Lipid encapsulated nanoparticles

Abstract

The present disclosure provides an ionizable lipid of Formula (I) or Formula (II).

Description

[0001] LIPID ENCAPSULATED NANOPARTICLES

[0002] Cross-Reference to Related Applications

[0003] This application claims priority to U. S. Provisional Application Serial Nos. 63 / 730,344, filed on December 10, 2024, and 63 / 821,318, filed on June 10, 2025, each of which is incorporated by reference it its entirety herein.

[0004] Sequence Listing

[0005] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled FMDL0019WOSEQ.xml created December 5, 2025, which is 3 kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

[0006] Field

[0007] The present disclosure provides ionizable lipids for use in formulating lipid nanoparticles (“LNPs”).

[0008] Background

[0009] Nucleic acids show promise as therapeutic modalities and have been used in a number of therapeutic applications, including RNA-modulating applications (e.g., antisense oligonucleotides and siRNA oligonucleotides) and mRNA-delivery applications (e.g., vaccines and gene editing). However, RNA and its mimics are susceptible to nuclease digestion in plasma, and many oligonucleotides and mRNA mimics do not biodistribute in an ideal manner. Thus, substantial difficulties have arisen due to the practical obstacles in delivering nucleotides and nucleic acids to target cellular compartments in their active forms.

[0010] Previous attempts to meet such difficulties resulted in the development of early lipid delivery platforms that could successfully deliver siRNA using LNPs formed from a combination of different lipid components. This knowledge was later transferred into successful delivery of self-replicating RNA and then mRNA.

[0011] Key to such success was development of the ionizable lipid. Having a pKa of between 4-7, the ionizable lipid is positively charged at acidic pH, thereby allowing for efficient complexing with a negatively charged nucleic acid. However, when the ionizable lipid is then delivered in vivo, it becomes neutral at physiological pH. Charge neutrality avoids attracting other charged molecules in vivo, such as serum proteins, which would otherwise lead to increased clearance and reduced delivery efficiency. Charge neutrality therefore improves in vivo circulation, and hence cellular uptake via endocytosis. Entrapment of the ionizable lipid within the endosome results in a pH drop back down to an acidic level, where the ionizable lipid again becomes protonated and positively charged. This then causes pairing of the ionizable lipid with endosomal membrane negatively charged lipids, followed by disruption of the endosomal membrane and release of the nucleic acid into the cytoplasm of the cell.

[0012] Lipid nanoparticles (“LNPs”) formed from ionizable lipids and other components can therefore serve as therapeutic cargo vehicles for delivery of biologically active agents, such as oligonucleotides and nucleic acids. LNPs can facilitate delivery of such agents across cell membranes and can be used to introduce components and compositions into living cells. However, challenges remain with existing systems, such as efficient delivery of cargo to target organs or target cell types, and reducing immunogenicity triggered by innate immune responses after administration. For example, administration of LNPs has previously been shown to induce production of natural IgM and / or IgG antibodies, mediated by activation of B cells. These responses can contribute to accelerated blood clearance (ABC) and dose-limiting toxicity such as acute phase response (APR) and complement activation-related pseudoallergy (CARPA). Thus, there remains a need for improved lipid nanoparticle systems for the delivery of nucleotides and nucleic acids, including oligonucleotides and nucleic acid mimics, as well as the delivery of ribonucleoproteins and / or other types of cargo.

[0013] Summary

[0014] The present disclosure provides ionizable lipids, which can be used in combination with at least one other lipid component, such as non-cationic helper lipids, sterols, and / or polymer conjugated lipids, to form lipid nanoparticles (LNPs).

[0015] In certain embodiments, an ionizable lipid has Formula I:

[0016]

[0017] Formula I

[0018] wherein

[0019] X is -OR1, -NR2R3, -Q3-(CH2)s-OR1, or -Q3-(CH2)s-NR2R3;

[0020] Llaand L2aare each independently Ci to Cis alkyl, or -(CH2)p-Q4-R4;

[0021] Llband L2bare each independently H, or Ci to Cis alkyl, or -(CH2)p-Q4-R4;

[0022] Q1and Q2are each independently -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -N(R5)C(=O)-, -C(=O)N(R5)-, -N(R5)-O-, -O-N(R5)-, -O-, -S-, -S-S-, -N=N-, -C(=O)-N-N=CH-, -CH=N-N-C(=O)-, -O-P(=O)(OCH3)-N-, -O-Si(OCH3)2-O-, or -C=C-;

[0023] Q3is -C=C-; each instance of Q4is independently -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -C(=S)O-, -OC(=S)-, -N(R5)C(=O)-, -C(=O)N(R5)-, -N(R5)-O-, -O-N(R5)-, -O-, -S-, -S-S-, or - C=C-;

[0024] R1is H, or Ci-Ce alkyl;

[0025] R2and R3are each independently H, or Ci to C3 alkyl, or both R2and R3together are linked by a shared Ci to C3 alkyl to form a 4- to 6-membered nitrogen-containing ring;

[0026] each instance of R4is independently H, Ci to Ci6 alkyl, or Ci to Ci6 alkenyl;

[0027] each instance of R5is independently H, or Ci to Ci6 alkyl;

[0028] m is 1 to 10;

[0029] n is 2 to 10;

[0030] each instance of p is independently 1 to 18;

[0031] r and s are each independently 0 to 10.

[0032] In certain embodiments, an ionizable lipid has Formula la, lb, Ic, Id, le, If, or Ig:

[0033] IPL-001, Formula la

[0034] HO.

[0035]

[0036] IPL-002, Formula lb IPL-003, Formula Ic

[0037]

[0038]

[0039] IPL-006, Formula Ig.

[0040] Also disclosed herein are LNPs comprising ionizable lipids. In certain embodiments, the LNPs comprise an ionizable lipid, a non-cationic helper lipid, a sterol, and a polymer lipid. In certain embodiments, the LNPs may be used to facilitate the intracellular delivery of therapeutic cargo in vitro and / or in vivo. Also disclosed herein are pharmaceutical compositions comprising ionizable lipids.

[0041] Additionally disclosed herein are methods of use of ionizable lipids. In certain embodiments, the use comprises delivery of nucleic acids such as oligonucleotides, siRNA, self-replicating RNA, or exogenous mRNA to a cell. In certain embodiments, the use comprises delivery of a ribonucleoprotein complex (RNP) to a cell.

[0042] Additionally disclosed herein are methods of preparing an LNP. In certain embodiments, the methods comprise formulation of ionizable lipids into LNPs.

[0043] Further disclosed herein are LNPs prepared by the above methods.

[0044] Further disclosed herein are kits and devices comprising ionizable lipids. In certain embodiments, the kits and devices comprise LNPs comprising ionizable lipids. Detailed Description

[0045] It is to be understood that both the foregoing background and summary, and the following detailed description, are exemplary and explanatory only and are not restrictive of the embodiments, as claimed.

[0046] Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and / or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting.

[0047] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0048] All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and GenBank and NCBI reference sequence records, including any drawings and appendices, are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety for all purposes to the same extent as if each was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0049] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that prior art forms part of the common general knowledge of the person skilled in the art.

[0050] As used herein, numerical ranges are provided for certain quantities. It is to be understood that these ranges comprise all subranges therein. Thus, the range “from 50 to 80” includes all possible ranges therein (e.g., 51-79, 52-78, 53-77, 54-76, 55-75, 60-70, etc.). Furthermore, all values within a given range may be an endpoint for the range encompassed thereby (e.g., the range 50-80 includes the ranges with endpoints such as 55-80, 50-75, etc.).

[0051] As used herein, “a” or “an” refers to one or more of that entity; for example, “a lipid nanoparticle” refers to one or more lipid nanoparticles or at least one lipid nanoparticle. As such, the terms “a” (or “an”), “one or more” and “at least one” are used interchangeably herein. In addition, reference to “a lipid nanoparticle” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the lipid nanoparticles is present, unless the context clearly requires that there is one and only one lipid nanoparticle.

[0052] As used herein, "administration" or "administering" refers to routes of introducing a compound or composition provided herein to a subject to perform its intended function. Examples of routes of administration that can be used include, but are not limited to, administration by inhalation, parenteral routes (e.g. subcutaneous injection, intramuscular injection, intravenous infusion, intraarterial infusion, intrathecal injection), topical administration and oral administration.

[0053] As used herein, “antisense activity” means any detectable and / or measurable change attributable (whether directly and / or indirectly) to hybridization of an antisense agent to a target nucleic acid. As used herein, “antisense agent” means an oligomeric agent comprising an oligonucleotide having at least one region that is complementary to a target nucleic acid. An antisense oligonucleotide may be paired with a second oligonucleotide that is complementary to the antisense oligonucleotide, may be an unpaired antisense oligonucleotide, or may be a “hairpin oligonucleotide” that has at least one region that is self-complementary. An antisense agent includes, but is not limited to, an RNAi agent and an RNase H agent.

[0054] As used herein, “cargo” means an agent that is encapsulated in a lipid nanoparticle and at least partially released after the lipid nanoparticle is taken up by a cell. Cargo may include, but is not limited to, nucleic acids, oligonucleotides, peptides, proteins, and small molecules. Cargo may include oligomeric agents. An LNP may contain one or more types of cargo.

[0055] As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other component known in the art.

[0056] As used herein, “Cas enzyme” means a guided nucleic acid binding agent that is capable of cleaving a target nucleic acid. The target nucleic acid may be a double-stranded “target DNA”. A Cas enzyme may be a “Cas nickase” that cleaves one strand of a target DNA or may be capable of cleaving both strands of a target DNA.

[0057] As used herein, a “Cas fusion protein” means a protein comprising a polypeptide sequence corresponding to that of a guided nucleic acid binding agent and a polypeptide sequence corresponding to at least one additional protein domain.

[0058] As used herein, a “Cas protein” means a “Cas enzyme” a “dead Cas protein”, or a “Cas fusion protein”.

[0059] As used herein, a “cationic lipid” is a lipid molecule that carries a nonexchangeable net positive charge.

[0060] As used herein, “cholesterol” means:

[0061]

[0062] As used herein, “complementary” in reference to an oligonucleotide means that at least 70% of the nucleobases of such oligonucleotide or one or more regions thereof and the nucleobases of another nucleic acid or one or more regions thereof are capable of hydrogen bonding with one another when the nucleobase sequence of the oligonucleotide and the other nucleic acid are aligned in opposing directions. Complementary nucleobases are nucleobase pairs that are capable of forming hydrogen bonds with one another. Complementary nucleobase pairs include adenine (A) and thymine (T); adenine (A) and uracil (U); cytosine (C) and guanine (G); and 5-methyl cytosine (mC) and guanine (G).

[0063] As used herein, “dead Gas protein”, “dead Gas” or “dCas” means a guided nucleic acid binding agent that does not have any nucleic acid cleavage activity.

[0064] As used herein, “editing system” is a system comprising at least one guided nucleic acid binding agent and at least one guide.

[0065] As used herein, “effective amount” means the amount of a formulation according to the invention that, when administered to an animal, is sufficient to effect desired treatment. The “effective amount” will vary depending on the active ingredient, the state, disorder, or condition to be treated and its severity, and the age, weight, physical condition and responsiveness of the animal to be treated.

[0066] As used herein, “endonuclease” and “nuclease” are used interchangeably to refer to an enzyme which possesses endonucleolytic catalytic activity for polynucleotide cleavage.

[0067] As used herein, “exogenous mRNA” means any mRNA that is introduced into an organism or cell and that is not synthesized by the recipient organism or cell itself. An exogenous mRNA can be isolated or purified from an organism or cell, can be transcribed in vitro, or can be produced by synthetic means. An exogenous mRNA comprises a coding region (e.g., an open reading frame (ORF)) encoding a polypeptide sequence.

[0068] As used herein, “fragment” when used in relation to a polypeptide or nucleic acid, means a polypeptide or nucleic acid sequence that is at least one amino acid shorter than a reference sequence but otherwise identical to the reference sequence.

[0069] As used herein, “guide” means an oligonucleotide (a “single guide” )or a complex consisting of two or more oligonucleotides that are partially hybridized to one another (e.g. a “dual guide”), in both cases comprising a “spacer”, which is a target-recognition region, and a “scaffold”, which is a protein-recognition region. In embodiments in which a guide is a single guide, the spacer and the scaffold are regions of the one oligonucleotide that constitutes the single guide. In embodiments in which a guide is a dual guide, the spacer is a region of a first oligonucleotide and the scaffold is a portion of the complex that includes regions of each of the two oligonucleotides. In certain embodiments, a guide directs a guided nucleic acid binding agent to a target sequence of a target nucleic acid.

[0070] As used herein, “guided nucleic acid binding agent” means a polypeptide comprising (1) a region that interacts with the scaffold of a guide; and (2) a region that interacts with the protospacer adjacent motif (PAM) of a target nucleic acid. In certain embodiments, the spacer of the guide causes the guided nucleic acid binding agent to specifically interact with a target nucleic acid. A guided nucleic acid binding agent may comprise one or more active or inactive nuclease domains. A guided nucleic acid binding agent may comprise a heterologous domain. In certain embodiments, the guided nucleic acid binding agent is a Gas protein, including Cas enzymes, dead Cas proteins, and Cas fusion proteins.

[0071] As used herein, a “helper lipid” or “non-cationic helper lipid” is a non-cationic lipid that is not a sterol or a polymer lipid. A “helper lipid” may also be described as a “structural lipid”.

[0072] As used herein, "hybridization", “hybridizing” or “hybridize” means the act or process of two complementary regions of strands of linked oligomeric subunits e.g., oligonucleotides, nucleic acids) annealing together to form a double-stranded region. While not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.

[0073] As used herein, “identity” refers to a relationship between the sequences of two or more polypeptides or nucleic acids, as determined by comparing respective sequences. Identity measures the percent of identical matches between the smaller of two or more sequences when the sequences are aligned for maximal similarity. Identity of related polypeptides or nucleic acids can be calculated by known methods. “Percent (%) identity” refers to the percentage of amino acids or nucleobases in a candidate amino acid or nucleic acid sequence that are identical with the amino acids or nucleobases in a second sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation. Methods and computer programs for sequence alignment are well known in the art, and include the BLAST suite (Stephen F. Altschul, et al (1997), " Gapped BLAST and PSLBLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402), the Smith-Waterman algorithm (Smith, T. F. & Waterman, M. S. (1981) “Identification of common molecular subsequences.” J. Mol. Biol. 147:195-197), the Needleman-Wunsch algorithm (Needleman, S. B. & Wunsch, C. D. (1970) “A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol. 48:443-453) and the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) for global alignment of amino acid or nucleic acid sequences.

[0074] As used herein, an “ionizable lipid” is a lipid molecule which is substantially protonated (e.g. it becomes ‘cationic’) at or below physiological pH (e.g., pH 7-7.5, or pH 7.4). An ionizable lipid containing an amine (for example, a tertiary amine), is an “ionizable amino lipid”.

[0075] As used herein, the term “lipid” refers to a hydrophobic organic compound, including but not limited to fatty acids. A lipid may be an “ionizable lipid”, a “cationic lipid”, or a “non-cationic lipid”.

[0076] As used herein, “lipid nanoparticle” is used interchangeably with “LNP” and in some embodiments refers to a composition comprising an ionizable and / or a cationic lipid, a non-cationic lipid, a sterol and a polymer lipid. In some embodiments, the non-cationic lipid is a helper lipid. In some embodiments, the noncationic lipid is a neutral or zwitterionic lipid. In some embodiments, the sterol is cholesterol. In some embodiments, the polymer lipid is a pegylated (“PEG”) lipid. As used herein, “modulating” refers to changing or adjusting a feature in a cell, tissue, organ or organism.

[0077] As used herein, “messenger RNA” (“mRNA”) is any ribonucleic acid (RNA) or modified ribonucleic acid that encodes at least one protein, including naturally-occurring, non-naturally-occurring, or modified polymers of amino acids, and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo. An mRNA may contain one or more modified nucleotides.

[0078] As used herein, “non-cationic lipid” is a lipid molecule that is not a “cationic lipid” or an “ionizable lipid”. A “non-cationic lipid” includes neutral and zwitterionic lipids, negatively charged lipids, sterols, and polymer lipids.

[0079] As used herein, “oligomeric agent” means a compound or complex comprising or consisting of at least one modified oligonucleotide and optionally one or more additional associated features selected from: (a) one or more additional modified or unmodified oligonucleotides, each of which may be hybridized to or covalently linked to the at least one modified oligonucleotide and / or to each other; (b) one or more conjugate groups, which may be covalently attached directly or indirectly to any oligonucleotide of such oligomeric agent; and (c) one or more terminal groups. As used herein, oligomeric agents include, but are not limited to, guides, antisense agents, antisense oligonucleotides, siRNA, and nicked hairpins.

[0080] As used herein, “oligonucleotide” means a strand of linked nucleosides connected via internucleoside linkages, wherein each nucleoside and internucleoside linkage may be modified or unmodified.

[0081] As used herein, a “polymer” is a molecule comprising repeating chemical subunits.

[0082] As used herein, a “polymer lipid” is a molecule comprising both a hydrophobic lipid portion and a hydrophilic polymer portion. A pegylated or PEG lipid is a polymer lipid wherein the polymer portion is a polyethylene glycol (PEG).

[0083] As used herein, a “pharmaceutically acceptable carrier or diluent” can include, but is not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents.

[0084] As used herein “pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of compounds, i.e., salts that retain the desired biological activity of the compound. The term “pharmaceutically acceptable salts” includes both acid and base addition salts. Pharmaceutically acceptable salts include those obtained by reacting the active compound functioning as a base. Those skilled in the art will further recognize that acid addition salts may be prepared by reaction of the compounds with the appropriate inorganic or organic acid via any of a number of known methods.

[0085] As used herein “pharmaceutical composition” means a mixture of substances suitable for administering to a subject. For example, in one embodiment a pharmaceutical composition may comprise one or more cargo agents encapsulated in an LNP comprising a lipid of Formula I. As used herein, a “phospholipid” is a lipid that includes a phosphate moiety and one or more carbon chains, such as saturated or unsaturated fatty acid chains. A phospholipid is a type of “helper lipid” and a type of “non-cationic lipid”.

[0086] As used herein “prodrug” means a therapeutic agent in a first form outside the body that is converted to a second form within a subject or cells thereof. Typically, conversion of a prodrug within the subject is facilitated by the action of an enzyme (e.g., endogenous or viral enzyme) or chemicals present in cells or tissues and / or by physiologic conditions. In certain embodiments, the first form of the prodrug is less active than the second form.

[0087] As used herein, a “scaffold region” is a region of a guide that has a “scaffold sequence”. A “scaffold region” may adopt a tertiary structure that interacts with a Cas protein.

[0088] As used herein, a “spacer region” is a region of a guide that has a “spacer sequence” that is complementary to a target nucleic acid.

[0089] As used herein, “stereoisomer” refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. The present disclosure contemplates various stereoisomers and mixtures thereof and includes “enantiomers”, which refers to two stereoisomers whose molecules are nonsuperimposable mirror images of one another.

[0090] As used herein, “subject” refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

[0091] As used herein, “target nucleic acid,” “target DNA”, “target RNA,” “and “nucleic acid target” means a nucleic acid that an oligonucleotide or ribonucleoprotein complex is designed to affect. In certain embodiments, an oligonucleotide has a nucleobase sequence that is complementary to more than one RNA or DNA, only one of which is the target RNA or DNA of the oligonucleotide. In certain embodiments, the target RNA or target DNA is an RNA or DNA present in the species to which an oligonucleotide is administered.

[0092] As used herein, “target sequence” refers to the 12-30 linked nucleoside portion of a nucleic target that is complementary to the “spacer” of a guide. The target sequence is within the “complementary strand” of a target DNA.

[0093] As used herein, “tautomer” refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present disclosure includes tautomers of any said compounds unless otherwise indicated.

[0094] As used herein, “therapeutically effective” applied to dose or amount refers to that quantity of a compound or composition that is sufficient to result in a desired clinical benefit after administration to an animal in need thereof.

[0095] As used herein, “treating” refers to administering a compound or pharmaceutical composition to an animal in order to effect an alteration or improvement of a disease, disorder, or condition in the animal. In some embodiments, “treating” means one or more of relieving, alleviating, delaying, reducing, improving, ameliorating or managing at least one symptom of a condition in an animal. The term “treating” may also mean one or more of arresting, delaying the onset (i.e., the period prior to clinical manifestation of the condition) or reducing the risk of developing or worsening a condition.

[0096] As used herein, “vector” refers to a delivery system used to transmit genetic material to a host cell. Vectors can be, for example, viruses, plasmids, cosmids, or phage. A vector as used herein can be used to deliver either DNA or RNA. An “expression vector” is a vector that is capable of directing the expression of a protein encoded by one or more genes carried by the vector when it is present in the appropriate environment. Vectors are preferably capable of autonomous replication. Typically, an expression vector comprises a transcription promoter, a gene, and a transcription terminator. Protein expression is usually placed under the control of a promoter, and an open reading frame is said to be “operably linked to” the promoter.

[0097] As used herein, "alkyl" refers to a saturated straight or branched hydrocarbon substituent group containing up to twenty-four carbon atoms. Examples of alkyl groups include without limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like. Alkyl groups typically include from 1 to 20 carbon atoms (“C1-C20 alkyl”), more typically from 1 to 12 carbon atoms (“C1-C12 alkyl”) with from 1 to 6 carbon atoms (“Ci-Ce alkyl”) being more preferred. Alkyl groups as used herein may optionally include one or more further substituent groups.

[0098] As used herein, “alkylene” refers to a fully saturated, straight or branched divalent hydrocarbon chain radical, and typically having from one to twelve or more carbon atoms. Non-limiting examples of C1-C12 alkylene include methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, and the like. The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically, an alkylene chain can be optionally substituted.

[0099] As used herein, "alkenyl," refers to a straight or branched hydrocarbon chain substituent group containing up to twenty-four carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include without limitation, ethenyl, propenyl, butenyl, l-methyl-2-buten-l-yl, dienes such as 1,3-butadiene and the like. Alkenyl groups typically include from 2 to 20 carbon atoms, more typically from 2 to 12 carbon atoms with from 2 to 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally include one or more further substituent groups.

[0100] As used herein, “alkenylene” refers to a straight or branched divalent hydrocarbon chain radical, and typically having from two to twelve or more carbon atoms, and having one or more carbon-carbon double bonds. Non-limiting examples of C2-C12 alkenylene include ethene, propene, butene, and the like. The alkenylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkenylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically, an alkenylene chain can be optionally substituted.

[0101] As used herein, “alkylamino” refers to a radical of the formula -NHRaor -NRaRawhere each Rais, independently, an alkyl, alkenyl or alkynyl radical as defined herein typically containing one to twelve or more carbon atoms. Unless stated otherwise specifically, an alkylamino group can be optionally substituted.

[0102] As used herein, "alkynyl", refers to a straight or branched hydrocarbon substituent group containing up to twenty-four carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically include from 2 to 20 carbon atoms, more typically from 2 to 12 carbon atoms with from 2 to 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally include one or more further substituent groups.

[0103] As used herein, "alkoxy" refers to an alkyl-O- substituent group, where alkyl is as defined herein. Examples of alkoxy groups include without limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, secbutoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include further substituent groups.

[0104] As used herein, “amino” refers to the -Nib radical.

[0105] As used herein, "aryl" refers to a carbocyclic ring system substituent group having one or more aromatic rings. The aryl may be monocyclic or may include two or more fused rings. Examples of aryl groups include without limitation, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl and the like.

[0106] Preferred aryl ring systems have from 6 to 10 ring atoms. Aryl groups as used herein may optionally include further substituent groups.

[0107] As used herein, “aralkyl” or “arylalkyl” refers to a radical of the formula -Rb-Rc where Rb is an alkylene group as defined herein and Rc is one or more aryl radicals as defined herein, for example, benzyl, diphenylmethyl and the like. Unless stated otherwise specifically, an aralkyl group can be optionally substituted.

[0108] As used herein, “azido” refers to the -N3 group.

[0109] As used herein, “carbocyclyl,” “carbocyclic ring” or “carbocycle” refers to a ring structure, wherein the atoms which form the ring are each carbon. Carbocyclic rings can typically comprise from 3 to 20 or more carbon atoms in the ring. Carbocyclic rings include aryls and cycloalkyl, cycloalkenyl and cycloalkynyl as defined herein. Unless stated otherwise specifically, a carbocyclyl group can be optionally substituted.

[0110] As used herein, “cyano” refers to the -CN radical.

[0111] As used herein, "cycloalkyl" refers to a saturated or unsaturated carbocyclic ring system substituent group that does not include an aromatic ring. The cycloalkyl may be monocyclic or may include two or more fused rings. Examples of cycloalkyl groups include without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, and the like. Preferred cycloalkyl ring systems have from 3 to 10 ring atoms (“C3-C10 cycloalkyl”). Cycloalkyl groups as used herein may optionally include further substituent groups.

[0112] As used herein, “cycloalkenyl” refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, having one or more carbon-carbon double bonds, which can include fused or bridged ring systems, typically having from three to twenty or more carbon atoms, preferably having from three to ten carbon atoms, and which is attached to the rest of the molecule by a single bond. Monocyclic cycloalkenyl radicals include, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl, cycloctenyl, and the like. Polycyclic cycloalkenyl radicals include, for example, bicyclo[2.2.1]hept-2-enyl and the like. Unless otherwise stated specifically, a cycloalkenyl group can be optionally substituted.

[0113] As used herein, “cycloalkynyl” refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, having one or more carbon-carbon triple bonds, which can include fused or bridged ring systems, typically having from three to twenty or more carbon atoms, preferably having from three to ten carbon atoms, and which is attached to the rest of the molecule by a single bond. Monocyclic cycloalkynyl radicals include, for example, cycloheptynyl, cyclooctynyl, and the like. Unless otherwise stated specifically, a cycloalkynyl group can be optionally substituted.

[0114] As used herein, “haloalkyl” refers to an alkyls, as defined herein, that is substituted by one or more halos, as defined herein, e.g., trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl,

[0115] 1,2-difluoroethyl, 3-bromo-2 -fluoropropyl, 1,2-dibromoethyl, and the like. Unless stated otherwise specifically, a haloalkyl group can be optionally substituted.

[0116] As used herein, "halo" or "halogen" refers to a substituent group selected from fluoride, chloride, bromide and iodide.

[0117] As used herein, "heteroaryl" refers to a substituent group comprising a ring system in which at least one of the rings is aromatic, and at least one ring includes one or more ring heteroatoms. The heteroaryl may be monocyclic or may include two or more fused rings. Heteroaryl groups include at least one ring atom selected from sulfur, nitrogen or oxygen, wherein the sulfur is optionally present as a sulfoxide or sulfone, and wherein the nitrogen is optionally present as an N-oxide. Examples of heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, thiophenyl, furanyl, quinolinyl, and the like. Heteroaryl groups as used herein may optionally include further substituent groups.

[0118] As used herein, "heteroalkyl" refers to an alkyl substituent group as defined herein in which one or more CH2 units are replaced with a heteroatom independently selected from O, NH, N(Ci-6alkyl), S, SO, and SO2, except that heteroalkyl does not encompass groups defined herein as alkoxy. Examples of heteroalkyl groups include without limitation, methoxypropyl, ethoxymethyl, propylsulfonyl, l-(methylthio)propan-2-yl, methyl(methylthio)amino, N-propylamino, 2-(methylamino)ethyl, and the like. Heteroalkyl groups typically include from 1 to 20 carbon atoms (“C1-C20 heteroalkyl”), more typically from 1 to 12 carbon atoms (“C1-C12 heteroalkyl”) with from 1 to 6 carbon atoms (“Ci-Ce heteroalkyl”) being more preferred. Heteroalkyl groups as used herein may optionally include one or more further substituent groups.

[0119] As used herein, "heterocyclyl" refers to a substituent group comprising a ring system in which none of the rings are aromatic, and at least one ring includes one or more ring heteroatoms. Heterocyclyl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms. Heterocyclyl groups include at least one ring atom selected from sulfur, nitrogen or oxygen, wherein the sulfur is optionally present as a sulfoxide or sulfone. Examples of heterocyclyl groups include without limitation, morpholino, oxirane, tetrahydropyranyl, tetrahydrothienyl, sulfolanyl, and the like.

[0120] Heterocyclyl groups as used herein may optionally include further substituent groups.

[0121] As used herein, “hydroxy” or “hydroxyl” refers to the -OH radical.

[0122] As used herein, “imino” refers to the =NH substituent.

[0123] As used herein, “nitro” refers to the -NO2 radical.

[0124] As used herein, “oxo” refers to the =0 substituent.

[0125] As used herein, “ring” refers to a cyclic group which can be fully saturated, partially saturated, or fully unsaturated. A ring can be monocyclic, bicyclic, tricyclic, or tetracyclic. Unless stated otherwise specifically, a ring can be optionally substituted.

[0126] The term “substituted”, with respect to chemical groups means, unless otherwise indicated, a group is substituted with 1, 2, 3, 4, or 5 or more substituent groups selected from halo (e.g., perhalo), hydroxy, azido, SH, CN, OCN, nitro, C1-C20 alkyl (e.g., C1-C2 alkyl), C1-C10 substituted alkyl (e.g., CF3), C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 heteroalkyl, C1-C10 alkoxy, C1-C10 substituted alkoxy (e.g., OCF3), S-alkyl, N(Rm)-alkyl, O-alkenyl, S-alkenyl, N(Rm)-alkenyl, O-alkynyl, S-alkynyl, N(Rm)-alkynyl, O-alkylenyl-O-alkyl, aralkyl, O-aralkyl, Cs-iocycloalkyl, Ce-ioaryl, heterocyclyl, heteroaryl, N(Rm)(Rn), C(O)N(Rm)(Rn), N(Rm)(Rn)C(0)Rm, S(O)2N(Rm)(Rn), N(Rm)(Rn)S(O)2Rm, OC(O)N(Rm)(Rn), N(Rm)C(O)N(Rm)(Rn), N(Rm)C(O)ORn, C(O)ORm, and OC(O)Rm, where each Rmand Rnis, independently, H, OH, an amino protecting group, or substituted or unsubstituted C1-C10 alkyl. Substituent groups of this paragraph can be unsubstituted or further substituted at a carbon atom with one or more groups independently selected from: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, cyano, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl.

[0127] As used herein, “thioalkyl” refers to a radical of the formula -SRawhere Rais an alkyl, alkenyl, or alkynyl as defined herein typically containing one to twelve or more carbon atoms. Unless stated otherwise specifically, a thioalkyl group can be optionally substituted.

[0128] Certain Embodiments

[0129] The present disclosure provides the following non-limiting embodiments:

[0130] Embodiment 1. An ionizable lipid having Formula I:

[0131]

[0132] I

[0133] or a pharmaceutically acceptable salt, tautomer, stereoisomer or prodrug thereof

[0134] wherein

[0135] X is -OR1, -NR2R3, -Q3-(CH2)s-OR1, or -Q3-(CH2)s-NR2R3;

[0136] Llaand L2aare each independently Ci to Cis alkyl, or -(CH2)p-Q4-R4;

[0137] Llband L2bare each independently H, or Ci to Cis alkyl, or -(CH2)p-Q4-R4;

[0138] Q1and Q2are each independently -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -N(R5)C(=O)-, -C(=O)N(R5)-, -N(R5)-O-, -O-N(R5)-, -O-, -S-, -S-S-, -N=N-, -C(=O)-N-N=CH-, -CH=N-N-C(=O)-, -O-P(=O)(OCH3)-N-, -O-Si(OCH3)2-O-, or -C=C-; Q3is -C=C-;

[0139] each instance of Q4is independently -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -C(=S)O-, -OC(=S)-, -N(R5)C(=O)-, -C(=O)N(R5)-, -N(R5)-O-, -O-N(R5)-, -O-, -S-, -S-S-, or -C=C-;

[0140] R1is H, or Ci-Ce alkyl;

[0141] R2and R3are each independently H, or Ci to C3alkyl, or both R2and R3together are linked by a shared Ci to C3alkyl to form a 4- to 6-membered nitrogen-containing ring; each instance of R4is independently H, Ci to Ci6 alkyl, or Ci to Ci6 alkenyl;

[0142] each instance of R5is independently H, or Ci to Ci6 alkyl;

[0143] m is 1 to 10;

[0144] n is 2 to 10;

[0145] each instance of p is independently 1 to 18;

[0146] r and s are each independently 0 to 10.

[0147] Embodiment 2. The ionizable lipid of embodiment 1, wherein m is 1.

[0148] Embodiment 3. The ionizable lipid of embodiment 1, wherein m is 2.

[0149] Embodiment 4. The ionizable lipid of embodiment 1, wherein m is 3.

[0150] Embodiment 5. The ionizable lipid of embodiment 1, wherein m is 5.

[0151] Embodiment 6. The ionizable lipid of embodiment 1, wherein n is 6.

[0152] Embodiment 7. The ionizable lipid of embodiment 1, wherein Llbis H and Llais Ci3alkyl.

[0153] Embodiment 8. The ionizable lipid of embodiment 1, wherein m is 1, Llbis H and Llais Ci3alkyl. Embodiment 9. The ionizable lipid of embodiment 7 or 8, wherein Q1is -OC(=O)-. Embodiment 10. The ionizable lipid of embodiment 1, wherein Llbis H and Llais Cn alkyl.

[0154] Embodiment 11. The ionizable lipid of embodiment 1, wherein m is 3, Llbis H and Llais Cn alkyl. Embodiment 12. The ionizable lipid of embodiment 10 or 11, wherein Q1is -OC(=O)-.

[0155] Embodiment 13. The ionizable lipid of embodiment 1, wherein Llbis H and Llais C12 alkyl.

[0156] Embodiment 14. The ionizable lipid of embodiment 1, wherein m is 2, Llbis H and Llais C12 alkyl. Embodiment 15. The ionizable lipid of embodiment 13 or 14, wherein Q1is -C(=O)O-.

[0157] Embodiment 16. The ionizable lipid of embodiment 1, wherein Llais Cs alkyl and Llbis Cs alkyl. Embodiment 17. The ionizable lipid of embodiment 1, wherein m is 5, Llais Cs alkyl and Llbis Cs alkyl.

[0158] Embodiment 18. The ionizable lipid of embodiment 16 or 17, wherein Q1is -C(=O)O-.

[0159] Embodiment 19. The ionizable lipid of any of embodiments 1-18, wherein and L2bis Ce alkyl. Embodiment 20. The ionizable lipid of embodiment 19, wherein L2ais Cs alkyl.

[0160] Embodiment 21. The ionizable lipid of embodiment 19, wherein n is 6.

[0161] Embodiment 22. The ionizable lipid of embodiment 19, wherein n is 6 and L2ais Cs alkyl.

[0162] Embodiment 23. The ionizable lipid of any of embodiments 19-22, wherein Q2is -OC(=O)-.

[0163] Embodiment 24. The ionizable lipid of any of embodiments 1-23, wherein r is 0 and X is OH. Embodiment 25. An ionizable lipid having Formula la, Formula lb, Formula Ic, or Formula Id:

[0164] Formula la

[0165] HO.

[0166]

[0167] Formula lb Formula Ic

[0168]

[0169] Formula Id.

[0170] Embodiment 26. A lipid nanoparticle comprising an ionizable lipid according to any of embodiments 1-25.

[0171] Embodiment 27. The lipid nanoparticle of embodiment 26, comprising a non-cationic lipid.

[0172] Embodiment 28. The lipid nanoparticle of embodiment 27, wherein the non-cationic lipid is a sterol. Embodiment 29. The lipid nanoparticle of embodiment 28, wherein the sterol is cholesterol.

[0173] Embodiment 30. The lipid nanoparticle of any of embodiments 26-29, comprising a cationic lipid. Embodiment 31. The lipid nanoparticle of embodiment 27, wherein the non-cationic lipid is a helper

[0174] Embodiment 32. The lipid nanoparticle of embodiment 31, wherein the helper lipid is a phospholipid. Embodiment 33. The lipid nanoparticle of any of embodiments 26-32, comprising a polymer lipid. Embodiment 34. The lipid nanoparticle of embodiment 33, wherein the polymer lipid is a pegylated (polyethylene glycol) lipid (PEG lipid).

[0175] Embodiment 35. A lipid nanoparticle comprising:

[0176] (i) an ionizable lipid having Formula la, Formula lb, Formula Ic, or Formula Id: Formula la

[0177] Formula Ic

[0178]

[0179] Formula Id;

[0180] (ii) a helper lipid; (iii) cholesterol; and

[0181] (iv) a PEG lipid;

[0182] and optionally a cargo.

[0183] Embodiment 36. The lipid nanoparticle of embodiment 35, wherein the cargo comprises an oligonucleotide, optionally wherein the oligonucleotide comprises a guide.

[0184] Embodiment 37. A method of preparing a lipid nanoparticle, wherein the method comprises combining the ionizable lipid of any of embodiments 1-25 with a helper lipid, a sterol and a polymer lipid.

[0185] Embodiment 38. A lipid nanoparticle prepared by the method of embodiment 37.

[0186] Embodiment 39. A composition comprising the lipid nanoparticle of any of embodiments 26-36 or 38. Embodiment 40. A method of delivering a cargo to a cell, tissue, organ or subject comprising encapsulating the cargo in a lipid nanoparticle of any of embodiments 26-36 or 38, or a composition of embodiment 37, and administering the lipid nanoparticle or composition to the cell, tissue, organ or subject.

[0187] Embodiment 41. The method of embodiment 40, wherein the cargo comprises a nucleic acid.

[0188] Embodiment 42. The method of embodiment 41, wherein the nucleic acid comprises an exogenous mRNA.

[0189] Embodiment 43. The method of embodiment 40, wherein the cargo comprises an oligonucleotide. Embodiment 44. The method of embodiment 43, wherein the oligonucleotide comprises a guide. Embodiment 45. A kit comprising the ionizable lipid of any of embodiments 1-25, the lipid nanoparticle of any of embodiments 26-36 or 38, or the composition of embodiment 37, optionally wherein the kit is for, or when used for, delivering a cargo to a cell, tissue, organ or subject.

[0190] Embodiment 46. An ionizable lipid having Formula II:

[0191]

[0192] or a pharmaceutically acceptable salt, tautomer, stereoisomer or prodrug thereof, wherein:

[0193] X is -OR1, -NR2R3, -Q3-(CH2)s-OR1, or -Q3-(CH2)s-NR2R3;

[0194] Llaand L2aare each independently Ci to Cis alkyl, or -(CH2)P-Q4-R4;

[0195] Llband L2bare each independently H, Ci to Cis alkyl, or -(CH2)P-Q4-R4; Q1and Q2are each independently -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -N(R5)C(=O)-, -C(=O)N(R5)-, -N(R5)-O-, -O-N(R5)-, -O-, -S-, -S-S-, -N=N-, -C(=O)-N-N=CH-, -CH=N-N-C(=O)-, -O-P(=O)(OCH3)-N-, -O-Si(OCH3)2-O-, or -C=C-; Q3is selected from -C=C-, -C(=O)O-, and -OC(=O)-;

[0196] each instance of Q4is independently -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -C(=S)O-, -OC(=S)-, -N(R5)C(=O)-, -C(=O)N(R5)-, -N(R5)-O-, -O-N(R5)-, -O-, -S-, -S-S-, or -C=C-;

[0197] R1is H, or Ci-Ce alkyl;

[0198] R2and R3are each independently H, or Ci to C3alkyl, or both R2and R3together are linked by a shared Ci to C3alkyl to form a 4- to 6-membered nitrogen-containing ring; each instance of R4is independently H, Ci to Ci6 alkyl, or Ci to Ci6 alkenyl;

[0199] each instance of R5is independently H, or Ci to Ci6 alkyl;

[0200] m is 1 to 10;

[0201] n is 2 to 10;

[0202] u is 0 to 3;

[0203] v is 0 to 3.

[0204] each instance of p is independently 1 to 18; and

[0205] r and s are each independently 0 to 10.

[0206] Embodiment 47. The ionizable lipid of embodiment 46, wherein m is 1.

[0207] Embodiment 48. The ionizable lipid of embodiment 46, wherein m is 2.

[0208] Embodiment 49. The ionizable lipid of embodiment 46, wherein m is 3.

[0209] Embodiment 50. The ionizable lipid of embodiment 46, wherein m is 5.

[0210] Embodiment 51. The ionizable lipid of embodiment 46, wherein n is 6.

[0211] Embodiment 52. The ionizable lipid of embodiment 46, wherein Llbis H and Llais Ci3alkyl.

[0212] Embodiment 53. The ionizable lipid of embodiment 46, wherein m is 1, Llbis H and Llais Ci3alkyl. Embodiment 54. The ionizable lipid of embodiment 52 or 53, wherein Q1is -OC(=O)-.

[0213] Embodiment 55. The ionizable lipid of embodiment 46, wherein Llbis H and Llais Cn alkyl.

[0214] Embodiment 56. The ionizable lipid of embodiment 46, wherein m is 3, Llbis H and Llais Cn alkyl. Embodiment 57. The ionizable lipid of embodiment 55 or 56, wherein Q1is -OC(=O)-.

[0215] Embodiment 58. The ionizable lipid of embodiment 46, wherein Llbis H and Llais C12 alkyl.

[0216] Embodiment 59. The ionizable lipid of embodiment 46, wherein m is 2, Llbis H and Llais C12 alkyl. Embodiment 60. The ionizable lipid of embodiment 58 or 59, wherein Q1is -C(=O)O-.

[0217] Embodiment 61. The ionizable lipid of embodiment 46, wherein Llais Cs alkyl and Llbis Cs alkyl. Embodiment 62. The ionizable lipid of embodiment 46, wherein m is 5, Llais Cs alkyl and Llbis Cs alkyl. Embodiment 63. The ionizable lipid of embodiment 61 or 62, wherein Q1is -C(=O)O-.

[0218] Embodiment 64. The ionizable lipid of embodiment 46, wherein Llais Cs alkyl and Llbis C7 alkyl. Embodiment 65. The ionizable lipid of embodiment 46, wherein m is 5, Llais Cs alkyl and Llbis C7 alkyl

[0219] Embodiment 66. The ionizable lipid of embodiment 64 or 65, wherein Q1is -C(=O)O-.

[0220] Embodiment 67. The ionizable lipid of any of embodiments 46-66, wherein L2bis Ce alkyl.

[0221] Embodiment 68. The ionizable lipid of any of embodiments 46-66, wherein L2bis C5 alkyl.

[0222] Embodiment 69. The ionizable lipid of embodiment 67 or 68, wherein L2ais Cs alkyl.

[0223] Embodiment 70. The ionizable lipid of embodiment 67 or 68, wherein n is 6.

[0224] Embodiment 71. The ionizable lipid of embodiment 67 or 68, wherein n is 6 and L2ais Cs alkyl. Embodiment 72. The ionizable lipid of any of embodiments 46-71, wherein Q2is -OC(=O)-.

[0225] Embodiment 73. The ionizable lipid of any of embodiments 46-71, wherein Q2is -C(=O)O-.

[0226] Embodiment 74. The ionizable lipid of any of embodiments 46-73, wherein u is 0.

[0227] Embodiment 75. The ionizable lipid of any of embodiments 46-73, wherein u is 1.

[0228] Embodiment 76. The ionizable lipid of any of embodiments 46-75, wherein v is 0.

[0229] Embodiment 77. The ionizable lipid of any of embodiments 46-75, wherein v is 1.

[0230] Embodiment 78. The ionizable lipid of any of embodiments 46-77, wherein r is 0 and X is OH.

[0231] Embodiment 79. The ionizable lipid of embodiment 46, wherein Llaand L2aare each independently Ci to Cis alkyl, and Llband L2bare each independently H, or Ci to Cis alkyl.

[0232] Embodiment 80. An ionizable lipid having Formula III:

[0233]

[0234] Ill

[0235] or a pharmaceutically acceptable salt, tautomer, stereoisomer or prodrug thereof wherein

[0236] Llaand L2aare each independently Ci to Cis alkyl, or -(CH2)P-Q4-R4;

[0237] Llband L2bare each independently H, Ci to Cis alkyl, or -(CH2)P-Q4-R4;

[0238] Q1and Q2are each independently -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -N(R5)C(=O)-, -C(=O)N(R5)-, -N(R5)-O-, -O-N(R5)-, -O-, -S-, -S-S-, -N=N-, -C(=O)-N-N=CH-, -CH=N-N-C(=O)-, -O-P(=O)(OCH3)-N-, -O-Si(OCH3)2-O-, or -C=C-; each instance of Q4is independently -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -C(=S)O-, -OC(=S)-, -N(R5)C(=O)-, -C(=O)N(R5)-, -N(R5)-O-, -O-N(R5)-, -O-, -S-, -S-S-, or -C=C-;

[0239] each instance of R4is independently H, Ci to Ci6 alkyl, or Ci to Ci6 alkenyl;

[0240] each instance of R5is independently H, or Ci to Ci6 alkyl;

[0241] m is 1 to 10;

[0242] n is 2 to 10;

[0243] u is 0 to 3;

[0244] v is 0 to 3; and

[0245] each instance of p is independently 1 to 18.

[0246] Embodiment 81. The ionizable lipid of embodiment 80, wherein m is 1.

[0247] Embodiment 82. The ionizable lipid of embodiment 80, wherein m is 2.

[0248] Embodiment 83. The ionizable lipid of embodiment 80, wherein m is 3.

[0249] Embodiment 84. The ionizable lipid of embodiment 80, wherein m is 5.

[0250] Embodiment 85. The ionizable lipid of any of embodiments 80-84, wherein n is 6.

[0251] Embodiment 86. The ionizable lipid of any of embodiments 80-85, wherein Llbis H and Llais C13 alkyl.

[0252] Embodiment 87. The ionizable lipid of any of embodiments 80-85, wherein Llbis H and Llais Cn alkyl.

[0253] Embodiment 88. The ionizable lipid of any of embodiments 80-85, wherein Llais Cs alkyl and Llbis Cs alkyl.

[0254] Embodiment 89. The ionizable lipid of any of embodiments 80-85, wherein Llais Cs alkyl and Llbis C7alkyl.

[0255] Embodiment 90. The ionizable lipid of any of embodiments 80-85, wherein L2ais Ce alkyl and L2bis Cs alkyl.

[0256] Embodiment 91. The ionizable lipid of any of embodiments 80-85, wherein L2ais C5 alkyl and L2bis Cs alkyl.

[0257] Embodiment 92. The ionizable lipid of any of embodiments 80-81, wherein u is 1.

[0258] Embodiment 93. The ionizable lipid of any of embodiments 80-81, wherein u is 0.

[0259] Embodiment 94. The ionizable lipid of any of embodiments 80-93, wherein v is 1.

[0260] Embodiment 95. The ionizable lipid of any of embodiments 80-93, wherein v is 0.

[0261] Embodiment 96. The ionizable lipid of any of embodiments 80-95, wherein Q1is -OC(=O)-. Embodiment 97. The ionizable lipid of any of embodiments 80-95, wherein Q1is -C(=O)O-. Embodiment 98. The ionizable lipid of any of embodiments 80-97, wherein Q2is -OC(=O)-. Embodiment 99. The ionizable lipid of any of embodiments 80-97, wherein Q2is -C(=O)O-. Embodiment 100. An ionizable lipid having Formula la:

[0262]

[0263] Formula la.

[0264] Embodiment 101. An ionizable lipid having Formula lb:

[0265] HO.

[0266] Formula lb.

[0267]

[0268] Formula Ic. Formula Id. Formula le.

[0269]

[0270] Embodiment 106. An ionizable lipid having Formula Ig:

[0271]

[0272] Formula Ig.

[0273] Embodiment 107. A lipid nanoparticle comprising an ionizable lipid according to any of embodiments 46-106.

[0274] Embodiment 108. The lipid nanoparticle of embodiment 107, comprising a non-cationic lipid.

[0275] Embodiment 109. The lipid nanoparticle of embodiment 108, wherein the non-cationic lipid is a sterol. Embodiment 110. The lipid nanoparticle of embodiment 109, wherein the sterol is cholesterol.

[0276] Embodiment 111. The lipid nanoparticle of any of embodiments 107-110, comprising a cationic lipid. Embodiment 112. The lipid nanoparticle of embodiment 108, wherein the non-cationic lipid is a helper lipid.

[0277] Embodiment 113. The lipid nanoparticle of embodiment 112, wherein the helper lipid is a phospholipid.

[0278] Embodiment 114. The lipid nanoparticle of any of embodiments 107-113, comprising a polymer lipid. Embodiment 115. The lipid nanoparticle of embodiment 114, wherein the polymer lipid is a pegylated (polyethylene glycol) lipid (PEG lipid).

[0279] Embodiment 116. A lipid nanoparticle comprising:

[0280] (i) an ionizable lipid having Formula la, Formula lb, Formula Ic, Formula Id, Formula le, Formula If, or Formula Ig: Formula la

[0281] Formula Ic

[0282]

[0283] Formula Id

[0284]

[0285] 5

[0286] 10

[0287] additionally a cargo

[0288]

[0289] Embodiment 117. The lipid nanoparticle of embodiment 116, wherein the cargo comprises an oligomeric agent.

[0290] Embodiment 118. The lipid nanoparticle of embodiment 117, wherein the oligomeric agent comprises an oligonucleotide, optionally wherein the oligonucleotide comprises a guide.

[0291] Embodiment 119. The lipid nanoparticle of embodiment 116, wherein the cargo comprises a nucleic acid, optionally wherein the nucleic acid comprises an exogenous mRNA.

[0292] Embodiment 120. A method of preparing a lipid nanoparticle, wherein the method comprises combining the ionizable lipid of any of embodiments 46-106 with a helper lipid, a sterol and a polymer lipid.

[0293] Embodiment 121. A lipid nanoparticle prepared by the method of embodiment 120.

[0294] Embodiment 122. A composition comprising the lipid nanoparticle of any of embodiments 107-118 or 120.

[0295] Embodiment 123. A method of delivering a cargo to a cell, tissue, organ or subject comprising encapsulating the cargo in a lipid nanoparticle of any of embodiments 107-118 or 120, or a composition of embodiment 119, and administering the lipid nanoparticle or composition to the cell, tissue, organ or subject.

[0296] Embodiment 124. The method of embodiment 123, wherein the cargo comprises a nucleic acid.

[0297] Embodiment 125. The method of embodiment 124, wherein the nucleic acid comprises an exogenous mRNA.

[0298] Embodiment 126. The method of embodiment 123, wherein the cargo comprises an oligomeric agent. Embodiment 127. The method of embodiment 126, wherein the oligomeric agent comprises an oligonucleotide.

[0299] Embodiment 128. The method of embodiment 127, wherein the oligonucleotide comprises a guide. Embodiment 129. A kit comprising the ionizable lipid of any of embodiments 46-106, the lipid nanoparticle of any of embodiments 107-119 or 121, or the composition of embodiment 122, optionally wherein the kit is for, or when used for, delivering a cargo to a cell, tissue, organ or subject. Certain Compounds

[0300] Lipids and Lipid Nanoparticles

[0301] As provided herein, ionizable and / or cationic lipids may be used in combination with other lipids to form lipid nanoparticles (LNPs). In certain embodiments, the LNPs comprise (1) ionizable and / or cationic lipids in combination with (2) non-cationic lipids such as neutral, zwitterionic or negatively charged lipids, also known as helper lipids, (3) sterols such as cholesterol and (4) polymer lipids such as PEG lipids.

[0302] I. Lipids

[0303] A. Ionizable and / or Cationic Lipids

[0304] In certain embodiments, provided herein is an ionizable lipid.

[0305] In certain embodiments, the ionizable lipid comprises an ionizable head group. In some embodiments, the ionizable head group is a tertiary amine head group.

[0306] In certain embodiments, the ionizable lipid comprises a hydrophobic tail. In some embodiments, the hydrophobic tail has two fatty acid chains.

[0307] In some embodiments, the head group and the tail are joined by a linker group.

[0308] In certain embodiments, provided herein is an ionizable lipid having Formula I:

[0309]

[0310] I

[0311] wherein

[0312] X is -OR1or -NR2R3, -Q3-(CH2)S-OR1, or -Q3-(CH2)S-NR2R3;

[0313] Llaand L2aare each independently Ci to Cis alkyl, or -(CH2)P-Q4-R4;

[0314] Llband L2bare each independently H, Ci to Cis alkyl, or -(CH2)P-Q4-R4;

[0315] Q1and Q2are each independently -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, N(R5)C(=O)-, -C(=O)N(R5)-, -N(R5)-O-, -O-N(R5)-, -O-, -S-, -S-S-, -N=N-, -C(=O)-N-N=CH-, -CH=N-N-C(=O)-, -O- P(=O)(OCH3)-N-, -O-Si(OCH3)2-O-, or -C=C-;

[0316] Q3is -C=C-;

[0317] each instance of Q4is independently -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -C(=S)O-, -OC(=S)-, -N(R5)C(=O)-, -C(=O)N(R5)-, -N(R5)-O-, -O-N(R5)-, -O-, -S-, -S-S-, or -C=C-;

[0318] R1is H, or Ci-Ce alkyl;

[0319] R2and R3are each independently H, or Ci to C3alkyl, or both R2and R3together are linked by a shared Ci to C3alkyl to form a 4- to 6-membered nitrogen-containing ring; each instance of R4is independently H, Ci to Ci6 alkyl, or Ci to Ci6 alkenyl;

[0320] each instance of R5is independently H, or Ci to Ci6 alkyl;

[0321] m is 1 to 10;

[0322] n is 2 to 10;

[0323] each instance of p is independently 1 to 18;

[0324] r and s are each independently 0 to 10.

[0325] In certain embodiments, m is 1.

[0326] In certain embodiments, m is 2.

[0327] In certain embodiments, m is 3.

[0328] In certain embodiments, m is 5.

[0329] In certain embodiments, n is 6.

[0330] In certain embodiments, Llbis H and Llais C13 alkyl.

[0331] In certain embodiments, m is 1, a is 0 and Llais C13 alkyl. In certain such embodiments, Qi is -OC(=O)-.

[0332] In certain embodiments, Llbis H and Llais Cn alkyl.

[0333] In certain embodiments, m is 3, Llbis H and Llais Cn alkyl. In certain such embodiments, Qi is -OC(=O)-.

[0334] In certain embodiments, Llbis H and Llais C12 alkyl.

[0335] In certain embodiments, m is 2, Llbis H and Llais C12 alkyl. In certain such embodiments, Qi is -C(=O)O-.

[0336] In certain embodiments, Llais Cs alkyl and Llbis Cs alkyl.

[0337] In certain embodiments, m is 5, Llais Cs alkyl and Llbis Cs alkyl. In certain such embodiments, Qi is -C(=O)O-. In certain embodiments, n is 6, L2ais Cs alkyl and L2bis Ce alkyl. In certain such embodiments, Q2 is -OC(=O)-.

[0338] In certain embodiment, r is 0, and X is -OH.

[0339] In certain embodiments, provided herein is an ionizable lipid having Formula I, wherein Fragment A and / or Fragment B is represented by a structure provided in Table 1.

[0340]

[0341] L2b

[0342] Fragment A Fragment B Table 1

[0343] Lipid Fragments

[0344] Ionizable Fragment A Fragment B

[0345] cationic

[0346] lipid

[0347] IPL-001 0

[0348] 0

[0349] IPL-002 0

[0350] 0

[0351] IPL-003 0

[0352] 0

[0353] IPL-004

[0354]

[0355] In certain embodiments, provided herein is an ionizable lipid having Formula II:

[0356]

[0357] wherein

[0358] X is -OR1, -NR2R3, -Q3-(CH2)s-OR1, or -Q3-(CH2)s-NR2R3;

[0359] Llaand L2aare each independently Ci to Cis alkyl, or -(CH2)p-Q4-R4;

[0360] Llband L2bare each independently H, or Ci to Cis alkyl, or -(CH2)p-Q4-R4;

[0361] Q1and Q2are each independently -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -N(R5)C(=O)-, -C(=O)N(R5)-, -N(R5)-O-, -O-N(R5)-, -O-, -S-, -S-S-, -N=N-, -C(=O)-N-N=CH-, -CH=N-N-C(=O)-, -O-P(=O)(OCH3)-N-, -O-Si(OCH3)2-O-, or -C=C-; Q3is selected from -C=C-, -C(=O)O-, and -OC(=O)-;

[0362] each instance of Q4is independently -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -C(=S)O-, -OC(=S)-, -N(R5)C(=O)-, -C(=O)N(R5)-, -N(R5)-O-, -O-N(R5)-, -O-, -S-, -S-S-, or -C=C-;

[0363] R1is H, or Ci-Ce alkyl; R2and R3are each independently H, or Ci to C3 alkyl, or both R2and R3together are linked by a shared Ci to C3 alkyl to form a 4- to 6-membered nitrogen-containing ring;

[0364] each instance of R4is independently H, Ci to Ci6 alkyl, or Ci to Ci6 alkenyl;

[0365] each instance of R5is independently H, or Ci to Ci6 alkyl;

[0366] m is 1 to 10;

[0367] n is 2 to 10;

[0368] u is 0 to 3;

[0369] v is 0 to 3;

[0370] each instance of p is independently 1 to 18; and

[0371] r and s are each independently 0 to 10.

[0372] In certain embodiments, m is 1.

[0373] In certain embodiments, m is 2.

[0374] In certain embodiments, m is 3.

[0375] In certain embodiments, m is 5.

[0376] In certain embodiments, n is 6.

[0377] In certain embodiments, Llbis H and Llais C13 alkyl.

[0378] In certain embodiments, m is 1, a is 0 and Llais C13 alkyl. In certain such embodiments, Qi is -OC(=O)-.

[0379] In certain embodiments, Llbis H and Llais Cn alkyl.

[0380] In certain embodiments, m is 3, Llbis H and Llais Cn alkyl. In certain such embodiments, Qi is -OC(=O)-.

[0381] In certain embodiments, Llbis H and Llais C12 alkyl.

[0382] In certain embodiments, m is 2, Llbis H and Llais C12 alkyl. In certain such embodiments, Qi is -C(=O)O-.

[0383] In certain embodiments, Llais Cs alkyl and Llbis Cs alkyl.

[0384] In certain embodiments, m is 5, Llais Cs alkyl and Llbis Cs alkyl. In certain such embodiments, Qi is -C(=O)O-.

[0385] In certain embodiments, Llais Cs alkyl and Llbis C7 alkyl.

[0386] In certain embodiments, m is Llais Cs alkyl and Llbis C7 alkyl. In certain such embodiments, Qi is -C(=O)O-.

[0387] In certain embodiments, n is 6, L2ais Cs alkyl and L2bis Ce alkyl. In certain such embodiments, Q2 is -OC(=O)-. In certain such embodiments, Q2 is -C(=O)O-.

[0388] In certain embodiments, n is 6, L2ais Cs alkyl and L2bis C5 alkyl. In certain such embodiments, Q2 is -C(=O)O-.

[0389] In certain embodiment, r is 0, and X is -OH. In certain embodiments, u is 0 and v is 0.

[0390] In certain embodiments, u is 1 and v is 1.

[0391] In certain embodiments, u is 1 and v is 0.

[0392] In certain embodiments, provided herein is an ionizable lipid having Formula II, wherein Fragment C and / or Fragment D is represented by a structure provided in Table 2.

[0393]

[0394] Fragment C Fragment D

[0395] Table 2

[0396] Lipid Fragments

[0397] Ionizable Fragment C Fragment D

[0398] cationic

[0399] lipid

[0400] IPL-001 0

[0401] 0

[0402] IPL-002 0

[0403] 0

[0404] IPL-003 0

[0405] 0

[0406] IPL-004

[0407] IPL-004.1 o

[0408] 0k / xzx IPL-005 0 0

[0409] IPL-006 0

[0410] 0

[0411]

[0412] In certain embodiments, an ionizable lipid provided herein is an ionizable lipid having Formula III:

[0413] L1b

[0414] N

[0415]

[0416] L2b

[0417] Formula III

[0418] wherein

[0419] Llaand L2aare each independently Ci to Cis alkyl, or -(CH2)p-Q4-R4;

[0420] Llband L2bare each independently H, or Ci to Cis alkyl, or -(CH2)p-Q4-R4;

[0421] Q1and Q2are each independently -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -N(R5)C(=O)-, -C(=O)N(R5)-, -N(R5)-O-, -O-N(R5)-, -O-, -S-, -S-S-, -N=N-, -C(=O)-N-N=CH-, -CH=N-N-C(=O)-, -O-P(=O)(OCH3)-N-, -O-Si(OCH3)2-O-, or -C=C-; each instance of Q4is independently -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -C(=S)O-, -OC(=S)-, -N(R5)C(=O)-, -C(=O)N(R5)-, -N(R5)-O-, -O-N(R5)-, -O-, -S-, -S-S-, or -C=C-;

[0422] each instance of R4is independently H, Ci to Ci6 alkyl, or Ci to Ci6 alkenyl;

[0423] each instance of R5is independently H, or Ci to Ci6 alkyl;

[0424] m is 1 to 10;

[0425] n is 2 to 10;

[0426] u is 0 to 3;

[0427] v is 0 to 3; and

[0428] each instance of p is independently 1 to 18.

[0429] In certain embodiments, m is 1.

[0430] In certain embodiments, m is 2.

[0431] In certain embodiments, m is 3.

[0432] In certain embodiments, m is 5.

[0433] In certain embodiments, n is 6.

[0434] In certain embodiments, u is 0.

[0435] In certain embodiments, u is 1.

[0436] In certain embodiments, v is 0.

[0437] In certain embodiments, v is 1.

[0438] In certain embodiments, Llbis H and Llais C13 alkyl. In certain such embodiments, Q1is -OC(=O)-. In certain embodiments, Llbis H and Llais Cn alkyl. In certain such embodiments, Q1is -OC(=O)-. In certain embodiments, Llais Cs alkyl and Llbis Cs alkyl. In certain such embodiments, Q1is - OC(=O)-.

[0439] In certain embodiments, Llais Cs alkyl and Llbis C7 alkyl. In certain such embodiments, Q1is - OC(=O)-.

[0440] In certain embodiments, L2ais Cs alkyl and L2bis Ce alkyl. In certain such embodiments, Q2is - OC(=O)-. In certain such embodiments, Q2is -C(=O)O-.

[0441] In certain embodiments, L2ais Cs alkyl and L2bis Ce alkyl. In certain such embodiments, Q2is - C(=O)O-.

[0442] In certain embodiments, an ionizable lipid has Formula la, lb, Ic, Id, le, If, or Ig:

[0443] IPL-001, Formula la

[0444] HO,

[0445]

[0446] IPL-003, Formulaic

[0447]

[0448] IPL-005, Formula If

[0449]

[0450] IPL-006, Formula Ig

[0451] In certain embodiments, provided herein is an LNP comprising a cationic lipid.

[0452] When used in an LNP, an ionizable lipid may enhance endosomal drug escape compared to an LNP with a cationic lipid. In vivo, delivery capacity of an LNP may benefit from electrostatic charge interactions with the endosome and subsequent LNP destabilization to release the cargo. An ionizable lipid can aid delivery by either (i) protonating in mildly acidic environments to acquire positive charges and / or (ii) incorporating pH-labile groups that cleave or change conformation in a low pH environment. Such pH-sensitive lipids therefore help enhance the charge-based uptake of liposomes in target cells or trigger cargo release by destabilizing liposome membranes.

[0453] Fusogenicity is a property which describes the ability of a lipid or multi -lipid construct to join with a lipid layer. See, e.g., Hashiba et al., Small Science, 3(1), 2023, 2200071; V. Gyanani, and R. Goswami, Pharmaceutics, 2023, 15(4): 1184. Lipid designs that are believed to impart fusogenic character to a LNP are (i) unsaturation in the lipid tails, (ii) branching in the lipid carbon chains, (iii) multi-tail lipids, e.g., 7C1 and G0-C14 lipids, see, e.g., Dahlman et al., Nat Nanotechnol. 2014 Aug; 9(8): 648-655, and (iv) incorporation of polymer lipids.

[0454] Ionizable and / or cationic lipids comprising hydrocarbon chains including unsaturation may provide LNPs having higher fluidity. Example publications describing ionizable and / or cationic lipids include U. S, Patent Publication Nos. 2006 / 0083780 and 2006 / 0240554; 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 each of which are herein incorporated by reference in their entirety.

[0455] In certain embodiments, the ionizable and / or cationic lipids may include an ionizable or cationic head group, optionally including one or more linear hydrocarbon chains of 10-20 carbon atoms, and optionally one or more branched, optionally unsaturated, hydrocarbon chains of 12-30 carbon atoms, each of which is optionally interrupted by one or more functionalities selected from ester, ether, amine (e.g., tertiary amine), amide, carbonate, carbamate, urea, and disulfide. The ionizable and / or cationic lipid may comprise a branching moiety. In certain embodiments the branching moiety comprises a tertiary carbon atom, a quaternary carbon atom, a tertiary amine, a vicinal diol, an amide, a carbamate, or an acetal. In certain embodiments, an ionizable lipid contains an amine (“ionizable amino lipid”), for example, a tertiary amine. It is believed that proton cycling of an amino group at disparate pH may provide additional stabilization of a negatively charged cargo (e.g., an exogenous mRNA or an oligonucleotide), while promoting cargo release inside a low pH compartment in vivo (e.g., an endosome).

[0456] In certain embodiments, the ionizable lipid has a pKa of the ionizable (e.g., amino) group in the range of about 4 to about 7. Such ionizable lipids have a positive charge in an acidic buffer, thereby allowing for efficient encapsulation of a negatively charged nucleic acid during formulation, and wherein upon in vivo administration at physiological pH, the ionizable lipid is neutral. In certain embodiments, the pKa is in a range of X to Y, wherein X represents the lowest number in the range and Y represents the highest number in the range, wherein X and Y are each independently selected from 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0; provided that X< Y. In certain embodiments, the pKa is in a range of 6.2-6.6, or 6.6-6.9 or 6.2-6.9. Because such lipids will be largely surface neutralized at physiological pH, it is believed this will reduce susceptibility to clearance. pKa measurements of lipids within lipid particles can be performed, for example, by using the fluorescent probe 2-(p-toluidino)-6-napthalene sulfonic acid (TNS), using methods described in Cullis et al., (1986) Chem Phys Lipids 40, 127-144. See also Hassett et al. (2019) Mol Ther Nucleic Acids 2019 Apr 15:15:1-11.

[0457] In certain embodiments, the ionizable lipid is formulated in an LNP together with an additional ionizable and / or cationic lipid. In certain embodiments, the additional ionizable and / or cationic lipid may comprise a single amine group. Such lipids are described herein and known in the art, e.g., as described in WO 2013 / 126803, WO 2021 / 163339, WO 2021 / 188389, and WO 2022 / 060871, the disclosure of each of which is herein incorporated by reference in its entirety.

[0458] In certain embodiments, the additional ionizable or cationic lipid may comprise a plurality of amine groups. Such lipids are described herein and known in the art, e.g., as described in WO 2010 / 129709, WO 2015 / 199952, WO 2018 / 191657, WO 2019 / 036008, WO 2019 / 036030, WO 2019 / 036030, WO 2019 / 036000, WO 2020 / 081938, and WO 2020 / 146805, the disclosure of each of which is herein incorporated by reference in its entirety.

[0459] In certain embodiments, the additional ionizable or cationic lipid is l,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2- dilinolenyloxy-N, N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-K-C2-DMA;“XTC2”), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[l,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[l,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[l,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[l,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), l,2-dilinoleylcarbamoyloxy-3 -dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA), l,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), l,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1 -linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), l,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA. Cl), l,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP. Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N, N-dilinoleylamino)- 1.2-propanediol (DLinAP), 3-(N, N-dilinoleylamino)-l,2-propanediol (DOAP), l,2-dilinoleyloxo-3-(2-N, N-dimethylamino)ethoxypropane (DLin-EG-DMA), N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), 1.2-dioleyloxy-N, N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N, N-dimethylaminopropane (DSDMA), N-(l-(2,3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTMA), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTAP), 3-(N-(N', N'-dimethylaminoethane)-carbamoyl)cholesterol (DC -Choi), N-(l,2-dimyristyloxyprop-3-yl)-N, N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N, N-dimethyl-l-propanaminiumtrifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N, N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), l,2-N, N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), or 1,2-N, N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), or mixtures thereof. In certain embodiments, the additional ionizable lipid is DLinDMA, DLin-K-C2-DMA (“XTC2”), or mixtures thereof.

[0460] In certain embodiments, the additional ionizable or cationic lipid is N, N -dioleyl-N, N -dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N, N- dimethylaminopropane (DODMA), 1,2-distearyloxy-N, N-dimethylaminopropane (DSDMA), N- (l-(2,3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTMA), N, N-distearyl-N, N- dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N, N, N- trimethylammonium chloride (DOTAP), 3-(N — (N', N'-dimethylaminoethane)- carbamoyl)cholesterol (DC -Choi), N-( 1,2-dimyristyloxyprop-3-yl)-N, N-dimethyl-N- hydroxyethyl ammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N, N-dimethyl-l-propanaminiumtrifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan- 4-oxy)-l-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3.beta.-oxy)- 3 '-oxapentoxy)-3 -dimethyl- 1 -(cis,cis-9', 1 -2'-octadecadienoxy)propane (CpLinDMA), N, N- dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N, N'-dioleylcarbamyl-3- dimethylaminopropane (DOcarbDAP), 1,2-N, N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), l,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), and mixtures thereof. A number of these lipids and related analogs have been described in U. S. Patent Publication Nos. 20060083780 and 20060240554; U. S. Pat. Nos. 5,208,036; 5,264,618; 5,279,833;

[0461] 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO 96 / 10390, the disclosures of which are each herein incorporated by reference in their entirety. In certain embodiments, the additional ionizable or cationic lipid comprises multiple sites of protonation. See, e.g., Qin et al., Signal Transduction and Targeted Therapy (2022) 7:166, which is incorporated by reference herein in its entirety. In certain embodiments, the additional ionizable or cationic lipid comprises 2, 3, 4, 5 or 6 sites of protonation (e.g., 2, 3, 4, 5, or 6 amino groups). See, e.g., WO 2010 / 053572.

[0462] Further additional ionizable or cationic lipids include those disclosed in, e.g., WO 2011 / 068810, WO 2012 / 000104, WO 2012 / 170930, WO 2013 / 086354, WO 2018 / 006052, WO 2020 / 097520, WO 2021 / 000041, WO 2022 / 266032, WO 2021 / 026647, WO 2022 / 173531, the disclosure of each of which is herein incorporated by reference in its entirety.

[0463] An ionizable or cationic lipid can be synthesized according to methods known in the art. The synthesis of lipids such as DLin-K-C2-DMA (“XTC2”), DLin-K-C3-DMA, DLin-K-C4-DMA, DLin-K6-DMA, and DLin-K-MPZ, as well as other ionizable or cationic lipids, is described in WO 2010 / 042877, the disclosure of which is herein incorporated by reference in its entirety. The synthesis of lipids such as DLin-K-DMA, DLin-C-DAP, DLin-DAC, DLin-MA, DLinDAP, DLin- S-DMA, DLin-2-DMAP, DLin-TMA. Cl, DLin-TAP. Cl, DLin-MPZ, DLinAP, DOAP, and DLin-EG-DMA, as well as other lipids, is described in WO 2009 / 086558, the disclosure of which is herein incorporated by reference in its entirety. The synthesis of lipids such as CLinDMA, as well as other lipids, is described in U. S. Patent Publication No. 2006 / 0240554, the disclosure of which is herein incorporated by reference in its entirety.

[0464] In certain embodiments, the ionizable lipid is DLin-MC3-DMA, ALC-0315, SM-102, or LP-01:

[0465] CH3O

[0466] H3C...,.. CH3DLin-MC3-DMA

[0467]

[0468] ALC-0315

[0469]

[0470] LP-01.

[0471] B. Polymer lipids

[0472] Polymer lipids such as PEG lipids may be used as a component in an LNP to modify the surface of the LNP. Such modification can stabilize an LNP in the presence of serum and therefore assist in extending circulation in vivo due to reduced protein absorption. In addition, the amount of polymer lipid in the LNP influences overall LNP size, which can affect the rate of cellular uptake.

[0473] In certain embodiments the ionizable lipids disclosed herein are combined with a polymer lipid to form an LNP. In certain embodiments, the polymer lipid is a pegylated or PEG lipid. When formulated as part of an LNP, PEG lipids can mask or cloak the cargo in vivo, thereby reducing immunogenicity and antigenicity of the cargo.

[0474] The polymer lipid may be a polymer-functionalized lipid, in which the polymer and lipid (e.g., a hydrocarbon chain optionally interrupted by one or more intervening functionalities) are joined by covalent bonds with optional intervening atoms. The polymer lipid optionally includes a branching moiety. The lipid may be, e.g., a hydrocarbon chain optionally interrupted by one or more intervening functionalities. The polymer lipid generally includes an uncharged, hydrophilic moiety which is believed to limit aggregation, such as PEG, GMI, or ATTA, during formulation of an LNP. Thus, it is believed that a polymer lipid may also reduce aggregation when included in an LNP. In certain embodiments, the content of the polymer lipid in the LNP is selected to reduce particle aggregation.

[0475] Examples of polymer lipids include polyethylene glycol (PEG)-modified lipids, monosialoganglioside GMI, and polyamide oligomers (" PAG") such as described in U. S. Pat. No. 6,320,017. ATTA-lipids are described, e.g., in U. S. Patent No. 6,320,017, and PEG-functionalized lipid are described, e.g., in U. S. Patent Nos. 5,820,873, 5,534,499 and 5,885,613. In certain embodiments, the polymer lipid comprises one or more hydrocarbon chains that are interrupted by a biodegradable functional group, e.g., an ester. The polymer lipid may comprise a branching moiety. In certain embodiments the branching moiety comprises a tertiary carbon atom, a quaternary carbon atom, a tertiary amine, a vicinal diol, an amide, a carbamate, or an acetal.

[0476] The lipid and optional branching moiety is believed to influence associative strength of a polymer lipid with the LNP. It is believed that at least three characteristics influence the rate of exchange: length of lipid chain, rigidity (e.g., as determined by saturation) of lipid chain, and size of the steric-barrier head group (see, e.g., U. S. Patent No. 5,820,873). For some therapeutic applications it may be preferable for the PEG-functionalized lipid to be rapidly lost from the LNP in vivo, and thus the PEG-lipid would have relatively short lipid anchors. In other therapeutic applications it may be preferable for a nucleic acid-lipid particle to exhibit a longer plasma circulation lifetime and hence the PEG-lipid will possess relatively longer lipid anchors. It is believed that mPEG (mw2000)-diastearoylphosphatidylethanolamine (PEG-DSPE) will remain associated with an LNP for days in vivo. Other conjugates, such as PEG-CerC20 have similar staying capacity. PEG-CerC14, however, is believed to more rapidly exchange out of the formulation upon exposure to serum.

[0477] In certain embodiments, the LNP may be formulated with free PEG or free ATTA (see, e.g., U. S. Patent Nos. 6,320,017 and 6,586,559) in a surrounding carrier solution. In such embodiments, the PEG or ATTA can be subsequently removed (e.g., by dialysis). Other polymer lipids include polyoxazoline (POZ)-lipid (e.g. POZ-DAA conjugates; see, e.g., WO 2010 / 006282), polyamide oligomers (e.g., ATTA-lipid conjugates), and those described in WO 2008 / 011561, WO 2009 / 140427, W02015 / 017519, WO 2016 / 118697, WO2016 / 094342, US 9,517,270 and US 9,801,944.

[0478] In certain embodiments, the polymer lipid is non-cationic. In certain embodiments, the non-cationic lipid is charge neutral. In certain embodiments, the polymer lipid is Zwitterionic.

[0479] The polymer may be a hydrophilic polymer, for example, a poly(ethylene glycol), also referred to as a poly(ethylene oxide) or PEG (“PEG-lipid”). In certain embodiments, PEG is an optionally substituted linear polymer of ethylene glycol or ethylene oxide. In certain embodiments, the PEG moiety is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In certain embodiments, the PEG moiety includes PEG copolymer such as PEG-polyurethane or PEG-polypropylene (see, e.g., J. Milton Harris, Polyethylene glycol) chemistry: biotechnical and biomedical applications (1992)). In certain embodiments, the PEG moiety is a PEG homopolymer.

[0480] Examples of polymer lipids include PEG-functionalized phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20 which are described in U. S. Patent No.

[0481] 5,820,873), PEG-modified dialkylamines, PEG-modified l,2-diacyloxypropan-3-amines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols. The lipid chain may vary according to known determinants in the art and may be, for example, a hydrocarbon of 10 to 30 carbon atoms in length. For example, certain embodiments provide a pegylated diacylglycerol (PEG-DAG) such as 1- (monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG- dilauroylglycerol, PEG-distearoylglycerol (PEG-DSPE), or 4-O-(2’, 3 ’-di (tetradecanoy loxy)propyl-l -0-(co- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG); a pegylated phosphatidylethanolamine (PEG-PE); a PEG-glycamide ((N-acyl-N-alkyl-glycamine) such as PEG- dimyristylglycamide, PEG- dipalmitoylglycamide, or PEG-disterylglycamide; a PEG dialkoxypropylcarbamate such as co- methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(co- methoxy(polyethoxy)-ethyl)carbamate; PEG-choles terol; PEG-dialkyloxypropyls (e.g., PEG-DAA), PEG- phosphatidylethanolamine, and PEG-ceramide (see, e.g., U. S. Pat. No. 5,885,613). For example, a polymer lipid may be PEG-DMG, PEG-c-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE, or a combination thereof.

[0482] In general, the length of the PEG chain may be represented by either a number indicating the number of repeating ethyl oxide units, or by a molecular weight in Daltons of the PEG portion of the polymer lipid. In a particular example, PEG-DMG 2000 has the following structure:

[0483]

[0484] where 2000 is the approximate molecular weight in Daltons of the PEG portion of the molecule. The length of PEG chain may vary between individual lipids in a sample and thus may be expressed as an average number of repeating units or average molecular weight.

[0485] A polymer lipid such as a PEG-lipid described herein may comprise a methyl (methoxy) or a hydroxyl group at the terminus of the PEG chain. In certain embodiments, the polymer lipid comprises an methoxy group at the terminus of a PEG chain. In certain embodiments, the polymer lipid is a PEG-OH lipid and comprises an -OH group at the terminus of a PEG chain. A polymer lipid can comprise a defined number of ethylene glycol units, for example, at least 1, at least 2, at least 5, at least 10, 10-150, or 40-60 ethylene glycol units. In certain embodiments, a number average molecular weight of the polymer portion of a polymer lipid is from about 200 Da to about 5000 Da.

[0486] In certain embodiments, the polymer lipid has a molecular weight from about 1500 Da to about 3500 Da.

[0487] In certain embodiments, the LNP comprises two or more polymer lipids having distinct structures. In certain embodiments, the polymer lipid has a structure described in WO 2015 / 199952:

[0488]

[0489] R” wherein

[0490] R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and z has mean value ranging from 30 to 60.

[0491] In certain embodiments the polymer lipid is a compound having the following structure:

[0492] O

[0493]

[0494] R"

[0495] or a salt thereof, wherein: R' and R" are each independently a saturated alkyl having from 8 to 12 carbon atoms, provided that the total number of carbon atoms collectively in both of R' and R" is no more than 23; R'" is H or Ci-Ce alkyl; and n is an integer ranging from 30 to 60. In other embodiments, R'" is H or CH3. In various different embodiments, the total number of carbon atoms collectively in both of R' and R" ranges from 16 to 22, 16 to 21, 16 to 20, 18 to 23, 18 to 22, 18 to 21, 19 to 23, 19 to 22, 19 to 21, 20 to 23, or 20 to 22. In still more embodiments: a) R' and R" are each a saturated alkyl having 8-11 carbon atoms. In some embodiments, n is an integer from 40 to 50;

[0496] or wherein: R' and R" are each independently a saturated alkyl having from 8 to 12 carbon atoms; R'" is H or Ci-Ce alkyl; and n is an integer ranging from 30 to 60.

[0497] In certain embodiments the polymer lipid is:

[0498] O

[0499]

[0500] wherein the average n is about 49.

[0501] In certain embodiments the polymer lipid is a compound of formula:

[0502] R 1’D

[0503] or salts thereof, wherein: R3is -OR0; R° is hydrogen, optionally substituted alkyl, or an oxygen protecting group; r is an integer between 1 and 150, inclusive; L1is optionally substituted C O alkylene, wherein at least one methylene of the optionally substituted CMO alkylene is independently replaced with optionally substituted carbocyclyclene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -O-, -N(RN)-, -S-, -C(O)-, - C(O)N(RN)-, -NRNC(O )-, - C(O)O-, -OC(O)-, -OC (O)O-, -OC(O)N(RN) -, -NRNC(O)O -, or -NRNC(O)N(RN)-; D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is of the formula: L1— R2

[0504]

[0505] each instance of L2is independently a bond or optionally substituted Ci-6 alkylene, wherein one methylene unit of the optionally substituted Ci-6 alkylene is optionally replaced with -O-, -N(RN)-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(RN)-, -NRC(O)O-, or -NRNC(O)N(RN)-; each instance of R2is independently optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted C1-30 alkynyl; optionally wherein one or more methylene units of R2are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(RN)-, -O-, -S-, -C(O)-, -C(O)N(RN)-, - NRNC(O)-, -NRNC(O)N(RN)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(RN)-, - NRNC(O)O-, -C(O)S- -SC(O)-, -C(=NRN)-, -C(=NRN)N(RN)-, -NRNC(=N RN)-, - NRNC(=NRN)N(RN)-, - C(S)-, C(S)N(RN)-, -NRNC(S)-, -NRNC(S)N(RN)-, -S(O)-, - OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O -, -N(RN)S(O)-, -S(O)N(RN)-, -N(RN)S(O)N(RN)-, -OS(O)N(RN)-, -N(RN)S(O)O-, -S(O)2-, -N(RN)S(O)2-,- S(O)2N(RN)-, -N(RN)S(O)2N(RN)-, -OS(O)2N(RN)-, or -N(RN)S(O)2O-; each instance of RNis independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and p is 1 or 2.

[0506] In certain embodiments the polymer lipid is a pegylated (PEG) fatty acid. In certain embodiments the polymer lipid is a compound of formula:

[0507] O

[0508]

[0509] or salts thereof, wherein: R3is -OR0; R° is hydrogen, optionally substituted alkyl or an oxygen protecting group; r is an integer between 1 and 100, inclusive; R5is optionally substituted C10-40 alkyl, optionally substituted C10-40 alkenyl, or optionally substituted C10-40 alkynyl; and optionally one or more methylene groups of R5are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(RN)-, -O-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -NRNC(O)N(RN)-, - C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(RN)-, -NRNC(O)O-, -C(O)S- -SC(O)-, - C(=NRN)-, -C(=NRN)N(RN)-, -NRNC(=N RN)-, -NRNC(=NRN)N(RN)-, - C(S)-, C(S)N(RN)-, -NRNC(S)-, -NRNC(S)N(RN)-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, - OS(O)2-, -S(O)2O-, -OS(O)2O -, -N(RN)S(O)-, -S(O)N(RN)-, -N(RN)S(O)N(RN)-, - OS(O)N(RN)-, -N(RN)S(O)O-, -S(O)2-, -N(RN)S(O)2-,-S(O) 2N(RN)-, -N(RN)S(O) 2N(RN)-, -OS(O)2N(RN)-, or -N(RN)S(O)2O-; and each instance of RNis independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group.

[0510] In certain embodiments, the polymer lipid has a structure described in WO 2012 / 099755: R4R4'

[0511]

[0512] R2

[0513] or a pharmaceutically acceptable salt thereof, wherein: each of R1and R2, independently, is a Cio to C30 aliphatic group, where the aliphatic group is optionally substituted by one or more groups each independently selected from Ra; and where the aliphatic group is optionally interrupted by cycloalkylene, -O-, -S-, -C(O)-, -OC(O)-,-C(O)O-, -N(RC)-, -C(O)N(RC)-, or -N(RC)C(O)-; X is -(CRaRb)i-, -O-, -S-, -C(O)-, -N(RC)-, -OC(O)-, -C(O)O-, -OC(O)O-, -C(O)N(Rc)-,-N(Rc)C(O)-, -OC(O)N(RC)-, -N(RC)C(O)O-, -N(RC)C(O)N(RC)-, -SC(O)N(RC)-, or -N(RC)C(O)S-; Y is -(CRaRb)i-, -O-, -S-, -C(O)-, -N(RC)-, -OC(O)-, -C(O)O-, -OC(O)O-, -C(O)N(Rc)-,-N(Rc)C(O)-, -OC(O)N(RC)-, -N(RC)C(O)O-, -N(RC)C(O)N(RC)-, - SC(O)N(RC)-, or -N(RC)C(O)S-; L is -L'-Z'-(L2-Z2) -L3-; L1is a bond, -(CR5R5)i-, or -(CR5R5’)i-(C(Ra)=C(Rb))k-(CAC)k, -(CRaRb)j Z1is -O-, -S-, -N(RC)-, -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)N(RC)-, - N(RC)C(O)O-, -N(RC)C(O)-, -C(O)N(RC)-, -N=C(Ra)-, -C(Ra)=N-, -O-N=C(Ra)-, or -O- N(RC)-; L2is -(CRaRb)P- or -(CRaRb)r(C(Ra)=C(Rb))k-(CAC)k-(CRaR )j; Z2is -O-, -S-, -N(RC)-, -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)N(RC)-, -N(RC)C(O)O-, -N(RC)C(O)-, -C(O)N(RC)-, -N=C(Ra)-, -C(Ra)=N-, -O-N=C(Ra)-, or -O- N(RC)-; L3is -(CRaRb)i-; each A, independently, is -L4-, -NH-(L4)q-(CRaRb)r-C(O)- or -C(O)- (CRaRb)r-(L4)q-NH-; where each q, independently, is 0, 1, 2, 3, or 4; and each r, independently, is 0, 1, 2, 3, or 4; each L4, independently, is -(CRaRb)sO- or -O(CRaRb)s-; where each s, independently, is 0, 1, 2, 3, or 4; R3is -H, -R, or -OR; each of R4and R4’, independently, is -H, halo, cyano, hydroxy, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, or cycloalkoxy; each R5and each R5’, independently, is -H, halo, cyano, hydroxy, nitro, alkyl, alkenyl, alkynyl, or cycloalkyl; or R4and one R5, taken together, can form a 5- to 8-membered cycloalkyl or heterocyclic ring; each Ra, independently, is -H, halo, cyano, hydroxy, nitro, amino, alkylamino, dialkylamino, alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, heteroaryl, or heterocyclyl; each Rb, independently, is -H, halo, cyano, hydroxy, nitro, amino, alkylamino, dialkylamino, alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, heteroaryl, or heterocyclyl; each Rcis -H, alkyl, acyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl; a is 0 or 1; b is an integer from 1 to 1,000; c is 0 or 1; each occurrence of i, independently, is 1, 2, 3, 4, 5, or 6; each occurrence of j, independently, is 0, 1, 2, or 3; each occurrence of k, independently, is 0, 1, 2, or 3; and p is 1 to 10; with the proviso that (i) X and Y are not simultaneously -CH2-; and (ii) when a is 1 and L1is -CH2-, then (a) X and Y are not simultaneously -O-; and (b) X and Y are not simultaneously -C(O)O-.

[0514] In certain embodiments, the polymer lipid has a structure described in WO 2013 / 049328:

[0515] R1., L4A}R

[0516] ' 'b3

[0517]

[0518] R2 or a pharmaceutically acceptable salt thereof, wherein R1and R2are each, independently, a Cio to C30 aliphatic group, wherein each aliphatic group is optionally substituted by one or more groups each independently selected from Ra; L is -L1-Z1-(L2-Z2)c-L3-; L1is a bond, -(CR5R50)i-, or -(CR5R50)i-(C(Ra)=C(Rb))k-(C°C)k-(CRaRb)j-; Z1is -O-, -S-, -N(RC)-, -OC(O)-, -C(O)O-, -OC(O)O-, -N(RC)C(O)O-, -N(RC)C(O)N(RC)-, -N(RC)C(O)-, -C(O)N(RC)-, -N=C(Ra)-, -C(Ra)=N-, -O-N=C(Ra)-, - O-N(RC)-; heteroaryl, or heterocyclyl; L2is -(CRaRb)p- or -(CRaRb)j-(C(Ra)=C(Rb))k-(C°C)k-(CRaRb)j-; Z2is -O-, -S-, -N(RC)-, -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)N(RC)-, - N(RC)C(O)O-, -N(RC)C(O)-, -C(O)N(RC)-, -N=C(Ra)-, -C(Ra)=N-, -O-N=C(Ra)-, -O- N(RC)-, heteroaryl, or heterocyclyl; L3is -(CRaRb)i-; each A, independently, is -L4-, -NH-(L4)q-(CRaRb)r-C(O)-, or -C(O)- (CRaRb)r-(L4)q-NH-; wherein each q, independently, is 0, 1, 2, 3, or 4; and each r, independently, is 0, 1, 2, 3, or 4; each L4, independently, is -(CRaRb)sO- or -O(CRaRb)s-, wherein each s, independently, is 0, 1, 2, 3, or 4; R3is H, -Rc, or -ORC; each occurrence of R5and R5is, independently, H, halo, cyano, hydroxy, nitro, alkyl, alkenyl, alkynyl, or cycloalkyl; each occurrence of Raand Rbis, independently, H, halo, cyano, hydroxy, nitro, amino, alkylamino, dialkylamino, alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, aryl, heteroaryl, or heterocyclyl; each Rcis, independently, H, alkyl, acyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl; b ranges from 5 to about 500; c is 0 or 1; each i is, independently, 1, 2, 3, 4, 5, 6, 7, 8, or 9; each occurrence of j and k, independently, is 0, 1, 2, or 3; and p is an integer from 1 to 10; or R1and R2are each, independently Cio to C30 aliphatic group; L is -IJ-Z1-! -; L1is a bond or -(CR5R5)i-; Z1is -N(RC)-, -N(RC)C(O)O-, -N(RC)C(O)N(RC)-, -N(RC)C(O)-, or - N=C(Ra)-, wherein the leftmost nitrogen atom in Z1is bound to L1or if L1is a bond, then to the central tertiary carbon atom, or Z1is a nitrogen-containing heteroaryl or heterocyclyl, wherein the nitrogen atom of the heteroaryl or heterocyclyl is bound to L1or if L1is a bond, then to the central tertiary carbon atom; L3is -(CRaRb)i-; each A is, independently, -L4-; b ranges from about 5 to about 500; each L4, independently, is -OCH2CH2-, -CH2CH2O-, -OCH(CH3)CH2- or -OCH2CH(CH3)-; R3is -ORC; each occurrence of Ra, Rc, R5and R5is, independently, H or alkyl; and i is 2, 3, 4 or 5; or R1and R2are each, independently C12 to C20 alkyl or C12 to C2oalkenyl; L is -U-Zkl Z2-! -; L1is a bond or -(CR5R5)i-; Z1is -N(RC)-, -N(RC)C(O)O-, -N(RC)C(O)N(RC)-, -N(RC)C(O)-, or - N=C(Ra)-, wherein the leftmost nitrogen atom in Z1is bound to L1or if L1is a bond, then to the central tertiary carbon atom, or Z1is a nitrogen-containing heteroaryl or heterocyclyl, wherein the nitrogen atom of the heteroaryl or heterocyclyl is bound to L1or if L1is a bond, then to the central tertiary carbon atom; L2is -(CRaRb)P; Z2is -O-, -C(O)O-, -C(O)N(RC)-, or heteroaryl; L3is -(CRaRb)i-; each A is, independently, -L4-; b ranges from about 5 to about 500; each L4, independently, is -OCH2CH2-, -CH2CH2O-, -OCH(CH3)CH2- or -OCH2CH(CH3)-; R3is -ORC; each occurrence of Ra, Rb, Rc, R5and R5is, independently, H or alkyl; i is 2, 3, 4 or 5; and p is 1 to 10.

[0519] In certain embodiments the polymer lipid is:

[0520]

[0521] wherein n is an integer ranging from 30 to 60.

[0522] In certain embodiments, the polymer lipid has a structure described in WO2t020 / 219941 or WO 2022 / 133344.

[0523] In certain embodiments, the polymer lipid has a structure described in WO 2020 / 061284:

[0524]

[0525] or a pharmaceutically acceptable salt thereof; wherein: L1is a bond, optionally substituted C1-3 alkylene, optionally substituted C1-3 heteroalkylene, optionally substituted C2-3 alkenylene, optionally substituted C2-3 alkynylene; R1is optionally substituted C5-30 alkyl, optionally substituted C5-30 alkenyl, or optionally substituted C5-30 alkynyl; R° is hydrogen, optionally substituted alkyl, optionally substituted acyl, or an oxygen protecting group; and r is an integer from 2 to 100, inclusive.

[0526] In certain embodiments, polymer lipids useful in the present invention can be pegylated (PEG) lipids described in International Publication No. WO 2012 / 099755, the contents of which is herein incorporated by reference in its entirety. In certain embodiments the polymer lipid is PEG-DMG 2000.

[0527] In certain embodiments, the polymer lipid is:

[0528]

[0529] On= 45-50

[0530] 2-[(polyethylene glycol)-2000]-N, N-ditetradecylacetamide (ALC-159).

[0531] In certain embodiments, the polymer lipid has a formula described in WO 2020 / 061295, the contents of which is herein incorporated by reference in its entirety, including:

[0532]

[0533] or a salt thereof; wherein r is independently an integer from 35-55, inclusive. In some embodiments, r=45.

[0534] In some embodiments, the polymer lipid has a formula of:

[0535]

[0536] or a salt thereof, wherein:

[0537] R3is -OR0;

[0538] R° is hydrogen, optionally substituted alkyl or an oxygen protecting group;

[0539] r is an integer between 1 and 100, inclusive;

[0540] R5is optionally substituted Cio-4o alkyl, optionally substituted Cio-4o alkenyl, or optionally substituted Cio-4o alkynyl; and optionally one or more methylene groups of R5are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(RN)-, -O-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -NRNC(O)N(RN)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(RN)-, -NRNC(O)O-, -C(O)S-, -SC(O)-, -C(=NRN)-, -C(=NRN)N(RN)-, -NRNC(=NRN)-, -NRNC(=NRN)N(RN)-, -C(S)-, -C(S)N(RN)-, -NRNC(S)-, -NRNC(S)N(RN)-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(RN)S(O)-, -S(O)N(RN)-, -N(RN)S(O)N(RN)-, -OS(O)N(RN)-, -N(RN)S(O)O-, -S(O)2-, -N(RN)S(O)2-, -S(O)2N(RN)-, -N(RN)S(O)2N(RN)-, -OS(O)2N(RN)-, or -N(RN)S(O)2O-; and

[0541] each instance of RNis independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group.

[0542] C. Non-Cationic Lipids

[0543] Non-cationic lipids can help stabilize an LNP and form the basic outer layer structure of an LNP. In certain embodiments the ionizable and / or cationic lipids disclosed herein are combined with one or more noncationic lipid to form an LNP. In certain embodiments, the non-cationic lipid is a (charge) neutral or Zwitterionic lipid. The non-cationic lipid can generally be any lipid species which is uncharged or neutral zwitterionic form at physiological pH. Generally, the non-cationic lipid will include a polar head group and one or more (e.g., two) hydrophobic tail groups. Exemplary head groups include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and inositol.

[0544] In certain embodiments, the non-cationic lipid comprises two hydrocarbon groups which are each optionally interrupted with a biodegradable moiety. Non-cationic 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 certain embodiments, the non-cationic lipid comprises saturated fatty acids, or mono-or di-unsaturated fatty acids. Additionally, lipids having mixtures of saturated and unsaturated fatty acid chains can be used. In certain embodiments, a fatty acid is interrupted by a biodegradable moiety such as an ester.

[0545] In certain embodiments, the non-cationic lipid comprises a phosphatidylcholine (PC), phosphatidylethanolamine (PE), glycerophospholipid, sphingophospholipid, sphingolipid, phosphono lipids, natural lecithins, or hydrogenated phospholipid. Such lipids include, for example diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0546] The selection of non-cationic lipid is generally guided by consideration of, e.g., LNP particle size and stability in circulation.

[0547] In certain embodiments, the non-cationic lipid is a phospholipid. As used herein, a “phospholipid” refers to a lipid that includes a hydrophilic phosphate head group and one or more hydrophobic tail groups. In some embodiments, a phospholipid may facilitate fusion to a membrane. For example, the positive charge on a zwitterionic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane may allow delivery of the one or more components of the ENP, e.g., the cargo, through the membrane, e.g., into a cell.

[0548] In certain embodiments, the phospholipid is a phosphatidylcholine. Exemplary phosphatidylcholines include, but are not limited to, l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), dipalmitoyl phosphatidylcholine, dipalmitoyl -sn-glycero-3-phosphocholine (DPPC), 2-oleoyl-l-palmitoyl-sn-glycero-3-phosphocholine (POPC), dimyristoyl phosphatidylcholine (DMPC), and dioleoyl phosphatidylcholine (DOPC).

[0549] In certain embodiments, the phospholipid is a phosphatidylethanolamine. In certain embodiments, the phosphatidylethanolamine is distearoyl phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), 1,2- dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), dimyristoyl phosphoethanolamine (DMPE), 16:0-monomethyl phosphatidylethanolamine, 16:0-dimethyl phosphatidylethanolamine, 18:1 -trans phosphatidylethanolamine, palmitoyl oleoylphosphatidylethanolamine (POPE), or 1 -stearoyl -2 -oleoyl-phosphatidyl ethanolamine (SOPE).

[0550] In certain embodiments, the phospholipid comprises a glycerophospholipid. In certain embodiments, the glycerophospholipid is plasmalogen, phosphatidate, or phosphatidylcholine. In certain embodiments, the glycerophospholipid is phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, palmitoyl oleoyl phosphatidylglycerol (POPG), or lysophosphatidylcholine. In some embodiments, the phospholipid comprises a sphingophospholipid. In some embodiments, the sphingophospholipid is sphingomyelin, ceramide phosphoethanolamine, ceramide phosphoglycerol, or ceramide phosphoglycerophosphoric acid.

[0551] In certain embodiments, the phospholipid comprises a natural membrane lipid, e.g. a lecithin. In some embodiments, the natural lecithin is egg yolk lecithin or soybean lecithin. In some embodiments, the phospholipid comprises a hydrogenated phospholipid. In some embodiments, the hydrogenated phospholipid is hydrogenated soybean phosphatidylcholine.

[0552] In certain embodiments, the non-cationic lipid is selected from l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); 1,2-dilinoleoyl- sn-glycero-3 -phosphocholine (DLPC); 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC); 1,2 dioleoyl-sn-glycero-3-phosphocholine (DOPC); 1,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC); 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), l,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 2,3-dipalmitoyl-sn-glycero-1 -phosphocholine, dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16:0-monomethyl phosphatidylethanolamine, 16:0-dimethyl phosphatidylethanolamine, 18:1 -trans phosphatidylethanolamine, l-stearioyl-2-oleoylphosphatidyethanol amine (SOPE), l,2-dielaidoyl-sn-glycero-3- phophoethanolamine (transDOPE), 1,2-dioctadecenyl-sn-glycero-3 -phosphocholine (18:0 diether PC); l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC); 1-hexadecyl-sn- glycero-3 -phosphocholine (CI 6 Lyso PC); 1,2-dilinolenoyl-sn-glycero-3- phosphocholine; l,2-diarachidonoyl-sn-glycero-3-phosphocholine; 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine; l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine; 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine; 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine; 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine; l,2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG); sphingomyelins (SM); and ceramides. Such lipids may be synthetic or naturally derived.

[0553] In certain embodiments, the non-cationic lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.

[0554] In certain embodiments, the non-cationic lipid is l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0555] In certain embodiments, the LNP comprises a plurality of non-cationic lipids, for example, 2, 3, or 4 distinct phospholipids selected from those described herein or known in the art. D. Sterols and derivatives thereof

[0556] Sterols can also help stabilize an LNP. In certain embodiments the ionizable lipids disclosed herein are combined with a sterol and other lipids to form an LNP. In certain embodiments, the sterol is cholesterol.

[0557] Cholesterol is a ubiquitous structural membrane lipid and its role in a lipid composition depends on context. When combined with phospholipids with low gel-liquid crystalline phase transitions (Tm), cholesterol is believed to aid formation of a liquid-ordered phase which is characterized by increased bilayer thickness and membrane rigidity. It is believed that cholesterol and other linear and / or branched hydrocarbon-containing (e.g., low Tm) lipids combine such that the cross-sectional area of the lipid and cholesterol is lower than the sum of the individual cross-sectional areas. However, when combined with high Tm lipids, cholesterol is believed to increase membrane fluidity and provide narrower bilayers. In any case, cholesterol is believed to encourage a liquid-ordered phase. Additionally, cholesterol is believed to reduce the amount of surface-bound protein and improve circulation half-life. The amount of cholesterol in an LNP may be selected to match the in vivo membrane cholesterol content.

[0558] In some embodiments, provided is an LNP comprising cholesterol. In some embodiments, provided is an LNP comprising a cholesterol derivative.

[0559] Many isomers and derivatives of cholesterol are present in organisms throughout nature. In certain embodiments, the LNP comprises a cholesterol derivative. In certain embodiments, the cholesterol derivative has the formula:

[0560]

[0561] wherein G1, G2, G3, and G4are each independently 1-4 substituent(s) selected from halo, cyano, hydroxy, Ci-ealkyl optionally substituted with Ral, Ci-ehaloalkyl, Ci-ehydroxyalkyl, Ci-eheteroalkyl, Cs-iocycloalkyl optionally substituted with Ral, Cs-iocycloalkyl-Ci-ealkyl optionally substituted with Ral, Ce-ioaryl optionally substituted with Ral, Ce-ioaryl-Ci-ealkyl optionally substituted with Ral, heteroaryl optionally substituted with Ral, heteroaryl-Ci-6alkyl optionally substituted with Ral, heterocyclyl optionally substituted with Ral, heterocyclyl-Ci.6alkyl optionally substituted with Ral, ORa2, -NH2, -NHRa2, -N(Ra2)2, -Ci- 6alkylene-NH2, -Ci-6alkylene-NHRa2, -Ci-6alkylene-N(Ra2)2, -C(O)Ra3, -C(O)ORa3, -C(O)NHRa3, -C(O)N(Ci.4alkyl)Ra3, -S(O)2Ra3, -S(O)Ra3, -NHC(O)Ra3, -N(Ci.4alkyl)C(O)Ra3, -NHS(O)Ra3, -N(Ci.4alkyl)S(O)Ra3, -NIISrt))2Ra3, and -N(Ci.4alkyl )S(O)2Ra3; each Ra2is independently selected from Ci-ealkyl, Cs-iocycloalkyl, Ce-ioaryl, heteroaryl, and heterocyclyl; each Ra3is independently hydrogen, -OH, Ci-ealkyl, Ci-ehaloalkyl, C3-locycloalkyl, Ce-ioaryl, heteroaryl, or heterocyclyl; each Ralis independently halo, cyano, hydroxy, -NH2, -NHRa4, -N(Ra4)2, Ci-ealkyl, Ci-ehaloalkyl, ORa4, or Cs-iocycloalkyl; each Ra4is independently selected from Ci-ealkyl, Cs-iocycloalkyl, Ce-ioaryl, heteroaryl, and heterocyclyl, and each Ra4is optionally substituted with hydroxy, one to six halo, or (’^alkoxy.

[0562] In certain embodiments, the cholesterol derivative is selected from P-sitosterol, -sitosterol acetate, a P-sitosterol amino acid conjugate, fecosterol, ergosterol, 9,11 -dehydroergosterol, campesterol, stigmasterol, brassicasterol, fucosterol, tomatidine, ursolic acid, a-tocopherol, daucosterol, cholesterol, 5-heptadecylresorcinol, cholesterol hemisuccinate, 6-keto-5a-hydroxycholesterol, 7 a-hydroxy cholesterol, 7P-hydroxycholesterol, 7-ketocholesterol, 7p,25-dihydroxycholesterol, 27 -hydroxy cholesterol, 25-hydroxycholesterol, 20a-hydroxycholesterol, 5a-cholestanol, 5P-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, 6-ketocholestanol, cholesteryl -(4'-hydroxy)-butyl ether, 5a-cholestane, cholestenone, 5P-cholestanone, cholesteryl decanoate, vitamin D3, vitamin D2, calcipotriol, botulin, luperol, ursolic acid, oleanolic acid, DC-cholesterol, BHEM-cholesterol, cholesteryl oleate, or a combination thereof. See, e.g., Paunovska, K. et al., Adv Mater. 2019 April; 31(14); Patel et al., Nat. Comm., (2020) 11:983; Ni et al., Nat. Comm. 13, Article number: 4766 (2022); Kim et al., ACS Nano 2022, 16(9), 14792-14806.

[0563] In certain embodiments, the cholesterol derivative is a corticosteroid. In certain embodiments, the LNP comprises a corticosteroid selected from cortisone, cortisol, prednisolone, methylprednisolone, 20a-dihydroprednisolone, 20P-dihydroprednisolone, betamethasone, dexamethasone, prednisone, flumethasone, isoflupredone, eclomethasone, clobetasol, triamcinolone acetonide, and hydrocortisone.

[0564] II. Lipid Nanoparticles

[0565] A. Composition

[0566] In certain embodiments, ionizable lipids may be used in combination with other lipids to form lipid nanoparticles (LNPs). In certain embodiments, the LNPs comprise (1) ionizable lipids in combination with (2) non-cationic helper lipids such as neutral, zwitterionic or negatively charged lipids, (3) sterols such as cholesterol and (4) polymer lipids such as PEG lipids.

[0567] In certain embodiments, disclosed herein are methods of preparing ionizable lipids. In certain embodiments, the methods comprise formulation of ionizable lipids into LNPs.

[0568] In certain embodiments, an LNP at least partially encapsulates a cargo. In certain embodiments, the cargo comprises a nucleic acid, e.g., an exogenous mRNA. In certain embodiments, the cargo comprises an oligonucleotide. In certain embodiments, the cargo comprises an exogenous mRNA and an oligonucleotide. In certain embodiments, the LNP is provided as a suspension in an aqueous medium. In certain embodiments, a pharmaceutical composition comprises an LNP at least partially encapsulating a cargo in an aqueous medium.

[0569] In some embodiments, the LNP comprises 20-70 mol% ionizable and / or cationic lipid. For example, the LNP may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-70 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-70 mol%, 40-60 mol%, 40-50 mol%, 50-70 mol% or 50-60 mol% ionizable and / or cationic lipid. In some embodiments, the LNP comprises 20 mol%, 30 mol%, 40 mol%, 50, 60 mol% or 70 mol% ionizable and / or cationic lipid.

[0570] In some embodiments, the LNP comprises 5-30 mol% helper lipid. For example, the LNP may comprise 5-25 mol%, 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-30 mol%, 10-25 mol%, 10-20 mol%, 10-15 mol%, 15-30 mol%, 15-25 mol%, 15-20 mol%, 20-30 mol%, 20-25 mol% or 25-30 mol% helper lipid. In some embodiments, the LNP comprises 5 mol%, 10 mol%, 15 mol%, 20 mol%, 25 mol% or 30 mol% helper lipid.

[0571] In some embodiments, the LNP comprises 25-55 mol% sterol. For example, the LNP may comprise 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% sterol. In some embodiments, the LNP comprises 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% sterol.

[0572] In some embodiments, the LNP comprises 0.5-15 mol% polymer lipid. For example, the LNP may comprise 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol% polymer lipid. In some embodiments, the LNP comprises 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% polymer lipid.

[0573] In certain embodiments, the LNP comprises 30 to 70 mol % of an ionizable and / or cationic lipid; 5 to 30 mol % of a helper lipid; 20 to 50 mol % of cholesterol or a derivative thereof; and 1 to 10 mol % of a polymer lipid. In certain embodiments, the LNP comprises 40 to 60 mol % of an ionizable and / or cationic lipid; 5 to 15 mol % of a helper lipid; 30 to 50 mol % of cholesterol or a derivative thereof; and 2 to 4 mol % of a polymer lipid.

[0574] In some embodiments, the LNP comprises 20-60 mol% ionizable lipid, 5-25 mol% helper lipid, 25-55 mol% sterol, and 0.5-15 mol% PEG-lipid. In some embodiments, the LNP comprises 40-50 mol% ionizable amino lipid, 5-15 mol% helper lipid, 20-40 mol% cholesterol, and 0.5-3 mol% PEG-lipid. In some embodiments, the LNP comprises 45-50 mol% ionizable amino lipid, 9-13 mol% helper lipid, 35-45 mol% cholesterol, and 2-3 mol% PEG-lipid. In some embodiments, the LNP comprises 48 mol% ionizable amino lipid, 11 mol% helper lipid, 68.5 mol% cholesterol, and 2.5 mol% PEG-lipid.

[0575] The mol % of each of the ionizable and / or cationic lipid, a helper lipid, polymer lipid, and sterol lipid are determined together (“total lipid”) irrespective of any cargo or excipients.

[0576] The LNP may be characterized by a molar ratio or mass ratio of a cargo (such as an exogenous mRNA or oligomeric agent) to total lipid. In certain embodiments, the lipid to cargo ratio is 20:1 to 1:1, 10:1 to 1:1, or 5:1 to 1:1 (mass:mass). In certain embodiments, the polymer lipid is a PEG-lipid. In certain embodiments, the PEG-lipid is PEG-DMG 2000. In certain embodiments, the helper lipid is a Zwitterionic phospholipid. In certain embodiments, the Zwitterionic phospholipid is DSPC.

[0577] In certain embodiments, the LNP comprises 1 to 15 mol% of polymer lipid. The amount of polymer lipid in the LNP may be adjusted to alter the pharmacokinetics and / or biodistribution of the LNP. In certain embodiments, the LNP may comprise from 0.1 to 5.0, from 1.0 to 3.5, from 1.5 to 4.0, from 2.0 to 4.5, from 2.0 to 3.0, from 2.5 to 5.0, and / or from 3.0 to 6.0 mol% of the polymer lipid. In certain embodiments, the molar ratio of ionizable or cationic lipid to the polymer lipid ranges from about 35:1 to about 15:1. In some embodiments, the molar ratio of ionizable or cationic lipid to polymer lipid ranges from about 100:1 to about 10:1. In certain embodiments, the polymer lipid is a PEG-lipid. In certain embodiments, an LNP comprises 2 to 3% of the polymer lipid, wherein the polymer lipid is a PEG-lipid. Certain contents of PEG-lipid are believed to contribute to improved distribution and / or bioavailability in primates, perhaps due to providing smaller particle size LNPs. See, e.g., WO 2021 / 030701, the contents of which is herein incorporated by reference in its entirety.

[0578] In certain embodiments, the LNP has a mean diameter in a range from X nm to Y nm, wherein X represents the lowest number in the range and Y represents the highest number in the range, wherein X and Y are each independently selected from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200; provided that X< Y.

[0579] In certain embodiments, the LNP has a mean diameter of from 20 nm to 150 nm, from 30 nm to 100 nm, from 40 nm to 150 nm, from 50 nm to 150 nm, from 60 nm to 130 nm, from 70 nm to 110 nm, from 70 to 90 nm, from 30 nm to 60 nm, or from 50 nm to 60 nm. The size of LNPs can be measured using a variety of techniques known in the art such as dynamic light scattering, size exclusion chromatography, nuclear magnetic resonance spectroscopy, and / or microscopy.

[0580] A polydispersity index may be used to indicate the particle size distribution of a population of LNPs. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. In certain embodiments, a polydispersity index (PDI) of diameter of a population of LNPs is less than 2 or less than 1. In certain embodiments, a polydispersity index of diameter of a population of LNPs is 0 to 0.25. The surface charges of LNPs are related to lipid head groups. The surface potential, which depends on the surface charge density, affects the interactions between particles and the adsorption of counterions. Surface-charged particles repel each other, which is believed to slow or prevent aggregation, and is believed to affect stability of the LNP. Surface charge of LNPs is most often expressed by a zeta potentials, which is the electrical potential of a particle measured from a plane just outside the layer of fluid bound to the particle. Zeta potential is commonly calculated from electrophoretic mobility. LNP compositions with relatively low charges at physiological pH, positive or negative, are generally believed to be desirable. In some embodiments, the zeta potential of the LNP may be from about -10 mV to about +20 mV, from about -5 mV to about +5 mV.

[0581] In general, an LNP is a metastable system comprising a distal surface that contacts a surrounding medium and an internal cavity which carries the cargo. In certain embodiments, the LNP comprises a lipid monolayer or bilayer which may encompass a portion of the LNP, e.g., some or all of the distal surface of the LNP. The LNP may comprise a lipid monolayer or bilayer in an internal cavity, which optionally forms multiple vesicles in a single particle. The LNP may comprise a mixture of tightly associated lipids and cargo. The half-life of an LNP in vitro in aqueous suspension may be on the order of, e.g., one day, ten days, 100 days, 1000 days, or 10,000 days. The LNP may comprise solid phase lipids, liquid phase lipids, or liquidcrystalline lipids, or mixtures thereof. In certain embodiments, the LNP may be formulated as a nanoparticle such as a nucleic acid-lipid nanoparticle described in WO 2009 / 127060.

[0582] B. LNP Preparation

[0583] The LNP can be prepared by any method known in the art including, but not limited to, a continuous mixing method or a direct dilution process. LNPs can be generated according to methods known in the art, see for example PCT / US2016 / 052352; PCT / US2016 / 068300; PCT / US2017 / 037551; PCT / US2015 / 027400; PCT / US2016 / 047406; PCT / US2016 / 000129; PCT / US2016 / 014280; PCT / US2016 / 014280;

[0584] PCT / US2017 / 038426; PCT / US2014 / 027077; PCT / US2014 / 055394; PCT / US2016 / 052117;

[0585] PCT / US2012 / 069610; PCT / US2017 / 027492; PCT / US2016 / 059575 and PCT / US2016 / 069491 all of which are incorporated by reference herein in their entirety.

[0586] In certain embodiments, provided is a method for preparing an LNP by a continuous mixing method, e.g., a process that includes providing an aqueous solution comprising a nucleic acid in a first reservoir, providing a lipid solution in a second reservoir, and mixing the aqueous solution with the lipid solution such that the organic lipid solution mixes with the aqueous solution so as to rapidly produce an LNP encapsulating the cargo. The lipid solution comprises a lower alcohol such as ethanol. This process and the apparatus for carrying this process are described in detail in U. S. Patent Publication No. 2004 / 0142025, the disclosure of which is herein incorporated by reference in its entirety. By mixing the aqueous solution comprising a cargo with the organic lipid solution, the organic lipid solution undergoes a continuous stepwise dilution in the presence of the buffer solution (i.e., aqueous solution) to produce an LNP.

[0587] In certain embodiments, provided is a method for preparing an LNP by a direct dilution process that includes forming an LNP solution and directly introducing the LNP solution into a collection vessel containing a controlled amount of dilution buffer. The collection vessel may include one or more elements configured to stir the contents of the collection vessel to facilitate dilution. In one aspect, the amount of dilution buffer present in the collection vessel is substantially equal to the volume of liposome solution introduced thereto. As a non-limiting example, a liposome solution in about 45% ethanol when introduced into the collection vessel containing an equal volume of dilution buffer will advantageously yield smaller particles.

[0588] In certain embodiments, provided is a method for preparing an LNP by a direct dilution process in which a third reservoir containing dilution buffer is fluidly coupled to a second mixing region. In this embodiment, the LNP solution formed in a first mixing region is immediately and directly mixed with dilution buffer in the second mixing region. In preferred aspects, the second mixing region includes a T-connector arranged so that the LNP solution and the dilution buffer flows meet as opposing 180° flows; however, connectors providing shallower angles can be used. In one aspect, the flow rate of dilution buffer provided to the second mixing region is controlled to be substantially equal to the flow rate of liposome solution introduced thereto from the first mixing region. Such control of the dilution buffer flow rate may advantageously allow for small particle size formation at reduced concentrations. Processes and apparatuses for carrying out direct dilution processes are described in U. S. Patent Publication No. 2007 / 0042031, the disclosure of which is herein incorporated by reference in its entirety.

[0589] The particle size distribution of LNPs can be controlled using manufacturing methods such as extrusion, sonication, homogenization, and microfluidic methods. An LNP provided herein can be size-adjusted by a method known in the art. One sizing method is described in U. S. Pat. No. 4,737,323, the disclosure of which is herein incorporated by reference in its entirety. Sonicating a particle suspension either by bath or probe sonication may produce a size reduction down to particles of less than about 50 nm in size. Homogenization is another method which relies on shearing energy to fragment larger particles into smaller ones. In a typical homogenization procedure, particles are recirculated through a standard emulsion homogenizer until selected particle sizes are observed.

[0590] Particle size distribution can be monitored by conventional methods including laser-beam particle size discrimination, or QELS.

[0591] Extrusion of an LNP through a small-pore membrane (e.g., of polycarbonate) or an asymmetric ceramic membrane is also an effective method for reducing particle size or for producing LNPs of low polydispersion. Typically, the suspension is cycled through the membrane one or more times until the desired particle size distribution is achieved. The particles may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in size.

[0592] Before loading into the LNP, a cargo may be precondensed with a non-lipid polycation as described in, e.g., WO 2000 / 003683, the disclosure of which is herein incorporated by reference in its entirety.

[0593] Examples of suitable non-lipid poly cations include, hexadimethrine bromide (sold under the brand name POLYBRENE®, from Aldrich Chemical Co.) or other salts of hexadimethrine. Other suitable polycations include, for example, salts of poly-L-omithine, poly- L-arginine, poly-L-lysine, poly-D-lysine, polyallylamine, and polyethyleneimine.

[0594] Any suitable apparatus may be used in the preparation of the LNP. A multi-inlet vortex mixer (MIVM) may be suitable for use in formation of an LNP. See, e.g., Liu Y. et al. (2008) Chemical Engineering Science 63:2829-2842. The MIVM has been utilized in processes for preparing multicomponent composite nanoparticles, see, e.g., WO 2009 / 061406. In certain embodiments, the LNP is prepared using methods utilizing microfluidic mixers. Exemplary microfluidic mixers may include, but are not limited to a slit interdigital micromixer including, but not limited to those manufactured by Microinnova (Allerheiligen bei Wildon, Austria) and / or a staggered herringbone micromixer (SHM), (see Zhigaltsev, I. V. et al., Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing have been published Langmuir.2012.28:3633-40; Belliveau, N. M. et al., Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA. Molecular Therapy-Nucleic Acids.2012.1:e37; Chen, D. et al., Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation. J Am Chem Soc.2012.134(16):6948-51; each of which is herein incorporated by reference in its entirety).

[0595] In certain embodiments, methods of LNP generation comprising SHM further comprise the mixing of at least two input streams wherein mixing occurs by microstructure-induced chaotic advection (MICA). According to this method, fluid streams flow through channels present in a herringbone pattern causing rotational flow and folding the fluids around each other. This method may also comprise a surface for fluid mixing wherein the surface changes orientations during fluid cycling. Methods of generating LNPs using SHM include those disclosed in U. S. Application Publication Nos.2004 / 0262223 and 2012 / 0276209. The LNP may be prepared using a micromixer such as, but not limited to, a Slit Interdigital Microstructured Mixer (SIMM- V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging jet (IJMM) from the Institut fur Mikrotechnik Mainz GmbH, Mainz Germany). The LNP may be prepared using microfluidic technology (see Whitesides, George M. The Origins and the Future of Microfluidics. Nature, 2006442: 368-373; and Abraham et al. Chaotic Mixer for Microchannels. Science, 2002295: 647-651; each of which is herein incorporated by reference in its entirety). As anon-limiting example, controlled microfluidic formulation includes a passive method for mixing streams of steady pressure-driven flows in micro channels at a low Reynolds number (See e.g., Abraham et al. Chaotic Mixer for Microchannels. Science, 2002295: 647651; which is herein incorporated by reference in its entirety). Additionally, the LNP may be prepared using a micromixer chip such as, but not limited to, those from Harvard Apparatus (Holliston, Mass.) or Dolomite Microfluidics (Royston, UK). A micromixer chip can be used for rapid mixing of two or more fluid streams with a split and recombine mechanism.

[0596] In certain embodiments, the LNP may be formed by a method described in International Publication Nos. WO 2011 / 127255 or WO 2008 / 103276, the contents of each of which is herein incorporated by reference in their entirety. In certain embodiments, the LNP may be formulated by the methods described in US Patent Publication No US 2013 / 0156845 or International Publication No WO 2013 / 093648 or WO 2012 / 024526, each of which is herein incorporated by reference in its entirety.

[0597] In certain embodiments, an LNP may be formed by a method described in US 9,668,980.

[0598] The lipid nanoparticles described herein may be made in a sterile environment, e.g., using the method described in US Patent Publication No. US 2013 / 0164400, herein incorporated by reference in its entirety. The LNP may be sterilized by sterile filtration.

[0599] The efficiency of encapsulation of a cargo describes the amount of cargo that is encapsulated or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided. An exemplary method for determining encapsulation efficiency is comparing the amount of cargo in a solution containing the LNP before and after disintegrating the LNP, e.g., using one or more organic solvents or detergents. For example, fluorescence may be used to measure the amount of free cargo (e.g., exogenous mRNA) in a solution. In certain embodiments, the encapsulation efficiency of a cargo may be at least 50%, for example at least 90%.

[0600] In certain embodiments, a cargo may be loaded into an LNP following nanoparticle formation. See, e.g., WO 2018 / 089801.

[0601] In certain embodiments, a cargo may be encapsulated in the lipid portion of the LNP or in an aqueous space enveloped by some or all of the lipid portion of the LNP. The encapsulation can be full encapsulation, partial encapsulation, or both. In some embodiments, a cargo is fully encapsulated in the LNP.

[0602] In certain embodiments, one or more cargos may be associated with an LNP via a covalent bond or a non-covalent bond. In some embodiments, any of the cargos may be separately or together contained in an LNP.

[0603] C. Cargo

[0604] I. Certain Nucleic Acids Encoding Polypeptides

[0605] i. Certain Nucleic Acids

[0606] In certain embodiments, provided herein are LNPs comprising ionizable lipids. In certain embodiments, the LNPs comprise, encapsulate or are capable of encapsulating a cargo, wherein the cargo is one or more nucleic acid. In certain embodiments, the nucleic acid may be or may include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or modified nucleosides.

[0607] In certain embodiments, the nucleic acid is an exogenous mRNA.

[0608] In certain embodiments, an exogenous mRNA comprises a coding region (e.g., an open reading frame (ORF)) encoding a polypeptide sequence, a cap, and one or more non-coding regions. In certain embodiments, the exogenous mRNA comprises a cap, 5' UTR, 3' UTR, a coding region, a poly(A) tail, and optionally one or more introns. The mRNA may comprise nucleotides selected from adenosine, guanosine, cytosine, uridine, N1 -methylpseudouridine. In certain embodiments, the mRNA may comprise nucleotides selected from adenosine, guanosine, cytosine, and uridine. In certain embodiments, the mRNA may comprise one or more modified nucleotides.

[0609] Naturally -occurring eukaryotic mRNA molecules can contain non-coding regions, including, but not limited to untranslated regions (UTR) at their 5 '-end (5' UTR) and / or at their 3 '-end (3' UTR), a 5'-cap structure and a 3'-poly(A) tail. Exogenous mRNA may be configured to include such regions, which facilitate cellular processing and translation. In certain embodiments, a formulation, e.g., an ENP may be configured to deliver an exogenous mRNA having an open reading frame encoding a polypeptide. In certain embodiments, provided are methods in which an exogenous mRNA is translated in a cell.

[0610] An exogenous mRNA may comprise a repeat region of contiguous nucleosides such as adenosine nucleosides, e.g., a poly(A) tail. A poly(A) tail is a region that is 3’ of the 3' UTR that contains multiple, consecutive adenosine nucleosides. It is believed that apoly(A) tail may protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, and / or export of the mRNA from the nucleus, and in translation. A poly(A) tail may contain 10 to 300 contiguous adenosine nucleosides. For example, an exogenous mRNA may comprise at its 3’ terminus a repeat region of 50-100, 50-150, 50-200, 100-250, 120-160, or 200-300 adenosine nucleosides.

[0611] ii. Capping Groups

[0612] Eukaryotic mRNAs, as well as RNA viral genomes, include a 7-methylguanosine (m7G) cap at the 5' end of the mRNA sequence, attached to the 5’-most mRNA nucleotide through a 5',5'-triphosphate bridge (ppp) during mRNA transcription. The cap structure plays essential functions in mRNA translation by recruiting translation initiation factors, and different 5' caps can be incorporated into naturally occurring mRNAs. CapO protects endogenous mRNA from nuclease attack and is also involved in nuclear export and translation initiation. Both Capl and Cap2 are two 5' caps that contain additional methyl groups on the second or third ribonucleotide. The additional modification of Capl and Cap2 are believed to reduce immunogenicity compared to CapO. A 5 ’-cap may be introduced during in vitro transcription.

[0613] An exogenous mRNA may comprise a specific capping group such as described herein or as known in the art. Examples of 5’ capping groups include: “Cap 0”: m7G(5')ppp(5')N; “Cap 1”: m7G(5')ppp(5')(2'OMeN); and “Cap 2”: m7G(5')ppp(5')(2'OMeN)(2'OMeN); in which m7G indicates a guanosine nucleoside methylated at its 7-position and having a free 3 ’-OH, (5') indicates a 5' point of attachment, p is a phosphodiester internucleoside linkage, each N is independently a nucleoside, e.g. guanosine or adenosine, and (2'OMeN) is independently a 2-O-methyl nucleoside, e.g., 2’-O-methylguanosine or 2’-O-methyladenosine. A first nucleoside adenosine following the cap may also be methylated at its N6 position.

[0614] Specific capping groups include (m7(3'OMeG)(5')ppp(5')(2'OMeA)pG; 3'-O-Me-m7G(5')ppp(5’)G (“ARCA” cap); G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; and m7G(5')ppp(5')(2'OMeN)pG (“CleanCap™” ), where m7(3'OMeG) indicates a guanosine nucleoside methylated at its 7-position and having a 3’-O-methyl. Commercial sources are available for some group (e.g., New England BioLabs, Ipswich, MA, and TriLink Biotechnologies, San Diego, CA). 5'- Capping of exogenous mRNA may be completed during in vitro transcription, or can be completed post-transcriptionally, e.g., using Vaccinia Vims Capping Enzyme Cap 1 structure may be generated using both Vaccinia Vims Capping Enzyme and a 2'-0 methyl-transferase to generate. Cap 2 structure may be generated from the Cap 1 structure followed by the 2'-O-methylation of the 5'- third most nucleotide using a 2'-0 methyl-transferase. Cap 3 structure may be generated from the Cap 2 structure followed by the 2'-O-methylation of the 5'-fourthmost nucleotide using a 2'-0 methyl-transferase. Enzymes may be derived, for example, from a recombinant source.

[0615] iii. Poly(A) Tail

[0616] The 3 '-poly (A) tail is a region of contiguous adenosine nucleosides at the 3 '-end of the transcribed mRNA. In certain embodiments, the 3'-poly(A) tail comprises one to 400 adenosine nucleosides. In certain embodiments, the 3’-poly(A) tail comprises unmodified adenosine nucleosides linked by phosphodiester internucleoside linkages.

[0617] In some embodiments, the exogenous mRNA comprises a stabilizing element. A stabilizing element may be a histone stem-loop. A stem-loop binding protein (SLBP), a 32 kDa protein has been identified. It is associated with the histone stem-loop at the 3 '-end of the histone messages in both the nucleus and the cytoplasm, and is believed to promote efficient 3 '-end processing of histone pre-mRNA and stimulation of translation. In some embodiments, the histone stem-loop sequence comprises a length of 15 to 45 nucleotides.

[0618] In some embodiments, exogenous mRNA includes a coding region, at least one histone stem-loop, and optionally, apoly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal is believed to increase the expression of an encoded protein.

[0619] iv. Coding Region and Sequence Optimization

[0620] A coding region may comprise a contiguous sequence beginning with a start codon (e.g., methionine (AUG)), and ending with a stop codon (e.g., UAA, UAG or UGA), and comprising one or more nucleotide sequences encoding amino acids. Generally, a coding region encodes a polypeptide which forms a protein via translation, whether in vitro or in vivo.

[0621] In certain embodiments, a coding region comprises selected nucleotide codons to improve one or more properties of the exogenous mRNA or of the encoded protein, and may comprise a codon optimized ORF. For example, a coding region of an exogenous mRNA sequence may be codon optimized. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; add or remove post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or generally to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art - non limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and / or proprietary methods. In certain embodiments, a coding region sequence is optimized using an optimization algorithm as provided herein or as known in the art.

[0622] In certain embodiments, a coding region encodes a polypeptide. In certain embodiments, the encoded polypeptide is or is part of a guided nucleic acid binding agent. In certain embodiments, the encoded polypeptide comprises or consists of a guided nucleic acid binding agent. In certain embodiments, the guided nucleic acid binding agent is a Gas protein.

[0623] In certain embodiments, the encoded polypeptide is a variant that differs in amino acid sequence from a wild-type (naturally occurring), native, or reference protein sequence, and may possess substitutions, deletions, and / or insertions at certain positions within the amino acid sequence, as compared to a wild-type, native or reference sequence. In certain embodiments, the % identity to a wild-type, native or reference sequence is in a range of X to Y, wherein X represents the lowest number in the range and Y represents the highest number in the range, wherein X and Y are each independently selected from 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100; provided that X< Y.

[0624] In certain embodiments, variant polypeptides encoded by nucleic acids may contain amino acid changes that confer any one or more desirable properties, such as reducing clearance, enhancing immunogenicity, enhancing expression, and / or improving stability or pharmacokinetic / pharmacodynamic (“PK / PD”) properties. Variant polypeptides can be made using routine mutagenesis techniques and assayed to determine the presence of desired properties. In addition, assays to determine expression levels and immunogenicity are well known, and PK / PD properties of a variant polypeptide can also be measured using well known techniques, such as by determining protein expression in a subject over time. Stability of a variant polypeptide can be measured, for example, by assaying thermal stability, stability upon urea denaturation or using in silico prediction.

[0625] In certain embodiments, variant polypeptides encoded by nucleic acids may contain sequence tags or other additional amino acids at the N-terminal or C-terminal ends. These can be used for peptide detection, purification or localization. For example, lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acids located at the carboxy and amino terminal regions of the amino acid sequence of a polypeptide may be deleted, thereby providing for truncated sequences (fragments). In some embodiments, cavities in the core of proteins can be fdled to improve stability, e.g., by introducing larger amino acids. In other embodiments, buried hydrogen bond networks may be replaced with hydrophobic resides to improve stability. In yet other embodiments, glycosylation sites may be removed and replaced with appropriate amino acids. Such sequences are readily identifiable to one of skill in the art.

[0626] In certain embodiments, the encoded polypeptide is a fusion protein. The fusion protein may include two or more proteins or fragments thereof joined together. In certain embodiments, the fusion protein is a Cas fusion protein.

[0627] In certain embodiments, a coding region further encodes a linker located between various domains of a fusion protein. In some embodiments, the linker is a cleavable linker or protease-sensitive linker. In some embodiments, the linker is selected from the group consisting of F2A linker, P2A linker, T2A linker, E2A linker, and combinations thereof. This family of self-cleaving peptide linkers, referred to as 2A peptides, has been described in the art (see for example, Kim, J. H. et al. (2011) PLoS ONE 6:el8556 and WO2017 / 127750). In some embodiments, the linker is an F2A linker. In some embodiments, the linker is a GGGS linker.

[0628] In certain embodiments, a coding region comprises a signal peptide fused to a polypeptide of interest. Signal peptides, comprising the N-terminal 15-60 amino acids of proteins, are typically required for translocation across the membrane on the secretory pathway and, thus control the entry of most proteins to the secretory pathway. The signal peptide of a nascent precursor protein (pre-protein) directs the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates the transport of the growing peptide chain across it for processing. ER processing produces mature proteins, wherein the signal peptide is cleaved from precursor proteins, typically by an ER-resident signal peptidase of the host cell, or they remain uncleaved and function as a membrane anchor. A signal peptide may also facilitate targeting of a polypeptide to the cell membrane. Signal peptides from heterologous genes are known in the art and can be tested for desired properties and then incorporated into a polypeptide sequence.

[0629] In certain embodiments, a signal peptide may have a length of X-Y amino acids, wherein X represents the lowest number in the range and Y represents the highest number in the range, wherein X and Y are each independently selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60; provided that X< Y.

[0630] In certain embodiments, a coding region shares less than 95%, less than 90%, less than 80%, or less than 70% sequence identity to a naturally-occurring or wild-type sequence ORF (open reading frame; e.g., a naturally-occurring or wild-type mRNA sequence encoding a protein), or a range of values therebetween.

[0631] In some embodiments, an exogenous mRNA may be one in which the levels of G / C are enhanced. The G / C -content of nucleic acid molecules (e.g., mRNA) may influence the stability of the RNA. RNA having an increased amount of guanine (G) and / or cytosine (C) nucleobases may be more stable than RNA containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleobases. See, e.g., WO02 / 098443.

[0632] The coding region comprises at least one start codon (an AUG nucleotide triplet) among its 5 ’-most nucleotides and at least one stop codon (an UAG, UAA, or UGA nucleotide triplet) among its 3 ’-most nucleotides. In certain embodiments, an exogenous mRNA includes 2 or more stop codons, e.g., 2-10 stop codons.

[0633] Exemplary codons for specific amino acids are known in the art.

[0634] v. Chemical Modifications For Exogenous mRNA

[0635] In certain embodiments, provided herein are LNPs comprising ionizable lipids. In certain embodiments, the LNPs comprise, encapsulate or are capable of encapsulating a cargo, wherein the cargo is an exogenous mRNA comprising modified nucleotides or nucleosides (those other than A, C, G, and U joined by only phosphodiester linkages; exclusive of a 5’ cap). Such modified nucleotides and nucleosides can be naturally-occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include those described herein with respect to oligonucleotides.

[0636] In certain embodiments, a naturally-occurring modified nucleotide or nucleotide of the disclosure is one generally known or recognized in the art. Non-limiting examples of such naturally occurring modified nucleotides and nucleotides can be found, inter aha, in the widely recognized MODOMICS database. In certain embodiments, an exogenous mRNA comprises a modified nucleotide or modified nucleoside described in one or more of US application Nos. PCT / US2012 / 058519; PCT / US2013 / 075177;

[0637] PCT / US2014 / 058897; PCT / US2014 / 058891; PCT / US2014 / 070413; PCT / US2015 / 027400;

[0638] PCT / US2015 / 36773; PCT / US2015 / 36759; PCT / US2015 / 36771; or PCT / IB 2017 / 051367 all of which are incorporated by reference herein. Hence, an exogenous mRNA can comprise unmodified nucleosides, modified nucleosides, or a combination thereof. In certain embodiments, each nucleoside in an exogenous mRNA is modified similarly to other nucleosides of the same type; for example, all uridine nucleosides in a parent sequence are replaced by N1 -methylpseudouridine nucleosides. A modification may provide reduced degradation and / or reduced immunogenicity compared to an RNA comprising only unmodified nucleosides. In certain embodiments, an exogenous mRNA comprises a modified nucleoside selected from 1-methylpseudouridine, 1 -ethylpseudouridine, 5-methoxyuridine, 5-methylcytidine, and pseudouridine. In certain embodiments, an exogenous mRNA comprises a modified nucleoside selected from 5-methoxymethyl uridine, 5-methylthiouridine, 1 -methoxymethyl pseudouridine, 5-methylcytidine, and 5-methoxycytidine. In certain embodiments, an exogenous mRNA comprises a modified nucleoside selected from a combination of two or more (e.g., 2, 3, 4) of any of the modified nucleobases of this paragraph.

[0639] In certain embodiments, an exogenous mRNA comprises 1 -methylpseudouridine in place of one more, e.g. all uridine nucleosides of a parent sequence. In certain embodiments, an exogenous mRNA comprises 1 -methylpseudouridine at one or more, e.g., all uridine positions of a parent sequence, and 5-methylcytidine at one or more, e.g., all cytidine positions of a parent sequence.

[0640] In certain embodiments, an exogenous mRNA comprises pseudouridine at one or more, e.g. all uridine nucleosides of a parent sequence. In certain embodiments, an exogenous mRNA comprises pseudouridine at one or more, e.g., all uridine positions of a parent sequence, and 5-methylcytidine at one or more, e.g., all cytidine positions of a parent sequence.

[0641] In certain embodiments, an exogenous mRNA comprises unmodified uridine at one or more, e.g., all uridine positions of a parent sequence.

[0642] In certain embodiments, all nucleotides of a particular type in an exogenous mRNA (or in a sequence region thereof) are modified nucleotides compared to a parent sequence, wherein the nucleotides modified from the parent sequence may be any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

[0643] In certain embodiments, a nucleic acid may contain from X%-Y% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i. e., any one or more of A, G, U or C), wherein X represents the lowest number in the range and Y represents the highest number in the range, wherein X and Y are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100; provided that X< Y.

[0644] vi. Untranslated Regions (UTRs)

[0645] In certain embodiments, provided herein are LNPs comprising ionizable lipids. In certain embodiments, the LNPs comprise, encapsulate or are capable of encapsulating a cargo, wherein the cargo is an exogenous mRNA comprising an untranslated region. In wild-type mRNA, certain regions of a nucleic acid may be transcribed into RNA but not translated. In exogenous mRNA as described herein, a 5' UTR may start at the transcription start site and continue to a start codon at the beginning of the coding region, but does not include the start codon. The 3' UTR following a stop codon may include a transcription termination signal. The 5' UTR and the 3' UTR do not encode protein (are non-coding regions).

[0646] In certain embodiments, the presence of a UTR enhances the stability of the exogenous mRNA, or induces downregulation of the exogenous mRNA in undesirable sites or tissues. A variety of 5' UTR and 3' UTR sequences are known and available in the art.

[0647] A 5' UTR is a region of an mRNA that is directly upstream (5') from the start codon (the first codon of an mRNA transcript translated by a ribosome). In certain embodiments, the 5' UTR may provide translation initiation, and / or form secondary structures which are involved in elongation factor binding. The 5' UTR may include an initiation sequence such as a Kozak sequence. The Kozak sequence, also called the Kozak consensus sequence, is believed to be involved in ribosomal initiation of translation. In certain embodiments, the Kozak sequence comprises AUGG. In certain embodiments, the Kozak sequence is GCCRCCAUGG (SEQ ID NO: 1) or CCRCCAUGG, where R is a purine nucleoside (adenosine or guanosine).

[0648] In certain embodiments of the disclosure, a 5' UTR is an unmodified UTR, i.e., one found in nature. In another embodiment, a 5' UTR is a modified UTR, i.e., does not occur in nature. In certain embodiments, a modified UTR increases gene expression relative to an unmodified counterpart. Exemplary 5' UTRs include Xenopus laevis or human derived a-globin or b-globin (e.g., US Patent No. 9012219), human cytochrome b-245 a polypeptide, and hydroxysteroid (17b) dehydrogenase, and Tobacco etch virus (e.g., US9012219), CMV immediate-early 1 (IE1) gene (US2014 / 0206753, WO2013 / 185069), the sequence GGGAUCCUACC (SEQ ID NO: 2) (WO2014 / 144196), and a 5' UTR described in US Patent Application Publication No. 2010 / 0293625 or WO2015085318. In another embodiment, 5' UTR of a TOP gene is a 5' UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract) (e.g., WO / 2015 / 101414, W02015 / 101415, WO / 2015 / 062738, WO2015 / 024667, WO2015 / 024667); 5' UTR element derived from ribosomal protein Large 32 (L32) gene (WO / 2015 / 101414, W02015 / 101415, WO / 2015 / 062738), 5' UTR element derived from the 5' UTR of an hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (WO2015 / 024667), or a 5' UTR element derived from the 5' UTR of ATP5A1 (WO2015 / 024667) can be used. In some embodiments, an internal ribosome entry site (IRES) is used instead of a 5' UTR.

[0649] In general, an exogenous mRNA may include a UTR from any suitable gene. The naturally occurring UTR or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5' or 3' UTR may be inverted, shortened, or lengthened. A reference UTR, e.g., a naturally occurring UTR, may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping, or transposition of nucleotides or sequences thereof.

[0650] In certain embodiments, the 3' UTR sequences may include adenosine- and uridine-containing repeats. Such AU rich sequences are believed to provide high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I ARBs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I ARBs. Class II ARBs possess two or more overlapping UUAUUUASS nonamers, where S is adenosine or uridine. Molecules containing this type of ARBs include GM-CSB and TNB-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. It is believed that including the HuR specific binding sites into the 3' UTR will provide stabilization of the message in vivo. In certain embodiments, a 3' UTR includes a repeated ARE.

[0651] Unmodified (natural) and modified (non-natural) 3' UTR sequences are known in the art. UTRs known in the art include globin UTRs, including Xenopus b-globin UTRs and human b-globin UTRs (9012219, US2011 / 0086907), a modified b-globin construct (US2012 / 0195936, WO2014 / 071963), a2-globin, al-globin, UTRs (W02015 / 101415, WO2015 / 024667), CYBA (Eerizi et al, 2015) and albumin (Thess et al, 2015), bovine or human growth hormone (wild type or modified) (WO2013 / 185069, US2014 / 0206753, WO2014152774), rabbit b globin and hepatitis B virus (HBV), a-globin 3' UTR and Viral VEEV 3' UTR sequences, the sequence UUUGAAUU (WO2014 / 144196), human and mouse ribosomal protein, rps93UTR (W02015 / 101414), FIG4 (W02015 / 101415), and human albumin 7 (W02015 / 101415).

[0652] The untranslated region may also include an upregulation motif, e.g., a translation enhancer element (TEE). As a non-limiting example, the TEE may include those described in WO1999024595, W02012009644, W02009075886, W02007025008, WO1999024595, European Patent Publication No. EP2610341A1 and EP2610340A1, US Patent No. US6310197, US6849405, US7456273, US7183395, US Patent Publication No. US20090226470, US20110124100, US20070048776, US20090093049, or US20130177581 each of which is herein incorporated by reference in its entirety.

[0653] In certain embodiments, the 5’ UTR or 3’ UTR may comprise one or more additional functional regions selected from an upregulation region or a ribosome binding region. In certain embodiments, the functional region is an upregulation region (e.g., TEE). In certain embodiments, the TEE is one known in the art, e.g., in US Application No. 2009 / 0226470. In certain embodiments, an exogenous mRNA comprises a plurality of upregulation motifs which may be the same or different from each other and which number, e.g., 2, 3, 4, 5, or more.

[0654] In certain embodiments, the exogenous mRNA comprises a ribosome binding region, e.g., an internal ribosome entry site (IRES). The IRES may be, for example, one described in US Patent No. US7468275 and International Patent Publication No. W02001055369, each of which is herein incorporated by reference in its entirety. In certain embodiments, the IRES is one known in the art, e.g., in W02014 / 081507.

[0655] In certain embodiments, an exogenous mRNA may comprise a double, triple or quadruple UTR such as a 5' UTR or 3' UTR. As used herein, a “double” UTR is one in which two copies of the same UTR are included contiguously or substantially contiguously. For example, an exogenous mRNA may comprise a double beta-globin 3' UTR as described in US Patent publication No. 2010 / 0129877. In certain embodiments, a 5’ UTR or 3’ UTR may comprise a repeated set of functional sequences, e.g., as AA, AB AB or AABB or ABCABC or variants thereof, in which each letter, A, B, and C represent a different functional region. The pattern may be repeated once, twice, or 3 or more times.

[0656] Generally, any 5' UTR sequence and any 3' UTR sequence may be combined in a particular exogenous mRNA. Those of ordinary skill in the art will understand that 5’ UTRs that are heterologous or synthetic may be used with any desired 3’ UTR sequence. For example, a heterologous 5’ UTR may be used with a synthetic 3’ UTR or with a heterologous 3’ UTR.

[0657] Other non-coding sequences may also be used as regions or subregions within an exogenous mRNA. For example, introns or portions of introns sequences may be incorporated into regions of nucleic acid of the disclosure. In certain embodiments, inclusion of intronic sequences may increase protein production as well as nucleic acid levels.

[0658] Combinations of features may be included in flanking regions and may be contained within other features.

[0659] vii. Length

[0660] In certain embodiments, an exogenous mRNA includes 200 to 3,000 nucleotides. For example, an exogenous mRNA may include 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides. Unless otherwise indicated, the exogenous mRNA comprises contiguous nucleosides.

[0661] viii. Preparation

[0662] In some embodiments, the exogenous mRNA is generated using a non-amplified, linearized DNA template in an in vitro transcription reaction to generate the exogenous mRNA. In some embodiments, the template DNA is isolated DNA. In some embodiments, the template DNA is cDNA. In some embodiments, the cDNA is formed by reverse transcription of an RNA polynucleotide.

[0663] An in vitro transcription system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase. The NTPs may be purchased from a supplier or may be synthesized as described herein according to methods known in the art.

[0664] In vitro transcription of RNA is known in the art and is described in International Publication WO 2014 / 152027, which is incorporated by reference herein in its entirety. In certain embodiments, the exogenous mRNA is prepared in accordance with any one or more of the methods described in WO 2015 / 164674, WO 2018 / 053209, WO 2019 / 036682 and WO 2022 / 221440, each of which is incorporated by reference herein.

[0665] In some embodiments, the exogenous mRNA is generated using solid phase chemical synthesis, liquid phase chemical synthesis, a combination of synthetic methods or via ligation. The use of solid phase or liquid phase chemical synthesis in combination with enzymatic ligation may provide efficient generation of long chain RNA transcripts that is difficult to obtain by chemical synthesis alone.

[0666] Solid phase chemical synthesis can be an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution. Solid-phase synthesis is useful in sitespecific introduction of chemical modifications.

[0667] Liquid phase chemical synthesis can also be an automated method involving sequential addition of monomer building in a liquid phase.

[0668] DNA or RNA ligases can also be used to promote intermolecular ligation of the 5’ and 3’ ends of polynucleotide chains through the formation of a phosphodiester bond. Nucleic acids such as chimeric polynucleotides and / or circular nucleic acids may be prepared by ligation of one or more regions or subregions. DNA fragments can be joined by a ligase catalyzed reaction to create recombinant DNA with different functions. Two oligodeoxynucleotides, one with a 5’ phosphoryl group and another with a free 3’ hydroxyl group, serve as substrates for a DNA ligase. The DNA can then be used as a template in an in vitro transcription reaction to generate the exogenous mRNA.

[0669] ix. Purification

[0670] Purification of the exogenous mRNA described herein may comprise steps including nucleic acid clean-up, quality assurance, and quality control. Clean-up may be performed by methods known in the arts such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term “purified” when used in relation to exogenous mRNA refers to one that is separated from at least one contaminant. A “contaminant” is any substance that makes another unfit, impure or inferior. Thus, a purified nucleic acid (e.g., DNA and RNA) is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment or purification method.

[0671] A quality assurance and / or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.

[0672] In some embodiments, the nucleic acids may be sequenced by methods including, but not limited to reverse-transcriptase-PCR.

[0673] II. Oligonucleotides

[0674] i. Certain Oligonucleotides and Oligomeric Agents

[0675] In certain embodiments, provided herein are LNPs comprising ionizable lipids. In certain embodiments, the LNPs comprise, encapsulate or are capable of encapsulating a cargo, wherein the cargo is an oligonucleotide consisting of linked nucleosides. In certain embodiments, the LNPs comprise, encapsulate or are capable of encapsulating a cargo, wherein the cargo is an oligomeric agent comprising an oligonucleotide consisting of linked nucleosides.

[0676] In certain embodiments, oligonucleotides can be used as a guide to target a target nucleic acid, such as RNA or DNA. The targeting of DNA using the RNA-guided, DNA-targeting principle of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) systems has been previously validated. CRISPR-Cas systems can be divided in two classes, with Class 1 systems utilizing a complex of multiple Cas proteins (such as type I, III, and IV CRISPR-Cas systems) and Class 2 systems utilizing a single Cas protein (such as type II, V, and VI CRISPR-Cas systems). Type II CRISPR-Cas-based systems have been used for genome editing, and require a Cas protein or variant thereof guided by a customizable guide for programmable DNA targeting.

[0677] In certain embodiments, a single guide comprises an oligonucleotide. In certain embodiments, a dual guide comprises two oligonucleotides. Oligonucleotides may be unmodified oligonucleotides or may be modified oligonucleotides. Modified oligonucleotides comprise at least one modification relative to unmodified nucleic acids. That is, modified oligonucleotides comprise at least one modified nucleoside (comprising a modified sugar moiety and / or a modified nucleobase) and / or at least one modified internucleoside linkage.

[0678] In certain embodiments, the oligomeric agent comprising one or more oligonucleotides is capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity; such oligomeric agents are antisense agents. In certain embodiments, antisense agents are capable of hybridizing to one or more target nucleic acid, resulting in one or more desired antisense activity. In certain embodiments, antisense agents selectively affect one or more target nucleic acid, resulting in one or more desired antisense activity.

[0679] In certain antisense activities, hybridization of an antisense agent to a target nucleic acid results in recruitment of a protein, e.g., RNase H or Argonaute, that cleaves the target nucleic acid. In certain embodiments, oligomeric agents are antisense agents that are sufficiently “DNA-like” to elicit RNase H activity. In certain embodiments, one or more non-DNA-like nucleosides in the antisense agent are tolerated and RNase H activity is retained.

[0680] In certain antisense activities, an antisense agent is loaded into an RNA-induced silencing complex (RISC), ultimately resulting in cleavage of the target nucleic acid. For example, certain antisense agents result in cleavage of the target nucleic acid by Argonaute. Antisense agents that comprise an antisense agent that is loaded into RISC may be double-stranded (siRNA or dsRNAi), single-stranded (ssRNA), or a hairpin oligonucleotide that has a double-stranded region.

[0681] In certain antisense activities, hybridization of an antisense agent to a target nucleic acid results in inhibition of a binding interaction between the target nucleic acid and a protein or other nucleic acid (e.g., miRNA, IncRNA, sncRNA). In certain embodiments, hybridization of an antisense agent to a target nucleic acid results in modulation of translation of the target nucleic acid. In certain activities, hybridization of an antisense agent to a target nucleic acid results in alteration of splicing of the target nucleic acid (e.g. exon inclusion, exon exclusion, intron retention, or retained intron exclusion).

[0682] a. Certain Modified Nucleosides

[0683] Modified nucleosides comprise a modified sugar moiety or a modified nucleobase or both a modified sugar moiety and a modified nucleobase.

[0684] 1. Certain Sugar Moieties

[0685] In certain embodiments, modified sugar moieties are non-bicyclic modified sugar moieties. In certain embodiments, modified sugar moieties are bicyclic or tricyclic sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of other types of modified sugar moieties.

[0686] In certain embodiments, modified sugar moieties are non-bicyclic modified furanosyl sugar moieties comprising one or more substituent groups including, but not limited to, substituents at the 2', 3', 4', and / or 5' positions, as numbered below:

[0687] \ 5'

[0688] 4'^ ^1

[0689] 3'| 2'

[0690]

[0691] In certain embodiments, the modified furanosyl sugar moiety is a ribosyl sugar moiety that is not an unmodified sugar moiety (z.e., an unmodified RNA or unmodified DNA moiety). In certain embodiments, the modified furanosyl sugar moiety is a xylosyl, lyxosyl, or arabinosyl sugar moiety.

[0692] In certain embodiments, non-bicyclic modified sugar moieties are 2'-substituted sugar moieties and comprise a substituent group at the 2'-position. In certain embodiments one or more non-bridging substituent of non-bicyclic modified sugar moieties is branched.

[0693] In certain embodiments, a 2'-substituted sugar moiety comprises a non-bridging 2'-substituent group selected from: F, NH2, N3, OCF3, OCH3(“OMe”), O(CH2)3NH2, CH2CH=CH2, OCH2CH=CH2, OCH2CH2OCH3(“MOE”), O(CH2)2SCH3, O(CH2)2ON(CH3)2(“DMAOE”), O(CH2)2O(CH2)2N(CH3)2(“DMAEOE”), OCH2C(=O)-N(H)CH3(“NMA”), O(CH2)2ON(Rm)(Rn), and N-substituted acetamide (OCH2C(=O)-N(Rm)(Rn)), where each Rmand Rnis, independently, H, an amino protecting group, or substituted or unsubstituted Ci-Cio alkyl.

[0694] In certain embodiments, non-bicyclic modified sugar moieties comprise a substituent group at the 'position, the 5 ’-position, or both the 2’ and 5 ’-position.

[0695] Certain modified sugar moieties are bicyclic sugar moieties and comprise a substituent that bridges two atoms of the furanosyl ring to form a second ring. In certain embodiments, the bicyclic sugar moiety comprises a bridge between the 4' and the 2' furanose ring atoms. Examples of such 4' to 2' bridging sugar substituents include, but are not limited to: 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2' (“LNA”), 4'-CH2-S-2', 4'-(CH2)2-O-2' (“ENA”), 4'-CH(CH3)-O-2' (referred to as “constrained ethyl” or “cEt” when in the S configuration), 4'-CH2-O-CH2-2', 4'-CH2-N(R)-2', 4'-CH(CH2OCH3)-O-2' (“constrained MOE” or “cMOE”) and analogs thereof, 4'-C(CH3)(CH3)-O-2' and analogs thereof, 4'-CH2-N(OCH3)-2' and analogs thereof, 4'-CH2-O-N(CH3)-2', 4'-CH2-C(H)(CH3)-2', 4'-CH2-C(=CH2)-2' and analogs thereof, 4'-C(RaRb)-N(R)-O-2', 4'-C(RaRb)-O-N(R)-2', 4'-CH2-O-N(R)-2', and 4'-CH2-N(R)-O-2', wherein each R, Ra, and Rbis, independently, H, a protecting group, or C1-C12 alkyl. Representative U. S. patents that teach the preparation of such bicyclic sugar moieties include, but are not limited to: Imanishi et al., U. S. 7,427,672; Swayze et al., U. S. 7,741,457; Swayze et al., U. S. 8,022,193; Seth et al., U. S. 8,278,283; Prakash et al., U. S. 8,278,425; and Seth et a / ., U. S. 8,278,426.

[0696] 2. Certain Modified Nucleobases

[0697] In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising an unmodified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleoside that does not comprise a nucleobase, referred to as an abasic nucleoside. In certain embodiments, modified oligonucleotides comprise one or more inosine nucleosides (i.e., nucleosides comprising a hypoxanthine nucleobase). An “unmodified nucleobase” is adenine (A), thymine (T), cytosine (C), uracil (U), or guanine (G). A modified nucleobase is a group of atoms other than unmodified A, T, C, U, or G capable of pairing with at least one other nucleobase. A 5 -methylcytosine is an example of a modified nucleobase. A universal base is a modified nucleobase that can pair with any one of the five unmodified nucleobases.

[0698] In certain embodiments, modified nucleobases are selected from: 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and 0-6 substituted purines. In certain embodiments, modified nucleobases are selected from: 5 -methylcytosine, 2-aminopropyladenine, 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (-C=C-CH3) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5 -ribosyluracil (pseudouracil), N1 -methylpseudouracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo (particularly 5-bromo), 5 -trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3 -deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one and 9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T., Ed., CRC Press, 2008, 163-166 and 442-443.

[0699] Publications that teach the preparation of certain of the above noted modified nucleobases, as well as other modified nucleobases include without limitation, Rogers et al., U. S. 5,134,066; Benner et al., U. S. 5,432,272; Matteucci et al., U. S. 5,502,177; Froehler et al., U. S. 5,594,121; and Cook et al., U. S. 5,681,941.

[0700] 3. Certain Modified Internucleoside Linkages

[0701] The naturally occurring internucleoside linkage of RNA and DNA is a 3' to 5' phosphodiester linkage. In certain embodiments, nucleosides of modified oligonucleotides may be linked together using one or more modified internucleoside linkages. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiesters, which contain a phosphodiester bond (also referred to as unmodified or naturally occurring linkages), phospho triesters, methylphosphonates, phosphoramidates (such as mesyl phosphoramidate), phosphorothioates, phosphonoacetates (“PACE”), thiophosphonoacetates (“Thio-PACE”) and phosphorodithioates. Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH₂-N(CH₃)-O-CH₂-), thiodiester, thionocarbamate (-O-C(=O)(NH)-S-); siloxane (-O-Si(R₂)-O-); and N, N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). In certain embodiments, each internucleoside linkage is selected from a phosphodiester, a phosphorothioate, and a mesyl phosphoramidate.

[0702] b. Certain Motifs

[0703] In certain embodiments, guides (modified oligonucleotides) comprise one or more modified nucleoside(s) comprising a modified sugar moiety. In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more modified internucleoside linkage. In such embodiments, the modified, unmodified, and differently modified sugar moieties, nucleobases, and / or internucleoside linkages of a modified oligonucleotide define a pattern or motif. In certain embodiments, the patterns of sugar moieties, nucleobases, and internucleoside linkages are each independent of one another. Thus, a modified oligonucleotide may be described by its sugar motif, nucleobase motif and / or internucleoside linkage motif (as used herein, “nucleobase motif’ describes the modifications to the nucleobases independent of the sequence of nucleobases). 1. Certain Sugar Motifs

[0704] In certain embodiments, oligonucleotides comprise one or more type of modified sugar and / or unmodified sugar moiety arranged along the oligonucleotide or region thereof in a defined pattern or sugar motif. In certain instances, such sugar motifs include but are not limited to any of the sugar modifications discussed herein. Generally, the guide comprises unmodified RNA nucleosides, and optionally one or more modified nucleosides as described herein. In certain embodiments, the guide comprises or consists of a modified oligonucleotide.

[0705] In certain embodiments, the 5 ’-most nucleoside of a modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, the 3 ’-most nucleoside of a modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, at least 1, 2, 3, 4, or 5 of the 5’-most five nucleosides of a modified oligonucleotide comprise a modified sugar moiety. In certain embodiments, at least 1, 2, 3, 4, or 5 of the 3 ’-most five nucleosides of a modified oligonucleotide comprise a modified sugar moiety.

[0706] 2. Certain Nucleobase Motifs

[0707] In certain embodiments, oligonucleotides comprise modified and / or unmodified nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases are modified. In certain embodiments, each purine or each pyrimidine is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each uracil is modified. In certain embodiments, each cytosine is modified. In certain embodiments, some or all of the cytosine nucleobases in a modified oligonucleotide are 5 -methylcytosines. In certain embodiments, all of the cytosine nucleobases are 5-methylcytosines and all of the other nucleobases of the modified oligonucleotide are unmodified nucleobases.

[0708] In certain embodiments, the modified nucleobase is selected from pseudouracil, 2-6 diamino purine, 2-thiouracil, 4-thiouracil, 2-aminoadenine, 6-methyladenine, hypoxanthine, or 5 -methylcytosine.

[0709] 3. Certain Internucleoside Linkage Motifs

[0710] In certain embodiments, oligonucleotides comprise modified and / or unmodified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or motif.

[0711] In certain embodiments, the 5 ’-most internucleoside linkage of a modified oligonucleotide is a modified internucleoside linkage. In certain embodiments, the 3 ’-most internucleoside linkage of the guide is a modified internucleoside linkage. In certain embodiments, at least 1, 2, 3, 4, or 5 of the 5’-most five internucleoside linkages of a modified oligonucleotide are modified internucleoside linkages. In certain embodiments, at least 1, 2, 3, 4, or 5 of the 3’-most five internucleoside linkages of a modified oligonucleotide are modified internucleoside linkages. In certain embodiments, the modified internucleoside linkage is a phosphorothioate internucleoside linkage. 4. Certain Guide Chemical Modification Motifs

[0712] A guide can comprise any of the modifications commonly applied to modified oligonucleotides described herein, including modified sugar moieties, modified internucleoside linkages, and modified nucleobases. In certain embodiments, a guide is modified at the 3 ’-end, the 5 ’-end, or both the 3 ’-end and o’¬ end. In certain embodiments, the three nucleosides at the 3 ’-end and the three nucleosides at the 5 ’-end are 2’-0Me nucleosides, and the remainder of the nucleosides of the guide are unmodified RNA nucleosides. In certain embodiments, each nucleoside within the 5’-most stem loop is an unmodified RNA nucleoside.

[0713] In certain embodiments, each internucleoside linkage of the guide is an unmodified phosphodiester linkage. In certain embodiments, the guide comprises one or more modified internucleoside linkages. In certain embodiments, the guide comprises one or more phosphorothioate internucleoside linkages. In certain embodiments, the ’-most one, two, three, four, or five internucleoside linkages are modified internucleoside linkages. In certain embodiments, the 5’ -most one, two, three, four, or five internucleoside linkages are modified internucleoside linkages. In certain embodiments, the 3 ’-most and 5 ’-most one, two, three, four, or five internucleoside linkages are modified internucleoside linkages, and the remainder of the linkages are unmodified phosphodiester internucleoside linkages. In certain embodiments, the 3 ’-most one, two, three, four, or five internucleoside linkages are phosphorothioate internucleoside linkages. In certain embodiments, the 5 ’-most one, two, three, four, or five internucleoside linkages are phosphorothioate internucleoside linkages. In certain embodiments, the 3 ’-most and 5’ -most one, two. three, four, or five internucleoside linkages are phosphorothioate internucleoside linkages, and the remainder of the linkages are unmodified phosphodiester internucleoside linkages. In certain embodiments, the three 3 ’-most and the three 5 ’-most internucleoside linkages are phosphorothioate internucleoside linkages, and the remainder of the linkages are unmodified phosphodiester internucleoside linkages.

[0714] c. Certain Lengths

[0715] In certain embodiments, oligonucleotides (including guides) can have any of a variety of ranges of lengths. In certain embodiments, oligonucleotides consist of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180; provided that X< Y. For example, in certain embodiments, oligonucleotides consist of 50-180, 80-180, 90-180, 100-180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 160-170, 90-100,100-110, 110-120, 120-130, 130-140, 140-150, 100-140, 110-140, 110-130, 120-140, or 120-150 linked nucleosides.

[0716] In certain embodiments, a single guide oligonucleotide consists of 50-180, 80-180, 90-180, 100-180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 160-170, 90-100,100-110, 110-120, 120-130, 130-140, 140-150, 100-140, 110-140, 110-130, 120-140, or 120-150 linked nucleosides. In certain embodiments, the spacer of a guide consists of 15-30, 18-26, 18-24, 18-22, 20-26, 20-24, 20-22, 21-23, 22-23, 20, 21, 22, or 23 linked nucleosides.

[0717] In certain embodiments, the scaffold consists of 60-130, 60-120, 60-110, 60-100, 70-130, 70-120, 70-110, 70-100, 70-90, 70-80, 80-130, 80-120, 80-110, 80-100, 80-90, 90-130, 90-120, 90-110, 90-100, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 linked nucleosides. In certain embodiments, the scaffold represents a truncated version of a native guide for the corresponding Cas protein. In certain embodiments, the guide is truncated at the 3’ end. In certain embodiments, the guide is shortened by removing nucleotides from linker, stem loop and / or hairpin structures, such as removing one base pair from a hairpin to shorten the hairpin. In certain embodiments, the truncated version is at least 5, at least 10, at least 15, or at least 20 nucleotides shorter than the native guide. In certain embodiments, the scaffold of a guide represents a length-extended version of a native guide for the corresponding Cas protein. In certain embodiments, additional nucleotides are added at the 3’ end. In certain embodiments, additional nucleotides are inserted within linkers, stem loops and / or hairpins of the native guide.

[0718] d. Nucleobase Sequence

[0719] In certain embodiments, oligonucleotides (unmodified or modified oligonucleotides) are further described by their nucleobase sequence. In certain embodiments oligonucleotides have a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid. In certain such embodiments, a region of an oligonucleotide has a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid. In certain embodiments, the nucleobase sequence of a region or entire length of an oligonucleotide is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the second oligonucleotide or nucleic acid, such as a target nucleic acid.

[0720] e. Secondary Structure

[0721] In certain embodiments, oligonucleotides or portions thereof adopt a defined secondary structure. In certain embodiments, secondary structure is determined by Watson-Crick base pairing, Hoogsteen base pairing, and / or non-canonical base pairing interactions. The secondary structure of an oligonucleotide sequence can be predicted using standard software, such as the Vienna RNA package RNAfold (rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi; see Lorenz, et al., Algorithms for Molecular Biology, 6:1 26, 2011). In certain embodiments, an oligonucleotide may have more than one predicted secondary structure.

[0722] A guide oligonucleotide has secondary structure, at least some portion of which is important in forming contacts with its cognate Cas protein.

[0723] In certain embodiments, the guide is a two-piece or dual guide comprising a CRISPR RNA (“crRNA”) and a transactivating CRISPR RNA (“tracrRNA”). In certain embodiments, the crRNA and the tracrRNA form a duplex, with the crRNA and the tracrRNA annealed together.

[0724] In certain embodiments, the guide is a “single guide” In certain embodiments, the single guide comprises a crRNA and a tracrRNA. In certain embodiments, the crRNA and the tracrRNA form a duplex, with the crRNA and the tracrRNA annealed together. In certain embodiments, the crRNA and the tracrRNA are connected by a linker, hairpin or stem loop.

[0725] In certain embodiments, the crRNA comprises a region comprising a spacer sequence that is complementary to a target sequence in a target nucleic acid, and a region comprising a repeat sequence. In certain embodiments, the tracrRNA comprises a region comprising a tracrRNA anti-repeat sequence and a region comprising a 3’ tracrRNA sequence. In some embodiments, the 3’ end of the crRNA repeat region is linked to the 5’ end of the tracrRNA anti-repeat region, e.g., by a tetraloop, wherein the crRNA repeat region and the tracrRNA anti-repeat region hybridize to form a single guide. In some embodiments, the single guide comprises 5’ to 3’: a spacer region, a crRNA repeat region, a tetraloop, a tracrRNA anti -repeat region, and a 3’ tracrRNA region. In some embodiments, the single guide comprises a 5’ spacer extension region. In some embodiments, the single guide comprises a 3’ tracrRNA extension region. The 3’ tracrRNA can comprise, or consist of, one or more linkers, hairpins and / or stem loops, for example one, two, three, or more linkers, hairpins and / or stem loops.

[0726] A “spacer region” of a guide has a spacer sequence. In certain embodiments, the spacer sequence is a sequence that is complementary to the target sequence of a target nucleic acid. In some embodiments, the spacer sequence ranges from X to Y nucleobases, wherein X represents the lowest number in the range and Y represents the highest number in the range, wherein X and Y are each independently selected from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49 or 50; provided that X< Y. In some embodiments, a spacer sequence contains 18, 19 or 20 nucleobases. In some embodiments, the guide hybridizes, or is capable of hybridizing, to the forward strand of the target nucleic acid. In some embodiments, the guide hybridizes, or is capable of hybridizing, to the reverse strand of the target nucleic acid.

[0727] In certain embodiments, a target sequence is in a region of the target nucleic acid that on the opposite strand of a DNA from a PAM sequence. The spacer hybridizes to the complementary region located in the non-PAM strand of the target nucleic acid. The spacer region interacts with a target nucleic acid of interest in a sequence-specific manner via hybridization. The nucleotide sequence of the spacer thus varies depending on the target sequence of the target nucleic acid.

[0728] In certain embodiments, the spacer region is designed to hybridize to a region of the target nucleic acid that is complementary to a region located 5' of a PAM recognizable by guided nucleic acid binding agent. In certain embodiments, the guided nucleic acid binding protein is a Cas protein. The spacer sequence can perfectly match the target sequence or can have mismatches. Each guided nucleic acid binding agent has a particular PAM sequence that it recognizes in a target nucleic acid, though some guided nucleic acid binding agents can recognize PAM sequences with variation at one or more positions.

[0729] In some embodiments, the target sequence ranges from X to Y nucleotides, wherein X represents the lowest number in the range and Y represents the highest number in the range, wherein X and Y are each independently selected from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49 or 50; provided that X< Y. In some embodiments, a target sequence contains 18, 19 or 20 nucleotides.

[0730] In some embodiments, the percent complementarity between the spacer sequence and the target n sequence can be about, at least, at least about, at most or at most about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the spacer sequence and the target nucleic acid sequence is 100% complementary. In some embodiments, the percent complementarity between the spacer sequence and the target sequence is 100% over the six contiguous 5 '-most nucleotides of the target sequence. In some embodiments, the percent complementarity between the spacer sequence and the target nucleic acid is at least 60% over about 20 contiguous nucleotides. In other embodiments, the spacer sequence and the target sequence can contain up to 10 mismatches, e.g., up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 mismatch.

[0731] (ii ) Combinations of Certain Oligonucleotides with Certain Polypeptides

[0732] In certain embodiments, provided herein are LNPs comprising ionizable lipids. In certain embodiments, the LNPs comprise, encapsulate or are capable of encapsulating a cargo, wherein the cargo is or includes an oligonucleotide consisting of linked nucleosides. In certain embodiments, the oligonucleotide is a guide. In certain embodiments, the cargo includes a guided nucleic acid binding agent or an exogenous mRNA encoding a guided nucleic acid binding agent.

[0733] In certain embodiments, the guide combines, or is capable of combining, with a guided nucleic acid binding agent to form a ribonucleoprotein (“RNP”). In certain embodiments, the guided nucleic acid binding agent is a Cas protein.

[0734] In certain embodiments, the guided nucleic acid binding agent is naturally occurring or non-naturally occurring. In certain embodiments, the guided nucleic acid binding agent is selected from a Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas12, CasX, CasY, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cpfl (Casl2a) protein, and functional derivatives thereof.

[0735] In certain embodiments, the guided nucleic acid binding agent is a Cas protein. In certain embodiments, the Cas protein is a Cas enzyme, dead Cas protein, or Cas fusion protein.

[0736] In certain embodiments, the Cas protein is a Cas9 protein selected from Streptococcus pyogenes (SpyCas9), Staphylococcus lugdunensis (SluCas9), P. pneumotropica Cas9 (PpCas9), Staphylococcus auricularis Cas9 (SauriCas9), Staphylococcus lugdunensis Cas9 (SlugCas9), Staphylococcus lutrae Cas9 (SlutrCas9) Staphylococcus haemolyticus Cas9 (ShaCas9), Campylobacter jejuni (CjCas9), Staphylococcus aureus (SaCas9), or a variant thereof.

[0737] In certain embodiments, the guided nucleic acid binding agent is a variant of Cas9, including but not limited to, a small Cas9, a dead Cas9 (dCas9), and a Cas9 nickase. In certain embodiments, the Cas protein comprises a RuvC or RuvC-like nuclease domain (e.g., Cpfl) and / or a HNH or HNH-like nuclease domain (e.g., Cas9).

[0738] In certain embodiments, the Cas9 protein is S. pyogenes Cas9, S. aureus Cas9, N. meningitides Cas9, S. thermophilus Cas9, S. thermophilus 3 Cas9, T. denticola Cas9, or a variant thereof.

[0739] In certain embodiments, the guided nucleic acid binding agent is a Cas 12 protein or a variant of a Casl2 protein.

[0740] In certain embodiments, the guided nucleic acid binding agent comprises a small RNA-guided endonuclease. The small RNA-guided endonuclease can be engineered from portions of guided nucleic acid binding agents derived from any of the RNA-guided endonucleases described herein and known in the art. The small RNA-guided endonuclease can be, e.g., a small Cas protein.

[0741] In certain embodiments, the guided nucleic acid binding agent is a mutant guided nucleic acid binding agent. For example, the guided nucleic acid binding agent can be a mutant of a naturally occurring guided nucleic acid binding agent. The mutant guided nucleic acid binding agent can also be a mutant guided nucleic acid binding agent with altered activity compared to a naturally occurring guided nucleic acid binding agent, such as altered activity (e.g., altered or abrogated DNA endonuclease activity without substantially diminished binding affinity to a target nucleic acid). Such modification can allow for the sequence-specific nucleic acid targeting of the mutant guided nucleic acid binding agent for the purpose of transcriptional modulation (e.g., activation or repression); epigenetic modification or chromatin modification by methylation, demethylation, acetylation or deacetylation, or any other modifications of target nucleic acid binding and / or modifying proteins known in the art. In some embodiments, the mutant guided nucleic acid binding agent has no DNA endonuclease activity.

[0742] In certain embodiments, the guided nucleic acid binding agent is a nickase that cleaves the complementary strand of a target DNA but has reduced ability to cleave the non-complementary strand of the target DNA, or that cleaves the non-complementary strand of the target DNA but has reduced ability to cleave the complementary strand of the target DNA. In some embodiments, the guided nucleic acid binding agent has a reduced ability to cleave both the complementary and the non-complementary strands of the target DNA.

[0743] In certain embodiments, the guided nucleic acid binding agent binds to a guide to form a ribonucleoprotein, or RNP. In certain embodiments, the RNP binds, or is capable of binding, to a target nucleic acid. Upon binding, the RNP may create a break in the target nucleic acid, such as a double strand break or a single strand break (e.g., a nick). In certain embodiments, the break may be repaired by a process of non-homologous end-joining (“NUEJ”) or homology -directed repair (“UDR”). In certain embodiments, repair of a break can result in, for example, a gene knockout or a gene knock-in.

[0744] In certain embodiments, the guide combines, or is capable of combining, with a dead Cas protein to form a RNP that binds, or is capable of binding, to a target nucleic acid but does not break the target nucleic acid, wherein binding of the RNP prevents transcription or translation, thereby silencing expression of a target nucleic acid. In certain embodiments, the dead Cas protein is a fusion protein that includes a transcriptional repressor domain. Such embodiments encompass methods of CRISPR interference (“CRISPRi”). See, for example, Qi LS, Larson MH, et al. (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression, Cell 152(5): 1173-83, the contents of which are incorporated herein by reference in their entirety.

[0745] In certain embodiments, the guide combines, or is capable of combining, with a dead Cas protein to form a RNP that binds, or is capable of binding, to a target nucleic acid but does not break the target nucleic acid, wherein binding of the RNP activates transcription or translation, thereby activating expression of or over-expressing a target nucleic acid. In certain embodiments, the dead Cas protein is a fusion protein that includes a transcriptional activator. Such embodiments encompass methods of CRISPR activation (“CRISPRa”). See, for example, Polstein LR, Gersbach CA. (2015) A light inducible CRISPR-Cas9 system for control of endogenous gene activation, Nat Chem Biol, 11 (3):198-200; and Zalatan JG, Lee ME, et al. (2015) Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds, Cell 15;160(1 -2):339-50, the contents of which are incorporated herein by reference in their entirety.

[0746] In certain embodiments, the guide combines, or is capable of combining, with a dead Cas protein to form a RNP that binds, or is capable of binding, to a target nucleic acid but does not break the target nucleic acid, wherein binding of the RNP enables visualization of a target nucleic acid. In certain embodiments, the dead Cas protein is a fusion protein that includes a fluorescent protein. Such embodiments encompass methods of gene visualization. See, for example, Ma H, Naseri A, et al. (2015) Multicolor CRISPR labeling of chromosomal loci in human cells, Proc Natl Acad Sci USA. 10;l 12(10):3002- 7; and Ma H, Tu LC, et al. (2016) Multiplexed labelling of genomic loci with dCas9 and engineered sGRNAs using CRISPRainbow, Nat Biotechnol 34(5):528-30; and Carlson-Stevermer, J., Kelso, R., Kadina, A., Joshi, S., Rossi, N., Walker, J., Stoner, R., & Mau res, T. (2020) CRISPRoff enables spatio-temporal control of CRISPR editing, Nature Communications, 11(1), 1-7, the contents of which are incorporated herein by reference in their entirety.

[0747] In certain embodiments, the guide combines, or is capable of combining, with a Cas protein to form a RNP that binds, or is capable of binding, to a target nucleic acid, wherein binding of the RNP enables introduction of point mutations into a target nucleic acid. In certain embodiments, the Cas protein is a fusion protein that includes a nucleobase deaminase. In certain embodiments, the nucleobase deaminase is a cytosine base editor that can introduce C> T or T> C transitions. In certain embodiments, the nucleobase deaminase is an adenine base editor that can introduce A> G or G> A transitions. Such embodiments encompass methods of base editing.

[0748] In certain embodiments, the guide is prime editing guide and combines, or is capable of combining, with a Cas nickase protein to form a RNP that binds, or is capable of binding, to a target nucleic acid, wherein binding of the RNP enables introduction of point mutations into a target nucleic acid. In certain embodiments, the Cas nickase protein is a fusion protein that includes a reverse transcriptase. Such embodiments encompass methods of prime editing. See, for example, Gao P, Lyu Q, et al. (2021) Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression, Genome Biol. 22(1 ):83, the contents of which are incorporated herein by reference in their entirety.

[0749] Guide secondary structure features for binding to spCas9 have been described in detail based on both empirical evidence (see, e.g., Zhang, et al., ChemPlusChem, 2021 and Dong, et al., Curr. Opinion in Biotech., 2022) and the crystal structures of a guide-SpCas9-target DNA complex (see Nishimasu, et al., Cell, 2014). By adding a tetraloop to the duplex that forms between the native crRNA and native tracrRNA of spCas9, the functions of the crRNA and tracrRNA are combined into a single guide. The same tetraloop can be added to join the crRNA and tracrRNA of a novel type II Cas protein. A single guide for spCas9 has conserved structural features within its protein-binding region, including the lower stem, the upper stem, the tetraloop, the bulge, the nexus region (a hairpin with a 5 nucleotide loop), and two hairpins at the 3 ’-end. The guide for certain novel type II Cas proteins have been shown to have unique secondary structures with common features in the protein-binding region, including a duplex region (dual guides) or a stem loop (single guides) and one or more hairpins at the 3’ end. The 5 ’-most stem loop feature of a guide is also known as the Repeat-Anti-repeat region (R-AR).

[0750] III. Other Cargos

[0751] Additional exemplary cargos include, but are not limited to, sterols, peptides, therapeutic polypeptides including antibodies, small molecule therapeutics, vitamins, antigenic polypeptides, enzymatic nucleic acids, allozymes, aptamers, ribozyme, and plasmids. Certain Compositions and Methods for Formulating Certain Compositions

[0752] In certain embodiments, the ionizable lipids are provided as compositions. In certain embodiments, the compositions comprise LNPs comprising the ionizable lipids. In certain embodiments, the LNPs comprise, encapsulate or are capable of encapsulating a cargo. In certain embodiments, the cargo is or includes an oligonucleotide consisting of linked nucleosides. In certain embodiments, the cargo is or includes an exogenous mRNA. In certain embodiments, the compositions are pharmaceutical compositions.

[0753] Also provided are compositions comprising a plurality of LNPs having distinct compositions and / or cargos. See, e.g., WO 2014 / 144196, WO 2016 / 197133. In certain embodiments, provided is a pharmaceutical composition comprising a first LNP comprising a first cargo and having a composition defined herein, and a second LNP comprising a second cargo and having a composition different from the first LNP, wherein the first cargo and the second cargo are different. In certain embodiments, provided is a pharmaceutical composition comprising a first LNP comprising a first cargo and having a composition defined herein, and a second LNP comprising a second cargo and having a composition substantially identical to the first LNP, wherein the first cargo and the second cargo are different.

[0754] In certain embodiments, the pharmaceutical composition can have one or more additional reagents, wherein such additional reagents are selected from a buffer, a buffer for introducing a polypeptide or polynucleotide into a cell, a wash buffer, a control reagent, a control vector, a control RNA polynucleotide, a reagent for in vitro production of a polypeptide from DNA, adaptors for sequencing and the like. A buffer can be a stabilization buffer, a reconstituting buffer, a diluting buffer, or the like. In some embodiments, the pharmaceutical composition can also include one or more components that can be used to facilitate or enhance on-target binding or cleavage of a target nucleic acid by an endonuclease, or improve specificity of targeting.

[0755] In certain embodiments, the pharmaceutical composition may contain one or more excipients to facilitate systemic delivery of an oligonucleotide. In certain embodiments, any components of a pharmaceutical composition are formulated with pharmaceutically acceptable excipients such as carriers, solvents, stabilizers, adjuvants, diluents, etc., depending upon the particular mode of administration and dosage form. Suitable excipients can include, for example, carrier molecules that include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, poly glycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Other exemplary excipients include antioxidants (for example and without limitation, ascorbic acid), chelating agents (for example and without limitation, EDTA), carbohydrates (for example and without limitation, dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose), stearic acid, liquids (for example and without limitation, oils, water, saline, glycerol and ethanol), wetting or emulsifying agents, pH buffering substances, and the like. In certain embodiments, the LNP composition may comprise one or more excipients, for example, a triglyceride, a surfactant, and / or a hydrophobic excipient such as a wax. Triglycerides include trimyristin (Dynasan 114), tripalmitin (Dynasan 116), or tristearin (Dynasan 118), Witeposol bases, glyceryl stearates (Imwitor 900), glyceryl behenates (Compritol 888 ATO), and glyceryl palmitostearates (Precirol ATO 5). Nonionic surfactants may include moieties such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters, nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers. Anionic surfactants include carboxylates, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. Specific surfactants include lecithin, Poloxamer 188, Poloxamer 407, Tyloxapol, Polysorbate 20, Polysorbate 60, Polysorbate 80, sodium cholate, sodium glycocholate, taurodeoxycholic acid sodium, butanol, butyric acid, cetylpyridinium chloride, sodium dodecyl sulfate, sodium oleate, polyvinyl alcohol, or Cremophor EL. Waxes include beeswax and cetyl palmitate. Other hydrophobic excipients include stearic acid, palmitic acid, behenic acid, Miglyol 812, and paraffin.

[0756] Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.

[0757] Certain embodiments provide pharmaceutical compositions comprising one or more nucleic acids, oligonucleotides, or salts thereof. In certain such embodiments, the pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises a sterile saline solution and one or more oligonucleotide. In certain embodiments, a pharmaceutical composition comprises one or more oligonucleotide and sterile water. In certain embodiments, the sterile water is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises or consists of one or more oligonucleotide and phosphate-buffered saline (PBS). In certain embodiments, the sterile PBS is pharmaceutical grade PBS. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

[0758] In certain embodiments, the pharmaceutical composition can include one or more guide(s) and a guided nucleic acid binding agent or an exogenous mRNA encoding the guided nucleic acid binding agent. In some embodiments, the pharmaceutical composition further comprises an oligonucleotide to be inserted (e.g., a donor template) to affect a desired sequence modification. In certain embodiments, guide compositions are generally formulated to achieve a physiologically compatible pH, and range from a pH of about 3 to a pH of about 11, about pH 3 to about pH 7, depending on the formulation and route of administration. In some embodiments, the pH is adjusted to a range from about pH 5.0 to about pH 8.

[0759] In certain embodiments, pharmaceutical compositions disclosed herein are prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration. In certain embodiments, a pharmaceutical composition is prepared for administration by injection e.g., intravenous, subcutaneous, intramuscular, intrathecal (IT), intracerebroventricular (ICV), etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi -dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0760] In certain embodiments, provided is a pharmaceutically acceptable salt of an oligonucleotide or nucleic acid described herein wherein the salt is sodium or potassium.

[0761] In certain embodiments, the composition comprises one or more antioxidants. In certain embodiments, the one or more antioxidants function to reduce a degradation of the LNPs or components thereof, such as the ionizable lipids, the cargo, or both. In certain embodiments, the antioxidant is a chelating agent such as EDTA, citrate, vitamin E, or a polyphenol.

[0762] Methods of Delivery

[0763] In certain embodiments, there are provided methods of use of the ionizable lipids. In certain embodiments, the use comprises delivery of nucleic acids such as oligonucleotides, siRNA, self-replicating RNA, and / or exogenous mRNA to a cell. In certain embodiments, the ionizable lipids are delivered as pharmaceutical compositions. In certain embodiments, the pharmaceutical compositions comprise LNPs comprising the ionizable lipids.

[0764] In certain embodiments, the LNPs and compositions disclosed herein (e.g., a guided nucleic acid binding agent or a nucleic acid encoding the guided nucleic acid binding agent and / or guide oligonucleotides) can be delivered via transfection such as calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, electrical nuclear transport, chemical transduction, electrotransduction, Lipofectamine-mediated transfection, Effectene-mediated transfection, LNP-mediated transfection, or any combination thereof. In some embodiments, the composition is introduced to cells via lipid-mediated transfection using an LNP.

[0765] In some embodiments, the pharmaceutical composition can be administered by aerosol delivery, nasal delivery, vaginal delivery, rectal delivery, buccal delivery, ocular delivery, local delivery, topical delivery, intracistemal delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous injection, subcutaneous injection, intranodal injection, intratumoral injection, intraperitoneal injection, and / or intradermal injection, or any combination thereof. The administration can be local or systemic. The systemic administration includes enteral and parenteral administration.

[0766] In certain embodiments, a subject is administered a pharmaceutical composition two or more times. In certain embodiments, multiple administrations of the pharmaceutical composition can be separated by a suitable period of time, such as one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, three months, four months, five months, six months, a year, eighteen months, two years, three years, five years, ten years, fifteen years, or more.

[0767] In some embodiments, the two or more administrations are about two weeks to about two months apart. The suitable time period between some administrations can be the same as or different from the suitable time period between other two administrations. In some embodiments, the method described herein comprises administration of a single dose of a pharmaceutical composition to the subject in a provided period of time, for example, one year, two years, three years, five years, six years, eight years, ten years, fifteen years, twenty years, or longer. In some embodiments, the method described herein comprises administration of a single dose of a pharmaceutical composition to a subject in the subject’s life time. In some embodiments, the method described herein comprises administration of a single dose of a pharmaceutical composition to the subject.

[0768] In certain embodiments, the pharmaceutical composition can be administered to a subject at a pharmaceutically effective amount. In some embodiments, the pharmaceutical composition is administered to the subject at a dose of about 0.01-5 mg / kg, for example 0.05-2 mg / kg, 0.5-3 mg / kg or 0.1-1 mg / kg, guide per administration. In some embodiments, the pharmaceutical composition is administered to the subject at a dose of about 0.01-5 mg / kg, for example 0.05-2 mg / kg, 0.5-3 mg / kg or 0.1-1 mg / kg, total nucleic acid (i.e., the total of the guide and mRNA encoding the guided nucleic acid binding agent) per administration.

[0769] Certain Mechanisms

[0770] In certain embodiments, provided herein are LNPs comprising ionizable lipids. In certain embodiments, the LNPs comprise, encapsulate or are capable of encapsulating a cargo, wherein the cargo is an oligonucleotide consisting of linked nucleosides, and / or an exogenous mRNA, and / or a ribonucleoprotein complex. Certain compounds described herein result in RNase H mediated cleavage of a target nucleic acid. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA: DNA duplex.

[0771] Cargo Delivery Efficiency

[0772] In certain embodiments, LNP compositions provided herein provide surprising and advantageous cargo delivery efficiency. In certain embodiments, the cargo is an editing system comprising a Cas protein and a guide or an expression system comprising an exogenous mRNA encoding a Cas protein and a guide, and target DNA cleavage is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In certain embodiments, the target DNA is in a liver cell, for example a hepatocyte. In certain embodiments, the subject is a mammal such as a human.

[0773] In certain embodiments, LNP compositions provided herein provide surprising and advantageous reductions in immunogenicity, thereby reducing blood clearance, reducing dose-limiting toxicity and reducing complement activation-related pseudoallergy (CARPA).

[0774] In certain embodiments, LNP compositions provided herein provide surprising and advantageous improvements in tolerability. In certain embodiments, tolerability is based on liver and kidney function, e.g., plasma levels of albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and / or total bilirubin (TBIL). Lower levels of ALB, ALT, BUN, and TBIL can indicate improved tolerability, for example, according to Example 7 and Example 9 herein. In certain embodiments, tolerability is determined based on plasma levels of markers of inflammation, such as for example, IFN-y, IL-10, IL-ip, IL-6, KC / GRO, TNF-a, and MIP-2, with lower levels indicating improved tolerability.

[0775] Kits and Devices

[0776] In certain embodiments, there are provided kits and devices comprising ionizable and / or cationic lipids. In certain embodiments, the kits and devices comprise LNPs comprising ionizable and / or cationic lipids.

[0777] Kits provided herein facilitate employment of the methods and uses of the invention. Typically, kits for carrying out a method or use of the invention contain all the necessary reagents and means to carry out the method. In one embodiment, the kit may comprise a composition of the present invention and, optionally, means to administer the composition such as devices for point of care methods.

[0778] In certain embodiments, kits comprise one or more containers. A compartmentalized kit includes any kit in which compositions are contained in separate containers, and may include small glass containers, plastic containers or strips of plastic or paper. Such containers may allow the efficient transfer of compositions from one compartment to another compartment whilst avoiding cross-contamination of compositions, and the addition of agents or solutions of each container from one compartment to another in a quantitative fashion. In certain embodiments, kits include instructions for using the kit to perform the appropriate methods and uses.

[0779] In certain embodiments, kits comprise a saline, a buffered solution and an LNP comprising an ionizable and / or cationic lipid. In one embodiment, the buffered solution may include sodium chloride, calcium chloride, phosphate and / or EDTA. In another embodiment, the buffer solution may include, but is not limited to, saline, saline with 2mM calcium, 5% sucrose, 5% sucrose with 2mM calcium, 5% Mannitol, 5% Mannitol with 2mM calcium, Ringer’s lactate, sodium chloride, sodium chloride with 2mM calcium and mannose (See e.g., U. S. Pub. No. 2012 / 0258046; herein incorporated by reference in its entirety).

[0780] In one embodiment, the buffered solution may be precipitated or it may be lyophilized. The amount of each component in the kit may be varied to enable consistent, reproducible higher concentration saline or simple buffered formulations.

[0781] In certain embodiments, the kits comprise one or more additional reagents selected from a buffer, a buffer for introducing a polypeptide or nucleic acid into a cell, a wash buffer, a control reagent, a control vector, a control RNA, a reagent for in vitro production of the polypeptide from DNA, adaptors for sequencing and the like. A buffer can be a stabilization buffer, a reconstituting buffer, a diluting buffer, or the like. The kits can also comprise one or more components that can be used to facilitate or enhance on-target binding or cleavage of a nucleic acid by an endonuclease, or improve specificity of targeting.

[0782] The components may also be varied in order to increase the stability of LNPs in the buffered solution over a period of time and / or under a variety of conditions.

[0783] In certain embodiments, the kits can further include instructions for using the components of the kit to practice the methods described herein. The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, etc. The instructions can be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with tire packaging or subpackaging), etc. The instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, flash drive, etc. In some instances, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the Internet), can be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.

[0784] Devices provided herein facilitate employment of the methods and uses of the invention. In certain embodiments, devices may incorporate an LNP comprising an ionizable lipid. In certain embodiments, devices contain in a stable formulation the reagents required to formulate an LNP to be immediately delivered to a subject in need thereof. Devices for administration may be employed to deliver LNPs according to single, multi- or splitdosing regimens. See, for example, PCT / US2013 / 30062, the contents of which are incorporated herein by reference in their entirety.

[0785] Devices known in the art for single or multi-administration to cells, organs and tissues can be used in conjunction with the methods and compositions disclosed herein. Examples of such devices include syringes, needles, devices having multiple needles, hybrid devices employing lumens or catheters, and devices utilizing heat, electric current or radiation driven mechanisms.

[0786] Multi-administration devices may be utilized to deliver single, multi- or split doses. See, for example, PCT / US2013 / 30062, the contents of which are incorporated herein by reference in their entirety.

[0787] Devices using catheters and lumens may be employed to administer LNPs on a single, multi- or split dosing schedule. Such methods and devices are described in PCT / US2013 / 30062, the contents of which are incorporated herein by reference in their entirety.

[0788] Devices utilizing electric current may be employed to deliver LNPs using single, multi- or split dosing regimens. Such methods and devices are described in PCT / US2013 / 30062, the contents of which are incorporated herein by reference in their entirety.

[0789] Also provided herein is an article of manufacture comprising an ionizable lipid as disclosed herein.

[0790] Nonlimiting Disclosure and Incorporation by Reference

[0791] Each of the literature and patent publications listed herein is incorporated by reference in its entirety. While certain compounds, compositions, and methods have been described herein with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references, GenBank accession numbers, ENSEMBL identifiers, and the like recited in the present application is incorporated herein by reference in its entirety.

[0792] The sequence listing accompanying this filing identifies each nucleic acid sequence as either “RNA” or “DNA” as required; however, one of skill in the art will readily appreciate that designation of “RNA” or “DNA” to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2’ -OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (i.e., 2’-OH in place of one 2’-H of DNA) or as an RNA having a modified base (i.e., thymine (5 -methyl uracil) in place of an uracil of RNA); and certain nucleic acid compounds described herein comprise one or more nucleosides comprising modified sugar moieties having 2’-substituent(s) that are neither OH nor H. One of skill in the art will readily appreciate that labeling such nucleic acid compounds “RNA” or “DNA” does not alter or limit the description of such nucleic acid compounds.

[0793] Herein, the description of compounds as having “the nucleobase sequence of’ a SEQ ID NO. describes only the nucleobase sequence. Accordingly, absent additional description, such description of compounds by reference to a nucleobase sequence of a SEQ ID NO. does not limit sugar or internucleoside linkage modifications or presence or absence of additional substituents such as a conjugate group. Further, absent additional description, the nucleobases of a compound “having the nucleobase sequence of’ a SEQ ID NO. include such compounds having modified forms of the identified nucleobases as described herein.

[0794] Herein, sugar, internucleoside linkage, and nucleobase modifications may be indicated within a nucleotide or nucleobase sequence or may be indicated in text accompanying a sequence (e.g., in separate text that appears within or above or below a table of compounds).

[0795] While effort has been made to accurately describe compounds in the accompanying sequence listing, should there be any discrepancies between a description in this specification and in the accompanying sequence listing, the description in the sequence listing is the accurate description.

[0796] Certain compounds described herein (e.g., lipids and nucleic acids such as modified oligonucleotides) may have one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), as a or P such as for sugar anomers, or as (D) or (L), such as for amino acids, etc. Compounds provided herein that are drawn or described as having certain stereoisomeric configurations are enriched (e.g., to at least about 50% ee) for the indicated stereochemistry. Compounds provided herein that are drawn or described with undefined stereochemistry include racemic, stereorandom, and optically enriched forms. The present disclosure includes all such possible isomers, as well as their racemic and optically pure forms whether or not they are specifically depicted herein. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. All tautomeric forms of the compounds provided herein are included unless otherwise indicated.

[0797] The compounds described herein include variations in which one or more atoms are replaced with a non-radioactive isotope or radioactive isotope of the indicated element. For example, compounds herein that comprise hydrogen atoms encompass all possible deuterium substitutions for each of the II hydrogen atoms. Isotopic substitutions encompassed by the compounds herein include but are not limited to:2H or3H in place of1H,13C or14C in place of12C,15N in place of14N,17O or18O in place of16O, and33S,34S,35S, or36S in place of32S. In certain embodiments, non-radioactive isotopic substitutions may impart new properties on the oligomeric compound that are beneficial for use as a therapeutic or research tool. In certain embodiments, radioactive isotopic substitutions may make the compound suitable for research or diagnostic purposes such as imaging. EXAMPLES

[0798] The following examples are intended to illustrate certain aspects of the invention and are not intended to limit the invention in any way.

[0799] Example 1: Synthesis of ionizable prolinol-based lipids

[0800] Ionizable prolinol-based lipids IPL-001, IPL-002, IPL-003, and IPL-004 were synthesized from commercially available starting materials, as shown in the schemes below. Synthesis of IPL-001

[0801] 4C Br p-TsOH (0.5 eq), Toluene (5 V), 110 °C, 12 h 4A 86% yield, 99% purity TBSCI (2 eq) TBSO„ / R) TBSQJR) Imidazole (2 eq) LiBH4(2 M, 1.5 eq)

[0802] OMe > / VS^OH DCM (10 V), THF(10 V), 0 °C, 2 h 20 °C, 1 h Cbz0Cbz 1 2 3

[0803] 79% yield, 100% purity 96% yield, 99% purity

[0804] TBSO,, (R) 4A(1.2 eq), Pd / C, H215 PSI K2CO3(2 eq) THF, 25 °C, 2 h DMF(10 V)

[0805] H 60 °C, 12 h 4 79% yield, 98% purity 70% yield, 97% purity HO

[0806] O

[0807] 5A DIPEA (2 eq), EDCI (2 eq), DMAP (0.2 eq), DCM (10 V), 20 °C, 12 h

[0808] 80% yield, 97% purity

[0809] HF-Py (2 eq) THF(10V), 0-20 °C, 2 h

[0810] IPL-001

[0811]

[0812] 330 mg, 39% yield, 99.46% purity Synthesis of IPL-002

[0813] (COCI)2(1.2 eq) TBSQ, Et3N (2 eq)

[0814] DMSO (0.1 eq) OH > 7A (1.5 eq) DCM (10 V) THF(10 V), 20 °C, 1 h Cbz 0-20 °C, 1 h 3 7 8

[0815] (89% yield, 90% purity) (97% yield, 99% purity)

[0816] LAH(2.5 M, 1 eq) TBSO1-< |^^OHPd / C, H2, 15 PSI VN, THF(10 V), -50 °C, 2 h Cbz THF, 25 °C, 1 h 9 10 (44% yield, 95% purity)

[0817] 4A(1.2 eq) K2CO3(2 eq) DMF(10V), 60 °C, 12 h

[0818] DIPEA (2 eq), EDCI (2 eq), DMAP(0.2 eq), DCM(10V), 20 °C, 5 h

[0819] HF»Py(1.2 eq) THF(10V), 0-20 °C, 2 h

[0820]

[0821] IPL-002

[0822] 300 mg, 50% yield, 99.6% purity Synthesis of IPL-003

[0823] 13 62% yield, 99% purity

[0824] DCM (10 V), 20 °C, 1 h 49% yield, 99% purity

[0825] 4A (1.2 eq) K2CO3(2 eq), DMF (10 V) 60 °C, 12 h

[0826] 16

[0827] HF-Py (2 eq) THF(10 V), 0-20 °C, 2 h

[0828]

[0829] IPL-003

[0830] 200 mg, 29% yield, 98.98% purity Synthesis ofIPL-004

[0831] 7B (1 eq) LiOH-H2O(2 eq) CS2CO3(2 eq), Tol.(10 V) THF: H2O(2:1, 10V) 30 °C, 12 h 20 °C, 5 h 17 68% yield, 80% purity

[0832] EDCI(1.2 eq), DMAP(0.1 eq),CbzDIPEA(2.5 eq) 18 DCM, 20 °C, 12 h 19 35% yield, 98% purity 66% yield, 96% purity

[0833] Pd / C (0.2 eq), H2(15 PSI) THF (10V), 25 °C, 1 h

[0834] 4A (1.2 eq) K2CO3(2 eq), DMF (10V) 60 °C, 12 h

[0835]

[0836] IPL-004

[0837] 23% yield, 99.95% purity Observed mass of synthesized ionizable prolinol-based lipids

[0838] The ionizable prolinol-based lipids IPL-001, IPL-002, IPL-003, IPL-004, described herein above were analyzed by LC-MS for identity and purity. The observed mass of each lipid is shown in the table below.

[0839] Table 3

[0840] Observed mass of synthesized ionizable prolinol-based lipids

[0841] Lipids Theoretical Mass Observed Mass Retention Time

[0842] [(M+H)+] (min)

[0843] IPL-001 680.6 680.6 4.468

[0844] IPL-002 680.6 680.9 11.909

[0845] IPL-003 680.6 680.9 12.735

[0846]

[0847] IPL-004 778.7 779.0 5.485

[0848] Example 2: Formulation of ionizable prolinol-based lipids in lipid nanoparticles

[0849] The ionizable prolinol-based lipids IPL-001, IPL-002, IPL-003, and IPL-004 described above were formulated into LNPs containing a firefly luciferase reporter mRNA.

[0850] The lipid mix was formed by mixing an ionizable lipid selected from the example above, with cholesterol (Sigma Aldrich), helper lipid, and DMG-PEG2K (Avanti Polar Lipids). The helper lipid consisted of either DSPC or DOPE (both from Avanti Polar Lipids). The ionizable lipid / cholesterol / helper lipid / DMG-PEG2K were mixed at a ratio of 0.5 / 0.385 / 0.1 / 0.015, respectively. The total lipid concentration was 5.82 pmol in 1 mL of ethanol.

[0851] 160 pg of fLuc mRNA from TriLink (L-7202) was added to 100 mM acetate buffer pH 4.0 for a final volume of 3mL.

[0852] LNPs were formulated on a NanoAssemblr Ignite (Precision Nanosystems) using a total flow rate of 12 mL / min with 3:1 ratio of RNA: Lipid. Immediately after formulation, LNPs were diluted with equi volume DI water then dialyzed overnight in 3 L of IX PBS buffer pH 7.4. LNPs were then concentrated using a 100kDa MWCO amicon filter (Millipore) to a final volume of approximately 0.5 mL. The final ionizable lipid nitrogen:nucleic acid phosphate molar (N: P) ratio was 6 for each LNP.

[0853] LNPs were characterized for total RNA concentration using Ribogreen (Thermofisher Scientific) to determine percentage of RNA successfully encapsulated within the LNP. Additionally, Z-averages and polydispersity index (PDI) for the LNP-mRNA formulated particles were determined using a Dynamic Light Scattering instrument (Malvern). Z-average measurement is the average particle size determined by the Malvern zeta-sizer. The Polydispersity Index (PDI) is a dimensionless value that measures the heterogeneity of particle sizes within a sample. Table 4

[0854] Lipid nanoparticle formulation and characterization

[0855] LNP Ionizable Helper Z-average Encapsulation PDI

[0856] Formulation lipid lipid (nm) (%)

[0857] 1 DSPC 75 0.06 92%

[0858] IPL-001

[0859] 2 DOPE 90 0.08 98% 3 DSPC 81 0.13 99%

[0860] IPL-002

[0861] 4 DOPE 93 0.13 99% 5 DSPC 82 0.11 98%

[0862] IPL-003

[0863] 6 DOPE 105 0.09 99% 7 DSPC 89 0.27 79%

[0864] IPL-004

[0865]

[0866] 8 DOPE 84 0.14 100%

[0867] Example 3: Evaluation of delivery of mRNA with lipid nanoparticles in Balb / c mice

[0868] Efficiency of delivery of mRNA using LNP-mRNA formulations described herein above was tested in vivo. Translation efficiency of reported mRNA was quantified at two timepoints using luminescence imaging.

[0869] Balb / c mice were divided into groups of 2, and each mouse received a single IV tail injection of LNPs at a concentration of 0.5mg / kg. At 5-hours and 28-hours post-LNP administration, mice were injected with 100 pL of 20 mg / mL D-luciferin substrate 15 -minutes prior to imaging with an IVIS Fluorescence Imaging system (PerkinElmer). fLuc luminescence was visualized and quantified using Living Image 4.8.2 (Revvity) software. The luminescence for each LNP formulation is presented in the table below as photons / second of Total Flux (xlO10p / s).

[0870] Table 5

[0871] Luminescence of fLuc in Balb / c mice

[0872] LNP Total Flux (xlO10p / s) Formulatio Ionizable Lipid Helper Lipid

[0873] n 5 hr 28 hr

[0874] 1 IPL-001 0.37 0.14

[0875] 3 IPL-002 4.67 1.34

[0876] DSPC

[0877] 5 IPL-003 16.3 7.59

[0878] 7 IPL-004 13.5 5.68 2 IPL-001 0.04 0.01 4 IPL-002 0.31 0.12

[0879] DOPE

[0880] 6 IPL-003 12.08 0.55

[0881]

[0882] 8 IPL-004 31.8 17.1 Example 4: Evaluation of lipid nanoparticle mediated delivery of a TTR guide sequence and Cas9 mRNA in Balb / c mice, in vivo

[0883] The ionizable prolinol-based lipids IPL-003 and IPL-004 described herein above were formulated into LNPs containing an M6 SpCas9 mRNA (Trilink Biotech, Catalog# L-8106) and a mouse TTR guide RNA (G211), previously described in Finn, et al. Cell Rep. 2018, 22 (9).

[0884] The lipid mix was formed by mixing an ionizable lipid selected from the example above, with cholesterol (Sigma Aldrich), helper lipid, and DMG-PEG2K (Avanti Polar Lipids). The helper lipid consisted of DSPC (from Avanti Polar Lipids) or DOPE (Avanti Polar Lipids). The ionizable lipid / cholesterol / helper lipid / DMG-PEG2K were mixed at a ratio of 0.5 / 0.385 / 0.1 / 0.015, respectively. The total lipid concentration ranged from 0.23mmol to 0.25mmol dissolved in varying amounts of ethanol for a final volume of 4mL. The RNA mixture, which contained 480pg of M6 SpCas9 mRNA and 480pg of G211 guide RNA, was added to 300 mM citrate buffer pH 4.0 for a final volume of 18mL and mixed with the lipid mixture for a final ionizable lipid nitrogen:nucleic acid phosphate molar (N: P) ratio of 6 for each LNP.

[0885] Balb / c mice were divided into groups of 4, and each mouse received a single IV tail vein injection of an LNP formulation prepared as described herein above at concentrations as indicated in the table below. One group of 4 mice received PBS and served as a negative control.

[0886] Blood plasma was collected from mice at 6 hour, 24 hour, and 7-day timepoints post injection to evaluate TTR editing. TTR levels were analyzed by ELISA using a prealbumin mouse ELISA kit from Abeam (Catalog #ab282297). The results were averaged for each group of mice and concentrations are presented in the table below in pg / ml.

[0887] Table 6

[0888] TTR levels in Balb / c mice

[0889] LNP Ionizable Helper Dose TTR (pg / ml)

[0890] Formulation Lipid Lipid (mg / kg) 6h 24h 7d

[0891] PBS - - 0 226 350 397 9 DSPC 0.3 277 275 189

[0892] IPL-003

[0893] 9 6 302 183 15

[0894] 10 DOPE 0.3 250 237 50

[0895] IPL-004

[0896]

[0897] 10 6 335 251 17

[0898] Example 5: Evaluation of lipid nanoparticle mediated delivery of a TTR guide sequence and Cas9 mRNA in Balb / c mice, in vivo

[0899] The ionizable prolinol-based lipids IPL-003 and IPL-004 described herein above were formulated into LNPs containing an M6 SpCas9 mRNA and a mouse TTR guide RNA (G211), previously described in Finn, et al. Cell Rep. 2018, 22 (9). The lipid mix was formed by mixing an ionizable lipid selected from the example above, with cholesterol (Sigma Aldrich), helper lipid, and DMG-PEG2K (Avanti Polar Lipids). The helper lipid consisted of DSPC (from Avanti Polar Lipids). The ionizable lipid / cholesterol / helper lipid / DMG-PEG2K were mixed at aratio of 0.5 / 0.385 / 0.1 / 0.015, respectively. The total lipid concentration ranged from 0.02mmol to 0.03mmol in 0.4mL to 0.7mL of ethanol for a total volume of 1 mL. The RNA mixture, which contained 80pg of M6 SpCas9 mRNA and 80pg of G211 guide RNA, was added to 300 mM citrate buffer pH 4.0 for a final volume of 3mL and mixed with the lipid mixture for a final ionizable lipid nitrogen:nucleic acid phosphate molar (N: P) ratio that ranged from 4 to 8 for each LNP as indicated in the table below.

[0900] Balb / c mice were divided into groups of 3, and each mouse received a single IV tail vein injection of 0.3 mg / kg of formulation, as indicated in the table below. One group of 3 mice received PBS and served as a negative control.

[0901] Blood plasma was collected from mice 7 days post treatment. TTR protein levels were analyzed by ELISA using a prealbumin mouse ELISA kit from Abeam (Catalog #ab282297). The results were averaged for each group of mice. Concentrations are presented in the table below in pg / ml. Reduction of TTR is presented as TTR protein relative to the amount of TTR protein in PBS treated control animals (% control).

[0902] Table 7

[0903] TTR protein and RNA levels in Balb / c mice

[0904] TTR protein

[0905] Helper TTR protein Formulation Ionizable N: P

[0906] levels

[0907] Lipid Ratio Lipid (% control)

[0908] (pg / ml)

[0909] PBS - - - 324 100 11 4 220 68 12 IPL-004 5 DSPC 44 13 13 105 32

[0910] 6

[0911] 10 DOPE 32 10 9 6 293 90 14 IPL-003 7 DSPC 174 54

[0912]

[0913] 15 8 149 46

[0914] Example 6: Determination of apparent pKavalues for lipid nanoparticles

[0915] The apparent pKavalues of LNPs described herein above were determined using a 6-(p-Toluidino)-2-naphthalenesulfonyl chloride (TNS) assay. The pH assay buffer (10 mM HEPES, 10 mM MES, 10 mM NIROAc, and 120 mM NaCl) was prepared and adjusted to exhibit pH values ranging from 3 to 11 in 0.5 pH unit increments. Each adjusted pH assay buffer was dispensed into 96-well assay plates (Thermo Fisher Scientific, Catalog No. 237108) at 190 pL per well, in triplicate. To each buffer-containing well, 5 pL of 4 mM LNPs and 5 pL of 40 pM TNS solution were added to yield final concentrations of 100 pM and 1 pM, respectively. The assay plate was read using a SpectraMax Gemini XPS plate reader (Molecular Devices) at λex=320 nm / λem=440 nm to obtain the fluorescence intensity values. The values were normalized to the highest fluorescence intensity value observed and plotted against pH values. Apparent pKavalues for LNPs were calculated as the pH at which the LNP showed 50% of the maximum normalized fluorescence using nonlinear least squares regression in GraphPad PRISM 10.

[0916] Table 8

[0917] Apparent pKavalues of lipid nanoparticles

[0918] Formulation Ionizable Helper Apparent

[0919] Lipid Lipid pKa

[0920] 1 DSPC 6.24

[0921] IPL-001

[0922] 2 DOPE 6.52

[0923] 3 DSPC 7.23

[0924] IPL-002

[0925] 4 DOPE 7.48

[0926] 5 IPL-003 DSPC 5.93

[0927] 7 DSPC 6.40

[0928] IPL-004

[0929]

[0930] 8 DOPE 6.86

[0931] Example 7: Tolerability of LNP formulations in Balb / c mice, in vivo

[0932] LNP formulations containing an M6 SpCas9 mRNA and a mouse TTR guide RNA (G211), described herein above, were evaluated in Balb / c mice for tolerability and inflammation markers.

[0933] As a comparator, an LNP was formulated with the ionizable lipid ALC-0315. The comparator LNP was formulated with ALC-0315 / cholesterol / DSPC helper lipid / DMG-PEG2K a ratio of 0.5 / 0.385 / 0.1 / 0.015, following methods disclosed herein above.

[0934] Balb / c mice were divided into groups of 4, and each mouse received a single IV tail vein injection of LNPs at a concentration indicated in the table below. One group of 4 mice received PBS and served as a negative control.

[0935] To evaluate the effect of the LNPs on liver function, blood plasma was collected from all mice at 6 hours, 24 hours, and 7 Days post-treatment, and plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400c, Melville, NY). The results were averaged for each group of mice and are presented in the table below. Table 9

[0936] Plasma Chemistry markers in wild type Balb / c mice

[0937] Plasma clinical chemistry

[0938] LNP Dose

[0939] Ionizabl Helper ALT AST ALT AST ALT AST (mg / kg

[0940] Formulation e Lipid Lipid (U / L) (U / L) (U / L) (U / L) (U / L) (U / L)

[0941] )

[0942] 6 hr 24 hr 7 days PBS - - - 42 110 34 85 22 53

[0943] 0.3 33 69 36 73 22 71 9 IPL-003 DSPC

[0944] 6 61 260 69$ 265$ 33 107 0.3 39 76 35 72 18 56 10 IPL-004 DOPE

[0945] 6 269 660 1118 2248 18 68 ALC- 0.3 33 73 58 90 48 269 16 DSPC

[0946] 0315

[0947]

[0948] 6 709 1547 4310 7585 24 70

[0949] $ indicates fewer than 4 samples available

[0950] To evaluate inflammatory cytokines, blood plasma was collected from the 0.6 mg / kg groups of mice at 4 hours post-treatment and plasma levels of inflammatory markers were measured by ELISA using a V-PLEX Mouse Cytokine 19-Plex Kit from Meso Scale Discovery (Catalog # K15255D).

[0951] After 24 hours, mice treated with 0.3 mg / kg and 6 mg / kg LNP formulations comprising IPL-003 or IPL-004 had significantly lower levels of inflammatory markers than those treated with an LNP comprising ALC-0315.

[0952] Example 8: Synthesis of ionizable prolinol-based lipids

[0953] Ionizable prolinol-based lipids IPL-004.1, IPL-005, and IPL-006 were synthesized from commercially available starting materials, as shown in the schemes below. Synthesis of IPL-004.1

[0954] 29C (0.95 eq) EDCI (1.15 eq), DMAP (0.95 eq), DCM (10 V)

[0955] 25 °C, 16 h 29A 37% yield, 90% purity

[0956] TBSQ TBSCI (1.5 eq), imidazole (2 eq) LiBH4(2 M, 1.5 eq) DCM (10 V), 20 °C, 10 h THF (10 V), 0-25 °C, 4 h 23 24 71% yield, 90% purity 65% yield, 90% purity

[0957] (COCI)2(2 eq), DMSO (2.5 eq) 7B (1.25 eq), K2CO3(2 eq) TEA (4 eq), DCM (24 V), -78-25 °C, 3 h ACN, 90 °C, 10 h 25 26 73% yield, 90% purity 54% yield, 80% purity

[0958] LiOH•H2O (5 eq) 18A (1.2 eq), EDCI (1.2 eq) THF: H2O (3:1, 12 V), 20 °C, 10 h DMAP (1 eq), DCM (10 V), 0-20 °C, 10 h 27 51% yield, 90% purity

[0959] 60% yield, 90% purity

[0960]

[0961] Synthesis of IPL-005

[0962] 1 -iodopentane (1.2 eq), NaH (1.1 eq), LDA (1.2 eq) BH3•Me2S (5 eq) HO THF (10 V), 0-45 °C,16.5 h THF (20 V), 0 °C,3 h 38B 38C 39% yield, 90% purity

[0963] 38E (1.2 eq) TEA (3 eq), DCM (20 V), 25 °C, 3 h 38A 64% yield, 90% purity 50% yield, 92% purity

[0964] TBSCI (1.5 eq), imidazole (2 eq) LiBH4(1.5 eq) DCM (10 V), 25 °C, 16 h THF(10 V), 0 °C, 16 h

[0965] 32 33 71% yield, 97% purity 65% yield, 100% purity

[0966] (COCI)2(1.5 eq), DMSO (3 eq), TEA (20 eq) 7B (1.25 eq), K2CO3(2 eq) DCM (10 V), -78-25 °C, 3 h MeCN (10 V), 90 °C, 16 h’

[0967] 34 35 67% yield, 92% purity 63% yield, 90% purity 1) DMF (0.1 eq), (COCI)2(1.5 eq) LiOH•H2O (3 eq) DCM (10 V), 0 °C, 1 h THF: H2O (3:1, 10 V), 50 °C, 2 h 2) 2-heptyldecan-1-ol (0.9 eq), TEA (5 eq),

[0968] DCM (10 V), 0 °C, 1 h 36 77% yield, 93% purity

[0969] 38A (1 eq), K2CO3(2 eq) DMF (10 V), 70 °C, 3 h

[0970]

[0971] 109 mg, 14% yield, 95.99% purity Synthesis of IPL-006

[0972] 29C (0.85 eq), EDCI (1.15 eq),

[0973] DMAP (0.95 eq)

[0974] DCM (10 V), 25 °C, 16 h

[0975] 40

[0976] 53% yield, 90% purity

[0977] HO

[0978] 38 (1 eq), K2CO3(2 eq)

[0979] DMF (10 V), 70 °C, 3 h

[0980] IPL-006

[0981]

[0982] 100 mg, 11% yield, 96.19% purity

[0983] Observed mass of synthesized ionizable prolinol-based lipids

[0984] The ionizable prolinol-based lipids IPL-004.1, IPL-005, and IPL-006 described herein above were analyzed by LC-MS for identity and purity. The observed mass of each lipid is shown in the table below.

[0985] Table 10

[0986] Observed mass of synthesized ionizable prolinol-based lipids

[0987] Lipid Theoretical Mass Observed Mass Retention Time

[0988] [(M+H)+] (min)

[0989] IPL-004.1 778.7 778.7 11.645 IPL-005 778.7 778.7 11.652

[0990]

[0991] IPL-006 778.7 778.7 11.622

[0992] Example 8: Formulation of ionizable prolinol-based lipids in lipid nanoparticles

[0993] The ionizable prolinol-based lipids IPL-004, IPL-004.1, IPL-005, and IPL-006 described herein above were formulated into LNPs containing a M6-capped Cas9 mRNA and G211 gRNA.

[0994] The lipid mix was formed by mixing an ionizable lipid described herein above, with cholesterol (Sigma Aldrich), DSPC helper lipid (Avanti Polar Lipids), and DMG-PEG2K (Avanti Polar Lipids). The ionizable lipid / cholesterol / DSPC / DMG-PEG2K were mixed at a ratio of 0.5 / 0.385 / 0.1 / 0.015, respectively. This mixture was prepared twice, each yielding a total lipid concentration of 17.45 pmol in 3 mL of ethanol. To each mixture, 240 pg of M6 spCas9 mRNA from TriLink (L-8106) and 240 pg of gRNA G211 from BioSpring (Lot No. 315693_A) were added to 300 mM citrate buffer pH 4.0 for a final volume of 9 mL.

[0995] LNPs were formulated on a NanoAssemblr Ignite (Precision Nanosystems) using a total flow rate of 12 mL / min with 3:1 ratio of RNA: Lipid. Immediately after formulation, LNPs were diluted with equi volume DI water then dialyzed overnight in 3 L of IX PBS buffer pH 7.4. The dialyzed LNPs were pooled together and concentrated using a 100kDa MWCO amicon filter (Millipore) to a final volume of approximately 0.5 mL. The final ionizable lipid nitrogen:nucleic acid phosphate molar (N: P) ratio was 6 for each LNP.

[0996] Total RNA concentration of the LNPs were characterized using RIBOGREEN® (Thermofisher Scientific) to determine the percentage of RNA successfully encapsulated within the LNP. Additionally, Z-averages and polydispersity index (PDI) for the LNP-mRNA formulated particles were determined using a Dynamic Light Scattering instrument (Malvern). Z-average measurement is the average particle size determined by the Malvern zeta-sizer. The PDI is a dimensionless value that measures the heterogeneity of particle sizes within a sample.

[0997] Table 11

[0998] Lipid nanoparticle formulation and characterization

[0999] LNP Helper Lipid

[1000] Formulat Ionizable Lipid Z-average (nm) PDI Encapsulation ion

[1001] 13 IPL-004 DSPC 86 0.15 98% 17 IPL-004.1 DSPC 88 0.17 98% 18 IPL-005 DSPC 91 0.18 99%

[1002]

[1003] 19 IPL-006 DSPC 93 0.19 99%

[1004] Example 9: Delivery efficacy and tolerability of LNP formulations in wild type mice, in vivo

[1005] The ionizable lipids described herein above were formulated into LNPs containing an M6 SpCas9 mRNA and a mouse TTR guide RNA (G211), previously described in Linn, et al. Cell Rep. 2018, 22 (9).

[1006] Delivery efficacy

[1007] Groups of 4 Balb / c mice each received a single IV tail vein injection of LNPs at a concentration of 0.1 mg / kg. One group of 4 Balb / c mice received PBS and served as a negative control. Blood plasma was collected 7 days post-treatment. TTR protein levels were analyzed by ELISA using a prealbumin mouse ELISA kit from Abeam (Catalog #ab282297). The results were averaged for each group of mice.

[1008] Concentrations of TTR are presented as averages for each group of min the table below in pg / ml. Reduction of TTR is presented as TTR protein relative to the amount of TTR protein in PBS treated control animals (% control). Table 12

[1009] TTR protein and RNA levels in Balb / c mice at a dose of 0.1 mg / kg

[1010] Formulation Ionizable Helper TTR protein TTR protein

[1011] Lipid Lipid levels (pg / ml) (% control)

[1012] PBS - - 265 100

[1013] 13 IPL-004 202 76

[1014] 18 IPL-005 235 89

[1015] DSPC

[1016] 19 IPL-006 202 76

[1017] 17

[1018]

[1019] IPL-004.1 236 89

[1020] Tolerability

[1021] Groups of 4 CD-1 mice each received a single IV tail vein injection of LNPs at a concentration of 6 mg / kg. One group of 4 CD-I mice received PBS and served as a negative control.

[1022] To evaluate the effect of the LNPs on liver function blood plasma was collected from the 6 mg / kg groups of CD-I mice at 4 hours and 7 Days post treatment and plasma levels of ALT and AST were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400c, Melville, NY). The results were averaged for each group of mice and are presented in the table below.

[1023] Table 13

[1024] Plasma Chemistry markers in wild type CD-I mice at a dose of 6 mg / kg

[1025] Plasma clinical chemistry Formulation ALT AST ALT AST Ionizable Lipid Helper Lipid (U / L) (U / L) (U / L) (U / L)

[1026] 4 hr 7 days

[1027] PBS - - 32 68 83 110 13 IPL-004 261 636 88 132

[1028] 18 IPL-005 199 454 45 57 DSPC

[1029] 19 IPL-006 375 739 49 71

[1030] 17

[1031]

[1032] IPL-004.1 414 767 35 74

[1033] To evaluate inflammatory cytokines blood plasma was collected from the 6 mg / ml groups of CD-I mice at 4 hours post-treatment and plasma levels of inflammatory markers were measured by ELISA using a V-PLEX Mouse Cytokine 19-Plex Kit from Meso Scale Discovery (Catalog # K15255D).

[1034] Example 10: Ionizable lipid degradation, in vitro pancreatin digest assay

[1035] Ionizable lipids described above were evaluated for stability in a pancreatin digest assay.

[1036] Bile salts (6 mg / mL) and pancreatin (40 mg / mL) were dissolved in DPBS and incubated at 37 °C and pH was adjusted to 8. Ionizable lipid (2 pmol) was incubated with 200 pl of pancreatin at 37 °C. The solution was sonicated, chloroform was added, centrifuged, and the organic phase was collected. Intact lipids were quantified on 0 hours (Baseline) and at 24 hours, and the results are presented as the amount of intact lipids relative to the amount of intact lipids at baseline (% Baseline).

[1037] Table 14

[1038] Pancreatin lipid Digest

[1039] Ionizable Lipid Intact Lipid (%

[1040] Baseline)

[1041] ALC-0315 87

[1042] IPL-003 92

[1043]

[1044] IPL-004 90

[1045] After 24 hours, assay samples of IPL-003 and IPL-004 showed 90% or more intact lipid, which was higher than for ALC-0315.

Claims

WHAT IS CLAIMED:

1. An ionizable lipid having Formula II:IIor a pharmaceutically acceptable salt, tautomer, stereoisomer or prodrug thereof,wherein:X is -OR1, -NR2R3, -Q3-(CH2)s-OR1, or -Q3-(CH2)s-NR2R3;Llaand L2aare each independently Ci to Cis alkyl or -(CH2)P-Q4-R4;Llband L2bare each independently H, Ci to Cis alkyl, or -(CH2)P-Q4-R4;Q1and Q2are each independently -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -N(R5)C(=O)-, -C(=O)N(R5)-, -N(R5)-O-, -O-N(R5)-, -O-, -S-, -S-S-, -N=N-, -C(=O)-N-N=CH-, -CH=N-N-C(=O)-, -O-P(=O)(OCH3)-N-, -O-Si(OCH3)2-O-, or -C=C-;Q3is selected from -C=C-, -C(=O)O-, and -OC(=O)-;each instance of Q4is independently -C(=O)O-, -OC(=O)-, -C(=O)S-,-SC(=O)-, -C(=S)O-, -OC(=S)-, -N(R5)C(=O)-, -C(=O)N(R5)-, -N(R5)-O-, -O-N(R5)-, -O-, -S-, -S-S-, or -C=C-;R1is H or Ci-Ce alkyl;R2and R3are each independently H or Ci to C3alkyl, or both R2and R3together are linked by a shared Ci to C3alkyl to form a 4- to 6-membered nitrogen-containing ring; each instance of R4is independently H, Ci to Ci6 alkyl, or Ci to Ci6 alkenyl;each instance of R5is independently H or Ci to Ci6 alkyl;m is 1 to 10;n is 2 to 10;u is 0 to 3;v is 0 to 3;each instance of p is independently 1 to 18; andr and s are each independently 0 to 10.

2. The ionizable lipid of claim 1, wherein m is 1.

3. The ionizable lipid of claim 1, wherein m is 2.

4. The ionizable lipid of claim 1, wherein m is 3.

5. The ionizable lipid of claim 1, wherein m is 5.

6. The ionizable lipid of claim 1, wherein n is 6.

7. The ionizable lipid of claim 1, wherein Llbis H and Llais C13 alkyl.

8. The ionizable lipid of claim 1, wherein m is 1, Llbis H and Llais C13 alkyl.

9. The ionizable lipid of claim 7 or 8, wherein Q1is -OC(=O)-.

10. The ionizable lipid of claim 1, wherein Llbis H and Llais Cn alkyl.

11. The ionizable lipid of claim 1, wherein m is 3, Llbis H and Llais Cn alkyl.

12. The ionizable lipid of claim 10 or 11, wherein Q1is -OC(=O)-.

13. The ionizable lipid of claim 1, wherein Llbis H and Llais C12 alkyl.

14. The ionizable lipid of claim 1, wherein m is 2, Llbis H and Llais C12 alkyl.

15. The ionizable lipid of claim 13 or 14, wherein Q1is -C(=O)O-.

16. The ionizable lipid of claim 1, wherein Llais Cs alkyl and Llbis Cs alkyl.

17. The ionizable lipid of claim 1, wherein m is 5, Llais Cs alkyl and Llbis Cs alkyl.

18. The ionizable lipid of claim 16 or 17, wherein Q1is -C(=O)O-.

19. The ionizable lipid of claim 1, wherein Llais Cs alkyl and Llbis C7 alkyl.

20. The ionizable lipid of claim 1, wherein m is 5, Llais Cs alkyl and Llbis C7 alkyl.

21. The ionizable lipid of claim 19 or 20, wherein Q1is -C(=O)O-.

22. The ionizable lipid of any of claims 1-21, wherein L2bis Ce alkyl.

23. The ionizable lipid of any of claims 1-21, wherein L2bis C5 alkyl.

24. The ionizable lipid of claim 22 or 23, wherein L2ais Cs alkyl.

25. The ionizable lipid of claim 22 or 23, wherein n is 6.

26. The ionizable lipid of claim 22 or 23, wherein n is 6 and L2ais Cs alkyl.

27. The ionizable lipid of any of claims 1-26, wherein Q2is -OC(=O)-.

28. The ionizable lipid of any of claims 1-26, wherein Q2is -C(=O)O-.

29. The ionizable lipid of any of claims 1-28, wherein u is 0.

30. The ionizable lipid of any of claims 1-28, wherein u is 1.

31. The ionizable lipid of any of claims 1-30, wherein v is 0.

32. The ionizable lipid of any of claims 1-30, wherein v is 1.

33. The ionizable lipid of any of claims 1-32, wherein r is 0 and X is OH.

34. The ionizable lipid of claim 1, wherein Llaand L2aare each independently Ci to Cis alkyl, and Llband L2bare each independently H or Ci to Cis alkyl.

35. An ionizable lipid having Formula III:T-k, L1aL1bNL2bor a pharmaceutically acceptable salt, tautomer, stereoisomer or prodrug thereofwhereinLlaand L2aare each independently Ci to Cis alkyl or -(CH2)P-Q4-R4;Llband L2bare each independently H, Ci to Cis alkyl, or -(CH2)P-Q4-R4;Q1and Q2are each independently -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-,-N(R5)C(=O)-, -C(=O)N(R5)-, -N(R5)-O-, -O-N(R5)-, -O-, -S-, -S-S-, -N=N-, -C(=O)-N-N=CH-, -CH=N-N-C(=O)-, -O-P(=O)(OCH3)-N-, -O-Si(OCH3)2-O-, or -C=C-;each instance of Q4is independently -C(=O)O-, -OC(=O)-, -C(=O)S-,-SC(=O)-, -C(=S)O-, -OC(=S)-, -N(R5)C(=O)-, -C(=O)N(R5)-, -N(R5)-O-, -O-N(R5)-, -O-, -S-, -S-S-, or -C=C-;each instance of R4is independently H, Ci to Ci6 alkyl, or Ci to Ci6 alkenyl;each instance of R5is independently H or Ci to Ci6 alkyl;m is 1 to 10;n is 2 to 10;u is 0 to 3;v is 0 to 3; andeach instance of p is independently 1 to 18.

36. The ionizable lipid of claim 35, wherein m is 1.

37. The ionizable lipid of claim 35, wherein m is 2.

38. The ionizable lipid of claim 35, wherein m is 3.

39. The ionizable lipid of claim 35, wherein m is 5.

40. The ionizable lipid of any of claims 35-39, wherein n is 6.

41. The ionizable lipid of any of claims 35-40, wherein Llbis H and Llais C13 alkyl.

42. The ionizable lipid of any of claims 35-40, wherein Llbis H and Llais Cn alkyl.

43. The ionizable lipid of any of claims 35-40, wherein Llais Cs alkyl and Llbis Cs alkyl.

44. The ionizable lipid of any of claims 35-40, wherein Llais Cs alkyl and Llbis C7 alkyl.

45. The ionizable lipid of any of claims 35-40, wherein L2ais Ce alkyl and L2bis Cs alkyl.

46. The ionizable lipid of any of claims 35-40, wherein L2ais C5 alkyl and L2bis Cs alkyl.

47. The ionizable lipid of any of claims 35-46, wherein u is 1.

48. The ionizable lipid of any of claims 35-46, wherein u is 0.

49. The ionizable lipid of any of claims 35-48, wherein v is 1.

50. The ionizable lipid of any of claims 35-48, wherein v is 0.

51. The ionizable lipid of any of claims 35-50, wherein Q1is -OC(=O)-.

52. The ionizable lipid of any of claims 35-50, wherein Q1is -C(=O)O-.

53. The ionizable lipid of any of claims 35-52, wherein Q2is -OC(=O)-.

54. The ionizable lipid of any of claims 35-52, wherein Q2is -C(=O)O-.

55. An ionizable lipid having Formula la:HO,Formula la.

56. An ionizable lipid having Formula lb:Formula lb.

57. An ionizable lipid having Formula Ic:Formula Ic.

58. An ionizable lipid having Formula Id:Formula Id.

59. An ionizable lipid having Formula le:

60. An ionizable lipid having Formula If:

61. An ionizable lipid having Formula Ig:

62. A lipid nanoparticle comprising an ionizable lipid according to any of claims 1-61.

63. The lipid nanoparticle of claim 62, comprising a non-cationic lipid.

64. The lipid nanoparticle of claim 63, wherein the non-cationic lipid is a sterol.

65. The lipid nanoparticle of claim 64, wherein the sterol is cholesterol.

66. The lipid nanoparticle of any of claims 62-65, comprising a cationic lipid.

67. The lipid nanoparticle of claim 63, wherein the non-cationic lipid is a helper lipid.

68. The lipid nanoparticle of claim 67, wherein the helper lipid is a phospholipid.

69. The lipid nanoparticle of any of claims 62-68, comprising a polymer lipid.

70. The lipid nanoparticle of claim 69, wherein the polymer lipid is a pegylated (polyethylene glycol) lipid (PEG lipid).

71. A lipid nanoparticle comprising:(i) an ionizable lipid having Formula la, Formula lb, Formula Ic, Formula Id, Formula le, Formula If, or Formula Ig:Formula laHO.Formula lbFormula Ig;(ii) a helper lipid;(iii) cholesterol; and(iv) a PEG lipid;and optionally a cargo.

72. The lipid nanoparticle of claim 71, wherein the cargo comprises an oligomeric agent.

73. The lipid nanoparticle of claim 72, wherein the oligomeric agent comprises an oligonucleotide, optionally wherein the oligonucleotide comprises a guide.

74. The lipid nanoparticle of claim 71, wherein the cargo comprises a nucleic acid, optionally wherein the nucleic acid comprises an exogenous mRNA.

75. A method of preparing a lipid nanoparticle, wherein the method comprises combining the ionizable lipid of any of claims 1-61 with a helper lipid, a sterol and a polymer lipid.

76. A lipid nanoparticle prepared by the method of claim 75.

77. A composition comprising the lipid nanoparticle of any of claims 62-74 or 76.

78. A method of delivering a cargo to a cell, tissue, organ or subject comprising encapsulating the cargo in a lipid nanoparticle of any of claims 62-74 or 76, or a composition of claim 77, and administering the lipid nanoparticle or composition to the cell, tissue, organ or subject.

79. The method of claim 78, wherein the cargo comprises a nucleic acid.

80. The method of claim 79, wherein the nucleic acid comprises an exogenous mRNA.

81. The method of claim 78, wherein the cargo comprises an oligomeric agent.

82. The method of claim 81, wherein the oligomeric agent comprises an oligonucleotide.

83. The method of claim 82, wherein the oligonucleotide comprises a guide.

84. A kit comprising the ionizable lipid of any of claims 1-61, the lipid nanoparticle of any of claims 62- 74 or 76, or the composition of claim 77, optionally wherein the kit is for, or when used for, delivering a cargo to a cell, tissue, organ or subject.

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