and substituted aryl."

[0042] In addition to the disclosure herein, the term “substituted,” when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below.

[0043] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for substituting for one or more hydrogens (any two hydrogens on a single carbon can be replaced with =O, =NR70, =N-OR70, =N2 or =S) on saturated carbon atoms in the specified group or radical are, unless otherwise specified, -R60, halo, =O, -OR70, -SR70, -NR80R80, trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R70, -SO2O–M+, -SO2OR70, -OSO2R70, -OSO2O–M+, -OSO2OR70, -P(O)(O–)2(M+)2, -P(O)(OR70)O–M+, -P(O)(OR70) 2, -C(O)R70, -C(S)R70, -C(NR70)R70, -C(O)O–M+, -C(O)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OC( O)O-M+, -OC(O)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2–M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R70is independently hydrogen or R60; each R80is independently R70or alternatively, two R80’s, taken together with the nitrogen atom to which they are bonded, form a 5-, 6- or 7-membered heterocycloalkyl which may optionally include from 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N and S, of which N may have -H or C1-C3alkyl substitution; and each M+is a counter ion with a net single positive charge. Each M+may independently be, for example, an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as+N(R60)4; or an alkaline earth ion, such as [Ca2+]0.5, [Mg2+]0.5, or [Ba2+]0.5 (“subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound of the invention and the other a typical counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound of the invention can serve as the counter ion for such divalent alkali earth ions). As specific examples, -NR80R80is meant to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N- methyl-piperazin-1-yl and N-morpholinyl.

[0044] In addition to the disclosure herein, substituent groups for hydrogens on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -R60, halo, -O-M+, -OR70, -SR70, -S–M+, -NR80R80, trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R70, -SO3–M+, -SO3R70, -OSO2R70, -OSO3–M+, -OSO3R70, -PO3-2(M+)2, -P(O)(OR70)O–M+, -P(O)(OR70)2, -C(O)R70, -C(S)R70, -C(NR70)R70, -CO2–M+, -CO2R70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OCO2–M+, -OCO2R70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2–M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60, R70, R80and M+are as previously defined, provided that in case of substituted alkene or alkyne, the substituents are not -O-M+, -OR70, -SR70, or -S–M+.

[0045] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for hydrogens on nitrogen atoms in “substituted” heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R60, -O-M+, -OR70, -SR70, -S-M+, -NR80R80, trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R70, -S(O)2O-M+, -S(O)2OR70, -OS(O)2R70, -OS( O)2O-M+, -OS(O)2OR70, -P(O)(O-)2(M+)2, -P(O)(OR70)O-M+, -P(O)(OR70)(OR70), -C(O)R70, - C(S)R70, -C(NR70)R70, -C(O)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OC(O)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70C(O)OR70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60, R70, R80and M+are as previously defined.

[0046] In addition to the disclosure herein, in a certain embodiment, a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0047] Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent “arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O-C(O)-.

[0048] As to any of the groups disclosed herein which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and / or synthetically non-feasible. In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.

[0049] In certain embodiments, a substituent may contribute to optical isomerism and / or stereo isomerism of a compound. Salts, solvates, hydrates, and prodrug forms of a compound are also of interest. All such forms are embraced by the present disclosure. Thus the compounds described herein include salts, solvates, hydrates, prodrug and isomer forms thereof, including the pharmaceutically acceptable salts, solvates, hydrates, prodrugs and isomers thereof. In certain embodiments, a compound may be a metabolized into a pharmaceutically active derivative.

[0050] The compounds of the disclosure may contain one or more chiral centers and / or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. The term “stereoisomers” when used herein consist of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols “R” or “S,” depending on the configuration of substituents around the stereogenic carbon atom. The present disclosure encompasses various stereoisomers of these compounds and mixtures thereof. Mixtures of enantiomers or diastereomers may be designated “(±)” in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. In some embodiments, an enantiomer or stereoisomer may be provided substantially free of the corresponding enantiomer.

[0051] Those skilled in the art will appreciate that a bond designated as in a small molecule structure, as used herein, refers to a bond that, in some embodiments, is a single (e.g., saturated) bond, and in some embodiments, is a double (e.g., unsaturated) bond. For example the following structure:is intended to encompass bothand

[0052] Unless otherwise specified, reference to an atom is meant to include isotopes of that atom. For example, reference to H is meant to include1H,2H (i.e., D) and3H (i.e., T), and reference to C is meant to include12C and all isotopes of carbon (such as13C).

[0053] Definitions of other terms and concepts appear throughout the detailed description. 4.2. Lipid Nanoparticle Compositions

[0054] Novel lipid nanoparticle compositions are provided for the delivery of nucleic acid to cells in vitro and in vivo with different and improved pharmacokinetic profiles ascompared to what is typically observed in the art. Also provided are methods for using the lipid nanoparticle compositions of this disclosure in research and as therapeutics.

[0055] A “lipid nanoparticle” refers to a lipid composition that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., DNA and / or RNA), a protein, a small molecule, and the like to a target site of interest. In the lipid nanoparticle, a nucleic acid agent may be encapsulated in the lipid, thereby protecting the agent from enzymatic degradation.

[0056] In general, the lipid nanoparticle includes several lipid components, including e.g., an ionizable lipid, one or more helper lipid(s) (e.g., non-cationic lipid(s)), and a lipid that prevents aggregation of the nanoparticle (also referred to as a coat lipid or conjugated lipid e.g., a PEG-lipid). In some aspects, this disclosure provides for a lipid nanoparticle (LNP) composition as described herein comprising a nucleic acid, where the nucleic acid is substantially encapsulated by the lipid components of the LNP. 4.3. Ionizable Lipids

[0057] The lipid nanoparticles (LNPs) of this disclosure include ionizable lipids. As summarized above, provided herein are novel ionizable lipids. The ionizable lipid is typically employed in lipid nanoparticles (LNPs) to condense its nucleic acid cargo, e.g., DNA or RNA, at low pH and to drive membrane association and fusogenicity. The term “ionizable lipid” refers to a lipid comprising an ionizable group that carries a net charge at a selected pH (e.g., a pH of 6.5 or less), but which can remain neutral at, e.g., a higher pH, such as physiological pH. The pH-sensitivity of such ionizable lipids can be desirable to provide for intracellular delivery of nucleic acid cargo. Ionizable lipids can have less interactions with cell membranes when neutral, and then become charged when internalized into endosomes in a target cell, where the pH is lower than in the extracellular environment. Ionizable lipids which are protonated and, therefore, become positively charged, may promote membrane destabilization and facilitate endosomal escape of the nanoparticle.

[0058] In some embodiments, the ionizable lipid is a cationic lipid. The term “cationic lipid” refers to a lipid that carries a net positive charge at a selected pH (e.g., a pH of 6.5 or less). In some embodiments, the ionizable lipids are cationic lipids including at least one ionizable amino group that is positively charged or becomes protonated at a selected pH, for example at pH of 6.5 or lower. In some embodiments, the cationic lipid includes one or more tertiary amino groups, e.g., a trialkyl amino group.

[0059] As disclosed herein, the ionizable lipid includes an ionizable headgroup (e.g., an ionizable amino group) connected to the lipid tails via a linear alkyl core. The linear alkyl core can have n carbon atoms, where n-1 carbon atoms in the linear alkyl core are linked to lipid tails. In some embodiments, the linear alkyl core has 3 carbon atoms and 2 lipid tails. In some embodiments, the linear alkyl core has 4 carbon atoms and 3 lipid tails. In some embodiments, the linear alkyl core has 5 carbon atoms and 4 lipid tails. In some embodiments, the linear alkyl core has 6 carbon atoms and 5 lipid tails.

[0060] In some embodiments, the cationic lipid comprises a protonatable tertiary amine (e.g., pH titratable) head group, a linear alkyl core, hydrocarbon chains (e.g., C8-C20 carbon chains, such as C18alkyl chains), ether linkages between the linear alkyl core and hydrocarbon chains, and 0 to 3 double bonds per hydrocarbon chain. In some embodiments, the cationic lipid comprises the same number of hydrocarbon chains as ether linkages. In some embodiments, the cationic lipid comprises a protonatable tertiary amine headgroup, a linear alkyl core, hydrocarbon chains (e.g., as described herein), and ester linkages between the linear alkyl core and hydrocarbon chains. In some embodiments, the cationic lipid comprises the same number of hydrocarbon chains as ester linkages. In some embodiments, the cationic lipid comprises a protonatable tertiary amine headgroup, a linear alkyl core, hydrocarbon chains (e.g., as described herein), and carbonate linkages between the linear alkyl core and hydrocarbon chains. In some embodiments, the cationic lipid comprises the same number of hydrocarbon chains as carbonate linkages. In some embodiments, the cationic lipid comprises the same number of hydrocarbon chains as ester linkages. In some embodiments, the cationic lipid comprises 2 or more hydrocarbon chains, such as 3 or more hydrocarbon chains, or 4 or more hydrocarbon chains. 4.4. Compounds

[0061] In some embodiments, the present disclosure provides preparations and uses of a compound. In some embodiments, the compound is of Formula I:Formula I or is a pharmaceutically acceptable salt thereof; wherein: Zais H or an optionally substituted ionizable group;Zbis H or an optionally substituted ionizable group; Lais a bond or an optionally substituted, saturated or partially unsaturated, straight or branched divalent aliphatic C1-12 hydrocarbon chain, wherein one or more carbon atoms of the chain are optionally replaced by a divalent group independently selected from -O-, -NRe-, -S-, - S(O)-, -S(NRe)-, -S(O)2-, -S(O)(NRe)-, -S(NRe)2-, and -C(O)-; Lbis a bond or an optionally substituted, saturated or partially unsaturated, straight or branched divalent aliphatic C1-12hydrocarbon chain, wherein one or more carbon atoms of the chain are optionally replaced by a divalent group independently selected from -O-, -NRe-, -S-, - S(O)-, -S(NRe)-, -S(O)2-, -S(O)(NRe)-, -S(NRe)2-, and -C(O)-; each Reis independently hydrogen or an optionally substituted C1-6aliphatic group; Wnis an optionally substituted linear alkylene of n carbon atoms, wherein n is 2 to 6; Wmis an optionally substituted linear alkylene of m carbon atoms, wherein m is 2 to 6; p is an integer from 1 to 3; q is an integer from 1 to 3; each Xais independently a bond or an optionally substituted, saturated or partially unsaturated, straight or branched divalent aliphatic C1-20hydrocarbon chain, wherein one or more carbon atoms of the chain are optionally replaced by a divalent group independently selected from - O-, -NRe-, -S-, -S(O)-, -S(NRe)-, -S(O)2-, -S(O)(NRe)-, -S(NRe)2-, and -C(O)-; each Xbis independently a bond or an optionally substituted, saturated or partially unsaturated, straight or branched divalent aliphatic C1-20hydrocarbon chain, wherein one or more carbon atoms of the chain are optionally replaced by a divalent group independently selected from - O-, -NRe-, -S-, -S(O)-, -S(NRe)-, -S(O)2-, -S(O)(NRe)-, -S(NRe)2-, and -C(O)-; each Rais independently H or an optionally substituted, saturated or partially unsaturated, straight or branched aliphatic C1-50hydrocarbon group, wherein one or more carbon atoms of the chain are optionally replacedby a divalent group independently selected from -O-, -S-, -S(O)-, - S(NRe)-, -S(O)2-, -S(O)(NRe)-, -S(NRe)2-, -C(O)- and -CyA-; each Rbis independently H or an optionally substituted, saturated or partially unsaturated, straight or branched aliphatic C1-50hydrocarbon group, wherein one or more carbon atoms of the chain are optionally replaced by a divalent group independently selected from -O-, -S-, -S(O)-, - S(NRe)-, -S(O)2-, -S(O)(NRe)-, -S(NRe)2-, -C(O)- and -CyA-; and each CyAindependently comprises an optionally substituted ring system selected from a 3- to 12-membered saturated or partially unsaturated carbocyclene; a phenylene; a 3- to 12-membered saturated or partially unsaturated heterocyclene having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; a 5- to 6-membered heteroarylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a combination of any of these ring systems including fused variants thereof.

[0062] In some embodiments, Zaand / or Zbis each independently H or an optionally substituted ionizable group selected from amine, guanidine, nitrogen-containing heteroaryl, carboxylic acid, sulfonic acid, and phosphonic acid. In some embodiments, Zais H or an optionally substituted ionizable group selected from amine, guanidine, nitrogen-containing heteroaryl, carboxylic acid, sulfonic acid, and phosphonic acid. In some embodiments, Zbis H or an optionally substituted ionizable group selected from amine, guanidine, nitrogen- containing heteroaryl, carboxylic acid, sulfonic acid, and phosphonic acid.

[0063] In some embodiments, Zaand / or Zbis each independently H or an optionally substituted ionizable group selected from amine, guanidine, and imidazole. In some embodiments, Zais H or an optionally substituted ionizable group selected from amine, guanidine, and imidazole. In some embodiments, Zbis H or an optionally substituted ionizable group selected from amine, guanidine, and imidazole.

[0064] In some embodiments, Zaand / or Zbis each independently H or an ionizable group selected from -N(Re)2,. In some embodiments, Zais H or anionizable group selected from -N(Re)2,, . In some beembodiments, Z is H or an ionizable group selected from -N(R )2, , and. In some embodiments, Zais . In some embodiments, Zais . In some embodiments, Zbis. In some embodiments, Zbis.

[0065] In some embodiments, Zaand / or Zbis each independently H or -N(Re)2.

[0066] In some embodiments, Zais -N(Re)2. In some embodiments, Zais -N(CH3)2.

[0067] In some embodiments, Zbis -N(Re)2. In some embodiments, Zbis -N(CH3)2.

[0068] In some embodiments, Zais H.

[0069] In some embodiments, Zbis H.

[0070] In some embodiments, Laand / or Lbis each independently a bond or an optionally substituted, saturated or partially unsaturated, straight or branched divalent aliphatic C1-12hydrocarbon chain, wherein one or more carbon atoms of the chain are optionally replaced by a divalent group independently selected from -O-, -NRe-, -S-, -S(O)-, -S(NRe)-, -S(O)2-, - S(O)(NRe)-, -S(NRe)2-, and -C(O)-. In some embodiments, Laand / or Lbis each independently a bond or an optionally substituted, saturated or partially unsaturated, straight or branched divalent aliphatic C1-12 hydrocarbon chain, wherein one or more carbon atoms of the chain are optionally replaced by a divalent group independently -O- or -C(O)-. In some embodiments, Lais a bond or an optionally substituted, saturated or partially unsaturated, straight or branched divalent aliphatic C1-12hydrocarbon chain, wherein one or more carbon atoms of the chain are optionally replaced by a divalent group independently selected from -O- or -C(O)-. In some embodiments, Lbis a bond or an optionally substituted, saturated or partially unsaturated, straight or branched divalent aliphatic C1-12 hydrocarbon chain, wherein one ormore carbon atoms of the chain are optionally replaced by a divalent group independently selected from -O- or -C(O)-.

[0071] In some embodiments, Laand / or Lbis each independently a bond or an optionally substituted C1-6 hydrocarbon chain, wherein one or more carbon atoms of the chain are optionally replaced by a divalent group independently selected from -O-, -NRe-, -S-, -S(O)-, - S(O)2-, and -C(O)-. In some embodiments, Lbis a bond or an optionally substituted C1-6 hydrocarbon chain, wherein one or more carbon atoms of the chain are optionally replaced by a divalent group independently selected from -O-, -NRe-, -S-, -S(O)-, -S(O)2-, and -C(O)-. In some embodiments, Lais a bond or an optionally substituted C1-6hydrocarbon chain, wherein one or more carbon atoms of the chain are optionally replaced by a divalent group independently selected from -O-, -NRe-, -S-, -S(O)-, -S(O)2-, and -C(O)-.

[0072] In some embodiments, Laand / or Lbis each independently a bond or an optionally substituted C1-6 hydrocarbon chain, wherein one or more carbon atoms of the chain are optionally replaced by a divalent group independently selected from -O- or -C(O)-. In some embodiments, Lbis a bond or an optionally substituted C1-6 hydrocarbon chain, wherein one or more carbon atoms of the chain are optionally replaced by a divalent group independently selected from -O- or -C(O)-. In some embodiments, Lais a bond or an optionally substituted C1-6hydrocarbon chain, wherein one or more carbon atoms of the chain are optionally replaced by a divalent group independently selected from -O- or -C(O)-.

[0073] In some embodiments, Laand / or Lbis each independently a bond or an optionally substituted C1-6hydrocarbon chain selected from methylene, ethylene, propylene, butylene, pentylene, and hexylene. In some embodiments, Laand / or Lbis each independently a bond or an optionally substituted C1-6hydrocarbon chain selected from methylene, ethylene, propylene, and butylene. In some embodiments, Lais a bond or an optionally substituted C1-6 hydrocarbon chain selected from methylene, ethylene, propylene, butylene, pentylene, and hexylene. In some embodiments, Lbis a bond or an optionally substituted C1-6 hydrocarbon chain selected from methylene, ethylene, propylene, butylene, pentylene, and hexylene.

[0074] In some embodiments, Laand / or Lbis each independently selected from a bond, - CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-, and - CH2-CH2-CH2-CH2-CH2-CH2-.

[0075] In some embodiments, Lais selected from a bond, -CH2-, -CH2-CH2-, -CH2-CH2-CH2- , -CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-, and -CH2-CH2-CH2-CH2-CH2-CH2-. In some embodiments, Lais a bond.

[0076] In some embodiments, Lbis selected from a bond, -CH2-, -CH2-CH2-, -CH2-CH2- CH2-, -CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-, and -CH2-CH2-CH2-CH2-CH2-CH2-. In some embodiments, Lbis a bond.

[0077] In some embodiments, Lais selected from a bond, -CH2-CH2-, -CH2-CH2-CH2, and - CH2-CH2-CH2-CH2-. In some embodiments, Lais -CH2-CH2-. In some embodiments, Lais - CH2-CH2-CH2. In some embodiments, Lais -CH2-CH2-CH2-CH2-.

[0078] In some embodiments, Lbis selected from a bond, -CH2-CH2-, -CH2-CH2-CH2, and - CH2-CH2-CH2-CH2-. In some embodiments, Lbis -CH2-CH2-. In some embodiments, Lbis - CH2-CH2-CH2. In some embodiments, Lbis -CH2-CH2-CH2-CH2-.

[0079] In some embodiments, m is 2 to 4.

[0080] In some embodiments, n is 2 to 4.

[0081] In some embodiments, m is 2 or 3.

[0082] In some embodiments, n is 2 or 3.

[0083] In some embodiments, W is independently selected from -CH2-CH2-, -CH2-CH2-CH2- , -CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-, and -CH2-CH2-CH2-CH2-CH2-CH2-.

[0084] In some embodiments, W is -CH2-CH2-. In some embodiments, W is -CH2-CH2-CH2. In some embodiments, W is -CH2-CH2-CH2-CH2-.

[0085] In some embodiments, p is an integer from 1 to 3. In some embodiments, p is 1.

[0086] In some embodiments, q is an integer from 1 to 3. In some embodiments, q is 1.

[0087] In some embodiments, each Xais independently selected from -C(=O)O-, -OC(=O)-, and -OC(=O)O-. In some embodiments, each Xais independently -C(=O)O- or -OC(=O)-. In some embodiments, each Xais -C(=O)O-.

[0088] In some embodiments, each Xais independently -C(=O)O-.

[0089] In some embodiments, each Xbis independently selected from -C(=O)O-, -OC(=O)-, and -OC(=O)O-. In some embodiments, each Xbis independently -C(=O)O- or -OC(=O)-. In some embodiments, each Xbis -C(=O)O-.

[0090] In some embodiments, each Xbis independently -C(=O)O-.

[0091] In some embodiments, the compound is represented by Formula II:Formula II; wherein Za, La, Wn, Wm, Xa, Xb, Ra, Rb, p, and q are defined herein.

[0092] In some embodiments, each Raand / or Rbis independently an optionally substituted C1-30 aliphatic group. In some embodiments, each Rais independently an optionally substituted C1-30aliphatic group. In some embodiments, each Rbis independently an optionally substituted C1-30 aliphatic group.

[0093] In some embodiments, each Raand Rbis independently selected from:,

[0094] In some embodiments, each Rais independently. , entlyn some embodiments, each Rais independently. In some embodiments, each Rais independentlysome embodiments, each Rais independentlye embodiments, each Rais independently. In some embodiments, each Rais independentlysome embodiments, each Rais independentlysome embodiments, each Rais independentlyeembodiments, each Rais independently. In some embodiments, each Rais independently. In some embodiments, each Rais independently. In some embodiments, each Rais independently. In some embodiments, each Rais independentlysome embodiments, each Rais independentlyn some embodiments, each Rais independentlysome embodiments, each Rais independently. In some embodiments, each Rais independently . In somee i i i iindependently soame embodiments, each R isi i i a

[0095] In some embodiments, each Rbis independentlysome embodiments, each Rbis independently. In some embodiments, each Rbis independentlysome embodiments, each Rbis independentlye embodiments, each Rbis independently. In some embodiments, each Rbis independentlysome embodiments, each Rbis independentlysome embodiments, each Rbis independentlye embodiments, each Rbis independently. In some embodiments, each Rbisindependently. In some embodiments, each Rbis independentlye embodiments, each Rbis independently. In some embodiments, each Rbis independentlysome embodiments, each Rbis independentlyn some embodiments, each Rbis independentlysome embodiments, each Rbis independently. In some embodiments, each Rbis independently. In some embodiments, each Rbis independently. In some embodiments, each Rbisi s i i i iindependently sbome embodiments, each R isi i a

[0096] In some embodiments, provided herein is a compound chosen from the compounds listed in Table 1 or a tautomer, stereoisomer, geometric isomer, or a mixture of stereoisomers, or a pharmaceutically acceptable salt, or hydrate, or deuterated derivative of any of the foregoing.

[0097] In certain embodiments, the lipid is selected from a compound of Table 1: Table 1: Exemplary ionizable lipids

[0098] In some embodiments, the lipid nanoparticle compositions can include one or more additional ionizable lipid components in addition to the ionizable lipid of Formula (I) (e.g., as described above). Any convenient lipid that carries a net positive charge at or around physiological pH may find use as an additional ionizable lipid in the compositions described herein.

[0099] Non-limiting examples of cationic lipids are described in detail herein. Cationic lipids and related analogs, which are useful in the lipid nanoparticles of the present disclosure, include but are not limited to, those lipids described in U.S. Patent Publication Nos.20060083780 and 20060240554; U.S. Pat. Nos.5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO 96 / 10390, the disclosures of which are herein incorporated by reference in their entirety for all purposes. Additional cationic lipids of interest include, but are not limited to, 1,2-distearyloxy-N,N- dimethyl-3-aminopropane (DSDMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), 1,2- dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), and heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), N,N-dioleyl-N,N- dimethylammonium chloride (“DODAC”); N-(2,3-dioleyloxy)propyl-N,N—N- triethylammonium chloride (“DOTMA”); N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”); 1,2-Dioleyloxy-3-trimethylaminopropane chloride salt (“DOTAP.Cl”); 3β-(N—(N′,N′- dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”), N-(1-(2,3-dioleyloxy)propyl)-N- 2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (“DOSPA”), dioctadecylamidoglycyl carboxyspermine (“DOGS”), 1,2-dioleoyl-3-dimethylammoniumpropane (“DODAP”), N,N-dimethyl-2,3-dioleyloxy)propylamine (“DODMA”), and N-(1,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (“DMRIE”). Additionally, a number of commercial preparations of cationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMA and DOPE, available from GIBCO / BRL), and LIPOFECTAMINE (comprising DOSPA and DOPE, available from GIBCO / BRL). In particular embodiments, a cationic lipid is an amino lipid including one or two fatty acyl or fatty alkyl chains.

[0100] Further exemplary ionizable lipids which can be adapted for use in the lipid nanoparticles of the present disclosure are described in International PCT Application No. PCT / US2023 / 079923 and International PCT patent publications WO2015 / 095340, WO2015 / 199952, WO2018 / 011633, WO2017 / 049245, WO2015 / 061467, WO2012 / 040184, WO2012 / 000104, WO2015 / 074085, WO2016 / 081029, WO2017 / 004143, WO2017 / 075531, WO2017 / 117528, WO2011 / 022460, WO2013 / 148541, WO2013 / 116126, WO2011 / 153120, WO2012 / 044638, WO2012 / 054365, WO2011 / 090965, WO2013 / 016058, WO2012 / 162210, WO2008 / 042973, WO2010 / 129709, WO2010 / 144740, WO2012 / 099755, WO2013 / 049328, WO2013 / 086322, WO2013 / 086373, WO2011 / 071860, WO2009 / 132131, WO2010 / 048536, WO2010 / 088537, WO2010 / 054401, WO2010 / 054406, WO2010 / 054405, WO2010 / 054384, WO2012 / 016184, WO2009 / 086558, WO2010 / 042877, WO2011 / 000106, WO2011 / 000107, WO2005 / 120152, WO2011 / 141705, WO2013 / 126803, WO2006 / 007712, WO2011 / 038160, WO2005 / 121348, WO2011 / 066651, WO2009 / 127060, WO2011 / 141704, WO2006 / 069782, WO2012 / 031043, WO2013 / 006825, WO2013 / 033563, WO2013 / 089151, WO2017 / 099823, WO2015 / 095346, and WO2013 / 086354, and US patent publications US2016 / 0311759, US2015 / 0376115, US2016 / 0151284, US2017 / 0210697, US2015 / 0140070, US2013 / 0178541, US2013 / 0303587, US2015 / 0141678, US2015 / 0239926, US2016 / 0376224, US2017 / 0119904, US2012 / 0149894, US2015 / 0057373, US2013 / 0090372, US2013 / 0274523, US2013 / 0274504, US2013 / 0274504, US2009 / 0023673, US2012 / 0128760, US2010 / 0324120, US2014 / 0200257, US2015 / 0203446, US2018 / 0005363, US2014 / 0308304, US2013 / 0338210, US2012 / 0101148, US2012 / 0027796, US2012 / 0058144, US2013 / 0323269, US2011 / 0117125, US2011 / 0256175, US2012 / 0202871, US2011 / 0076335, US2006 / 0083780, US2013 / 0123338, US2015 / 0064242, US2006 / 0051405, US2013 / 0065939, US2006 / 0008910, US2003 / 0022649, US2010 / 0130588, U52013 / 0116307, US2010 / 0062967, US2013 / 0202684, US2014 / 0141070, US2014 / 0255472, US2014 / 0039032, US2018 / 0028664, U52016 / 0317458, and US2013 / 0195920.4.4 Helper Lipids

[0101] LNPs of this disclosure can also include one or more helper lipid(s), in addition to the ionizable lipid component described herein. In some embodiments, the helper lipid is a neutral lipid. In some embodiments, the neutral lipid is zwitterionic, e.g., has an overall net zero charge.

[0102] Neural lipids include, for example, phospholipids, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids for use in the compositions described herein is generally guided by consideration of, e.g., LNP size and stability of the LNPs in the bloodstream. In general, the LNPs of this disclosure includes a helper lipid component that includes a neutral lipid that is a phospholipid. Non-limiting examples of phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatdylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine. In some embodiments, the neutral lipid component is a lipid having two acyl groups, (i.e., diacylphosphatidylcholine and diacylphosphatidylethanolamine). Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well- known techniques. In one embodiment, the neutral lipids include saturated fatty acids with carbon chain lengths in the range of C10 to C30. In one embodiment, neutral lipids with mono or diunsaturated fatty acids with carbon chain lengths in the range of C10 to C30 are used. Additionally, lipids having mixtures of saturated and unsaturated fatty acid chains can be used. The neutral lipids may also be composed of sphingomyelin, or dihydrosphingomyeline.

[0103] In some embodiments, the phospholipid is selected from a phosphatidylcholine (PC), a phosphatidylethanolamine (PE), a phosphatidylserine (PS), a phosphatidylinositol (PI), and a phosphatidylglycerol (PG).

[0104] In some embodiments, the phospholipid has a hydrocarbon chain, or “tail” having 12-24 carbons, e.g., 16-20 carbons, 18-22 carbons, 12-18 carbons. In some embodiments, phospholipid has a carbon tail of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbons. In some embodiments, the phospholipid tail comprises no double bonds, i.e. the bonds are saturated bonds. In some embodiments, the phospholipid tail is unsaturated, that is, it comprises one or more double bonds, e.g., 1, 2, 3, 4 or 5 double bonds. In some embodiments, the phospholipid tail is unsaturated, that is, it comprises one or more triplebonds, e.g.1, 2, 3, 4 or 5 triple bonds. In some embodiments, the phospholipid tail comprises one or more ring structures. In some embodiments, the one or more ring structures is selected from 3- to 7-membered saturated or partially unsaturated monocyclic carbocyclyl; 5- to 6- membered aryl; 7- to 10-membered saturated or partially unsaturated bicyclic carbocyclyl; and 7- to 10-membered bicyclic aryl wherein each ring structure is independently substituted with 0-7 RAgroups; each RAis independently halogen or an optionally substituted group selected from C1-12 aliphatic, phenyl, or 3- to 7-membered saturated or partially unsaturated monocyclic carbocyclyl. In some such instances, the ring structure is a cholesterol or cholesterol derivative. In some embodiments, the phospholipid is symmetric, i.e., all tails of the phospholipid are the same. In other embodiments, the phospholipid is asymmetric, i.e., the phospholipid comprises two different hydrocarbon chains.

[0105] In some embodiments, a helper lipid is or comprises symmetric or asymmetric aliphatic phospholipid moieties that are each independently optionally substituted, branched or straight, partially unsaturated or saturated C9-C24aliphatic.

[0106] In some embodiments, the helper lipid includes a phosphatidylethanolamine (PE). Phosphatidylethanolamines (PE) are a class of phospholipids that incorporate ethanolamine as a headgroup. In some embodiments, the phosphatidylethanolamine selected from the group consisting of phosphatidylethanolamine, dioleoylphosphatidylethanolamine (1,2- dioleyl-sn-glycero-3-phosphoethanolamine) (Δ9-Cis PE, or DOPE), palmitoyl- oleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) (DPPE), dimyristoylphosphoethanolamine (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) (DMPE), (1,2-distearoyl-sn-glycero-3-phosphoethanolamine) (DSPE), monomethyl- phosphatidyl-ethanolamine (e.g.16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1 -trans PE, l-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), dielaidoyl-phosphatidylethanolamine (DEPE), lysophosphatidylethanolamine, 1,2- dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), and 1,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (DiPPE). In certain embodiments, the phosphatidylethanolamine is dioleoylphosphatidylethanolamine (also referred to as 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine, or (Δ9-Cis) PE, or DOPE), having a tail of 18 carbons and one saturated bond (“18-1”) as shown below:

[0107] In some embodiments, the helper lipid includes a phosphatidylcholine (PC). Phosphatidylcholines (PC) are a class of phospholipids that incorporate choline as a headgroup. In some embodiments, phosphatidylcholine is selected from the group consisting of phosphatidylcholine, distearoylphosphatidylcholine (1,2-distearoyl-sn-glycero-3- phosphocholine) (DSPC), dioleoylphosphatidylcholine (1,2-dioleoyl-sn-glycero-3- phosphocholine) (Δ9-Cis PC, or DOPC), dipalmitoylphosphatidylcholine (1,2-dipalmitoyl- sn-glycero-3-phosphocholine) (DPPC), hydrogenated soy phosphatidylcholine (HSPC), palmitoyloleoylphosphatidylcholine (POPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (“20-1 PC” or “20:1 PC”), egg phosphatidylcholine (EPC), dimyristoyl phosphatidylcholine (DMPC), dierucoylphosphatidylcholine (DEPC), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, 1,2-dicholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (DChemsPC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-palmitoyl-2-cholesterylcarbonoyl-sn-glycero-3-phosphocholine (PChcPC), and 1-palmitoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (PChemsPC). In certain embodiments, the phosphatidylcholine is distearoylphosphatidylcholine (DSPC) (also referred to as 1,2-distearoyl-sn-glycero-3-phosphocholine), having a tail of 18 carbons and no saturated bonds (“18-0”) as shown below:

[0108] In certain embodiments, the phosphatidylcholine is dioleoylphosphatidycholine (also referred to as 1,2-dioleoyl-sn-glycero-3-phosphocholine, (Δ9-Cis) PC or DOPC), having a tail of 18 carbons and one saturated bond (“18-1”) as shown below:

[0109] In certain embodiments, the phosphatidylcholine is 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (delta9-Cis PC), having a tail of 16 carbons and one saturated bond (“16-1”) as shown below:

[0110] In certain embodiments, the phosphatidylcholine is an asymmetric lipid, having one tail of 16 carbons and a second tail of 18 carbons. In some such instances, the tail of the phosphatidylcholine having 18 carbons has one saturated bond, e,g, it is 1-palmitoyl-2- oleoyl-glycero-3-phosphocholine (also referred to as “16-0 / 18-1 PC”, “16:0 / 18:1 PC” or POPC) as shown below:

[0111] In certain embodiments, the phosphatidylcholine is 1,2- dicholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (DChemsPC), as shown below:

[0112] In certain embodiments, the phosphatidylcholine is 1-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), as shown below:

[0113] In certain embodiments, the phosphatidylcholine is 1-palmitoyl-2- cholesterylcarbonoyl-sn-glycero-3-phosphocholine (PChcPC), as shown below:

[0114] In certain embodiments, the phosphatidylcholine is 1-palmitoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (PChemsPC), as shown below:

[0115] In some embodiments, the helper lipid includes a phosphatidylglycerol selected from the group consisting of phosphatidylglycerol, dioleoylphosphatidylglycerol (1,2- dioleoyl-sn-glycero-3- phospho-(l’-rac-glycerol) (DOPG), dipalmitoylphosphatidylglycerol, (DPPG), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), and palmitoyloleyolphosphatidylglycerol (POPG).

[0116] In some embodiments, the helper lipid includes a phosphatidylserine, e.g. phosphatidylserine or dioleoylphosphatidylserine (DOPS).

[0117] In some embodiments, the helper lipid includes a lecithin, e.g. lecithin or lysolecithin.

[0118] In some embodiments, the helper lipid includes a sphingomyelin (SM), e.g. egg sphingomyelin (ESM).

[0119] In some embodiments, the helper lipid is cephalin, cardiolipin, phosphatidic acid, cerebrosides, or dicetylphosphate.

[0120] In some aspects, the LNP can further comprise a component, such as a sterol, to provide membrane integrity. One exemplary sterol that can be used in the lipid nanoparticle is cholesterol and derivatives thereof. Non-limiting examples of cholesterol derivatives include polar analogues such as 5α-cholestanol, 5β-coprostanol, cholesteryl-(2′-hydroxy)- ethyl ether, cholesteryl-(4′-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5α-cholestane, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryldecanoate; and mixtures thereof. Exemplary cholesterol derivatives are described in International application WO2009 / 127060 and US patent publication US2010 / 0130588. The component providing membrane integrity, such as a sterol, can comprise 0-50% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, such a component is 20- 50% (mol) 30-40% (mol) of the total lipid content of the lipid nanoparticle.

[0121] Accordingly, the neutral lipid component of the LNPs can further include cholesterol or a derivative or analog thereof. A variety of cholesterol analogs and derivatives can be adapted form use in the LNPs of this disclosure. In some embodiments, the helper lipid component includes cholesterol.

[0122] In some embodiments, the LNP includes a neutral lipid component that includes a mixture of one or more phospholipids and cholesterol or a derivative or analog thereof.

[0123] In some embodiments, the LNP includes a neutral lipid component that includes a phosphatidylethanolamine phospholipid and cholesterol or a derivative or analog thereof.

[0124] In some embodiments, the LNP includes a neutral lipid component that includes DOPE phospholipid and cholesterol. In some embodiments, the LNP includes a neutral lipid component that includes DSPC phospholipid and cholesterol. In some embodiments, the LNP includes a neutral lipid component that includes DOPC phospholipid and cholesterol. 4.5 Other Components

[0125] LNPs of this disclosure can also include one or more additional lipid components. Such lipids can be selected to provide for a desirable profile of nanoparticle properties, such as particle stability, delivery efficacy, tolerability and biodistribution.

[0126] In some aspects, the LNP can further comprise a non-cationic lipid. Non-ionic lipids include amphipathic lipids, neutral lipids and anionic lipids. Accordingly, the non- cationic lipid can be a neutral uncharged, zwitterionic, or anionic lipid. Non-cationic lipids are typically employed to enhance fusogenicity. Exemplary non-cationic lipids envisioned for use in the methods and compositions are described in International Application PCT / US2018 / 050042 published as WO2019051289A1. Exemplary non-cationic lipids are described in International application Publication WO2017 / 099823 and US patent publication US2018 / 0028664.

[0127] Non-limiting examples of non-cationic lipids include, nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers,triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, sphingomyelin, and the like.

[0128] In some embodiments, the LNP includes one or more lipids capable of reducing aggregation. In general, a lipids capable of reducing aggregation includes at least a hydrocarbon tail or chain linked to a hydrophilic group which is capable of being configured at the surface of the LNP and provide for reduced LNP aggregation. Thus, the lipid capable of reducing aggregation is sometimes referred to as a conjugated lipid or coat lipid.

[0129] A lipid capable of reducing aggregation of particles may comprise a conjugated lipid molecule, such as a polyethylene glycol (PEG). Generally, these are used to inhibit aggregation of lipid nanoparticles and / or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, polyethyleneglycol (PEG)-lipid conjugate, polyoxazoline (POZ)-lipid conjugates, a polyamide (ATTA)-lipid conjugate, a cationic- polymer-lipid conjugates (CPLs), or mixtures thereof. In one embodiment, the LNPs comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate. In certain embodiments, the PEG-lipid conjugate or ATTA-lipid conjugate is used together with a CPL.

[0130] In some embodiments, the lipid capable of reducing aggregation is a PEG-lipid. A PEG-lipid refers to a lipid having one or more hydrocarbon tail(s) linked to one or more polyethylene glycol (PEG) moiety(ies) via an optional linker.

[0131] It is understood that the PEG moieties may include terminal modification(s) to provide for e.g., conjugation to the lipid tails via the optional linker. The PEG moiety may be terminated in as a hydroxyl group, or an alkyl ether (e.g., a methoxy terminal group). In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)-conjugated lipid. PEG-lipids of interest include, but are not limited to, a PEG-diacylglycerol (DAG), a PEG dialkyloxypropyl (DAA), a PEG- phospholipid, a PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate may be PEG-dilauryloxypropyl (C12), a PEG-dimyristyloxypropyl (C14), a PEG- dipalmityloxypropyl (C16), a PEG-distearyloxypropyl (C18), or mixtures thereof.

[0132] Exemplary PEG-lipid conjugates include, but are not limited to, PEG- diacylglycerol (DAG) (such as 1-(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-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(w- methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N- (carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or a mixture thereof. Additional exemplary PEG-lipid conjugates are described, for example, in U.S. Pat. Nos.5,885,613, 6,287,591, US2003 / 0077829, US2003 / 0077829, US2005 / 0175682, US2008 / 0020058, US2011 / 0117125, US2010 / 0130588, US2016 / 0376224, and US2017 / 0119904. In some embodiments, a PEG- lipid is a compound disclosed in US2018 / 0028664. In some embodiments, a PEG-lipid is disclosed in US20150376115 or in US2016 / 0376224. The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of PEG-DMG, PEG- dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG- cholesterol (1-[8'-(Cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl- omegal-methyl-poly(ethylene glycol), PEG-DMB (3,4-Ditetradecoxylbenzyl-[omega]- methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine- N-[methoxy(polyethylene glycol)-2000]. In some examples, the PEG-lipid can be selected from the group consisting of PEG-DMG, 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], PEG-DSG.

[0133] As described above, 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 (CPL) conjugates can be used in place of or in addition to the PEG-lipid. Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and cationic polymer-lipids are described in the International patent application publications WO1996 / 010392, WO1998 / 051278, WO2002 / 087541, WO2005 / 026372, WO2008 / 147438, WO2009 / 086558, WO2012 / 000104, WO2017 / 117528, WO2017 / 099823, WO2015 / 199952, WO2017 / 004143, WO2015 / 095346, WO2012 / 000104, WO2012 / 000104, and WO2010 / 006282, US patent application publications US2003 / 0077829, US2005 / 0175682, US2008 / 0020058, US2011 / 0117125, US2013 / 0303587, US2018 / 0028664, US2015 / 0376115, US2016 / 0376224, US2016 / 0317458, US2013 / 0303587, US2013 / 0303587, and US20110123453, and US patents U.S. Pat. Nos.5,885,613, 6,287,591, 6,320,017, and 6,586,559. 4.6 Targeting ligand

[0134] In some embodiments, it may be desirable to limit transfection of the nucleic acids to certain cells or tissues. For example, the liver can be a target organ of interest in part due toits central role in metabolism and production of proteins and accordingly diseases which are caused by defects in liver-specific gene products (e.g., the urea cycle disorders) and may benefit from specific targeting of cells (e.g., hepatocytes).

[0135] In some embodiments, the LNP further includes a component including a targeting ligand. The targeting ligand can be selected as desired based on a target sell or tissue to which it is desired to direct the LNPs of this disclosure. In some embodiments, the targeting ligand is a ligand of a cell surface receptor. In some embodiments, the cell surface receptor is asialoglycoprotein receptor (ASGPR). The ASGPR is expressed on the surface of hepatocyte cells.

[0136] In some embodiments, the targeting ligand is a ligand for ASGPR, such as a N- acetylgalactosamine (GalNAc) containing ligand. A variety of GalNAc containing ligands and ligands, including multivalent GalNAc ligands are available for use in the LNP of this disclosure, including, e.g. those disclosed in WO2021178725, the full disclosure of which is incorporated herein by reference

[0137] In some embodiments, the PEG-lipid is linked to the targeting ligand. In some embodiments, the targeting ligand of interest (e.g., as described herein) is linked to a terminal of the PEG moiety. For example, a trisGalNac ligand conjugated to a PEG-lipid can provide for binding of the LNP to the ASGPR receptor of a target cell and result in endocytosis of the LNP. 4.7 Lipid Nanoparticles including lipids of Formula (I)

[0138] In some embodiments, the LNPs include an ionizable lipid of Formula (I) (e.g., as described herein); a nucleic acid cargo (e.g., as described herein); an additional ionizable lipid (e.g., as described herein); a phospholipid (e.g., as described herein), cholesterol (e.g., as described herein); and a lipid capable of reducing aggregation (e.g., as described herein).

[0139] In some embodiments of the LNP, the nucleic acid cargo comprises DNA, e.g., an oligonucleotide, a plasmid DNA, a doggybone DNA, a minicircle DNA, a covalently closed circular DNA, a ceDNA, or a chemically modified derivative thereof. In certain cases, the nucleic acid consists essentially of DNA. In some embodiments of the LNP, the nucleic acid cargo comprises RNA, e.g., an siRNA, a gRNA, an mRNA, a circular RNA, or a chemically modified derivative thereof. In certain cases, the nucleic acid consists essentially of RNA. In certain embodiments of the LNP, the nucleic acid cargo includes DNA, e.g,. an oligonucleotide, a plasmid DNA, a doggybone DNA, a minicircle DNA, a covalently closed circular DNA, a ceDNA, or a chemically modified derivative thereof, and further includesRNA, e.g., an siRNA, a gRNA, an mRNA, a circular RNA, and the like, or a chemically modified derivative thereof.

[0140] In some embodiments of the LNP, the phospholipid is selected from a phosphatidylcholine (PC), a phosphatidylethanolamine (PE), a phosphatidylserine (PS), a phosphatidylinositol (PI), and a phosphatidylglycerol (PG), and derivatives thereof. In certain cases, the phospholipid is phosphatidylethanolamine (PE). In certain cases, the phospholipid is a phosphatidylcholine (PC). In certain embodiments of the LNP, the phospholipid includes hydrocarbon chains each independently having 12-24 carbons. In some cases, the hydrocarbon chains each independently have 16-20 carbons. In certain cases, the hydrocarbon chains are saturated. In certain cases, the hydrocarbon chains are unsaturated. In certain cases, the hydrocarbon chains each independently comprise 1-4 double bonds. In certain cases, the phospholipid comprises two different hydrocarbon chains. In certain embodiments of the LNP, the phospholipid includes dioleoylphosphatidylethanolamine (DOPE, 18:1 PE). In certain cases, the phospholipid includes 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In certain cases, the phospholipid includes 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In certain cases, the phospholipid includes 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (delta9-Cis PC). In certain cases, the phospholipid includes 1-stearoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (SOPE). In certain cases, the phospholipid includes a mixture of dioleoylphosphatidylethanolamine (DOPE, 18-1) and dioleoylphosphatidycholine (DOPC, 18-1).

[0141] In certain embodiments of the LNP, the lipid capable of reducing aggregation is a PEG-lipid. In certain cases, the PEG lipid is 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (PEG-DMG[2K]) or PEG-1,2-distearoyl-rac-glycero-3- methylpolyoxyethylene 2000 (PEG-DSG[2K]).

[0142] In certain embodiments, the LNP further comprises a targeting ligand (e.g., as described herein). In certain cases, the targeting ligand comprises GalNac. In certain embodiments, the targeting ligand is linked to the ligand capable of reducing aggregation. In certain cases, the lipid capable of reducing aggregation linked to the targeting ligand is PEG- 1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG-DSG[2K]).

[0143] In some embodiments, the LNPs include an ionizable lipid of Formula (I) (e.g., as described herein); a phospholipid that is DOPE, cholesterol, and a lipid capable of reducing aggregation that is PEG-DMG.

[0144] In some embodiments, the LNPs include an ionizable lipid that is a cationic lipid comprising a tertiary amino ionizable group; a phospholipid that is a phosphatidylethanolamine, (e.g. DOPE), cholesterol, and a lipid capable of reducing aggregation that is PEG-DMG, and / or PEG-DSG-GalNAc or PEG-DSPE-GalNac.

[0145] In some embodiments, the LNPs include an ionizable lipid that is a cationic lipid comprising a tertiary amino ionizable group, a phospholipid that is a phosphatidylcholine (e.g.1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), cholesterol and a coat lipid (polyethylene glycol-dimyristolglycerol, PEG-DMG), for example as disclosed by Tam et al. (2013). Advances in Lipid Nanoparticles for siRNA delivery. Pharmaceuticals 5(3): 498-507.

[0146] Generally, the lipid particles are prepared that include a total lipid to DNA (mass or weight) ratio of from about 5:1 to 50:1. This is also referred to as the ratio of positively- chargeable polymer amine (N = nitrogen) groups to negatively-charged nucleic acid phosphate (P) groups, or N / P ratio. In some embodiments, the N / P ratio (mass / mass ratio; w / w ratio) can be in the range of from about 1:1 to about 50:1, from about 7:1 to about 25:1, 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 lipids and DNA can be adjusted to provide a desired N / P ratio, for example, N / P ratio of 3:1 (“3”), 4:1 (“4”), 5:1 (“5”), 6:1 (“6”), 7:1 (“7”), 8:1 (“8”), 9:1 (“9”), 10:1 (“10”), 11:1 (“11”), 12:1 (“12”), 13:1 (“13”), 14:1 (“14”) or higher. Generally, the lipid particle formulation's overall lipid content can range from about 5 mg / mL to about 30 mg / mL.

[0147] In some embodiments, the N / P ratio is from 5 to 30. In certain cases, the N / P ratio is 7. In certain cases, the N / P ratio is 14. In certain cases, the N / P ratio is 28.

[0148] In some embodiments, a lipid nanoparticle has a mean diameter between about 10 and about 1000 nm. In some embodiments, a lipid nanoparticle has a diameter that is less than 300 nm. In some embodiments, a lipid nanoparticle has a diameter between about 10 and about 300 nm. In some embodiments, a lipid nanoparticle has a diameter that is less than 200 nm. In some embodiments, a lipid nanoparticle has a diameter between about 25 and about 200 nm. In some embodiments, a lipid nanoparticle preparation (e.g., composition comprising a plurality of lipid nanoparticles) has a size distribution in which the mean size (e.g., diameter) is about 70 nm to about 200 nm, and more typically the mean size is about 100 nm or less.

[0149] In some embodiments, an LNP has a mean diameter of 25 to 250 nm, 25 to 240 nm, 25 to 230 nm, 25 to 220 nm, 25 to 210 nm, 25 to 200 nm, 25 to 190 nm, 25 to 180 nm,25 to 170 nm, 25 to 160 nm, 25 to 150 nm, 25 to 140 nm, 25 to 130 nm, 25 to 120 nm, 25 to 110 nm, 25 to 100 nm, 25 to 90 nm, 25 to 80 nm, 25 to 70 nm, 25 to 60 nm, or 25 to 50 nm.

[0150] In some embodiments, an LNP has a mean diameter of 60 to 250 nm, 70 to 250 nm, 80 to 250 nm, 90 to 250 nm, 100 to 250 nm, 110 to 250 nm, 120 to 250 nm, 130 to 250 nm, 140 to 250 nm, 150 to 250 nm, 160 to 250 nm, 170 to 250 nm, 180 to 250 nm, 190 to 250 nm, 200 to 250 nm, 210 to 250 nm, 220 to 250 nm, 230 to 250 nm, or 240 to 250 nm

[0151] In some embodiments, an LNP has a mean diameter of 60 to 250 nm, 70 to 240 nm, 80 to 230 nm, 90 to 220 nm, 100 to 210 nm, 110 to 200 nm, 120 to 190 nm, 130 to 180 nm, 140 to 170 nm, or 150 to 160 nm.

[0152] In some embodiments, the structural characteristics of the target tissue may be exploited to direct the distribution of the LNPs to such target tissues. For example, to target hepatocytes a LNP may be sized such that its dimensions are smaller than the fenestrations of the endothelial layer lining hepatic sinusoids in the liver; accordingly, the LNP can readily penetrate such endothelial fenestrations to reach the target hepatocytes. In some embodiments, a LNP may be sized such that the dimensions of the particles are of a sufficient diameter to limit or expressly avoid distribution into certain cells or tissues. For example, a LNP may be sized such that its dimensions are larger than the fenestrations of the endothelial layer lining hepatic sinusoids to thereby limit distribution of the LNPs to hepatocytes. In such an embodiment, large LNPs will not easily penetrate the endothelial fenestrations, and would instead be cleared by the macrophage Kupffer cells that line the liver sinusoids. In some embodiments, the size of the LNPs is within the range of about 25 to 250 nm or 25nm to 100nm, preferably less than 250 nm, less than 175 nm, less than 150 nm, less than 125 nm, or less than 100 nm.

[0153] Without limitations, ionizable lipid can comprise 20-90% (mol) of the total lipid present in the lipid nanoparticle. For example, ionizable lipid molar content can be 20-70% (mol), 30-60% (mol) or 40-50% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, ionizable lipid comprises from about 50 mol % to about 90 mol % of the total lipid present in the lipid nanoparticle. In some embodiments, the ionizable lipid comprises from about 50 mol % to about 85 mol %, from about 50 mol % to about 80 mol %, from about 50 mol % to about 75 mol %, from about 50 mol % to about 70 mol %, from about 50 mol % to about 65 mol %, from about 50 mol % to about 60 mol %, from about 55 mol % to about 65 mol %, or from about 55 mol % to about 70 mol % (or any fraction thereof or range therein) of the total lipid present in the particle. In particular embodiments, the cationic lipid comprises 40 mol %, 41 mol %, 42 mol %, 43 mol %, 44 mol %, 45 mol %, 46mol %, 47 mol %, 48 mol %, 49 mol %, 50 mol %, 51 mol %, 52 mol %, 53 mol %, 54 mol %, 55 mol %, 56 mol %, 57 mol%, 58 mol% or 60 mol% (or any fraction thereof) of the total lipid present in the particle.

[0154] The neutral lipid components can comprise 10-60% (mol) of the total lipid present in the lipid nanoparticle. For example, the non-cationic lipid content is 10-50% (mol) or 20- 55% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the non- cationic lipid comprises from about 10 mol % to about 60 mol %, from about 20 mol % to about 55 mol %, from about 20 mol % to about 45 mol %, from about 20 mol % to about 40 mol %, from about 25 mol % to about 50 mol %, from about 25 mol % to about 45 mol %, from about 30 mol % to about 50 mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %, from about 35 mol % to about 45 mol %, from about 37 mol % to about 42 mol % (or any fraction thereof or range therein) of the total lipid present in the particle. In particular embodiments, the non-cationic lipid comprises 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41 mol %, 42 mol %, 43 mol %, 44 mol %, 45 mol %, 46%, 47%, 48%, 49%, or 50% (or any fraction thereof or range therein) of the total lipid present in the particle.

[0155] In embodiments where the lipid particles contain a mixture of phospholipid and cholesterol or a cholesterol derivative, the mixture may comprise up to about 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % of the total lipid present in the particle. In particular embodiments, the mixture of phospholipid and cholesterol or a cholesterol derivative comprises up to 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41 mol %, 42 mol %, 43 mol %, 44 mol %, 45 mol %, 46%, 47%, 48%, 49%, or 50% (or any fraction thereof or range therein) of the total lipid present in the particle.

[0156] In some embodiments, the LNP comprises a phospholipid component in the mixture in an amount of from about 2 mol % to about 20 mol %, from about 2 mol % to about 15 mol %, from about 2 mol % to about 12 mol %, from about 4 mol % to about 15 mol %, or from about 4 mol % to about 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle. In some embodiments, the phospholipid component in the mixture comprises from about 5 mol % to about 10 mol %, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol %, or 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.

[0157] In some embodiments, the LNP includes a cholesterol component in the mixture in an amount of from about 25 mol % to about 45 mol %, from about 25 mol % to about 40mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %, from about 27 mol % to about 37 mol %, from about 25 mol % to about 30 mol %, or from about 35 mol % to about 40 mol % (or any fraction thereof or range therein) of the total lipid present in the particle. In some embodiments, the cholesterol component in the mixture comprises from about 25 mol % to about 35 mol %, from about 27 mol % to about 35 mol %, from about 29 mol % to about 35 mol %, from about 30 mol % to about 35 mol %, from about 30 mol % to about 34 mol %, from about 31 mol % to about 33 mol %, or 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34 mol %, 35 mol %, 36%, 37%, 38%, or 39% (or any fraction thereof or range therein) of the total lipid present in the particle.

[0158] It should be understood that the mol percentage of components described herein in the LNP is a target amount, and that the actual amount of each lipid component present in the formulation may vary, for example, by ±5 mol %.

[0159] In some embodiments, the LNP includes a lipid capable of reducing aggregation (e.g., a PEG-lipid conjugate) in an amount of about 1.5% to about 4%, for example about 1.5% to about 3%, about 2% to about 3%, about 2.5% to about 3%, about 1.5% to about 2.75%, about 1.5% to about 2.5%, about 1.5% to about 2.25%, about 1.5% to about 2%, about 1.5% to about 1.75%, about 2% to about 3%, about 2% to about 2.75%, about 2% to about 2.5%, about 2% to about 2.25% (or any fraction thereof or range therein) of the total lipid present in the particle. According to some embodiments, the lipid capable of reducing aggregation is present at 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3% or any fraction thereof or range therein) of the total lipid present in the particle.

[0160] In various embodiments, the molar ratio of ionizable lipid to the neutral lipid ranges from about 2:1 to about 8:1. In some embodiments, the lipid nanoparticles do not comprise any phospholipids.

[0161] In certain embodiments, the LNP comprises: a) an ionizable lipid at 40 to 60 mol % of the total lipid present; b) a phospholipid at 6 to 20 mol % of the total lipid present; c) cholesterol at 35 to 45 mol % of the total lipid present; and d) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present. In certain embodiments, the LNP comprises:a) an ionizable lipid at 40 to 60 mol % of the total lipid present; b) a phospholipid at 10 to 20 mol % of the total lipid present; c) cholesterol at 35 to 45 mol % of the total lipid present; and d) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present. In certain embodiments, the LNP comprises: a) an ionizable lipid at 40 to 49 mol % of the total lipid present; b) a phospholipid at 10 to 20 mol % of the total lipid present; c) cholesterol at 35 to 45 mol % of the total lipid present; and d) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.

[0162] In some embodiments, the ratio of ionizable lipid:phospholipid:cholesterol:PEG- lipid (as a percentage of total lipid content) is A:B:C:D, wherein: a. A = 40% - 60%, B = 5% - 20%, C = 25% - 50%, and D = 1.5% - 3.0% and wherein A+B+C+D = 100% b. A = 40% - 60%, B = 6% - 20%, C = 35% - 45%, and D = 1.5% - 2.5% and wherein A+B+C+D = 100%; c. A = 40% - 60%, B = 10% - 20%, C = 35% - 45%, and D = 1.5% - 2.5% and wherein A+B+C+D = 100%; d. A = 40% - 49%, B = 10% - 20%, C = 35% - 45%, and D = 1.5% - 2.5% and wherein A+B+C+D = 100%; e. A = 40% - 49%, B = 10% - 20%, C = 35% - 45%, and D = 1.5% - 2.5% and wherein A+B+C+D = 100%; f. A = 39% - 60%, B = 10% - 25%, C = 20% - 30%, and D = 0% - 3% and wherein A+B+C+D = 100%;g. A = 40% - 60%, B = 10% - 25%, C = 20% - 30%, and D = 0% - 3% and wherein A+B+C+D = 100%; h. A = 45% - 50%, B = 20% - 25%, C = 25% - 30%, and D = 0% - 1% and wherein A+B+C+D = 100% i. A = 40% - 60%, B = 10% - 30%, C = 20% - 45%, and D = 0% - 3% and wherein A+B+C+D = 100%; j. A = 40% - 60%, B = 10% - 30%, C = 25% - 45%, and D = 0% - 3% and wherein A+B+C+D = 100%; k. A = 45% - 55%, B = 10% - 20%, C = 30% - 40%, and D = 1% - 2% and wherein A+B+C+D = 100%; l. A = 45% - 50%, B = 10% - 15%, C = 35% - 40%, and D = 1% - 2% and wherein A+B+C+D = 100%; m. A = 45% - 65%, B = 5% - 20%, C = 20% - 45%, and D = 0% - 3% and wherein A+B+C+D = 100%; n. A = 45%, B = 15%, C = 37.5%, and D = 2.5%; o. A = 57%, B = 12%, C = 28.5%, and D = 2.5% p. A = 50% - 60%, B = 5% - 15%, C = 30% - 45%, and D = 0% - 3% and wherein A+B+C+D = 100%; q. A = 55% - 60%, B = 5% - 15%, C = 30% - 40%, and D = 1% - 2% and wherein A+B+C+D = 100%; or r. A = 55% - 60%, B = 5% - 10%, C = 30% - 35%, and D = 1% - 2% and wherein A+B+C+D = 100%. 4.8 Nucleic Acid Cargo

[0163] In many embodiments, a given lipid nanoparticle of the present disclosure can include a cargo, or payload, to be delivered to cells. Of particular interest in someembodiments are cargos that comprise a polynucleotide. In some embodiments, the polynucleotide is a DNA. DNA nucleic acid compositions of any structure may be included in the LNPs of the present disclosure. For example, the DNA may be circular, e.g., a plasmid, a nanoplasmid (npDNA), a minicircle, a covalently closed circular DNA, a circular viral genome, and the like. As another example, the DNA may be linear, e.g., a doggybone or other closed-end DNA, a linear viral genome, and the like. As another example, the DNA may be multivalent, e.g., a 3DNA. The DNA may be single stranded or double stranded or a hybrid of single and double stranded. The DNA may be chemically modified. In some embodiments, the polynucleotide is an RNA. RNA nucleic acid compositions of any structure may be included in the LNPs of the present disclosure. For example, the RNA may be linear or it may be circular. The RNA can be an mRNA, an siRNA, an shRNA, a guide RNA (gRNA), a microRNA (miRNA), or a circular RNA (circRNA). The RNA may be chemically modified.

[0164] The one or more additional compounds can be a therapeutic agent. The therapeutic agent can be selected from any class suitable for the therapeutic objective. In other words, the therapeutic agent can be selected according to the treatment objective and biological action desired. For example, if the DNA within the LNP is useful for treating cancer, the additional compound can be an anti-cancer agent (e.g., a chemotherapeutic agent, a targeted cancer therapy including, but not limited to, a small molecule, an antibody, or an antibody-drug conjugate). In another example, if the LNP containing the DNA is useful for treating an infection, the additional compound can be an antimicrobial agent (e.g., an antibiotic or antiviral compound). In yet another example, if the LNP containing the DNA is useful for treating an immune disease or disorder, the additional compound can be a compound that modulates an immune response (e.g., an immunosuppressant, immunostimulatory compound, or compound modulating one or more specific immune pathways). In some embodiments, different cocktails of different lipid nanoparticles containing different compounds, such as a DNA encoding a different protein or a different compound, such as a therapeutic may be used in the compositions and methods of the invention. In some embodiments, the additional compound is an immune modulating agent. For example, the additional compound is an immunosuppressant. In some embodiments, the additional compound is immune stimulatory agent.4.9 Pharmaceutical Compositions

[0165] Also provided herein is a pharmaceutical composition comprising the lipid nanoparticle-encapsulated nucleic acid (e.g., DNA) and a pharmaceutically acceptable carrier or excipient. In some aspects, the disclosure provides for a lipid nanoparticle formulation further comprising one or more pharmaceutical excipients. In some embodiments, the lipid nanoparticle formulation further comprises sucrose, tris, trehalose and / or glycine.

[0166] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0167] As used herein “pharmaceutically acceptable carrier, diluent or excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, to sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and any other compatible substances employed in pharmaceutical formulations.

[0168] “Pharmaceutically acceptable salt” includes both acid and base addition salts. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5- disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, ptoluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2- dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. 4.10 Methods of Preparation

[0169] Any convenient methods can be used to prepare the LNPs of this disclosure. The LNP compositions can be prepared by high energy mixing of ethanolic lipids with aqueous DNA at low pH which protonates the ionizable lipid and provides favorable energetics for DNA / lipid association and nucleation of particles. The particles can be further stabilized through aqueous dilution and removal of the organic solvent. The particles can be concentrated to the desired level.4.11 Methods of Use

[0170] As illustrated in the working examples and figures here, the LNPs and LNP pharmaceutical composition of the present disclosure, when formulated with nucleic acids, are less toxic in vivo as compared to an industry standard LNP (comprising 50% ionizable lipid ALC-0315, 10% DSPC, 38.5% cholesterol, and 1.5% PEG lipid) administered at the same dose, e.g. at least 2-fold less toxic, e.g.3-fold, 4-fold or 5-fold less toxic, in some instances 10-fold, 20-fold, or 50-fold less toxic, in certain instances 100-fold less toxic. By “less toxic”, it is meant eliciting a reduced immune response, e.g., characterized in a reduced amount of one or more cytokines upon administration to an organism.

[0171] At the same time, the LNPs and LNP pharmaceutical composition of the present disclosure have been observed to be efficacious at delivering their nucleic acid cargo to the target cell of interest, including where LNPs and LNP pharmaceutical composition of the present disclosure are equally or more efficacious at delivering their nucleic acid cargo to the target cell of interest as that same industry standard LNP administered at the same dose, e.g. having 2-fold the efficacy or more, e.g.3-fold, 4-fold or 5-fold the efficacy or more, in some instances 10-fold, 20-fold or 50-fold the efficacy, in certain instances 100-fold more efficacious or more. By “more efficacious”, it is meant able to deliver more nucleic acid cargo to the cell, resulting in an increase in the amount of mRNA transcribed from that nucleic acid cargo or an increase in the amount of protein translated, for example a 2-fold increase or more, e.g. a 3-fold, 4-fold, 5-fold increase, e.g.10-fold, 20-fold, 50-fold increase, in some instances a 100-fold increase or more.

[0172] Put another way, the LNPs of the present disclosure demonstrate an improved pharmacokinetics (PK) profile that broadens the therapeutic index of the composition. By a therapeutic index, or therapeutic ratio, it is meant the range of doses at which a medication is effective without unacceptable adverse events, calculated as the ratio that compares the blood concentration at which a drug becomes toxic and the concentration at which the drug is effective. This improvement over the art makes them more amenable to delivering nucleic acids, including DNA, to cells in vitro and in vivo, and accordingly they find many uses in many applications, including in the delivery of nucleic acids, including DNA, to cells for research and for therapeutic applications.

[0173] In performing such methods, the cells are typically contacted with the composition, e.g., LNP or pharmaceutical composition thereof, in amount effective to deliver the agent into the cytoplasm of the cell. In some embodiments, the contacting is in vitro. Inother embodiments, the contacting is in vivo. In some embodiments, the method further comprises measuring the amount of protein produced.

[0174] The present disclosure further provides methods of treating or preventing diseases in a subject in need thereof wherein an effective amount of the therapeutic compositions described herein is administered to the subject. The route of administration will vary, naturally, with the location and nature of the disease being treated, and may include, for example intradermal, transdermal, subdermal, parenteral, nasal, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration. The encapsulated polynucleotide compositions described herein are useful in the treatment of any of any indication in which it is beneficial to deliver a therapeutic cargo into the target cell.

[0175] The present disclosure further provides methods of immunizing a subject against a disease wherein an effective amount of a therapeutic composition described herein is administered to the subject. The route of administration will vary, naturally, with the location and nature of immunization agent, and may include, for example intradermal, transdermal, subdermal, parenteral, nasal, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration.

[0176] The present disclosure further provides a particle of the disclosure, a vector of the disclosure, a recombinant DNA of the disclosure, or compositions thereof, for use as a medicament. In some embodiments, the medicament is for expressing a protein in a cell. In some embodiments, the expressing of a protein is for the treatment of a disease in which the cell is deficient for the protein. In some embodiments, the expressing of a protein is for the treatment of a disease in which another cell is deficient for the protein. In some embodiments, the medicament is for the treatment of a cancer. In some embodiments, the medicament is for immunization against a disease. 4.12 Utility

[0177] The subject methods and compositions, e.g., as described above, can be used in any application where delivery of a cargo nucleic acid is desired. Applications of interest include both research and therapeutic applications. Applications of interest include, but are not limited to: research applications, diagnostic applications and therapeutic applications. In some instances, cargo nucleic acids that may be introduced into a cell, and subsequently a 61 of 94nucleus, via methods of the invention include those encoding research proteins, diagnostic proteins and therapeutic proteins.

[0178] Research proteins are proteins whose activity finds use in a research protocol. As such, research proteins are proteins that are employed in an experimental procedure. The research protein may be any protein that has such utility, where in some instances the research protein is a protein domain that is also provided in research protocols by expressing it in a cell from an encoding vector. Examples of specific types of research proteins include, but are not limited to: transcription modulators of inducible expression systems, members of signal production systems, e.g., enzymes and substrates thereof, hormones, prohormones, proteases, enzyme activity modulators, perturbimers and peptide aptamers, antibodies, modulators of protein-protein interactions, genomic modification proteins, such as CRE recombinase, meganucleases, Zinc-finger nucleases, CRISPR / Cas-9 nuclease, TAL effector nucleases, etc., cellular reprogramming proteins, such as Oct 3 / 4, Sox2, Klf4, c-Myc, Nanog, Lin-28, etc., and the like.

[0179] Diagnostic proteins are proteins whose activity finds use in a diagnostic protocol. As such, diagnostic proteins are proteins that are employed in a diagnostic procedure. The diagnostic protein may be any protein that has such utility. Examples of specific types of diagnostic proteins include, but are not limited to: members of signal production systems, e.g., enzymes and substrates thereof, labeled binding members, e.g., labeled antibodies and binding fragments thereof, peptide aptamers and the like.

[0180] Proteins of interest further include therapeutic proteins. Therapeutic proteins of interest include without limitation, hormones and growth and differentiation factors, fibrinolytic proteins, transcription factors, and enzymes.

[0181] Target cells to which nucleic acids may be delivered in accordance with embodiments of this disclosure may vary widely. Target cells of interest include, but are not limited to: cell lines, HeLa, HEK, CHO, 293 and the like, Mouse embryonic stem cells, human stem cells, mesenchymal stem cells, primary cells, tissue samples and the like. Some non-limiting examples of a mammalian cell include, without limitation, a mouse cell, a rat cell, hamster cell, a rodent cell, and a nonhuman primate cell. In some embodiments, the target cell is a human cell. It should also be appreciated that the target cell may be of any cell type. For example, the target cell may be a stem cell, which may include embryonic stem cells, induced pluripotent stem cells (iPS cells), fetal stem cells, cord blood stem cells, or adult stem cells (i.e., tissue specific stem cells). In other cases, the target cell may be any differentiated cell type found in a subject. Cells of interest include both dividing cells andnon-dividing cells. Examples of specific target cells of interest include, but are not limited to: hepatocytes, stellate cells, T lymphocytes, B lymphocytes, NK cells, skeletal muscle cells, cardiomyocytes, neurons, astrocytes, oligodendrocytes, dendritic cells, skin cells, etc.

[0182] Targeted cells may include the cells of a targeted location, such as, e.g., the liver, or cells near or adjacent to hepatocytes, e.g., hepatocytes, hepatic stellate cells (HSCs), Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), ductal cells, or combinations thereof.

[0183] In some instances, the application of interest is a therapeutic application, for example, in the treatment of a disease. For example, the compositions and methods of the present application may be used to deliver a nucleic acid sequence to a cell to complement a genetic deficiency. As one nonlimiting example, compositions of the present application may be used in the treatment of a genetic deficiency that impacts the function of hepatocytes, or in the treatment of a genetic deficiency elsewhere in the body that can be remedied by leveraging hepatocytes as a biofactory to secrete the deficient protein.

[0184] The following example(s) is / are offered by way of illustration and not by way of limitation. 5. EXAMPLES

[0185] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0186] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures inBiotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, cells, and kits for methods referred to in, or related to, this disclosure are available from commercial vendors such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., and the like, as well as repositories such as e.g., Addgene, Inc., American Type Culture Collection (ATCC), and the like. Materials and Methods

[0187] LNP formulation. LNPs encapsulating nucleic acid payloads are prepared by mixing an organic solution of lipids with an aqueous solution of nucleic acid (e.g., DNA only, mRNA only, or DNA / mRNA mixtures) as described in Prud’homme et al. (J Pharm Sci 2018). Briefly, the lipidic excipients mixture (ionizable lipid, helper lipid, cholesterol, PEG- lipid and potentially other targeting moieties) is dissolved in an organic solvent. An aqueous solution of the nucleic acid is prepared in a low pH buffer of range pH 3.0 – 4.0. The lipid mixture is then mixed with the aqueous nucleic acid solution at a flow ratio of 1:3 (V / V) using a commercially available mixer device. The resulting solution is immediately diluted with a buffer of acidic pH. The diluted LNP is subjected to dialysis purification against a secondary buffer having a neutral pH. The LNP solution is concentrated through Amicon Ultra centrifuge tubes (Millipore Sigma) followed by filtration through a PES sterilizing- grade filter. Particle size is determined by dynamic light scattering (Horiba nanoPartica SZ- 100). Encapsulation efficiency is calculated by using Quant-it RiboGreen assay kit.

[0188] EPO and cytokine detection in serum. Blood is collected via a retro-orbital bleed into serum separator tubes and processed to serum. The serum samples may be stored at -80C from collection until analysis. The serum levels of human EPO protein driven by expression from the DNA payload are quantified using the U-PLEX Human EPO Assay from MSD according to the manufacturer’s instructions. The serum levels of mouse cytokines resulting from exposure to DNA-LNPs were quantified using the Mouse ProInflammatory 7-Plex Tissue Culture Kit from MSD according to the manufacturer’s instructions.

[0189] FIX detection in plasma. Blood is collected via a retro-orbital bleed into K2EDTA tubes and processed to plasma. The plasma samples may be stored at -80C from collection until analysis. The plasma levels of human FIX following administration of LNPs were quantified using a U-Plex assay on the MSD platform. Briefly, a monoclonal mouse anti-human FIX antibody was conjugated to biotin and used as the capture reagent on streptavidin-coated plates. A polyclonal goat anti-human FIX antibody (Cedarlane) wasconjugated to Sulfo-TAG and used as the detection reagent with the standard setup for quantification of electrochemiluminescence (ECL) signal using the QuickPlex SQ 120MM instrument from MSD. Pooled normal human plasma (Affinity Biologicals), which is a pool of normal citrated human plasma collected from a minimum of 20 donors, was used to generate a standard curve and calculate % of normal human FIX levels. The assay was confirmed to be specific for human FIX and not to cross-react with mouse FIX, demonstrating very low levels of background in untreated mouse plasma samples. 5.1. Synthetic LNP Examples Example 1. Synthesis of di(pentadecane-8-yl) 5-(3 (dimethylamino)propylidene)nonanedioate (U-1)65 of 94

[0190] Synthesis of di(pentadecane-8-yl) 5-cyclopropylidenenonanedioate

[0191] Into a 100 mL round-bottom flask, was placed cyclopropyltriphenylphosphanium bromide (2.95 g, 7.704 mmol, 1.6 equiv) and THF (60 mL) at room temperature under N2 atmosphere. To this was added t-BuOK (7.22 mL, 7.223 mmol, 1.5 equiv into 1M THF) at 0°C and the mixture was stirred for 15 min at 0°C. To this was added 1,9-bis(pentadecan-8- yl) 5-oxononanedioate (prepared according to PCT / US2023 / 079923) (3 g, 4.815 mmol, 1 equiv into 10 ml THF) at 0°C. The mixture was stirred for 16 hours at room temperature and then quenched by the addition of water (40 ml) at 5ºC. The mixture was extracted with EA (2x80 ml) and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with Hep / EA (10 / 1) to afford 1,9-bis(pentadecan-8-yl) 5- cyclopropylidenenonanedioate (1.7 g, 52.05%) as a light-yellow oil.

[0192] Synthesis of 1,9-bis(pentadecan-8-yl) 5-(3-chloropropylidene) nonanedioate

[0193] In a 8 mL vial was placed 1,9-bis(pentadecan-8-yl) 5-cyclopropylidenenonanedioate (1.6 g, 2.473 mmol, 1 equiv) and dioxane (0.8 mL) at room temperature under N2atmosphere. To this was added HCl in 1,4-dioxane (4M, 0.80 mL, 26.337 mmol) at room temperature. The mixture was stirring for 12 min at 120ºC. The mixture was concentrated under reduced pressure to afford the crude 1,9-bis(pentadecan-8-yl) 5-(3-chloropropylidene) nonanedioate as a light-yellow oil. The product was used in the next step directly without further purification.

[0194] Synthesis of 1,9-bis(pentadecan-8-yl) 5-[3-(dimethylamino)propylidene]nonanedioate

[0195] In a 40 mL vial was placed 1,9-bis(pentadecan-8-yl) 5-(3 chloropropylidene)nonanedioate (1.2 g, 1.756 mmol, 1 equiv) and dimethylamine (2 M in MeOH) (6 mL) at room temperature under N2atmosphere. To this was added KI (0.06 g, 0.351 mmol, 0.2 equiv) at room temperature and the mixture was left to stir for 4 hours at 80°C. The resulting mixture was diluted with DCM (150 ml) and washed with water (2 x 50 ml). The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography, eluted with DCM / MeOH (10 / 1) to afford 1,9- bis(pentadecan-8-yl) 5-[3-(dimethylamino)propylidene]nonanedioate (600 mg, 86% in HPLC). The product (HPLC: 86%) was purified by reversed-phase flash chromatography with the following conditions: column, C18 silica gel; mobile phase, A: H2O (0.1% TFA), B: ACN, Gradient: 50-90% 15min, 90-90% 3 min, 95-95% 2min, Flow:90ml / min; Column Welch Phenyl; detector, UV 205 nm to afford 1,9-bis(pentadecan-8-yl) 5-[3- (dimethylamino)propylidene]nonanedioate 2,2,2-trifluoroacetic acid salt (500 mg). The product salt was dissolved in n-Heptane (50 mL) and washed with sat. Na2CO3 (3 x 50 mL), water (1 x 50 mL) and brine (1 x 50 mL) and dried with anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford 1,9-bis(pentadecan-8-yl) 5-[3-(dimethylamino)propylidene]nonanedioate (447.9 mg, HPLC: 98.0%，yield：36.12%) as light yellow oil. LCMS-PH : (ES, m / z): 691.65 [M+H]+.1H NMR (300 MHz, Chloroform-d) δ 5.183 (t, J = 6.9 Hz, 1H), 4.904-4.822 (m, 2H), 2.296-2.184 (m, 14 H), 2.081-1.999 (m, 4H), 1.761-1.631 (m, 4H), 1.631-1.496 (m, 8H), 1.305-1.185 (m, 40H), 0.876 (t, J = 6.3Hz, 12H). Example 2. Synthesis of 1,9-bis(pentadecan-8-yl) 5-[4- (dimethylamino)butylidene]nonanedioate (U-2)

[0196] Synthesis of 1,9-bis(pentadecan-8-yl) 5-(3-cyanopropylidene)nonanedioate

[0197] Into a 250 mL round-bottom flask, was place 1,9-bis(pentadecan-8-yl) 5-(3- chloropropylidene)nonanedioate (6 g, 8.778 mmol, 1 equiv), THF (60 mL) and TMSCN (6 mL) at 25°C. To this was added TBAF (6.58 mL, 13.160 mmol, 1.50 equiv) at 25°C under N2 atmosphere and the mixture was stirring for 16 hours at 100ºC. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with Heptane / EA gradient from 100:0 to 90:10, and collected product eluent Heptane / EA=94 / 6). Took sample for TLC analysis. (Heptane: EA = 10:10.2). To afford 1,9- bis(pentadecan-8-yl) 5-(3-cyanopropylidene)nonanedioate (4.6 g, 77.74%) as light yellow oil.

[0198] Synthesis of 1,9-bis(pentadecan-8-yl) 5-(4-aminobutylidene)nonanedioate

[0199] Into a 250 mL round-bottom flask, was place 1,9-bis(pentadecan-8-yl) 5-(3- cyanopropylidene)nonanedioate (4.5 g, 6.675 mmol, 1 equiv), MeOH (90 mL) and CoCl2^6H2O (2.38 g, 10.012 mmol, 1.5 equiv) at 0°C. To this was added NaBH4(1.52 g, 40.050 mmol, 6 equiv) in portions at 0°C under N2 atmosphere and the mixture was left to stir for 1 hour at 0ºC. The mixture was acidified to pH=7-8 with (0.8 M HCl aq.) and theaqueous layer was extracted with EA (3 x 150 mL). The organic phase dried over anhydrous Na2SO4and then concentrated under vacuum. The residue was purified by reversed-phase flash chromatography with the following conditions: column, CSH prep-C18 silica gel; mobile phase, 0.1% TFA in MeCN, 60% to 95% gradient in 30 min; detector, UV 200 nm to afford 1,9-bis(pentadecan-8-yl) 5-(4-aminobutylidene)nonanedioate (1.6 g, 33.15%) as light yellow oil.

[0200] Synthesis of 1,9-bis(pentadecan-8-yl) 5-[4-(dimethylamino)butylidene]nonanedioate

[0201] Into a 100 mL round-bottom flask, was placed 1,9-bis(pentadecan-8-yl) 5-(4- aminobutylidene)nonanedioate (1.5 g, 2.212 mmol, 1 equiv), formaldehyde (1.5 mL, 37%) and H2O (15 mL). To this was added HCOOH (1.5 mL) at 25ºC under N2atmosphere and the mixture was left stirring for 2 hours at 100ºC. The mixture was allowed to cool down to 25ºC. The residue was purified by reversed-phase flash chromatography with the following conditions: column, CSH prep-C18 silica gel; mobile phase, 0.1% TFA in ACN, 60% to 95% gradient in 30 min; detector, UV 200 nm. The mixture was based to pH =10 with Na2CO3(aq.) and was extracted with Heptane (2 x 300 mL). The organic phase was washed with 2 x 40 mL of (MeOH / H2O=4 / 1) and dried over anhydrous Na2SO4. The organic phase was concentrated under vacuum To afford 1,9-bis(pentadecan-8-yl) 5-[4- (dimethylamino)butylidene]nonanedioate (503.4 mg, 30.62%) as light yellow oil. LCMS: (ES, m / z): 705.66 [M+H]+.1H NMR (400 MHz, Chloroform-d) δ 5.183 (t, J = 7.2 Hz, 1H), 4.897-4.835 (m, 2H), 2.281-2.234 (m, 12 H), 2.061-1.994 (m, 6 H), 1.751-1.635 (m, 4 H), 1.541-1.486 (m, 8H), 1.305-1.185 (m, 40H), 0.894 (t, J = 7.2Hz, 12H). Example 3. Synthesis of 1,7-bis(pentadecan-8-yl) 4-[3- (dimethylamino)propylidene]heptanedioate (U-3)

[0202] Synthesis of 1,7-bis(pentadecan-8-yl) 4-oxoheptanedioate

[0203] To a stirred solution of 4-oxoheptanedioic acid (5 g, 28.711 mmol, 1 equiv) and DMF (0.10 g, 1.436 mmol, 0.05 equiv) in THF (150 mL) was added (COCl)2(18.22 g, 143.555 mmol, 5 equiv) dropwise at 0°C under nitrogen atmosphere. The resulting mixture was stirred for overnight at room temperature under nitrogen. The resulting mixture was concentrated under reduced pressure. To a stirred solution of pentadecan-8-ol (prepared according to 71 of 94PCT / US2023 / 079923) (13.77 g, 60.293 mmol, 2.1 equiv) and DMAP (7.02 g, 57.422 mmol, 2 equiv) was added the above mixture dissolved in THF (50 mL) dropwise at room temperature under nitrogen atmosphere, the resulting mixture was stirred overnight at room temperature under nitrogen atmosphere. The reaction was quenched by the addition of water / ice (100 mL, 20V) at room temperature and the resulting mixture was extracted with EA (2 x 200 mL, 40V). The organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford 1,7-bis(pentadecan-8- yl) 4-oxoheptanedioate (3.8 g, 22.25%) as light-yellow oil.

[0204] Synthesis of 1,7-bis(pentadecan-8-yl) 4-cyclopropylideneheptanedioate

[0205] Into a 100 ml round-bottom flask, was placed cyclopropyltriphenylphosphanium (3.30 g, 8.605 mmol, 1.6 equiv) and THF (66 mL) at room temperature under N2 atmosphere. To this was added t-BuOK (0.91 g, 8.110 mmol, 1.5 equiv) at 0°C and the mixture was stirring for 15 min at 0ºC. To this mixture was added 1,9-bis(pentadecan-8-yl) 5-oxononanedioate (3 g, 4.815 mmol, 1 equiv into 10 mL THF) at 0°C and the mixture was stirring for 16 hours at room temperature. The reaction was quenched by the addition of water (40 mL) at 5°C. The mixture was extracted with EA (2x80 mL) and the filtrate was concentrated under reduced pressure. To the filtrate was added 5 g of silica gel (type: ZCX-2, 100-200 mesh, 2.00 w. / w.). The mixture was concentrated to no fraction under vacuum while maintaining the temperature below 35°C.60 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w. / w.) was charged to the column, followed by the prepared dry silica gel which absorbed the reactionmixture of the last step. Using combi-flash to purify the product. Eluted with Hep / EA. (gradient from 100:0 to 95:5, collected every 200 ± 10 mL). Fractions were analyzed by TLC. (Hep / EA = 10:1) to afford1,7-bis(pentadecan-8-yl) 4-cyclopropylideneheptanedioate (1.9 g, 57.07%) as a light-yellow oil.

[0206] Synthesis of 1,7-bis(pentadecan-8-yl) 4-(3-chloropropylidene)heptanedioate

[0207] Into 8 mL vial, was place 1,7-bis(pentadecan-8-yl) 4-cyclopropylideneheptanedioate (2.0 g, 3.231 mmol, 1 equiv) and dioxane (3 mL) at room temperature under N2 atmosphere. To this was added HCl (gas) in 1,4-dioxane (3 mL) at room temperature. The mixture was stirring for 12 min at 120°C. The mixture was concentrated under reduced pressure to afford crude 1,7-bis(pentadecan-8-yl) 4-(3-chloropropylidene)heptanedioate (crude) as a light yellow oil. The product was used in the next step directly without further purification.

[0208] Synthesis of 1,7-bis(pentadecan-8-yl) 4-[3- (dimethylamino)propylidene]heptanedioate

[0209] Into a 40mL vial, was place 1,7-bis(pentadecan-8-yl) 4-(3- chloropropylidene)heptanedioate (1.8 g, 2.746 mmol, 1 equiv) and dimethylamine (2 M in MeOH) (7.2 mL) at room temperature under N2 atmosphere. To this was added KI (45.58 mg, 0.275 mmol, 0.1 equiv) at room temperature and the mixture was stirring for 4 hours at 80°C. The resulting mixture was diluted with DCM (150 mL) and washed with water (2x50 mL). The organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure and to the filtrate was added 5 g of silica gel (type: ZCX-2, 100-200 mesh, 2.00 w. / w.). The mixture was concentrated under vacuum while maintaining the temperature below 35°C.40 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w. / w.) was charged to the column, followed by the prepared dry silica gel which absorbed the reaction mixture of the last step. Using combi-flash to purify the product. Eluted with DCM / MeOH. (gradient from 100:0 to 95:5, collected every 200 ± 10 mL) and fractions were analyzed by TLC. (DCM / MeOH = 10:1) to afford 1,7-bis(pentadecan-8-yl) 4-[3- (dimethylamino)propylidene]heptanedioate (crude) as yellow oil. The crude product was dissolved with n-Heptane (200 mL), washed by 2 x 40 mL of MeOH / H2O (4 / 1) and the organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This resulted in 1,7-bis(pentadecan-8-yl) 4-[3- (dimethylamino)propylidene]heptanedioate (0.6874 g, HPLC: 96.6%) as yellow oil.14. LCMS (ES, m / z): 663.62 [M+H]+; 1H NMR (300 MHz, Chloroform-d) δ 5.203 (t, J = 6.6 Hz, 1H), 4.890-4.852 (m, 2H), 2.450-2.222 (m, 18 H), 1.522-1.510 (m, 8H), 1.305-1.185 (m, 40H), 0.894 (t, J = 6.6 Hz, 12H). Example 4. Synthesis of 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl]-9-pentadecan- 8-yl (5Z)-5-[4-(dimethyl amino)butylidene]nonanedioate

[0210] Synthesis of 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl] 9-pentadecan-8-yl 5- cyclopropylidene nonanedioate

[0211] Into a 500 mL 3-necked round bottom flask was added cyclopropyltriphenylphosphanium hydrobromidyl (6.6 g, 17.220 mmol, 1.61 equiv) and THF (156 mL) at room temperature under nitrogen atmosphere. To the above mixture was added t- BuOK (1M in THF, 16.1 mL, 16.100 mmol, 1.50 equiv) dropwise at 0 ºC over 20 min under nitrogen atmosphere. The resulting mixture was stirred for 30 min at 0ºC under nitrogen atmosphere. To the above mixture was added 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3- yl] 9-pentadecan-8-yl 5-oxononanedioate (prepared according to PCT / US2023 / 079923) (7.8 75 f 94g, 10.727 mmol, 1 equiv, in 50 mL of THF) dropwise at 0ºC. The resulting mixture was stirred for additional 18 h at room temperature. The reaction was quenched with sat. aqueous NH4Cl (200 mL) at 0ºC and then extracted with EtOAc (2 x 100 mL). The combined organic layers were washed with brine (1 x 100 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure and the residue was dissolved in n- heptane (150 mL). The organic layer was washed with sat. aqueous Na2CO3(5 %, 100 mL), MeOH / water (4:1, 3 x 100 mL), water (100 mL) and brine (1 x 100 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography, elution with petroleum ether / EtOAc (95:5) afforded 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl] 9-pentadecan-8-yl 5- cyclopropylidene nonanedioate (5.0 g, yield: 38.86%) as colorless oil.

[0212] Synthesis of 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl] 9-pentadecan-8-yl- (5Z)-5-(3-chloro propylidene)nonanedioate

[0213] Into a 40 mL vial was added 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl] 9- pentadecan-8-yl 5-cyclopropylidenenonanedioate (5 g, 6.656 mmol, 1 equiv) and 1,4-dioxane (7.5 mL) at room temperature under nitrogen atmosphere. To the above mixture was added HCl (4M in 1,4-dioxane, 7.5 mL, 30.000 mmol, 4.51 equiv) dropwise at room temperature. The resulting mixture was stirred for 15 min at 120ºC and then cooled down to room temperature. The resulting mixture was concentrated under reduced pressure. This resulted in 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl] 9-pentadecan-8-yl-(5Z)-5-(3-chloro propylidene)nonanedioate (5.2 g, crude) as light yellow oil. The product was used in the next step directly without further purification.

[0214] Synthesis of 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl]-9-pentadecan-8-yl- (5Z)-5-(3-cyanopropyl idene)nonanedioate

[0215] Into a 250 mL round-bottom flask were added 1-[1,5-bis(octahydro-1H-inden-2- yl)pentan-3-yl]-9-penta decan-8-yl (5Z)-5-(3-chloropropylidene)nonanedioate (5 g, 5.440 mmol, 1 equiv) and THF (100 mL) at room temperature under nitrogen atmosphere. To the above mixture was added TMSCN (5 mL, 50.399 mmol, 9.26 equiv) and TBAF (5.44 mL, 5.440 mmol, 1.00 equiv) dropwise at room temperature. The resulting mixture was stirred for 18 h at 100ºC. The mixture was allowed to cool down to room temperature and the resulting mixture was diluted with ethyl acetate (100 mL). The mixture was washed with 2 x 100 mL of water and 100 mL of brine, dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography, eluted with petroleum ether / EtOAc (94:6) to afford 1-[1,5-bis(octahydro- 1H-inden-2-yl)pentan-3-yl]-9-pentadecan-8-yl-(5Z)-5-(3-cyanopropyl idene)nonanedioate (3.0 g, yield: 64.41%) as colorless oil.

[0216] Synthesis of 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl]-9-pentadecan-8-yl- (5Z)-5-(3-cyano propylidene)nonanedioate

[0217] Into a 250 mL 3-necked round-bottom flask were added 1-[1,5-bis(octahydro-1H- inden-2-yl)pentan-3-yl]- 9-pentadecan-8-yl (5Z)-5-(3-chloropropylidene)nonanedioate (2.4 g, 2.770 mmol, 1 equiv), THF (24 mL), EtOH (72 mL) and CoCl2.6H2O (1.32 g, 5.548 mmol, 2.00 equiv) at room temperature under nitrogen atmosphere. To the above mixture was added NaBH4 (261.94 mg, 6.925 mmol, 2.5 equiv) in portions over 5 min at 0ºC. The resulting mixture was allowed to warm to room temperature with stirring for additional 3 h. The reaction was quenched by the addition of aqueous HCl (0.8 M, 10 mL) at 0ºC. The mixture basified pH to 9 with saturated Na2CO3(aqueous) under nitrogen atmosphere. The residue was diluted with water (100 mL) and EtOAc (100 mL) with stirring. The resulting mixture was filtered. The organic layer was separated and collected, washed with brine (50 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by Pre-HPLC with the following conditions: column, XBridge BEH Shield PR18 OBD Prep Column, 130 Å, 5μm, 19 mmX250mm, 1 / pk; mobile phase, MeCN / IPA(70:30, 0.1% TFA) in water (0.1% TFA), 90% to 91% gradient in 9 min; Flow rate: 20ml / min, Ms detector. The fraction (8.02 min) was collected and concentrated under reduced pressure. The residue was dissolved with water (80 mL). The mixture was basified pH to 9 with saturated Na2CO3 (aqueous). The resulting mixture was extracted with EtOAc (2 x 50 mL). The combined organic layers were washed with brine (1 x 100 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This resulted in 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl]-9-pentadecan-8-yl-(5Z)-5-(3- cyano propylidene)nonanedioate (830 mg, yield: 34.69%) as light yellow oil.

[0218] Synthesis of 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl]-9-pentadecan-8-yl (5Z)-5-[4-(dimethyl amino)butylidene]nonanedioate (U-4)Into a 50 mL round-bottom flask were added 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3- yl]-9-penta decan-8-yl (5Z)-5-(4-aminobutylidene)nonanedioate (1.05 g, 1.336 mmol, 1 equiv) and H2O (21 mL) at room temperature. To the above mixture was added formic acid (37% in water, 1.05 mL) and formaldehyde (1.05 mL) at room temperature. The resulting mixture was stirred for additional 2 h at 100ºC. The mixture was allowed to cool down to room temperature. The resulting mixture was diluted with water (50 mL). The mixture was basified pH to 9 with saturated aqueous Na2CO3. The resulting mixture was extracted with n- heptane (2 x 50 mL). The combined organic layers were washed with water (2 x 30 mL) and brine (50 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with DCM / MeOH (94:6) to afford 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl]- 9-pentadecan-8-yl-(5Z)-5-[4-(dimethylamino) butylidene]nonanedioate (530 mg, crude) as light yellow oil. The residue was purified by reversed-phase flash chromatography with the following conditions: column, C18 silica gel; mobile phase, water (0.05% TFA) in MeCN, 10% to 90% gradient in 10 min; detector, ELSD. The fractions were collected and concentrated under reduced pressure. The residue was dissolved in n-heptane (100 mL). The combined organic layers were washed with water (2 x 100 mL), sat. aqueous NaHCO3 (2 x 100 mL), water / MeOH (1:4, 3x200 mL), water (2 x 100 mL) and brine (1 x 100 mL), dried over anhydrous Na2SO4. The resulting mixture was filtered. The filtrate was concentrated under reduced pressure. This resulted in 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl]-9- pentadecan-8-yl (5Z)-5-[4-(dimethyl amino)butylidene]nonanedioate (504.6 mg, 46.63%) as light yellow oil. LC-MS (ES, m / z): 810.7 [M+H]+.1H NMR (400 MHz, Chloroform-d, ppm): δ 5.161 (t, J = 6.8 Hz, 1 H), 4.789-4.833 (m, 2H), 2.283-2.264 (m, 11H), 2.058-2.019 (m, 6H), 1.998-1.654 (m, 14H), 1.514-1.189 (m, 36H), 1.105-1.039 (m, 3H), 0.877 (t, J = 5.6 Hz, 6H).5.2. Biological Examples and Results Example 5. Preparation and analysis of lipid nanoparticle formulations comprising the ionizable lipid U-1

[0219] To determine the impact that the ionizable lipid of the present disclosure might have on the potency and toxicity of LNPs, DNA encoding an expression cassette for EPO under the control of a liver-specific promoter was formulated into lipid nanoparticles (LNPs) comprising ionizable lipid U-1 of the present disclosure and compared to the potency and toxicity of LNPs comprising ionizable lipids L-17 (PCT Application No. PCT / US2023 / 079923), L-21 (PCT Application No. PCT / US2023 / 079923), ALC-0315 (CAS No.2036272-55-4) or ARCT (CAS No.2230647-37-5, Rajappan et al., Org. Process Res. Dev 2021, 25, 6, 1383-1390), as described above and detailed in FIG.1A.

[0220] Adult wild type female BALB / c mice were dosed once by a single i.v. bolus injection into the tail vein at 5 mL / kg body weight. The DNA-LNPs were administered at 1 mg / kg and 0.3 mg / kg based on the weight of the DNA payload. Blood was collected via a retro-orbital bleed 4 hours, 3 days and 7 days after dosing, and IL-6 and EPO levels in serum determined as presented in FIG.1B through FIG.1D.

[0221] The DNA-LNP formulations with ionizable lipids U-1 generated serum levels of IL-6 that were comparable to or lower than LNPs comprising the exemplary ionizable lipid ALC- 0315 or exemplary ionizable lipid ARCT (FIG.1B). The DNA-LNP formulation with ionizable lipid U-1 produced robust EPO expression levels at day 3 (FIG.1C) and day 7 (FIG.1D), which by day 7 were comparable to levels achieved from LNPs formulated with ALC-0315 or ARCT in mice receiving the 1 mg / kg dose, and higher than levels achieved from the ALC-0315 LNP in mice receiving the 0.3 mg / kg dose. Example 6. Preparation and analysis of lipid nanoparticle formulations comprising the ionizable lipid U-3 To determine the impact that ionizable lipid U-3 of the present disclosure might have on the potency and toxicity of LNPs, DNA encoding an expression cassette for EPO under the control of a liver-specific promoter was formulated into lipid nanoparticles (LNPs) comprising U-3 and compared to the potency and toxicity of LNPs comprising ionizable lipids L-19 (PCT Application No. PCT / US2023 / 079923), L-20 (PCT Application No. PCT / US2023 / 079923), ALC-0315 (CAS No.2036272-55-4) or CL1 (CAS No.1450888-71-7), as described above and detailed in FIG 2A.

[0222] Adult wild type female BALB / c mice were dosed once by a single i.v. bolus injection into the tail vein at 5 mL / kg body weight. The DNA-LNPs were administered at 1 mg / kg and 0.3 mg / kg based on the weight of the DNA payload. Blood was collected via a retro-orbital bleed 4 hours, 3 days and 7 days after dosing, and IL-6 and EPO levels in serum determined as presented in FIG.2B through FIG.2D. The DNA-LNP formulation with ionizable lipid U-3 produced robust EPO expression levels at day 3 (FIG.2B) and day 7 (FIG.2D), which were comparable to the exemplary ionizable lipids ALC-0315 and CL1. The DNA-LNP formulations with ionizable lipids U-3 generated lower serum levels of IL-6 than the exemplary ionizable lipids ALC-0315 and CL1 (FIG.2D). Example 7. Preparation and analysis of lipid nanoparticle formulations comprising the ionizable lipid U-2

[0223] To determine the impact that ionizable lipid U-2 of the present disclosure has on the potency and toxicity of LNPs, LNPs comprising U-2 were formulated with nanoplasmid DNA (npDNA) encoding a human Factor IX (hFIX) expression cassette and an mRNA encoding an auxiliary protein designed to facilitate nuclear localization of the DNA payload (as disclosed in PCT Publication No. WO2024 / 091578) in a ratio of 1:2 DNA to mRNA. The performance of this LNP preparation was then compared to the performance of LNPs similarly formulated but with the ionizable lipid L-15 (PCT Application No. PCT / US2023 / 079923), L-17 (PCT Application No. PCT / US2023 / 079923), L-18 (PCT Application No. PCT / US2023 / 079923), or CL1 (CAS No.1450888-71-7), as described above and detailed in Fig.3A.

[0224] Adult wild type female BALB / c mice were dosed once by a single i.v. bolus injection into the tail vein at 5 mL / kg body weight. The LNPs were administered at 0.5 mg / kg based on the weight of the DNA payload (resulting in doses of 0.5 mg / kg DNA / 1.0 mg / kg mRNA), and the amount of hFIX in the plasma of the treated mice was detected by ELISA 7 days and 14 days later. In these experiments and below, the concentration of hFIX in plasma is reported relative to the amount of hFIX in plasma of normal human samples, i.e. “% normal human FIX. Given that the average concentration of hFIX in pooled normal human samples is 5 ug / ml, 100% normal human Factor IX levels is approximately 5 µg / mL as presented in FIG.3B and FIG.3C. The DNA-LNP formulation with ionizable lipid U-2 produced robust FIX expression levels that were higher than the exemplary ionizable lipid CL1.

[0225] To assess the potential toxicity of LNPs formulated with U-2, blood was collected 4 hours after dosing via a retro-orbital bleed and serum levels of multiple cytokines determined as presented in FIG.3B through FIG.3L. The DNA-LNP formulation made with U-2 elicited levels of all cytokines that were either comparable to or significantly lower than those elicited by LNPs made with any of the exemplary ionizable lipids. Example 8. Preparation and analysis of lipid nanoparticle formulations comprising the ionizable lipid U-4

[0226] To determine the impact that ionizable lipid U-4 of the present disclosure has on the potency and toxicity of LNPs, LNPs comprising U-4 were formulated with nanoplasmid DNA (npDNA) encoding a human Factor IX (hFIX) expression cassette and an mRNA encoding an auxiliary protein designed to facilitate nuclear localization of the DNA payload in a ratio of 1:2 DNA to mRNA. The performance of this LNP preparation was then compared to the performance of LNPs similarly formulated but with the ionizable lipid L-15 (PCT Application No. PCT / US2023 / 079923), L-18 (PCT Application No. PCT / US2023 / 079923), CL1 (CAS No.1450888-71-7), A6 (di(dec-3-yn-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate), A9 (bis(2-butyloctyl) 10-(N-(3- (dimethylamino)propyl)nonanamido)nonadecanedioate) or ARCT (CAS No.2230647-37-5), as described above and detailed in Fig.4A.

[0227] Adult wild type female BALB / c mice were dosed once by a single i.v. bolus injection into the tail vein at 5 mL / kg body weight. The LNPs were administered at 0.5 mg / kg based on the weight of the DNA payload (resulting in doses of 0.5mg / kg DNA / 1.0 mg / kg mRNA), and the amount of hFIX in the plasma of the treated mice was detected by ELISA 7 days and 14 days later. As in the prior experiment, the concentration of hFIX in plasma is reported relative to the amount of hFIX in plasma of normal human samples, i.e. “% normal human FIX”,as presented in FIG.4B and FIG.4C. The DNA-LNP formulation with ionizable lipid U-4 produced robust FIX expression levels, higher than LNPs made with exemplary ionizable lipids CL1, or A6, and comparable to LNPs made with the exemplary lipids ARCT and A9.

[0228] To assess the potential toxicity of LNPs formulated with U-4, blood was collected via a retro-orbital bleed 4 hours after dosing and the levels of IL-6 in serum determined as presented in FIG.4D. The DNA-LNP formulation made with U-4 elicited significant less IL- 6 than DNA-LNP formulations made with any of the exemplary ionizable lipids.

[0229] To evaluate the influence of ionizable lipids U-2, U-3, and U-4 described herein on lipid nanoparticle (LNP) potency, LNP formulations containing lipids U-2, U-3, or U-4 were prepared, as detailed in FIG.5A. Lipids U-2 and U3 were formulated with the helper lipid DSPC, Cholesterol, and PEG-DMG in a ratio of 50:10:38.5:1.5. Lipid U-4 was formulated with DSPC (U4:DSPC), DAPC (U-4:DAPC), or U-4:18:0 / 18:1 PE. These LNP formulations encapsulated nanoplasmid DNA (npDNA) comprising an expression cassette encoding human Factor IX (hFIX) together with mRNA encoding an auxiliary protein intended to enhance nuclear localization of the DNA payload. The npDNA and mRNA were incorporated at a 1:1 (w / w) ratio.

[0230] Adult wild type female BALB / c mice were dosed once by a single i.v. bolus injection into the tail vein at 5 mL / kg body weight. The LNPs were administered at 0.3 mg / kg based on the weight of the DNA payload (resulting in doses of 0.3 mg / kg DNA / 0.3 mg / kg mRNA), and the amount of hFIX in the plasma of the treated mice was detected by ELISA 7 days later. As in the prior experiment, the concentration of hFIX in plasma is reported relative to the amount of hFIX in plasma of normal human samples, i.e. “% normal human FIX”, as presented in FIG.5B. The DNA-LNP formulation with ionizable lipid U-2, U-3 and U-4 produced robust FIX expression levels, and U-4 formulated with 18-0 / 18-1 as the helper lipid resulted in substantially higher FIX expression compared to the same lipid formulated with DSPC. 6. EQUIVALENTS AND INCORPORATION BY REFERENCE

[0231] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0232] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examplesand conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0233] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0234] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.

[0235] All references, issued patents and patent applications mentioned or cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A compound, wherein the compound is of Formula I:or is a pharmaceutically acceptable salt thereof; wherein: Zaand Zbis each independently H or an optionally substituted ionizable group; Laand Lbis independently a bond or an optionally substituted, saturated or partially unsaturated, straight or branched divalent aliphatic C1-12 hydrocarbon chain, wherein one or more carbon atoms of the chain are optionally replaced by a divalent group independently selected from -O-, - NRe-, -S-, -S(O)-, -S(NRe)-, -S(O)2-, -S(O)(NRe)-, -S(NRe)2-, and -C(O)-; each Reis independently hydrogen or an optionally substituted C1-6aliphatic group; Wnis an optionally substituted linear alkylene of n carbon atoms, wherein n is 2 to 6; Wmis an optionally substituted linear alkylene of m carbon atoms, wherein m is 2 to 6; p is an integer from 1 to 3; q is an integer from 1 to 3; each Xaand Xbis independently a bond or an optionally substituted, saturated or partially unsaturated, straight or branched divalent aliphatic C1-20 hydrocarbon chain, wherein one or more carbon atoms of the chain are optionally replaced by a divalent group independently selected from -O-, - NRe-, -S-, -S(O)-, -S(NRe)-, -S(O)2-, -S(O)(NRe)-, -S(NRe)2-, and -C(O)-; each Raand Rbis independently H or an optionally substituted, saturated or partially unsaturated, straight or branched aliphatic C1-50hydrocarbon group, wherein one or more carbon atoms of the chain are optionallyreplaced by a divalent group independently selected from -O-, -S-, -S(O)-, -S(NRe)-, -S(O)2-, -S(O)(NRe)-, -S(NRe)2-, -C(O)- and -CyA-; and each CyAindependently comprises an optionally substituted ring system selected from a 3- to 12-membered saturated or partially unsaturated carbocyclene; a phenylene; a 3- to 12-membered saturated or partially unsaturated heterocyclene having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; a 5- to 6-membered heteroarylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and a combination of any of these ring systems including fused variants thereof.

2. The compound according to claim 1, wherein Zaand Zbis each independently H or an optionally substituted ionizable group selected from amine, guanidine, nitrogen- containing heteroaryl, carboxylic acid, sulfonic acid, and phosphonic acid.

3. The compound according to claim 1 or 2, wherein Zaand Zbis each independently H or an optionally substituted ionizable group selected from amine, guanidine, and imidazole.

4. The compound according to any one of the preceding claims, wherein Zaand Zbis eachindependently H or an ionizable group selected from -N(Re)2, , and.

5. The compound according to any one of the preceding claims, wherein Zaand Zbis each independently H or -N(Re)2.

6. The compound according to any one of the preceding claims, wherein Zais -N(Re)2.

7. The compound according to any one of the preceding claims, wherein Zbis -N(Re)2.

8. The compound according to any one of claims 1-5 or claim 7, wherein Zais H.

9. The compound according to any one of claims 1-6 or claim 8, wherein Zbis H.

10. The compound according to any one of the preceding claims, wherein Laand Lbis each independently a bond or an optionally substituted, saturated or partially unsaturated, straight or branched divalent aliphatic C1-12 hydrocarbon chain, wherein one or more carbon atoms of the chain are optionally replaced by a divalent group independently 86 of 94selected from -O-, -NRe-, -S-, -S(O)-, -S(NRe)-, -S(O)2-, -S(O)(NRe)-, -S(NRe)2-, and - C(O)-.

11. The compound according to any one of the preceding claims, wherein Laand Lbis each independently a bond or an optionally substituted C1-6 hydrocarbon chain, wherein one or more carbon atoms of the chain are optionally replaced by a divalent group independently selected from -O-, -NRe-, -S-, -S(O)-, -S(O)2-, and -C(O)-.

12. The compound according to any one of the preceding claims, wherein Laand Lbis each independently a bond or an optionally substituted C1-6 hydrocarbon chain selected from methylene, ethylene, propylene, butylene, pentylene, and hexylene.

13. The compound according to any one of the preceding claims, wherein Laand Lbis each independently selected from a bond, -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2- CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-, and -CH2-CH2-CH2-CH2-CH2-CH2-.

14. The compound according to any one of the preceding claims, wherein Lais selected from a bond, -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2- CH2-, and -CH2-CH2-CH2-CH2-CH2-CH2-.

15. The compound according to any one of the preceding claims, wherein Lbis selected from a bond, -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2- CH2-, and -CH2-CH2-CH2-CH2-CH2-CH2-.

16. The compound according to any one of the preceding claims, wherein Lais selected from a bond, -CH2-CH2-, and -CH2-CH2-CH2.

17. The compound according to any one of the preceding claims, wherein Lbis selected from a bond, -CH2-CH2-, and -CH2-CH2-CH2.

18. The compound according to any one of the preceding claims, wherein m is 2 to 4.

19. The compound according to any one of the preceding claims, wherein n is 2 to 4.

20. The compound according to any one of the preceding claims, wherein m is 2 or 3.

21. The compound according to any one of the preceding claims, wherein n is 2 or 3.

22. The compound according to any one of the preceding claims, wherein each Xais independently selected from -C(=O)O-, -OC(=O)-, and -OC(=O)O-.

23. The compound according to any one of the preceding claims, wherein each Xbis independently selected from -C(=O)O-, -OC(=O)-, and -OC(=O)O-.

24. The compound according to any one of the preceding claims, wherein each Xais - C(=O)O-.

25. The compound according to any one of the preceding claims, wherein each Xbis - C(=O)O-.

26. The compound according to any one of the preceding claims, wherein each Raand Rbis independently an optionally substituted C1-30aliphatic group.

27. The compound according to any one of the preceding claims, wherein each Raand Rbis independently selected from ,n each # represents the point of attachment to Xaor Xb.

28. The compound according to claim 1, wherein the compound is selected from Table 1.

29. A lipid nanoparticle comprising a compound according to any one of the preceding claims.

30. The lipid nanoparticle according to claim 29, further comprising a neutral lipid and a lipid capable of reducing aggregation.

31. The lipid nanoparticle according to claim 30, wherein the neutral lipid comprises a phospholipid.

32. The lipid nanoparticle according to claim 29-31, wherein the neutral lipid comprises cholesterol.

33. The lipid nanoparticle according to any one of claims 29-32, further comprising: a) one or more nucleic acids; b) an ionizable lipid; c) a phospholipid; d) cholesterol; and e) a lipid capable of reducing aggregation.

34. The lipid nanoparticle according to claim 33, wherein the nucleic acid is selected from a DNA and an RNA.

35. The lipid nanoparticle according to claim 33, wherein the nucleic acid is a DNA.

36. The lipid nanoparticle according to claim 34, wherein the nucleic acid is an RNA.

37. The lipid nanoparticle according to claim 34, wherein the lipid nanoparticle comprises a nucleic acid that is a DNA and a nucleic acid thalint is an RNA.

38. The lipid nanoparticle according to claim 36 or 37, wherein the RNA is selected from mRNA, gRNA, and siRNA.

39. The lipid nanoparticle according to claim 36, wherein the RNA is an mRNA and the DNA is a linear DNA.

40. The lipid nanoparticle according to any one of claims 33-35, 37 or 39 wherein the DNA is a therapeutic agent and the lipid nanoparticle further comprises an additional therapeutic agent.

41. The lipid nanoparticle according to any one of claims 33, 34, or 36-39, wherein the RNA is a therapeutic agent and the lipid nanoparticle further comprises an additional therapeutic agent.

42. The lipid nanoparticle according to any one of claims 33-41, wherein the phospholipid is selected from a phosphatidylcholine (PC), a phosphatidylethanolamine (PE), a phosphatidylserine (PS), a phosphatidylinositol (PI), and a phosphatidylglycerol (PG), and derivatives thereof.

43. The lipid nanoparticle according to claim 42, wherein the phospholipid is a phosphatidylethanolamine (PE).

44. The lipid nanoparticle according to claim 42, wherein the phospholipid is a phosphatidylcholine (PC).

45. The lipid nanoparticle according to any one of claims 33-44, wherein the phospholipid comprises hydrocarbon chains each independently having 12-24 carbons.

46. The lipid nanoparticle according to claim 45, wherein the phospholipid comprises hydrocarbon chains each independently having 16-20 carbons.

47. The lipid nanoparticle according to claim 45 or 46, wherein the hydrocarbon chains are saturated.

48. The lipid nanoparticle according to claim 45 or 46, wherein the hydrocarbon chains are unsaturated and / or further comprise a carbocyclyl.

49. The lipid nanoparticle according to claim 48, wherein the hydrocarbon chains each independently comprise 1-4 double bonds.

50. The lipid nanoparticle according to any one of claims 30-49, wherein the phospholipid comprises two different hydrocarbon chains.

51. The lipid nanoparticle according to claim 43, further comprising 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC).

52. The lipid nanoparticle according to claim 43, further comprising 1-stearoyl-2-oleoyl-sn- glycero-3-phosphoethanolamine (SOPE).

53. The lipid nanoparticle according to claim 44, further comprising 1,2-dipalmitoleoyl-sn- glycero-3-phosphocholine (Δ9Δ9-Cis PC).

54. The lipid nanoparticle according to claim 44, further comprising 1,2-dioleyl-sn-glycero- 3-phosphoethanolamine (DOPE).

55. The lipid nanoparticle according to claim 44, further comprising 1,2-dioleoyl-sn-glycero- 3-phosphocholine (DOPC).

56. The lipid nanoparticle according to any one of claims 42-55, wherein the lipid capable of reducing aggregation is a PEG-lipid.

57. The lipid nanoparticle according to claim 56, wherein the PEG-lipid is 1,2-dimyristoyl- rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG[2K]) or PEG-1,2- distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG-DSG[2K]). 91 of 9458. The lipid nanoparticle according to any one of claims 30-57, further comprising a targeting ligand.

59. The lipid nanoparticle according to claim 58, wherein the targeting ligand comprises GalNAc.

60. The lipid nanoparticle according to claim 58 or 59, wherein the targeting ligand is linked to the lipid capable of reducing aggregation.

61. The lipid nanoparticle according to claim 60, wherein the lipid capable of reducing aggregation is PEG-1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG- DSG[2K]).

62. The lipid nanoparticle according to any one of claims 30-61, wherein the N / P ratio (ratio of moles of the amine groups of cationic lipids to those of the phosphate ones of DNA) is from 5 to 30.

63. The lipid nanoparticle according to claim 62, wherein the N / P ratio is 7.

64. The lipid nanoparticle according to claim 62, wherein the N / P ratio is 14.

65. The lipid nanoparticle according to claim 62, wherein the N / P ratio is 28.

66. The lipid nanoparticle according to any one of claims 30-65, characterized by: a) an ionizable lipid at 40 to 60 mol % of the total lipid present; b) a phospholipid at 6 to 20 mol % of the total lipid present; c) cholesterol at 35 to 45 mol % of the total lipid present; and d) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.

67. The lipid nanoparticle according to any one of claims 30-65, characterized by: a) an ionizable lipid at 40 to 60 mol % of the total lipid present; b) a phospholipid at 10 to 20 mol % of the total lipid present; c) cholesterol at 35 to 45 mol % of the total lipid present; and d) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.

68. The lipid nanoparticle according to any one of claims 30-65, characterized by: a) an ionizable lipid at 40 to 49 mol % of the total lipid present; b) a phospholipid at 10 to 20 mol % of the total lipid present; c) cholesterol at 35 to 45 mol % of the total lipid present; andd) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.

69. A pharmaceutical composition comprising a lipid nanoparticle according to any one of claims 29-68 and a pharmaceutically acceptable excipient, carrier, or diluent.

70. A method for delivering a nucleic acid into a cell, the method comprising contacting the cell with a lipid nanoparticle according to any one of claims 29-68 or the pharmaceutical composition according to claim 69.

71. The method according to claim 70, wherein the cell is in vitro.

72. The method according to claim 70, wherein the cell is in vivo.

73. A method for delivering a nucleic acid for in vivo production of target protein, the method comprising: administering systemically to a subject in need thereof a pharmaceutical composition according to claim 69, wherein the nucleic acid encodes a target protein and is encapsulated within the lipid nanoparticles, and the administering of the pharmaceutical composition results in the prolonged stable expression of the target protein.

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