Lipid nanoparticles and lipid nanoparticle compositions

Abstract

Lipid nanoparticles comprising ionizable lipids, helper lipids, neutral lipids, structural PEG-lipids, and anchor PEG-lipids are provided herein, together with targeted lipid nanoparticle compositions, and uses thereof. Methods of administering targeted lipid nanoparticles and targeted lipid nanoparticle compositions for the delivery of biologically active agents, are also provided. Such compositions and methods can be used, for example, to deliver a cargo (e.g., a mRNA cargo), to a cell population of interest.

Description

NTLA-103PCT; 5640-104.PCT Lipid Nanoparticles and Lipid Nanoparticle Compositions CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the priority benefit of U.S. provisional application nos. 63 / 649,241 filed May 17, 2024 and 63 / 726,056 filed on November 27, 2024, the contents of which are incorporated herein by reference in their entireties. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 16, 2025, is named “5640-104PCT.xml” and is 53,248 bytes in size. FIELD

[0003] The instant disclosure relates generally to lipid nanoparticles and lipid nanoparticle compositions that facilitate the delivery of biologically active agents into a cell. BACKGROUND

[0004] Lipid nanoparticles (LNPs) encapsulating a cargo, for example a biologically active agent, can be used to deliver cargo to a cell. LNP cargo can include, for example, one or more small nucleic acid molecules, RNAi agents, short interfering nucleic acid (siNA), messenger ribonucleic acid (messenger RNA, mRNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules, peptide nucleic acid (PNA), a locked nucleic acid ribonucleotide (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), sisiRNA (small internally segmented interfering RNA), aiRNA (asymmetrical interfering RNA), and siRNA with 1, 2 or more mismatches between the sense and anti-sense strand to relevant cells and / or tissues, such as in a cell culture, subject or organism. Of particular interest is delivery of an mRNA and / or guide RNA.

[0005] Effective delivery of nucleic acids to appropriate sites within a cell or organism to affect a desired response in a biological system remains a significant challenge. There is a need for improved compositions for delivery of nucleic acids that can be used to introduce components and compositions, e.g., for gene editing, into particular cell populations in vivo. 1NTLA-103PCT; 5640-104.PCT SUMMARY OF THE INVENTION

[0006] Provided herein are lipid nanoparticle (LNP) compositions, including LNP compositions comprising a fifth lipid component to serve as a conjugation handle and anchor for a targeting ligand, and methods of administering lipid nanoparticle compositions. In addition, the present disclosure includes methods for genetically engineering a target cell using such LNP compositions. In certain aspects, a lipid nanoparticle is provided comprising: (i) a lipid component comprising: (a) an anchor PEG-lipid; (b) a helper lipid; (c) a neutral lipid; (d) a structural PEG-lipid; and (e) an ionizable lipid represented by any one of structural Formula I, or, wherein,bond, X1is a C1-5 alkylene, R1and R2are each independently a C1-3 alkyl, or R1taken together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X1form a 4-, 5-, or 6-membered ring, or R1taken together with R2and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, and 2NTLA-103PCT; 5640-104.PCT R3is H or C1-3alkyl, Z1and Z2are each independently a C1-5 alkylene, Z3and Z4are each independently a -C(=O)O- in either direction, Z5and Z6are each independently a direct bond or a C1-3alkylene, Y1is selected from H, a C1-10 alkyl, C3-10 alkenyl, and C3-10 alkynyl, Y2, Y3, and Y4are each independently selected from a C3-10alkyl, C3-10alkenyl, or C3-10alkynyl, and n is 0 or 1; and (ii) a first coupling moiety attached to the anchor PEG-lipid; and (iii) a cargo, wherein the cargo is encapsulated in the LNP.

[0007] In certain aspects, an LNP is provided comprising: (i) a lipid component comprising: (a) an anchor PEG-lipid; (b) a helper lipid; (c) a neutral lipid; (d) a structural PEG-lipid; and (e) an ionizable lipid comprising:d (iii) a cargo, wherein the cargo is encapsulated in the LNP.

[0008] In certain aspects, an LNP is provided comprising: (i) a lipid component comprising: (a) an anchor PEG-lipid; (b) a helper lipid; (c) a neutral lipid; (d) a structural PEG-lipid; and (e) an ionizable lipid represented by any one of structural Formula II, [Formula II] 3NTLA-103PCT; 5640-104.PCTwherein: Q is CH o A is O orX1is a C2-5 alkylene, R1and R2are each independently a C1-3alkyl, or R1taken together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X1form a 4-, 5-, or 6-membered ring, or R1taken together with R2and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, and Z1is a C2-9alkylene, Z2is a C2-3 alkylene or a direct bond, and Y1and Y2are each independently selected from a C3-10alkyl, C3-10alkenyl, or C3-10 alkynyl; (ii) a first coupling moiety attached to the anchor PEG-lipid; and (iii) a cargo, wherein the cargo is encapsulated in the LNP.

[0009] In certain aspects, an LNP is provided comprising: (i) a lipid component comprising: (a) an anchor PEG-lipid; (b) a helper lipid; (c) a neutral lipid; (d) a structural PEG-lipid; and (e) an ionizable lipid represented by any one of structural Formula III, [Formula III] 4NTLA-103PCT; 5640-104.PCT O R2 X1 N A O OY3 4 ,wherein: A is O or NH, X1is a C2-5 alkylene, R1and R2is each independently a C1-3alkyl, or R1taken together with R2and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, and Z1is a C2-5alkylene, Y1and Y2is each independently a C3-10 alkoxyl or O(C3-10 alkynyl), Z2is a C1-5alkylene or a direct bond, and Y3and Y4is each independently a C3-10 alkoxyl or O(C3-10 alkynyl), or Y3and Y4is each independently a C3-10alkyl or C3-10alkynyl, provided that if Y1, Y2, Y3, and Y4is each independently a C3-10 alkoxy, then R1and R2are not C2alkyl, and R1taken together with R2and the nitrogen atom to which they are attached do not form a 6-membered ring; (ii) a first coupling moiety attached to the anchor PEG-lipid; and (iii) a cargo, wherein the cargo is encapsulated in the LNP.

[0010] In certain aspects, an LNP is provided comprising: (i) a lipid component comprising: (a) an anchor PEG-lipid; (b) a helper lipid; (c) a neutral lipid; (d) a structural PEG-lipid; and (e) an ionizable lipid represented by any one of structural Formula IV, [Formula IV] 5NTLA-103PCT; 5640-104.PCT wurrence, X1is C5-11alkylene, Y1is C3-11 alkylene, bond to R1, Z1is C2-4 alkylene, Z2is selected from -OH, -NH2, -OC(=O)R3, -OC(=O)NHR3, -NHC(=O)NHR3, and - NHS(=O)2R3, 1 2 alkenyl,ly C4-12alkyl, and R3is C1-3 alkyl, or a salt thereof; (ii) a first coupling moiety attached to the anchor PEG-lipid; and (iii) a cargo, wherein the cargo is encapsulated in the LNP.

[0011] In certain aspects, an LNP is provided comprising: (i) a lipid component comprising: (a) an anchor PEG-lipid; (b) a helper lipid; (c) a neutral lipid; (d) a structural PEG-lipid; and (e) an ionizable lipid represented by any one of structural Formula V, [Formula V] 6NTLA-103PCT; 5640-104.PCT wX1is O, NR1, or a direct bond, X2is C2-5 alkylene, X3is C(=O) or a direct bond, R1is H or Me, R3is C1-3 alkyl, R2is C1-3 alkyl, or R2taken together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X2form a 4-, 5-, or 6-membered ring, or X1is NR1, R1and R2taken together with the nitrogen atoms to which they are attached form a 5- or 6-membered ring, or R2taken together with R3and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y1is C2-12 alkylene, Y2is selected from (entation),on), (n e t er orentation), 7NTLA-103PCT; 5640-104.PCT n is 0 to 3, R4is C1-15 alkyl, Z1is C1-6alkylene or a direct bond, ( ion) or absent, provided that if Z1is a direct bond, Z2is absent;R5is C5-9 alkyl or C6-10 alkoxy, R6is C5-9 alkyl or C6-10 alkoxy, W is methylene or a direct bond, and R7is H or Me, or a salt thereof, provided that if R3and R2are C2alkyls, X1is O, X2is linear C3alkylene, X3is C(=O), Y1is linear C6 alkylene, (Y2)n-R4is , 1Z is C2alkylene, Z2is absent, W is methylene, and R7is H, then R5and R6are not C8alkoxy; a first coupling moiety attached to the anchor PEG-lipid; and a cargo, wherein the cargo is encapsulated in the LNP.

[0012] In certain aspects, an LNP is provided comprising: (i) a lipid component comprising: (a) an anchor PEG-lipid; (b) a helper lipid; (c) a neutral lipid; (d) a structural PEG-lipid; and (e) an ionizable lipid represented by any one of structural Formulae I-V, above, or, an ionizable lipid comprising: ,NTLA-103PCT; 5640-104.PCT (ii) a first coupling moiety attached to the anchor PEG-lipid; and (iii) a cargo, wherein the cargo is encapsulated in the LNP, and wherein the LNP is stable following storage at room temperature, 4 ° C, and about -70 to -80 °C.

[0013] In certain aspects, the present disclosure includes any of the LNPs disclosed herein in which the neutral lipid is absent. In certain aspects, the present disclosure includes any of the LNPs disclosed herein comprising 0 to 25 mol% neutral lipid.

[0014] In certain aspects, compositions provided herein comprise the LNP, as described above, wherein the composition comprises a buffer, an excipient, or a combination thereof. In some aspects, the composition is provided in a kit.

[0015] In certain aspects, methods are provided for delivering a cargo into a target cell, comprising contacting the target cell with the LNP, as described above, or compositions thereof.

[0016] In certain aspects, methods are provided for expressing a cargo in a target cell comprising contacting the target cell with the LNP, as described above, or compositions thereof.

[0017] In certain aspects, methods are provided for administering a cargo to a subject in need thereof, comprising administering a composition comprising the LNP, as described above, or compositions thereof.

[0018] In certain aspects, methods are provided for genetically engineering a target cell comprising contacting the target cell with the LNP, as described above, or compositions thereof. In some aspects, the cargo is selected from a CRISPR / Cas system, a Tth Argonaute (TtAgo) system, a zinc finger nuclease (ZFN) system, ARCUS nuclease system, megaTALs, or a transcription activator-like effector nuclease (TALEN) system.

[0019] In certain aspects, a composition is provided comprising the LNP, as described above, or compositions thereof wherein the composition is stable following one or more freeze / thaw cycles. In some aspects, the composition is provided in a kit.

[0020] These and other aspects of the invention will be apparent upon reference to the following detailed description, claims, aspects, procedures, compounds, and / or compositions and associated background information and references, which are hereby incorporated in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS 9NTLA-103PCT; 5640-104.PCT

[0021] FIGs.1A-1B show the percentage of cells expressing GFP protein (%GFP+) and the Median Fluorescent Intensity (MFI) in GFP+ cells, at 6 or 14 hours after dosing. LNPs used in Experiment #1 were produced at large scale (25-1000mL).

[0022] FIGs.2A-2B show the percentage of cells expressing GFP protein (%GFP+) and the Median Fluorescent Intensity (MFI) in GFP+ cells, at 6 hours after dosing. LNPs used in Experiment #2 were produced at small scale (1-5mL).

[0023] FIGs.3A-3G show editing obtained from Experiment #3 using LNPs produced at large scale with Compound 3 as an ionizable lipid, and two different anchor PEG-lipid densities with the same targeting ligand conjugated to the anchor PEG-lipid with a SpyTag- SpyCatcher conjugation chemistry, where the targeting ligand is an antibody fragment, compared to an untargeted control LNP.

[0024] FIGs.4A-4D show editing obtained from Experiment #4 using LNPs produced at small scale with Compound 3 or Compound 4 as an ionizable lipid, different levels of total PEG lipid, and a targeting ligand conjugated to the anchor PEG-lipid with a SpyTag- SpyCatcher conjugation chemistry, where the targeting ligand is an antibody fragment, compared to untargeted control LNPs.

[0025] FIGs.5A-5H show editing obtained from Experiment #5 using LNPs produced at large scale with Compound 2 as an ionizable lipid, and different levels of anchor PEG-lipid density with a SpyTag-SpyCatcher conjugation chemistry, where the targeting ligand is an antibody fragment, compared to an untargeted control LNP.

[0026] FIGs.6A-6E show editing obtained from Experiment #6 using LNPs produced at small scale with Compound 3 as an ionizable lipid, and different levels of anchor PEG-lipid density with a SpyTag-SpyCatcher conjugation chemistry, where the targeting ligand is an antibody, compared to an untargeted control LNP.

[0027] FIGs.7A-7J show editing obtained from Experiment #7 in WT and ApoE KO mice, using LNPs produced at small scale with Compound 1 as an ionizable lipid, and different levels of anchor PEG-lipid density with an Azide-DBCO conjugation chemistry, where the targeting ligand is a peptide, compared to an untargeted control LNP.

[0028] FIGs.8A-8F show editing obtained from Experiment #8 in ApoE KO and LDLR KO mice, using a 4-component LNP, an untargeted 5-component LNP, and a peptide-conjugated 5-component LNP with an Azide-DBCO conjugation chemistry, all produced at small scale with Compound 1 as an ionizable lipid. 10NTLA-103PCT; 5640-104.PCT

[0029] FIGs.9A-9E show editing obtained from Experiment #9 in WT mice, using a 4- component LNP, an untargeted 5-component LNP, and a peptide-conjugated 5-component LNP with an Azide-DBCO conjugation chemistry, all produced at small scale with Compound 1 as an ionizable lipid.

[0030] FIGs.10A-10F show editing obtained from Experiment #10 using LNPs produced at large scale with Compound 1 as an ionizable lipid, and different levels of anchor PEG-lipid density with a Cysteine-Maleimide conjugation chemistry, where the targeting ligand is a peptide, compared to an untargeted control LNP.

[0031] FIGs.11A-11F show editing obtained from Experiment #11 using LNPs produced at small scale with Compound 3 as an ionizable lipid, and different levels of total PEG and anchor PEG-lipid densities with a Cysteine-Maleimide conjugation chemistry, where the targeting ligand is a peptide, compared to untargeted control LNPs.

[0032] FIGs.12A-12F show editing obtained from Experiment #12, using LNPs produced at small scale with Compound 3 as an ionizable lipid, and different levels of total PEG and anchor PEG-lipid densities with a Cysteine-Maleimide conjugation chemistry, where the targeting ligand is a peptide, compared to untargeted control LNPs.

[0033] FIGs.13A-13F show editing obtained from Experiment #13, using LNPs produced at small scale with Compound 1 or Compound 3 as an ionizable lipid, and different types and densities of anchor PEG-lipid with a Cysteine-Maleimide conjugation chemistry, where the targeting ligand is a peptide.

[0034] FIGS.14A-14F show editing obtained from Experiment #14, using LNPs produced at large scale with Compound 1 or Compound 3 as an ionizable lipid, and different densities of anchor PEG-lipid with a Cysteine-Maleimide conjugation chemistry, where the targeting ligand is a peptide, compared to untargeted control LNPs.

[0035] FIGs.15A-15E show editing obtained from Experiment #15, using LNPs produced at small scale with one of Compounds 3, 4, 7 or 8 as an ionizable lipid, a Cysteine-Maleimide conjugation chemistry, and a peptide as a targeting ligand.

[0036] FIGs.16A-16D show editing obtained from Experiment #16, using LNPs produced at small scale with one of Compounds 3, 5 or 6 as an ionizable lipid, and different structural PEG-lipids, having a peptide as a targeting ligand with a Cysteine-Maleimide conjugation chemistry, compared to an untargeted control LNP.

[0037] FIGs.17A-17D show editing obtained from Experiment #17, using LNPs produced at small or large scale with Compound 3 as an ionizable lipid, and different structural PEG- 11NTLA-103PCT; 5640-104.PCT lipids, having a peptide as a targeting ligand with a Cysteine-Maleimide conjugation chemistry, compared to an untargeted control LNP.

[0038] FIGs.18A-18D show editing obtained from Experiment #18, using a LNP produced at large scale with Compound 2 as an ionizable lipid, a Cysteine-Maleimide conjugation chemistry, and a peptide as a targeting ligand, compared to an untargeted control LNP.

[0039] FIGs.19A-19H show the percentage of cells expressing GFP protein (%GFP+) and the Median Fluorescent Intensity (MFI) in GFP+ cells, at 6 hours after dosing with targeted LNPs having varying ratios of neutral lipid.

[0040] FIGs.20A-20F show editing in wild-type mice, using LNPs produced at mid-scale with Compound 3 or Compound 7 as an ionizable lipid, and different lipid molar compositions, conjugated with a Fab 3 ligand, with a ligand density of 0.045%, through a SpyTag-SpyCatcher conjugation chemistry.

[0041] FIGs.21A-21B show mScarlet expression in the blood harvested from mice 24h post- injection with CD7 or CD8-targeted LNPs containing mScarlet mRNA versus non-targeted LNPs. MFI was normalized to the vehicle control group (FIG.21B).

[0042] FIGs.22A-22B show mScarlet expression in splenic cells harvested from mice 24h post-injection with CD7 or CD8-targeted LNPs containing mScarlet mRNA versus non- targeted LNPs. MFI was normalized to the vehicle control group (FIG.22B).

[0043] FIG.23 shows depletion of CD19+B cells in humanized mice 48 hours, one week, and two weeks after administration of CD7-targeted LNPs containing CD19 CAR mRNA.

[0044] FIG.24 shows the percent depletion of B cells 48 hours after the first dose and 48 hours after the second dose (on Day 21), relative to untreated control.

[0045] FIGs.25A-25B show in vivo editing efficacy of non-targeted LNPs with various ionizable lipids in lineage depleted / CD34(+) cells (FIG.25A) and Hematopoietic Stem Cells (HSCs) (FIG.25B).

[0046] FIGs.26A-26B show in vivo editing efficacy of non-targeted LNPs with varying DSPC amounts in lineage depleted / CD34(+) cells (FIG.26A) and Hematopoietic Stem Cells (HSCs) (FIG.26B).

[0047] FIGs.27A-27B show in vivo efficacy of non-targeted LNP formulations compared to targeted LNP formulations comprising an antibody fragment for editing (FIG.27A) and delivery (FIG.27B). DETAILED DESCRIPTION 12NTLA-103PCT; 5640-104.PCT

[0048] The disclosure relates to lipid nanoparticles (LNPs) with a lipid component comprising an ionizable lipid, a helper lipid, a neutral lipid, a structural PEG-lipid, and an anchor PEG-lipid. Targeted LNP compositions (t-LNPs) are also disclosed herein, including LNPs compositions comprising an anchor PEG-lipid conjugated to a targeting ligand that directs the t-LNP to a desired tissue or cell type. The disclosure also relates to lipid nanoparticle compositions and methods of administering lipid nanoparticles and lipid nanoparticle compositions. In addition, the present disclosure includes methods for genetically engineering a target cell.

[0049] While aspects of the subject matter of the present disclosure may be embodied in a variety of forms, the following description is merely intended to disclose some of these forms as specific examples of the subject matter encompassed by the present disclosure. Accordingly, the subject matter of this disclosure is not intended to be limited to the forms or aspects so described.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0051] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0052] Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of “about 0.01 to 2.0” should be interpreted to include not only the explicitly recited values of about 0.01 to about 2.0, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 0.5, 0.7, and 1.5, and sub-ranges such as from 0.5 to 1.7, 0.7 to 1.5, and from 1.0 to 1.5, etc. 13NTLA-103PCT; 5640-104.PCT Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described. Additionally, it is noted that all percentages are in weight, unless specified otherwise.

[0053] In understanding the scope of the present disclosure, the terms “including” or “comprising” and their derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of,” as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps. It is understood that reference to any one of these transition terms (i.e. “comprising,” “consisting,” or “consisting essentially”) provides direct support for replacement to any of the other transition term not specifically used. For example, amending a term from “comprising” to “consisting essentially of” or “consisting of” would find direct support due to this definition for any elements disclosed throughout this disclosure. Based on this definition, the present disclosure supports including or excluding any element disclosed herein or incorporated by reference from the claims.

[0054] As used herein, a plurality of compounds, elements, or steps may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary.

[0055] Although the present disclosure is described in detail below, it is to be understood that this disclosure is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to limit the scope of the 14NTLA-103PCT; 5640-104.PCT present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0056] While the invention is described in conjunction with the illustrated embodiments, it is understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, including equivalents of specific features, which may be included within the invention as defined by the appended claims.

[0057] The summary and detailed description, as well as the following examples, are exemplary and explanatory only and are not restrictive of the teachings. The section headings used herein are for organizational purposes only and are not to be construed as limiting the desired subject matter in any way. In the event that any literature incorporated by reference contradicts any term defined in this specification, this specification controls. All ranges given in the application encompass the endpoints unless stated otherwise. Definitions

[0058] The singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0059] Furthermore, the term “about” as used herein when referring to a measurable value such as an amount of the length of a polynucleotide or polypeptide sequence, dose, time, temperature, and the like, is meant to encompass variations of the number in context. In certain aspects, variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% may be defined. Embodiments in the specification that recite “about” various values are also contemplated as encompassing “at” the recited values.

[0060] Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0061] As used herein, “biological activity” refers to the ability of an LNP or LNP composition according to the present disclosure to elicit a measurable or observable response, which can be measured in vitro or in vivo. For example, biological activity may be a measurable or observable change in the genomic (e.g., a gene alteration) or transcriptomic (e.g., a change in expression level of a target gene) state. Such activity may also include, for example, a cellular response of a cell contacted with an LNP or LNP composition according to the present disclosure or a physiological response in an organism, including, without 15NTLA-103PCT; 5640-104.PCT limitation, a change in one or more biomarkers or physiological parameters relative to a control.

[0062] As used herein, a “buffer” refers to a solution that resists changes in pH when small amounts of an acid or base are added to the buffer solution.

[0063] “Cas nuclease”, as used herein, encompasses Cas cleavases, Cas nickases, and dCas DNA-binding agents. Cas cleavases / nickases and dCas DNA-binding agents include a Csm or Cmr complex of a type III CRISPR system, the Cas10, Csm1, or Cmr2 subunit thereof, a Cascade complex of a type I CRISPR system, the Cas3 subunit thereof, and Class 2 Cas nucleases, including Type II Cas nucleases.

[0064] As used herein, a “Class 2 Cas nuclease” is a single-chain polypeptide with RNA- guided DNA-binding activity. Class 2 Cas nucleases include Class 2 Cas cleavases and Class 2 Cas nickases (e.g., H840A, D10A, or N863A variants), which have RNA-guided DNA cleavase or nickase activity, and Class 2 dCas DNA-binding agents, in which cleavase / nickase activity is inactivated. Class 2 Cas nucleases that may be used with the LNP compositions described herein include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof. Cpf1 protein, Zetsche et al., Cell, 163: 1-13 (2015), is homologous to Cas9, and contains a RuvC-like nuclease domain. Cpf1 sequences of Zetsche are incorporated by reference in their entirety. See, e.g., Zetsche, Tables 2 and 4. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11): 722- 36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015). In some embodiments, a Class 2 Cas nuclease can be derived from S. pyogenes or N. meningitidis, e.g., a S. pyogenes Cas 9 (or “SpyCas9”), or a N. meningitidis Cas9 (or “NmeCas9”, e.g., Nme1Cas9 or Nme2Cas9).

[0065] As used herein, the term “Chimeric Antigen Receptor” or alternatively a “CAR” refers to a set of polypeptides, typically two in the simplest embodiments, which, when in an immune effector cell, provides the cell with specificity for a target cell and with intracellular signal generation. In some embodiments, a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain, and an intracellular domain comprising a functional signaling domain derived from a stimulatory molecule (e.g., an activation domain) and / or costimulatory molecule (e.g., a costimulatory domain). In some aspects, the set of polypeptides are contiguous with each other. 16NTLA-103PCT; 5640-104.PCT

[0066] As used herein, the terms “editing efficiency”, “editing percentage”, “indel efficiency,” “percent indels,” and “gene editing activity” refer to the total number of sequence reads with insertions or deletions relative to the total number of sequence reads. For example, editing efficiency at a target location in a genome may be measured by isolating and sequencing genomic DNA to identify the presence of insertions and deletions introduced by gene editing. In some embodiments, editing efficiency is measured as a percentage of cells that no longer contain a gene (e.g., CD3) after treatment, relative to the number of the cells that initially contained that gene (e.g., CD3+ cells).

[0067] As used herein, “encapsulation efficiency” refers to the amount of a therapeutic and / or prophylactic that becomes part of a nanoparticle composition, relative to the initial total amount of therapeutic and / or prophylactic used in the preparation of a nanoparticle composition. For example, if 97 mg of therapeutic and / or prophylactic are encapsulated in a nanoparticle composition out of a total 100 mg of therapeutic and / or prophylactic initially provided to the composition, the encapsulation efficiency may be given as 97%. As used herein, “encapsulation” may refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement of cargo.

[0068] The term “excipient” includes any ingredient other than the compound(s) of the disclosure, the other lipid component(s) and the biologically active agent. An excipient may impart either a functional (e.g. drug release rate controlling) and / or a non-functional (e.g. processing aid or diluent) characteristic to the compositions. The choice of excipient will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

[0069] As used herein “expression activity” refers to the ability of an LNP or LNP composition according to the present disclosure to elicit a measurable or observable change in expression of one or more genes in a cell or a tissue contacted with the LNP or the LNP composition. Expression activity includes translation of an mRNA to produce a protein encoded by an mRNA. For example, expression activity may be through LNP delivery of a cargo to a cell or tissue that results in the expression or translation of the cargo, such as a cargo comprising a plasmid DNA carrying an expression cassette capable of expressing a ribonucleic acid or a cargo comprising an mRNA encoding protein.

[0070] As used herein, “formulatability” means the ability of LNPs to meet one or more desired parameters, including but not limited to encapsulation efficiency of more than 50%, particle size of 80-120 nm, PDI less than 0.1, free-thaw stability, or a combination thereof. 17NTLA-103PCT; 5640-104.PCT

[0071] As used herein, “gene editing system” means an engineered cleavage systems to induce a double strand break (DSB) or a nick (e.g., a single strand break, or SSB) in a target DNA sequence. Cleavage or nicking can occur through the use of specific nucleases such as the engineered ZFN, TALENs, megaTALs, meganucleases (mns, also termed homing endonucleases), including an ARC nuclease of the ARCUS gene editing system, or using a guided system, for example, TtAgo or CRISPR / Cas system with an engineered guide DNA or RNA, respectively, to guide specific cleavage or nicking of a target DNA sequence.

[0072] As used herein, the term “genome editing tool” (or “gene editing tool”) is any component of “genome editing system” (or “gene editing system”) necessary or helpful for producing an edit in the genome of a cell. In some embodiments, the present disclosure provides for methods of delivering genome editing tools of a genome editing system a CRISPR / Cas system) to a cell (or population of cells).

[0073] Genome editing tools include, for example, nucleases capable of making single or double strand break in the DNA or RNA of a cell, e.g., in the genome of a cell. The genome editing tools, e.g. nucleases, may optionally modify the genome of a cell without cleaving the nucleic acid. A genome editing nuclease or nickase may be encoded by an mRNA. Such nucleases include, for example, RNA-guided DNA binding agents, and CRISPR / Cas components. Genome editing tools include fusion proteins, including e.g., a nickase fused to an effector domain such as an editor domain. Genome editing tools include any item necessary or helpful for accomplishing the goal of a genome edit, such as, for example, guide RNA, sgRNA, dgRNA, donor nucleic acid, and the like.

[0074] “Guide RNA”, “gRNA”, and “guide” are used herein interchangeably to refer to a cognate guide nucleic acid for an RNA-guided DNA-binding agent. Guide RNAs can include modified RNAs as described herein. A gRNA may be either a crRNA (also known as CRISPR RNA), or the combination of a crRNA and a trRNA (also known as tracrRNA). The crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). “Guide RNA” or “gRNA” refers to a sgRNA or a dgRNA. The trRNA may be a naturally-occurring sequence, or a trRNA sequence with modifications or variations compared to naturally- occurring sequences.

[0075] As used herein, a “guide RNA nucleic acid” may refer to a gRNA (e.g. an sgRNA or a dgRNA) or a gRNA expression cassette, which is a nucleic acid (e.g., plasmid DNA) that encodes one or more gRNAs. 18NTLA-103PCT; 5640-104.PCT

[0076] As used herein, a “guide sequence” refers to a sequence within a gRNA that is complementary to a target sequence and functions to direct a gRNA to a target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA-binding agent. A “guide sequence” may also be referred to as a “targeting sequence,” or a “spacer sequence.” A guide sequence can be 20 base pairs in length, e.g., in the case of Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs / orthologs. Shorter or longer sequences can also be used as guides, e.g., 15-, 16-, 17-, 18-, 19-, 21-, 22-, 23-, 24-, or 25-nucleotides in length. In some aspects, the target sequence is in a gene or on a chromosome, for example, and is complementary to the guide sequence. In some aspects, the degree of complementarity or identity between a guide sequence and its corresponding target sequence may be about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some aspects, the guide sequence and the target region may be 100% complementary or identical over a region of at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides. In other aspects, the guide sequence and the target region may contain at least one mismatch. For example, the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches, where the total length of the target sequence is at least 17, 18, 19, 20 or more base pairs. In some aspects, the guide sequence and the target region may contain 1-4 mismatches where the guide sequence comprises at least 17, 18, 19, 20 or more nucleotides. In some aspects, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches where the guide sequence comprises 20 nucleotides.

[0077] As used herein, the term “ionizable lipid” means a lipid with a basic amine in its structure, e.g., a cationic lipid, having at least one protonatable or deprotonatable group, such that the lipid is positively charged at a first pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will be understood by one of ordinary skill in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. In some embodiments, ionizable lipids have a pKa of the protonatable group in the range of about 4 to about 7. The pKa is the negative logarithm of the acid dissociation constant (Ka) of a protonatable group on a molecule (i.e., a group on a molecule capable of forming an acid and a conjugate base in solution), which is a measure of the acidic strength of the protonatable group in solution. When the solution pH is equal to the 19NTLA-103PCT; 5640-104.PCT pKa of the protonatable group, the concentration of acid form and conjugate base form of the protonatable group are equal.

[0078] As used herein, “lipid component,” refers to the lipid portion of the LNP, excluding a cargo encapsulated in the LNP, which form or contribute to the structure of the particle. For example, the lipid component may include an ionizable lipid, a neutral lipid, a helper lipid, and a PEG-lipid (e.g., a stealth or structural-PEG lipid, an anchor PEG-lipid, or a combination thereof).

[0079] As used herein, “LNP size distribution” means the distribution of the of LNP particle size by volume, mass, or particle number. Various techniques are known in the art for measuring particle size distribution, including, but not limited to, dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA).

[0080] The “mean diameter size,” also referred to as “mean size” of the LNPs represents the average diameter of all particles analyzed in the sample. The mean size may be determined by techniques known in the art, for example determined by Nanoparticles Tracking Analysis (NTA).

[0081] As used herein, the molar ratio (“mol %”) means a ratio of the number of moles a first component to the number of moles of a second component. Embodiments of the present disclosure provide lipid compositions described according to the respective molar ratios of the component lipids in the composition. All mol % numbers are given as a fraction of the lipid component of the lipid composition or, more specifically, the LNP compositions. In some embodiments, the lipid mol % of a lipid relative to the total lipid content (i.e., an ionizable lipid, a helper lipid, a neutral lipid, and a PEG- lipid) of a LNP will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the specified, nominal, or actual mol % of the lipid. In some embodiments, the lipid mol % of a lipid relative to the lipid component will be ±4 mol %, ±3 mol %, ±2 mol %, ±1.5 mol %, ±1 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.05 mol % of the specified, nominal, or actual mol % of the lipid component. In certain embodiments, the lipid mol % will vary by less than 15%, less than 10%, less than 5%, less than 1%, or less than 0.5% from the specified, nominal, or actual mol % of the lipid. In some embodiments, the mol % numbers are based on nominal concentration. As used herein, “nominal concentration” refers to concentration based on the input amounts of substances combined to form a resulting composition. For example, if 100 mg of solute is added to a 1 L solution, the nominal concentration is 100 mg / L. In some embodiments, the mol % numbers are based on actual concentration, e.g., concentration determined by an analytic method. In 20NTLA-103PCT; 5640-104.PCT some embodiments, actual concentration of the lipids of the lipid component may be determined, for example, from chromatography, such as liquid chromatography, followed by a detection method, such as charged aerosol detection. In some embodiments, actual concentration of the lipids of the lipid component may be characterized by lipid analysis, AF4-MALS, NTA, and / or cryo-EM. All mol % numbers are given as a percentage of the lipids of the lipid component.

[0082] “mRNA” refers to a polynucleotide and comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by a ribosome and amino-acylated tRNAs). mRNA can comprise a phosphate-sugar backbone including ribose residues or analogs thereof, e.g., 2’-methoxy ribose residues. In some embodiments, the sugars of an mRNA phosphate-sugar backbone consist essentially of ribose residues, 2’- methoxy ribose residues, or a combination thereof. In general, mRNAs do not contain a substantial quantity of thymidine residues (e.g., 0 residues or fewer than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% thymidine content). An mRNA can contain modified uridines at some or all of its uridine positions.

[0083] The term “PEG” as used herein means any polyethylene glycol or other polyalkylene ether polymer, such as an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In certain aspects, the PEG moiety is unsubstituted. Alternatively, the PEG moiety may be substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. For example, the PEG moiety may comprise a PEG copolymer such as PEG- polyurethane or PEG-polypropylene (see, e.g., J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)); alternatively, the PEG moiety may be a PEG homopolymer. In some embodiments, the term “PEG” does not include PEG copolymers. In some embodiments, the PEG has a molecular weight of from about 130 to about 50,000.

[0084] A “PEG lipid” is a lipid modified with a polyethylene glycol unit and unless otherwise specified may refer to both a structural PEG-lipid and an anchor PEG-lipid.

[0085] As used herein, the term “structural PEG-lipid” refers to a lipid comprising a polyethylene glycol “PEG” component that is not functionalized with a coupling moiety. For example, an anchor PEG-lipid and a structural PEG-lipid may both comprise a PEG2K- DSPE, except that the anchor PEG-lipid further comprises a coupling moiety, such as a maleimide functional group. “Structural PEG-lipid” may also be referred to as a “stealth 21NTLA-103PCT; 5640-104.PCT lipid.” As used herein, the term “stealth lipid” refers to lipids that alter the length of time the nanoparticles can exist in vivo (e.g., in the blood). Stealth lipids may assist in the formulation process by, for example, reducing particle aggregation and controlling particle size. Stealth lipids used herein may modulate pharmacokinetic properties of the LNP compositions or aid in stability of LNPs ex vivo. In some aspects, a stealth lipid is a PEG lipid.

[0086] As used herein, the term “anchor PEG-lipid” refers to a modified PEG-lipid that has an attached coupling moiety. The coupling moiety is a functional group that can interact (e.g., chemically react to form a covalent bond or interacting through a non-covalent bond) with a corresponding functional moiety on a separate molecule, such as a targeting ligand. For example, the anchor PEG-lipid may include a first coupling moiety and a targeting ligand may include a second coupling moiety, thereby allowing the anchor PEG-lipid to couple to the targeting ligand via the respective coupling moieties.

[0087] In some embodiments, the “anchor PEG-lipid” and the “structural PEG-lipid” may comprise the same PEG-lipid.

[0088] As used herein, the phrase “pharmaceutically acceptable carrier” refers to any and all solvents, dispersion media, coatings, antibacterial agents, antimicrobial agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers include, but are not limited to: water, saline, ringer’s solutions, dextrose solution, and about 5% human serum albumin. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0089] As used herein, the “polydispersity index” is a ratio that describes the homogeneity of the particle size distribution of a system. A small value, e.g., less than 0.1 indicates a narrow particle size distribution.

[0090] As used herein, “ribonucleoprotein” (RNP) or “RNP complex” refers to a gRNA together with an RNA-guided DNA-binding agent, such as a Cas nuclease, e.g., a Cas cleavase, Cas nickase, or dCas DNA-binding agent (e.g., Cas9). In some aspects, the gRNA guides the RNA-guided DNA-binding agent such as Cas9 to a target sequence, and the gRNA hybridizes with and the agent binds to the target sequence; in cases where the agent is a cleavase or nickase, binding can be followed by cleaving or nicking. 22NTLA-103PCT; 5640-104.PCT

[0091] As used herein, an “RNA-guided DNA-binding agent” means a polypeptide or complex of polypeptides having RNA and DNA-binding activity, or a DNA-binding subunit of such a complex, wherein the DNA-binding activity is sequence-specific and depends on the sequence of the RNA. Exemplary RNA-guided DNA-binding agents include Cas cleavases / nickases and inactivated forms thereof (“dCas DNA-binding agents”).

[0092] “Stability,” “stabilized,” and “stable” refers to the resistance of LNPs to changes in one or more physical properties, such changes to LNP size or percent encapsulation (%E). Stability may be determined using methods described in the art, e.g., fluorescence-based assays, such as a RiboGreen assay.

[0093] As used herein, “freeze / thaw stability” refers to the ability of LNPs to resist changes to physical properties (for example LNP size or the percent encapsulation (%E)) in response to freezing (at least overnight) followed by thawing. For example, freeze / thaw stability may be determined by comparing one or more physical properties of an LNP prior to freezing with one or more physical properties after freezing and thawing (i.e., a freeze / thaw cycle) using methods described in the art, e.g., fluorescence-based assays, such as a RiboGreen assay. Ionizable Lipids for Use in Lipid Nanoparticles (LNPs)

[0094] The disclosure provides ionizable lipids that can be used in LNP compositions.

[0095] The compounds of Formula (I)-(V) or Table 1 of the present disclosure may form salts depending upon the pH of the medium they are in. For example, in a slightly acidic medium, the compounds of Formula (I)-(V) or Table 1 may be protonated and thus bear a positive charge. Conversely, in a slightly basic medium, such as, for example, blood where pH is approximately 7.35, the compounds of Formula (I)-(V) or Table 1 may not be protonated and thus bear no charge. In some embodiments, the compounds of Formula (I)- (V) or Table 1 of the present disclosure may be predominantly protonated at a pH of at least about 9. In some embodiments, the compounds of Formula (I)-(V) or Table 1 of the present disclosure may be predominantly protonated at a pH of at least about 10.

[0096] The pH at which a compound of Formula (I)-(V) or Table 1 is predominantly protonated is related to its intrinsic pKa. In some embodiments, a salt of a compound of Formula (I)-(V) or Table 1 of the present disclosure has a pKa in the range of from about 5.1 to about 8.0, even more preferably from about 5.5 to about 7.6. In some embodiments, a salt of a compound of Formula (I)-(I) or Table 1 of the present disclosure has a pKa in the range of from about 5.7 to about 8, from about 5.7 to about 7.6, from about 6 to about 8, from about 6 to about 7.5, from about 6 to about 7, from about 6 to about 6.9, from about 6 to about 6.5, 23NTLA-103PCT; 5640-104.PCT from about 6.1 to about 6.9, or from about 6 to about 6.85. In some embodiments, a salt of a compound of Formula (I)-(V) or Table 1 of the present disclosure has a pKa of about 6.0, about 6.1, about 6.1, about 6.2, about 6.3, about 6.4, about 6.6, about 6.7, about 6.8, or about 6.9. Alternatively, a salt of a compound of Formula (I)-(V) or Table 1 of the present disclosure has a pKa in the range of from about 6 to about 8. The pKa of a salt of a compound of Formula (I)-(V) or Table 1 can be an important consideration in formulating LNPs, as it has been found that LNPs formulated with certain lipids having a pKa ranging from about 5.5 to about 7.0 are effective for delivery of cargo in vivo. Further, it has been found that LNPs formulated with certain lipids having a pKa ranging from about 5.3 to about 6.4 are effective for delivery in vivo, e.g. to tumors. See, e.g., WO 2014 / 136086. In some embodiments, the ionizable lipids are positively charged at an acidic pH but neutral in the blood.

[0097] In certain aspects, a lipid nanoparticle (LNP) is provided comprising (i) a lipid component comprising: a) an anchor PEG-lipid; b) a helper lipid; c) a neutral lipid; d) a structural PEG-lipid; and e) an ionizable lipid represented by any one of structural Formula I, ora sa t t ereo , wherein,, , t bond, X1is a C1-5alkylene, 24NTLA-103PCT; 5640-104.PCT R1and R2are each independently a C1-3alkyl, or R1taken together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X1form a 4-, 5-, or 6-membered ring, or R1taken together with R2and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, and R3is H or C1-3alkyl, Z1and Z2are each independently a C1-5 alkylene, Z3and Z4are each independently a -C(=O)O- in either direction, Z5and Z6are each independently a direct bond or a C1-3 alkylene, Y1is selected from H, a C1-10alkyl, C3-10alkenyl, and C3-10alkynyl, Y2, Y3, and Y4are each independently selected from a C3-10 alkyl, C3-10 alkenyl, or C3- 10alkynyl, and n is 0 or 1; and (ii) a first coupling moiety attached to the anchor PEG-lipid; and (iii) a cargo, wherein the cargo is encapsulated in the LNP.

[0098] In some aspects, the compound of Formula I is represented by structural formula Ia, a).

[0100] In some aspects, A is O. Alternatively, A is NH. Alternatively yet, A is a direct bond.

[0101] In some aspects, X1is a C1-4 alkylene, C1-3 alkylene, C1-5 alkylene, C1-2 alkylene, C2-5 alkylene, C2-4alkylene, or C2-3alkylene. For example, X1is a C2-3alkylene, such as a C2alkylene or a C3 alkylene. For example, X1is C2 alkylene.

[0102] In some aspects, Z1is a C1-4 alkylene, C1-3 alkylene, C1-5 alkylene, C1-2 alkylene, C2-5 alkylene, C2-4 alkylene, or C2-3 alkylene. For example, Z1is a C2-3 alkylene, such as a C2 alkylene or a C3 alkylene. 25NTLA-103PCT; 5640-104.PCT

[0103] In some aspects, Z2is a C1-4alkylene, C1-3alkylene, C1-5alkylene, C1-2alkylene, C2-5alkylene, C2-4 alkylene, or C2-3 alkylene. For example, Z1is a C2-3 alkylene, such as a C2 alkylene or a C3alkylene.

[0104] In some aspects, Z1and Z2is each independently C3alkylene.

[0105] In some aspects, Z1and Z2is each independently C5 alkylene.

[0106] In certain aspects, Z1is C2alkylene; and Z2is C3alkylene.

[0107] In some aspects, Z1and Z2is each independently C3 alkylene, A is NH, and X1is a C2 alkylene.

[0108] In some aspects, Z3and Z4is eac , wherein a indicates the point of attachment to Z1and Z2, respectively.O

[0109] In some aspects, Z3is b O , wherein b indicates the point of attachment to Z1;and Z4is , wherein b indicates the point of attachment to Z2.

[0110] Iects, Z5and Z6is each independently a direct bond.

[0111] In some aspects, Z5is C1alkylene; and Z6is a direct bond.

[0112] In some aspects, Y1is H. In some aspects, Y1, Y2, Y3, and Y4is each independently a C3-9alkyl. For example, Y1, Y2, Y3, and Y4 is each independently a C4-9 alkyl, C5-9 alkyl ̧C6-9alkyl, C7-9 alkyl ̧C8-9 alkyl, C3-8 alkyl, C3-7 alkyl, C3-6 alkyl, C3-5 alkyl¸ C3-4 alkyl, C4-8 alkyl,C4-7 alkyl, C4-6 alkyl, C4-5 alkyl ̧C5-8 alkyl, C5-7 alkyl, C5-6 alkyl, C6-8 alkyl ̧C6-7 alkyl, or C7-8alkyl. For example, Y1, Y2, Y3, and Y4is each independently a C7-9 alkyl.

[0113] In some aspects, Y1and Y2is each independently a C5-7alkyl and Y3and Y4is each independently a C3-5 alkyl.

[0114] In some aspects, R1and R2is each independently a C1-3alkyl. For example, R1and R2is each independently methyl ethyl, propyl, or isopropyl.

[0115] In some aspects, R1taken together with R2and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring. For example, R1taken together with R2and the nitrogen atom to which they are attached forms a 5-membered ring. Alternatively, R1taken together with R2and the nitrogen atom to which they are attached forms a 6-membered ring. Alternatively yet, R1taken together with R2and the nitrogen atom to which they are attached forms a 7-membered ring. 26NTLA-103PCT; 5640-104.PCT

[0116] In some aspects, the compound of Formula I is represented by one of the following structural formulae: , ,NTLA-103PCT; 5640-104.PCT , , ,NTLA-103PCT; 5640-104.PCT ,NTLA-103PCT; 5640-104.PCT

[0117] In some aspects, the compound of Formula I is represented by one of the following structural formulae: , ,NTLA-103PCT; 5640-104.PCT , , ,NTLA-103PCT; 5640-104.PCT nd oa sa e eo .

[0118] In certain aspects, an LNP is provided comprising: (i) a lipid component comprising: (a) an anchor PEG-lipid; (b) a helper lipid; (c) a neutral lipid; 32NTLA-103PCT; 5640-104.PCT (d) a structural PEG-lipid; and (e) an ionizable lipid comprising: (ii)(iii) a cargo, wherein the cargo is encapsulated in the LNP.

[0119] In certain aspects, an LNP is provided comprising: (i) a lipid component comprising: (a) an anchor PEG-lipid; (b) a helper lipid; (c) a neutral lipid; (d) a structural PEG-lipid; and (e) an ionizable lipid represented by any one of structural Formula II, [Formula II], wherein: Q is CH oA is O or NH, X1is a C1-5alkylene, R1and R2are each independently a C1-3 alkyl, or 33NTLA-103PCT; 5640-104.PCT R1taken together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X1form a 4-, 5-, or 6-membered ring, or R1taken together with R2and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, and Z1is a C2-9 alkylene, Z2is a C1-3alkylene or a direct bond, and Y1and Y2are each independently selected from a C3-10 alkyl, C3-10 alkenyl, or C3-10alkynyl; (ii) a first coupling moiety attached to the anchor PEG-lipid; and (iii) a cargo, wherein the cargo is encapsulated in the LNP.

[0120] In some aspects, the compound of Formula II is represented by structural Formula IIa, Ia).

[0122] In some aspects, X1is a C1-4 alkylene, C1-3 alkylene, C1-5 alkylene, C1-2 alkylene, C2-5 alkylene, C2-4alkylene, or C2-3alkylene. For example, X1is a C2-3alkylene, such as a C2alkylene or a C3 alkylene. For example, X1is a C3 alkylene.

[0123] In some aspects, Z1is a C3-9alkylene. For example, Z1is a C4-9alkylene, C5-9alkylene ̧C6-9 alkylene, C7-9 alkylene ̧C8-9 alkylene, C3-8 alkylene, C3-7 alkylene, C3-6 alkylene,C3-5alkylene¸ C3-4alkylene, C4-8alkylene, C4-7alkylene, C4-6alkylene, C4-5alkylene¸ C5-8alkylene, C5-7 alkylene, C5-6 alkylene, C6-8 alkylene¸ C6-7 alkylene, or C7-8 alkoxy. For example, Z1is a C3-5 alkylene, C5-7 alkylene, or C7-9 alkylene.

[0124] In some embodiment, Z2is a direct bond. In some embodiment, Z2is a C1-3alkylene.

[0125] In some aspects, Y1and Y2is each independently a C3-9 alkyl. For example, Y1and Y2is each independently a C4-9 alkyl, C5-9 alkyl ̧C6-9 alkyl, C7-9 alkyl¸ C8-9 alkyl, C3-8 alkyl, C3-7alkyl, C3-6 alkyl, C3-5 alkyl ̧C3-4 alkyl, C4-8 alkyl, C4-7 alkyl, C4-6 alkyl, C4-5 alkyl ̧C5-8 alkyl,C5-7 alkyl, C5-6 alkyl, C6-8 alkyl ̧C6-7 alkyl, or C7-8 alkyl. In some aspects, Y1and Y2is each independently a C3-5 alkyl, C5-7 alkyl, or C7-9 alkyl. 34NTLA-103PCT; 5640-104.PCT

[0126] In some aspects, the compound of Formula II is represented by one of the following structural formulae: , , , ,NTLA-103PCT; 5640-104.PCT , , , , , ,NTLA-103PCT; 5640-104.PCT , , ,

[0127] In certain aspects, an LNP is provided comprising: (i) a lipid component comprising: (a) an anchor PEG-lipid; (b) a helper lipid; (c) a neutral lipid; (d) a structural PEG-lipid; and (e) an ionizable lipid represented by any one of structural Formula III, [Formula III] O 2 1 37NTLA-103PCT; 5640-104.PCT or a salt thereof, wherein: A is O or NH, X1is a C1-5alkylene, R1and R2is each independently a C1-3 alkyl, or R1taken together with R2and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, and Z1is a C1-5alkylene, Y1and Y2is each independently a C3-10 alkoxyl or O(C3-10 alkynyl), Z2is a C1-5alkylene or a direct bond, and Y3and Y4is each independently a C3-10 alkoxyl or O(C3-10 alkynyl), or Y3and Y4is each independently a C3-10alkyl or C3-10alkynyl, provided that if Y1, Y2, Y3, and Y4is each independently a C3-10 alkoxy, then R1and R2are not C2 alkyl, and R1taken together with R2and the nitrogen atom to which they are attached do not form a 6-membered ring; (ii) a first coupling moiety attached to the anchor PEG-lipid; and (iii) a cargo, wherein the cargo is encapsulated in the LNP.

[0128] In some aspects, A is O. Alternatively, A is NH.

[0129] In some aspects, X1is a C1-4alkylene, C1-3alkylene, C1-5alkylene, C1-2alkylene, C2-5alkylene, C2-4 alkylene, or C2-3 alkylene. For example, X1is a C2-3 alkylene, such as a C2 alkylene or a C3alkylene.

[0130] In some aspects, Z1is a C1-4 alkylene, C1-3 alkylene, C1-5 alkylene, C1-2 alkylene, C2-5 alkylene, C2-4alkylene, or C2-3alkylene. For example, Z1is a C2-3alkylene, such as a C2alkylene or a C3 alkylene.

[0131] In some aspects, Z2is a C1-4alkylene, C1-3alkylene, C1-5alkylene, C1-2alkylene, C2-5alkylene, C2-4 alkylene, or C2-3 alkylene. For example, Z1is a C2-3 alkylene, such as a C2 alkylene or a C3 alkylene. In some aspects, Z2is a direct bond.

[0132] In some aspects, Z1is a C2-3alkylene and Z2is a direct bond.

[0133] In some aspects, Y1and Y2is each independently a C3-9 alkoxyl. For example, Y1andY2 is each independently a C4-9 alkoxyl, C5-9 alkoxyl ̧C6-9 alkoxyl, C7-9 alkoxyl ̧C8-9 alkoxyl,C3-8 alkoxyl, C3-7 alkoxyl, C3-6 alkoxyl, C3-5 alkoxyl¸ C3-4 alkoxyl, C4-8 alkoxyl, C4-7 alkoxyl, C4-6alkoxyl, C4-5alkoxyl¸ C5-8alkoxyl, C5-7alkoxyl, C5-6alkoxyl, C6-8alkoxyl¸ C6-7alkoxyl, or C7-8 alkoxy. For example, Y1and Y2is each independently a C6-9 alkoxyl. 38NTLA-103PCT; 5640-104.PCT

[0134] In some aspects, Y3and Y4is each independently a C3-9alkoxyl. For example, Y3and Y4 is each independently a C4-9 alkoxyl, C5-9 alkoxyl ̧C6-9 alkoxyl, C7-9 alkoxyl ̧C8-9 alkoxyl,C3-8alkoxyl, C3-7alkoxyl, C3-6alkoxyl, C3-5alkoxyl¸ C3-4alkoxyl, C4-8alkoxyl, C4-7alkoxyl, C4-6alkoxyl, C4-5alkoxyl¸ C5-8alkoxyl, C5-7alkoxyl, C5-6alkoxyl, C6-8alkoxyl¸ C6-7alkoxyl, or C7-8 alkoxy. For example, Y3and Y4is each independently a C6-9 alkoxyl.

[0135] In some aspects, Y3and Y4is each independently a C3-9alkyl. For example, Y3and Y4is each independently a C4-9 alkyl, C5-9 alkyl ̧C6-9 alkyl, C7-9 alkyl¸ C8-9 alkyl, C3-8 alkyl, C3-7alkyl, C3-6 alkyl, C3-5 alkyl ̧C3-4 alkyl, C4-8 alkyl, C4-7 alkyl, C4-6 alkyl, C4-5 alkyl ̧C5-8 alkyl,C5-7 alkyl, C5-6 alkyl, C6-8 alkyl ̧C6-7 alkyl, or C7-8 alkyl. For example, Y3is a C6-9 alkyl and Y4is a C3-5alkyl.

[0136] In some aspects, R1and R2is each independently a C1-3 alkyl. For example, R1and R2is each independently methyl ethyl, propyl, or isopropyl.

[0137] In some aspects, the compound of Formula III is represented by one of the following structural formulae: , ndNTLA-103PCT; 5640-104.PCT ,

[0138] In certain aspects, an LNP is provided comprising: (i) a lipid component comprising: (a) an anchor PEG-lipid; (b) a helper lipid; (c) a neutral lipid; (d) a structural PEG-lipid; and (e) an ionizable lipid represented by any one of structural Formula IV, [Formula IV] w, urrence, X1is C5-11 alkylene, Y1is C3-11alkylene,, a bond to R1, Z1is C2-4 alkylene, Z2is selected from -OH, -NH2, -OC(=O)R3, -OC(=O)NHR3, -NHC(=O)NHR3, and - NHS(=O)2R3, R1is C4-12alkyl or C3-12alkenyl, 40NTLA-103PCT; 5640-104.PCT each R2is independently C4-12alkyl, and R3is C1-3 alkyl, or a salt thereof; (ii) a first coupling moiety attached to the anchor PEG-lipid; and (iii) a cargo, wherein the cargo is encapsulated in the LNP.

[0139] In some aspects, X1is linear C5-11alkylene. In some aspects, X1is linear C6-10alkylene. In some aspects, X1is linear C6 alkylene, linear C7 alkylene, linear C8 alkylene, or linear C9alkylene.

[0140] In some aspects, Y1is linear C4-9 alkylene. In some aspects, Y1linear C6-8 alkylene. In some aspects, Y1is linear C7alkylene.

[0141] In some aspects, R1is C4-12 alkenyl. In some aspects, R1is C9 alkenyl.

[0142] In some aspects, Y2is .

[0143] In some aspects, Y1, Y, an are selected to form a linear chain of 16-21 atoms. In some aspects, Y1, Y2, and R1are selected to form a linear chain of 16-18 atoms.

[0144] In some aspects, Z1is linear C2-4 alkylene. In some aspects, Z1is C2 alkylene or C3 alkylene.

[0145] In some aspects, Z2is -OH. In some aspects, Z2is -NH2. In some aspects, Z2is - OC(=O)R3,-OC(=O)NHR3, -NHC(=O)NHR3, or -NHS(=O)2R3.

[0146] In some aspects, R3is methyl.

[0147] In some aspects, R1is a linear C4-12alkyl. In some aspects, R1is a linear C8-10alkyl. In some aspects, R1is a linear C9 alkyl. In some aspects, R1is a branched C6-12 alkyl. In some aspects, R1is a branched C8 alkyl, a branched C9 alkyl, or a branched C10 alkyl. In some aspects, each R2, independently, is a linear C5-12 alkyl. In some aspects, each R2, independently, is a linear C6-8 alkyl. In some aspects, each R2, independently, is a branched C5-12alkyl. In some aspects, each R2, independently, is a branched C6-8alkyl.

[0148] In some aspects, X1and one of the R2moieties are selected to form a linear chain of 16-18 atoms, including the carbon and oxygen atoms of the acetal.

[0149] In some aspects, the compound of Formula IV is represented by one of the following structural formulae: 41NTLA-103PCT; 5640-104.PCT42NTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCT , or a salte eo .

[0150] In certain aspects, an LNP is provided comprising: (i) a lipid component comprising: (a) an anchor PEG-lipid; (b) a helper lipid; (c) a neutral lipid; (d) a structural PEG-lipid; and (e) an ionizable lipid represented by any one of structural Formula V, [Formula V] 49NTLA-103PCT; 5640-104.PCTX1is O, NR1, or a direct bond, X2is C2-5 alkylene, X3is C(=O) or a direct bond, R1is H or Me, R3is C1-3 alkyl, R2is C1-3 alkyl, or R2taken together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X2form a 4-, 5-, or 6-membered ring, or X1is NR1, R1and R2taken together with the nitrogen atoms to which they are attached form a 5- or 6-membered ring, or R2taken together with R3and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y1is C2-12 alkylene, Y2is selected from (ientation),on), (n e t er orentation), 50NTLA-103PCT; 5640-104.PCT n is 0 to 3, R4is C1-15 alkyl, Z1is C1-6alkylene or a direct bond, on) or absent, provided that if Z1is a direct bond, Z2is absent;R5is C5-9 alkyl or C6-10 alkoxy, R6is C5-9 alkyl or C6-10 alkoxy, W is methylene or a direct bond, and R7is H or Me, or a salt thereof, provided that if R3and R2are C2alkyls, X1is O, X2is linear C3alkylene, X3is C(=O), Y1is linear C6 alkylene, (Y2)n-R4is l1, Z is C2 alkylene, Z2is absent, W is methylene, and R7is H, then R5and R6are not C8 alkoxy; (ii) a first coupling moiety attached to the anchor PEG-lipid; and (iii) a cargo, wherein the cargo is encapsulated in the LNP.

[0151] In some aspects, the ionizable compound is a compound of Formula Va

[0152] In some aspects, X is O, NR , or a direct bond, X2is C2-5alkylene, X3is C(=O) or a direct bond, R1is H or Me, R3is C1-3alkyl,

[0153] In some aspects, R2is C1-3 alkyl, or R2taken together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X2form a 4-, 5-, or 6-membered ring, or X1is NR1, R1and R2taken together with the nitrogen atoms to which they are attached form a 5- or 6- 51NTLA-103PCT; 5640-104.PCT membered ring, or R2taken together with R3and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring.

[0154] In some aspects, Y1is C2-12alkylene and Y2is selected from (in either, p , ;5-9alkyl or C6-10 alkoxy, R6is C5-9 alkyl or C6-10 alkoxy, W is methylene or a direct bond, and R7is H or Me, or a salt thereof, provided that if R3and R2are C2alkyls, X1is O, X2is linear C3alkylene, X3is C(=O), Y1is linear C6alkylene, Y2 is R4 is linear C4alkyl, Z1is C2 alkylene, Z2is absent, W is methylen6and R are not C8 alkoxy.

[0157] In some aspects, X2is linear C2 alkylene, a linear C3 alkylene, or a linear C4 alkylene.

[0158] In some aspects, R3is a C1alkyl or a C2alkyl. In some aspects, R2is C1alkyl or a C2alkyl.

[0159] In some aspects, R2taken together with the nitrogen atom and 1-2 carbon atoms of X2form a 5-membered ring. In some aspects, R2taken together with the nitrogen atom and 1-3 carbon atoms of X2form a 6-membered ring. In some aspects, R2and R3taken together with the nitrogen atom form a 5-membered ring.

[0160] In some aspects, X1is NH or a direct bond. In some aspects, Y1is a linear C3-10 alkylene.

[0161] In some aspects, Y1is a linear C4-8 alkylene. In some aspects, Y1is a linear C5-7 alkylene.

[0162] In some aspects, R4is a linear C4-14 alkyl. In some aspects, R4is a linear C6-12 alkyl.

[0163] In some aspects, Z1is a linear C2-4 alkylene.

[0164] In some aspects, R5and R6are each independently a linear C5-9 alkyl. In some aspects, R5and R6are each independently a linear C6-8 alkyl. In some aspects, R5and R6are each independently a linear C7-9alkoxy. 52NTLA-103PCT; 5640-104.PCT

[0165] In some aspects, Y2is 2 isso e aspecs, , , a ae seece o o a ea ca o - atoms, including the carbon and oxygen atoms of the ester and the acetal. In some aspects, Y1, Y2, and R4are selected to form a linear chain of 14-24 atoms, including the carbon and oxygen atoms of the ester(s), if present.

[0168] In some aspects, the compound of Formula V is represented by one of the following structural formulae:NTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCT, or a salt thereof.

[0169] In certain aspects, an LNP is provided comprising: (i) a lipid component comprising: (a) an anchor PEG-lipid; (b) a helper lipid; 73NTLA-103PCT; 5640-104.PCT (c) a neutral lipid; (d) a structural PEG-lipid; and (e) an ionizable lipid represented by any one of structural Formulae I-V, above, or, an ionizable lipid comprising: ,d (iii) a cargo, wherein the cargo is encapsulated in the LNP, and wherein the LNP is stable following storage at about -70 to -80 °C.

[0170] In some aspects, an LNP composition of the present disclosure is stable. For example, in some aspects, an LNP composition of the present disclosure can be stored at room temperature for more than 2 hours or at 4° C overnight without precipitation. In some aspects, an LNP of the present disclosure is stable following storage for at least one year at about -70 to -80° C. In some aspects, the LNP, after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 freeze / thaw cycles retains at least 80%, 85%, 90%, 95%, 98%, or 99% of the biological activity of the LNP before the freeze / thaw cycle(s). In some aspects, the LNP, after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 freeze / thaw cycles retains at least 80%, 85%, 90%, 95%, 98%, or 99% encapsulation efficiency of the LNP before the freeze / thaw cycle(s). In some aspects, the LNP, after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 freeze / thaw cycles retains at least 80%, 85%, 90%, 95%, 98%, or 99% LNP size distribution of the LNP before the freeze / thaw cycle(s). In some aspects, the LNP, after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 freeze / thaw cycles retains at least 80%, 85%, 90%, 95%, 98%, or 99% LNP mean size of the LNP before the freeze / thaw cycle(s). In some aspects, the LNP, after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 freeze / thaw cycles retains at least 80%, 85%, 90%, 95%, 98%, or 99% expression activity of the LNP before the freeze / thaw cycle(s). In some aspects, the LNP, after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 freeze / thaw cycles retains at least 80%, 85%, 90%, 95%, 98%, or 99% gene editing activity of the LNP before the freeze / thaw cycle(s). In some aspects, the LNP, after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 freeze / thaw cycles 74NTLA-103PCT; 5640-104.PCT has less than 20%, 15%, 10%, 8%, 5%, or 3% immunogenicity increase over the LNP before the freeze / thaw cycle(s). Lipid Compositions

[0171] Described herein are lipid compositions comprising at least one ionizable lipid of Formula (I)-(V) or disclosed in Table 1, a neutral lipid, a helper lipid, a structural PEG-lipid, and an anchor PEG-lipid. In some aspects, the lipid component includes a molar ratio of the ionizable lipid : neutral : helper : structural PEG-lipid : anchor PEG-lipid of 40 to 60 : 0 to 25 : 25 to 65 : 0.5 to 5 : 0.001 to 1.5. In some aspects, the lipid component includes a molar ratio of the ionizable lipid : DSPC : cholesterol : structural PEG-lipid : anchor PEG-lipid of 40 to 60 : 5 to 15 : 25 to 65 : 0.5 to 5 : 0.001 to 1.5. In some aspects, the lipid component includes a ratio of the ionizable lipid : DSPC : cholesterol : structural PEG-lipid : anchor PEG-lipid of 40 to 60 : 5 to 25 : 25 to 65 : 0.5 to 5 : 0.001 to 1.5. In some aspects, the DPSC is absent, and the targeted LNP is a 4-component formulation. In some aspects, the lipid component includes an anchor PEG-lipid, for example, included in the lipid composition in a range of 0.001 to 1.5.

[0172] The LNP composition may include a four component LNP formulation. In some aspects, the lipid component includes a ratio of the ionizable lipid : neutral lipid : helper lipid : structural PEG-lipid of 40 to 60 : 0 to 25 : 25 to 65 : 0.5 to 5, or any subranges according to any of the aspects described below. In some aspects, the four component formulation comprises an anchor PEG-lipid. For example, the neutral lipid may be absent such that the lipid component comprises ionizable lipid, helper lipid, structural PEG-lipid, and anchor PEG-lipid. For example, the neutral lipid may be absent such that the lipid component comprises a ratio of the ionizable lipid : helper lipid : structural PEG-lipid : anchor PEG-lipid of 40 to 60 : 25 to 65 : 0.5 to 5 : 0.01 to 1.5.

[0173] LNPs may also include a five component LNP formulation, including an ionizable lipid, a neutral lipid, a helper lipid, a structural PEG-lipid, and an anchor PEG-lipid, which can be conjugated to a targeting moiety capable of targeting the LNP to certain tissues. In some aspects, the lipid component includes a ratio of the ionizable lipid : neutral lipid : structural PEG-lipid of 40 to 60 : 0 to 25 : 25 to 65 : 0.5 to 5, or any subranges according to any of the aspects described above and below. The anchor PEG-lipid may be further included in the lipid component in a range of about 0.01 to about 1.5, or any subranges according to any of the aspects described below. The ratio of the structural PEG-lipid may be adjusted according to the amount of the anchor PEG-lipid. For example, the ratio of 75NTLA-103PCT; 5640-104.PCT structural PEG-lipid : anchor PEG-lipid may be a ratio of 1.5 to 2.99 : 0.01 to 1.5, a ratio of 2 to 2.985 : 0.015 to 1, a ratio of 2.1 to 2.09 : 0.02 to 0.9, a ratio of 2.2 to 2.975 : 0.025 to 0.8, a ratio of 2.3 to 2.97 : 0.03 to 0.7, a ratio of 2.4 to 2.965 : 0.035 to 0.6, a ratio of 2.5 to 0.296 : 0.04 to 0.5, a ratio of 2.52 to 2.958 : 0.042 to 0.48, or a ratio of about 2.955 : 0.045.

[0174] In some aspects, the lipid component comprises the anchor PEG-lipid in an amount from about 0.001 to about 1.5 mol % of the lipid component. In some aspects, the anchor PEG-lipid comprises about 0.05 to about 0.2 mol % of the lipid component. In some aspects, the anchor PEG-lipid comprises about 0.03 to about 0.1 mol % of the lipid component. In some aspects, the anchor PEG-lipid comprises about 0.25 to about 0.7 mol % of the lipid component. In some aspects, the anchor PEG-lipid comprises about 0.001 mol % to about 1.5 mol %, 0.002 mol % to about 1.5 mol %, 0.005 mol % to about 1.5 mol %, 0.0075 mol % to about 1.5 mol %, 0.01 mol % to about 1.5 mol %, 0.02 mol % to about 1.5 mol %, 0.03 mol % to about 1.5 mol %, 0.04 mol % to about 1.5 mol %, 0.05 mol % to about 1.5 mol %, 0.06 mol % to about 1.5 mol %, 0.07 mol % to about 1.5 mol %, 0.08 mol % to about 1.5 mol %, 0.09 mol % to about 1.5 mol %, 0.1 mol % to about 1.5 mol % of the lipid component. In some aspects, the anchor PEG-lipid comprises about 0.001 mol % to about 0.9 mol%, 0.001 mol % to about 0.8 mol%, 0.001 mol % to about 0.7 mol%, 0.001 mol % to about 0.6 mol%, 0.001 mol % to about 0.5 mol%, 0.001 mol % to about 0.4 mol%, 0.001 mol % to about 0.3 mol%, 0.001 mol % to about 0.2 mol%, or 0.001 mol % to about 0.1 mol% of the lipid component. In some aspects, the anchor PEG-lipid is included in an amount of about 0.001 mol %, 0.002 mol %, 0.005 mol %, 0.0075 mol %, 0.01 mol %, 0.02 mol %, 0.03 mol %, 0.04 mol %, 0.05 mol %, 0.06 mol %, 0.07 mol %, 0.08 mol %, 0.09 mol %, 0.1 mol %, 0.2 mol %, 0.3 mol %, 0.4 mol %, 0.5 mol %, 0.6 mol %, 0.7 mol %, 0.8 mol %, 0.9 mol %, 1.0 mol %, 1.1 mol %, 1.2 mol %, 1.3 mol %, 1.4 mol %, 1.5 mol % of the lipid component.

[0175] In some aspects and the anchor PEG-lipid is in an amount from about 0.005 to about 0.045 mol%, about 0.01 to about 0.045 mol%, about 0.015 to about 0.045 mol%, about 0.02 to about 0.045 mol% of the lipid component. In some aspects, the anchor PEG-lipid is in an amount from about 0.02 to about 0.045 mol%, about 0.02 mol% about 0.04 mol%, about 0.02 mol% to about 0.035 mol%, about 0.02 mol% to about 0.03 mol%, or about 0.02 to about 0.025 mol% of the lipid component. In some aspects, the anchor PEG-lipid is in an amount from about 0.005, 0.01, 0.02, 0.03, 0.04, or about 0.045 mol% of the lipid component.

[0176] In some aspects, the anchor PEG-lipid is in an amount from about 0.005 to about 0.075 mol%, about 0.01 to about 0.075 mol%, about 0.02 to about 0.075 mol%, about 0.03 to 76NTLA-103PCT; 5640-104.PCT about 0.075 mol% of the lipid component. In some aspects, the anchor PEG-lipid is in an amount from about 0.03 to about 0.07 mol%, 0.03 to about 0.065 mol%, 0.03 to about 0.06 mol%, 0.03 to about 0.055 mol%, 0.03 to about 0.05 mol%, 0.03 to about 0.045 mol%, or about 0.03 to about 0.04 mol% of the lipid component. In some aspects, the anchor PEG- lipid is in an amount from about 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, or about 0.075 mol% of the lipid component.

[0177] In some aspects, the LNP is conjugated to an antibody or fragment thereof. In some aspects, the LNP conjugated to an antibody or fragment thereof includes a molar ratio of the anchor PEG-lipid that is in an amount from about 0.005 to about 0.075 mol%, about 0.01 to about 0.075 mol%, about 0.02 to about 0.075 mol%, about 0.03 to about 0.075 mol% of the lipid component. In some aspects, the anchor PEG-lipid is in an amount from about 0.03 to about 0.07 mol%, 0.03 to about 0.065 mol%, 0.03 to about 0.06 mol%, 0.03 to about 0.055 mol%, 0.03 to about 0.05 mol%, 0.03 to about 0.045 mol%, or about 0.03 to about 0.04 mol% of the lipid component. In some aspects, the anchor PEG-lipid is in an amount from about 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, or about 0.075 mol% of the lipid component.

[0178] In some aspects, the anchor PEG-lipid is in an amount from about 0.1 to about 0.9 mol%, 0.2 to about 0.9 mol%, 0.3 to about 0.9 mol%, 0.4 to about 0.9 mol%, 0.5 to about 0.9 mol% of the lipid component. In some aspects, the anchor PEG-lipid is in an amount from about 0.5 to about 0.8 mol%, 0.5 to about 0.7 mol%, or 0.5 to about 0.6 mol% of the lipid component. In some aspects, the anchor PEG-lipid is in an amount from about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or about 0.9 mol% of the lipid component.

[0179] In some aspects, the LNP is conjugated to a peptide or small molecule. In some aspects, the LNP conjugated to peptide or small molecule includes a molar ratio of the anchor PEG-lipid that is in an amount from about 0.1 to about 0.9 mol%, 0.2 to about 0.9 mol%, 0.3 to about 0.9 mol%, 0.4 to about 0.9 mol%, 0.5 to about 0.9 mol% of the lipid component. In some aspects, the anchor PEG-lipid is in an amount from about 0.5 to about 0.8 mol%, 0.5 to about 0.7 mol%, or 0.5 to about 0.6 mol% of the lipid component. In some aspects, the anchor PEG-lipid is in an amount from about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or about 0.9 mol% of the lipid component.

[0180] In some aspects, the lipid component comprises the structural PEG-lipid in an amount from about 0.5 to about 5 mol % of the lipid component. In some aspects, the structural PEG-lipid comprises about 1.2 to about 2.2 mol% of the lipid component. In some aspects, the structural PEG-lipid comprises about 2.5 to about 3.5 mol% of the lipid component. In 77NTLA-103PCT; 5640-104.PCT some aspects, the structural PEG-lipid is about 1.5-3.5 mol %, about 2.0-2.7 mol %, about 2.0-3.5 mol %, about 2.3-3.5 mol %, about 2.3-2.7 mol %, about 2.5-3.5 mol %, about 2.5-2.7 mol %, about 2.9-3.5 mol %, or about 2.7 mol %. In additional embodiments, the amount of the structural PEG-lipid may be about 1.0-4.0 mol %, about 1.2-4.0 mol %, about 1.4-4.0 mol %, about 1.5-4.0 mol %, about 1.6-4.0 mol %, about 1.7-4.0 mol %, about 1.8-4.0 mol %, about 1.9-4.0 mol %, about 2.0-4.0 mol %, about 2.1-4.0 mol %, about 2.2-4.0 mol %, about 2.3-4.0 mol %, about 2.4-4.0 mol %, about 2.5-4.0 mol %, about 2.6-4.0 mol %, about 2.7-4.0 mol %, about 2.8-4.0 mol %, about 2.9-4.0 mol %, about 3.0-4.0 mol %, about 3.1-4.0 mol %, about 3.2-4.0 mol %, about 3.3-4.0 mol %, about 3.4-4.0 mol %, about 3.5-4.0 mol %, about 3.7-4.0 mol %, 1.0-3.7 mol %, about 1.2-3.7 mol %, about 1.4-3.7 mol %, about 1.5-3.7 mol %, about 1.6-3.7 mol %, about 1.7-3.7 mol %, about 1.8-3.7 mol %, about 1.9-3.7 mol %, about 2.0-3.7 mol %, about 2.1-3.7 mol %, about 2.2-3.7 mol %, about 2.3-3.7 mol %, about 2.4-3.7 mol %, about 2.5-3.7 mol %, about 2.6-3.7 mol %, about 2.7-3.7 mol %, about 2.8-3.7 mol %, about 2.9-3.7 mol %, about 3.0-3.7 mol %, about 3.1-3.7 mol %, about 3.2-3.7 mol %, about 3.3-3.7 mol %, about 3.4-3.7 mol %, about 3.5-3.7 mol %, 1.0-3.5 mol %, about 1.2-3.5 mol %, about 1.4-3.5 mol %, about 1.5-3.5 mol %, about 1.6-3.5 mol %, about 1.7-3.5 mol %, about 1.8-3.5 mol %, about 1.9-3.5 mol %, about 2.0-3.5 mol %, about 2.1-3.5 mol %, about 2.2-3.5 mol %, about 2.3-3.5 mol %, about 2.4-3.5 mol %, about 2.5-3.5 mol %, about 2.6-3.5 mol %, about 2.7-3.5 mol %, about 2.8-3.5 mol %, about 2.9-3.5 mol %, about 3.0-3.5 mol %, about 3.1-3.5 mol %, about 3.2-3.5 mol %, about 3.3-3.5 mol %, about 3.4-3.5 mol %, 1.0-3.4 mol %, about 1.2-3.4 mol %, about 1.4-3.4 mol %, about 1.5-3.4 mol %, about 1.6-3.4 mol %, about 1.7-3.4 mol %, about 1.8-3.4 mol %, about 1.9-3.4 mol %, about 2.0-3.4 mol %, about 2.1-3.4 mol %, about 2.2-3.4 mol %, about 2.3-3.4 mol %, about 2.4-3.4 mol %, about 2.5-3.4 mol %, about 2.6-3.4 mol %, about 2.7-3.4 mol %, about 2.8-3.4 mol %, about 2.9-3.4 mol %, about 3.0-3.4 mol %, about 3.1-3.4 mol %, about 3.2-3.4 mol %, about 3.3-3.4 mol %, 1.0-3.3 mol %, about 1.2-3.3 mol %, about 1.4-3.3 mol %, about 1.5-3.3 mol %, about 1.6-3.3 mol %, about 1.7-3.3 mol %, about 1.8-3.3 mol %, about 1.9-3.3 mol %, about 2.0-3.3 mol %, about 2.1-3.3 mol %, about 2.2-3.3 mol %, about 2.3-3.3 mol %, about 2.4-3.3 mol %, about 2.5-3.3 mol %, about 2.6-3.3 mol %, about 2.7-3.3 mol %, about 2.8-3.3 mol %, about 2.9-3.3 mol %, about 3.0-3.3 mol %, about 3.1-3.3 mol %, about 3.2-3.3 mol %, 1.0-3.2 mol %, about 1.2-3.2 mol %, about 1.4-3.2 mol %, about 1.5-3.2 mol %, about 1.6-3.2 mol %, about 1.7-3.2 mol %, about 1.8-3.2 mol %, about 1.9-3.2 mol %, about 2.0-3.2 mol %, about 2.1-3.2 mol %, about 2.2-3.2 mol %, about 2.3-3.2 mol %, about 78NTLA-103PCT; 5640-104.PCT 2.4-3.2 mol %, about 2.5-3.2 mol %, about 2.6-3.2 mol %, about 2.7-3.2 mol %, about 2.8- 3.2 mol %, about 2.9-3.2 mol %, about 3.0-3.2 mol %, about 3.1-3.2 mol %, 1.0-3.1 mol %, about 1.2-3.1 mol %, about 1.4-3.1 mol %, about 1.5-3.1 mol %, about 1.6-3.1 mol %, about 1.7-3.1 mol %, about 1.8-3.1 mol %, about 1.9-3.1 mol %, about 2.0-3.1 mol %, about 2.1-3.1 mol %, about 2.2-3.1 mol %, about 2.3-3.1 mol %, about 2.4-3.1 mol %, about 2.5-3.1 mol %, about 2.6-3.1 mol %, about 2.7-3.1 mol %, about 2.8-3.1 mol %, about 2.9-3.1 mol %, about 3.0-3.1 mol %, 1.0-3.0 mol %, about 1.2-3.0 mol %, about 1.4-3.0 mol %, about 1.5-3.0 mol %, about 1.6-3.0 mol %, about 1.7-3.0 mol %, about 1.8-3.0 mol %, about 1.9-3.0 mol %, about 2.0-3.0 mol %, about 2.1-3.0 mol %, about 2.2-3.0 mol %, about 2.3-3.0 mol %, about 2.4-3.0 mol %, about 2.5-3.0 mol %, about 2.6-3.0 mol %, about 2.7-3.0 mol %, about 2.8-3.0 mol %, about 2.9-3.0 mol %, 1.0-2.9 mol %, about 1.2-2.9 mol %, about 1.4-2.9 mol %, about 1.5-2.9 mol %, about 1.6-2.9 mol %, about 1.7-2.9 mol %, about 1.8-2.9 mol %, about 1.9-2.9 mol %, about 2.0-2.9 mol %, about 2.1-2.9 mol %, about 2.2-2.9 mol %, about 2.3-2.9 mol %, about 2.4-2.9 mol %, about 2.5-2.9 mol %, about 2.6-2.9 mol %, about 2.7-2.9 mol %, about 2.8-2.9 mol %, 1.0-2.8 mol %, about 1.2-2.8 mol %, about 1.4-2.8 mol %, about 1.5-2.8 mol %, about 1.6-2.8 mol %, about 1.7-2.8 mol %, about 1.8-2.8 mol %, about 1.9-2.8 mol %, about 2.0-2.8 mol %, about 2.1-2.8 mol %, about 2.2-2.8 mol %, about 2.3-2.8 mol %, about 2.4-2.8 mol %, about 2.5-2.8 mol %, about 2.6-2.8 mol %, about 2.7-2.8 mol %, 1.0-2.7 mol %, about 1.2-2.7 mol %, about 1.4-2.7 mol %, about 1.5-2.7 mol %, about 1.6-2.7 mol %, about 1.7-2.7 mol %, about 1.8-2.7 mol %, about 1.9-2.7 mol %, about 2.0-2.7 mol %, about 2.1-2.7 mol %, about 2.2-2.7 mol %, about 2.3-2.7 mol %, about 2.4-2.7 mol %, about 2.5-2.7 mol %, about 2.6-2.7 mol %, 1.0-2.6 mol %, about 1.2-2.6 mol %, about 1.4-2.6 mol %, about 1.5-2.6 mol %, about 1.6-2.6 mol %, about 1.7-2.6 mol %, about 1.8-2.6 mol %, about 1.9-2.6 mol %, about 2.0-2.6 mol %, about 2.1-2.6 mol %, about 2.2-2.6 mol %, about 2.3-2.6 mol %, about 2.4-2.6 mol %, about 2.5-2.6 mol %, 1.0-2.5 mol %, about 1.2-2.5 mol %, about 1.4-2.5 mol %, about 1.5-2.5 mol %, about 1.6-2.5 mol %, about 1.7-2.5 mol %, about 1.8-2.5 mol %, about 1.9-2.5 mol %, about 2.0-2.5 mol %, about 2.1-2.5 mol %, about 2.2-2.5 mol %, about 2.3-2.5 mol %, about 2.4-2.5 mol %, 1.0-2.4 mol %, about 1.2-2.4 mol %, about 1.4-2.4 mol %, about 1.5-2.4 mol %, about 1.6-2.4 mol %, about 1.7-2.4 mol %, about 1.8-2.4 mol %, about 1.9-2.4 mol %, about 2.0-2.4 mol %, about 2.1-2.4 mol %, about 2.2-2.4 mol %, about 2.3-2.4 mol %, 1.0-2.3 mol %, about 1.2-2.3 mol %, about 1.4-2.3 mol %, about 1.5-2.3 mol %, about 1.6-2.3 mol %, about 1.7-2.3 mol %, about 1.8-2.3 mol %, about 1.9-2.3 mol %, about 2.0-2.3 mol %, about 2.1-2.3 mol %, about 2.2-2.3 mol 79NTLA-103PCT; 5640-104.PCT %, 1.0-2.2 mol %, about 1.2-2.2 mol %, about 1.4-2.2 mol %, about 1.5-2.2 mol %, about 1.6-2.2 mol %, about 1.7-2.2 mol %, about 1.8-2.2 mol %, about 1.9-2.2 mol %, about 2.0-2.2 mol %, about 2.1-2.2 mol %, about 2.2-2.2 mol %, about 2.3-2.2 mol %, about 2.4-2.2 mol %, 1.0-2.1 mol %, about 1.2-2.1 mol %, about 1.4-2.1 mol %, about 1.5-2.1 mol %, about 1.6-2.1 mol %, about 1.7-2.1 mol %, about 1.8-2.1 mol %, about 1.9-2.1 mol %, about 2.0-2.1 mol %, 1.0-2.0 mol %, about 1.2-2.0 mol %, about 1.4-2.0 mol %, about 1.5-2.0 mol %, about 1.6-2.0 mol %, about 1.7-2.0 mol %, about 1.8-2.0 mol %, about 1.9-2.0 mol %, 1.0-1.9 mol %, about 1.2-1.9 mol %, about 1.4-1.9 mol %, about 1.5-1.9 mol %, about 1.6-1.9 mol %, about 1.7-1.9 mol %, about 1.8-1.9 mol %, 1.0-1.8 mol %, about 1.2-1.8 mol %, about 1.4-1.8 mol %, about 1.5-1.8 mol %, about 1.6-1.8 mol %, about 1.7-1.8 mol %, 1.0-1.7 mol %, about 1.2-1.7 mol %, about 1.4-1.7 mol %, about 1.5-1.7 mol %, about 1.6-1.7 mol %, 1.0-1.6 mol %, about 1.2-1.6 mol %, about 1.4-1.6 mol %, about 1.5-1.6 mol %, 1.0-1.5 mol %, about 1.2-1.5 mol %, about 1.4-1.5 mol %, about 1.5-1.5 mol %, about 1.6-1.5 mol %, about 1.7-1.5 mol %, about 1.8-1.5 mol %, about 1.9-1.5 mol %, 1.0-1.4 mol %, about 1.2-1.4 mol %, or 1.0-1.2 mol % of the lipid component.

[0181] In some aspects, the structural PEG-lipid is included in an amount of about 0.5 mol%, 0.6 mol% 0.7 mol%, 0.8 mol%, 0.9 mol%, 1.0 mol%, 1.2 mol%, 1.3 mol%, 1.4 mol%, 1.5 mol%, 1.6 mol%, 1.7 mol%, 1.8 mol%, 1.9 mol%, 2 mol%, 2.1 mol%, 2.2 mol%, 2.25 mol%, 2.5 mol%, 2.6 mol%, 2.7 mol%, 2.8 mol%, 2.9 mol%, 3 mol%, 3.1 mol%, 3.2 mol%, 3.3 mol%, 3.4 mol%, 3.5 mol%, 3.75 mol%, 4 mol%, 4.25 mol%, 4.5 mol%, 4.75 mol%, or about 5 mol% of the lipid component.

[0182] In some aspects, a molar ratio of the structural PEG-lipid to the anchor PEG-lipid is between about 2:1 to about 300:1, about 2:1 to about 275:1, about 2:1 to about 250:1, about 2:1 to about 225:1, about 2:1 to about 200:1, about 2:1 to about 175:1, about 2:1 to about 150:1, about 2:1 to about 125:1, about 2:1 to about 100:1, about 2:1 to about 75:1, about 2:1 to about 50:1, about 2.1 to about 40:1, about 2.1 to about 30:1, about 2.1 to about 25:1, about 2.1 to about 20:1, about 19:1, about 18:1, about 17:1, about 16:1, about 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, or about 2:1.

[0183] In some aspects, the anchor PEG-lipid is about 0.5 to about 50 mol%, about 0.5 to about 40 mol %, about 1 to about 40 mol %, about 1 to about 30 mol %, 1 to about 25 mol%, 1 to about 20 mol%, 1 to about 15 mol%, 1 to about 10 mol% relative to a total amount of structural PEG-lipids and anchor PEG-lipids together in the lipid component. In some 80NTLA-103PCT; 5640-104.PCT aspects, the anchor PEG-lipid is about 2 to about 10 mol %, 2 to about 10 mol%, 3 to about 10 mol%, 4 to about 10 mol%, 5 to about 10 mol%, 6 to about 10 mol%, 7 to about 10 mol%, 8 to about 10 mol%, or about 9 to about 10 mol% relative to a total amount of structural PEG-lipid and anchor PEG-lipids together in the lipid component. In some aspects, the anchor PEG-lipid is about 0.5, 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 or about 50 mol % relative to a total amount of structural PEG-lipid and anchor PEG-lipids together in the lipid component.

[0184] In some aspects, the lipid component comprises the helper lipid in an amount from about 25 to about 65 mol% of the lipid component. In some aspects, the helper lipid is included in an amount of about 30-50 mol %, about 30-65 mol %, about 30-55 mol %, about 33-50 mol %, about 32-55 mol %, about 32-65 mol %, about 35-50 mol %, about 35-55 mol %, about 35-40 mol %, about 35-45 mol %, or about 38 mol % of the lipid component. In some aspects, the helper lipid is about 30-50 mol %, about 30-65 mol %, about 30-55 mol %, about 33-50 mol %, about 32-55 mol %, about 32-65 mol %, about 35-50 mol %, about 35-55 mol %, about 35-40 mol %, about 35-45 mol %, or about 38 mol %. In additional embodiments, the amount of the helper lipid may be about 25-65 mol %, about 28-65 mol %, about 30-65 mol %, about 32-65 mol %, about 35-65 mol %, about 25-62 mol %, about 28-62 mol %, about 30-62 mol %, about 32-62 mol %, about 35-62 mol %, about 38-62 mol %, about 40-62 mol %, about 42-62 mol %, about 45-62 mol %, about 48-62 mol %, about 50-62 mol %, about 52-62 mol %, about 55-62 mol %, about 58-62 mol %, about 60-62 mol %, about 25-60 mol %, about 28-60 mol %, about 30-60 mol %, about 32-60 mol %, about 35-60 mol %, about 38-60 mol %, about 40-60 mol %, about 42-60 mol %, about 45-60 mol %, about 48-60 mol %, about 50-60 mol %, about 52-60 mol %, about 55-60 mol %, about 58-60 mol %, about 25-58 mol %, about 28-58 mol %, about 30-58 mol %, about 32-58 mol %, about 35-58 mol %, about 38-58 mol %, about 40-58 mol %, about 42-58 mol %, about 45-58 mol %, about 48-58 mol %, about 50-58 mol %, about 52-58 mol %, about 55-58 mol %, about 25-55 mol %, about 28-55 mol %, about 30-55 mol %, about 32-55 mol %, about 35-55 mol %, about 38-55 mol %, about 40-55 mol %, about 42-55 mol %, about 45-55 mol %, about 48-55 mol %, about 50-55 mol %, about 52-55 mol %, about 25-53 mol %, about 28-53 mol %, about 30-53 mol %, about 32-53 mol %, about 35-53 mol %, about 38-53 mol %, about 40-53 mol %, about 42-53 mol %, about 45-53 mol %, about 48-53 mol %, about 50-53 mol %, about 25-50 mol %, about 28-50 mol %, about 30-50 mol %, about 32-50 mol %, 81NTLA-103PCT; 5640-104.PCT about 35-50 mol %, about 38-50 mol %, about 40-50 mol %, about 42-50 mol %, about 45-50 mol %, about 48-50 mol %, about 25-48 mol %, about 28-48 mol %, about 30-48 mol %, about 32-48 mol %, about 35-48 mol %, about 38-48 mol %, about 40-48 mol %, about 42-48 mol %, about 45-48 mol %, about 25-45 mol %, about 28-45 mol %, about 30-45 mol %, about 32-45 mol %, about 35-45 mol %, about 38-45 mol %, about 40-45 mol %, about 42-45 mol %, about 25-43 mol %, about 28-43 mol %, about 30-43 mol %, about 32-43 mol %, about 35-43 mol %, about 38-43 mol %, about 40-43 mol %, about 25-40 mol %, about 28-40 mol %, about 30-40 mol %, about 32-40 mol %, about 35-40 mol %, about 38-40 mol %, about 25-38 mol %, about 28-38 mol %, about 30-38 mol %, about 32-38 mol %, about 35-38 mol %, about 25-35 mol %, about 28-35 mol %, about 30-35 mol %, about 32-35 mol %, about 25-33 mol %, about 28-33 mol %, about 30-33 mol %, about 35-45 mol %, or about 35-40 mol % of the lipid component. In some aspects, the helper lipid is included in an amount of about 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol% 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol% 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol%, 60 mol%, 61 mol%, 62 mol%, 63 mol%, 64 mol%, or about 65 mol% of the lipid component.

[0185] In some embodiments, the helper lipid mol % relative to the lipid component will be ±4 mol %, ±3 mol %, ±2 mol %, ±1.5 mol %, ±1 mol %, ±0.5 mol %, or ±0.25 mol % of the specified, nominal, or actual mol %. In certain embodiments, LNP inter-lot variability will be less than 15%, less than 10% or less than 5%. In some embodiments, the mol % numbers are based on nominal concentration. In some embodiments, the mol % numbers are based on actual concentration.

[0186] In some aspects, the lipid component comprises the neutral lipid in an amount from about 0 to about 25 mol%, about 1 to about 24 mol%, about 2 to about 23 mol%, about 4 to about 22 mol%, about 5 to about 21 mol%, about 5 to about 20 mol%, or about 5 to about 15 mol% of the lipid component. In some aspects, the neutral lipid is included in an amount of about 5 to about 15 mol%, 6 to about 15 mol%, 7 to about 15 mol%, 8 to about 15 mol%, 9 to about 15 mol%, 10 to about 15 mol%, 11 to about 15 mol%, or about 12 to about 15 mol% of the lipid component. In some aspects, the neutral lipid is included in an amount of about 15 to about 25 mol%, 16 to about 24 mol%, 17 to about 23 mol%, 18 to about 22 mol%, 19 to about 21 mol%, 20 to about 25 mol%, 21 to about 24 mol%, or about 22 to about 23 mol% of 82NTLA-103PCT; 5640-104.PCT the lipid component. In some aspects, the neutral lipid is included in an amount of about 5 to about 12 mol%, about 5 to about 11 mol%, about 5 to about 10 mol%, about 5 to about 9 mol%, about 5 to about 8 mol%, about 5 to about 7 mol%, or about 5 to about 6 mol% of the lipid component. In certain aspects, the lipid component comprises the neutral lipid in an amount from about 12 mol% to about 25 mol% or about 15 mol% to about 22.5 mol%. In some aspects, if present, the neutral lipid is included in an amount of about 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%, 15 mol%, 20 mol%, 22.5 mol%, or about 25 mol% of the lipid component. In some embodiments, the neutral lipid mol % relative to the lipid component will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the specified, nominal, or actual neutral lipid mol %. In some embodiments, the neutral lipid mol % relative to the lipid component will be ±4 mol %, ±3 mol %, ±2 mol %, ±1.5 mol %, ±1 mol %, ±0.5 mol %, or ±0.25 mol % of the specified, nominal, or actual mol %. In certain embodiments, LNP inter- lot variability will be less than 15%, less than 10% or less than 5%. In some embodiments, the mol % numbers are based on nominal concentration. In some embodiments, the mol % numbers are based on actual concentration.

[0187] In some aspects, the ionizable lipid is about 40 mol% to about 60 mol%, 41 mol% to about 60 mol%, 42 mol% to about 60 mol%, 43 mol% to about 60 mol%, 44 mol% to about 60 mol%, 45 mol% to about 60 mol%, 46 mol% to about 60 mol%, 47 mol% to about 60 mol%, 48 mol% to about 60 mol%, 49 mol% to about 60 mol%, 50 mol% to about 60 mol% of the lipid component. In some aspects, the ionizable lipid is about 40 mol% to about 60 mol%, about 40 mol% to about 59 mol%, about 40 mol% to about 58 mol%, about 40 mol% to about 57 mol%, about 40 mol% to about 56 mol%, about 40 mol% to about 55 mol%, about 40 mol% to about 54 mol%, about 40 mol% to about 53 mol%, about 40 mol% to about 52 mol%, about 40 mol% to about 51 mol%, about 40 mol% to about 50 mol% of the lipid component. In some aspects, the lipid component comprises the ionizable lipid in an amount of from about 40 to about 60 mol% of the lipid component. In some aspects, the ionizable lipid is included in an amount of about 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol%, or about 60 mol% of the lipid component.

[0188] In some aspects, the ionizable lipid mol % relative to the lipid component will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the specified, nominal, or actual mol 83NTLA-103PCT; 5640-104.PCT %. In some embodiments, the ionizable lipid mol % relative to the lipid component will be ±4 mol %, ±3 mol %, ±2 mol %, ±1.5 mol %, ±1 mol %, ±0.5 mol %, or ±0.25 mol % of the specified, nominal, or actual mol %. In certain embodiments, LNP inter-lot variability of the ionizable lipid mol % will be less than 15%, less than 10% or less than 5%. In some embodiments, the mol % numbers are based on nominal concentration. In some embodiments, the mol % numbers are based on actual concentration.

[0189] In some aspects, the ionizable lipids have a pKa of about 5 to about 8. In one some aspects, the preferred range of pKa is about 6 to about 7. In some aspects, the pKa is about 6.0 to about 6.9. In one embodiment, the pKa of the ionizable lipid is about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or about 6.9.

[0190] Ionizable lipids, as described herein, may be synthesized according to methods known in the art, for example as described in US63 / 610,628, WO2022221695A1, WO2022221697A1, WO2020118041A1, WO2020072605A1, WO2020219876, or WO2019067992A1, which are incorporated herein by reference in their entireties.

[0191] In some aspects, the structural PEG-lipid is selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG) (e.g., 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (PEG2K-DMG) (e.g., catalog # GM-020 from NOF, Tokyo, Japan)), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE) (e.g., catalog # DSPE-020CN, NOF, Tokyo, Japan), PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, and PEG-distearoylglycamide, PEG-cholesterol (1-[8’-(Cholest- 5-en-3[beta]-oxy)carboxamido-3’,6’-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol)ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2K-DMPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (PEG2K-DSPE) (e.g., cat. #880120C from Avanti Polar Lipids, Alabaster, Alabama, USA), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG2K-DSG; GS-020, NOF Tokyo, Japan), 1,2,-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol (PEG2K-DPG; GP-020, NOF Tokyo, Japan), poly(ethylene glycol)-2000-dimethacrylate (PEG2K-DMA), 1,2-distearyloxypropyl-3-amine-N- [methoxy(polyethylene glycol)-2000] (PEG2K-DSA), and methoxy-PEG2000-carbamoyl- 1,2-tridecyoxypropylamine (C13 Ether), and methoxy-PEG2000-carbamoyl-1,2- tetradecyoxypropylamine (C14 Ether). In certain such embodiments, the PEG lipid may be PEG2K-DMG. In some embodiments, the PEG lipid may be PEG2K-DSG. In other 84NTLA-103PCT; 5640-104.PCT embodiments, the PEG lipid may be PEG2K-DSPE. In some embodiments, the PEG lipid may be PEG2K-DMA. In yet other embodiments, the PEG lipid may be PEG2K-C-DMA. In certain embodiments, the PEG lipid may be compound S027, disclosed in WO2016 / 010840 (paragraphs

[0240] to

[0244] ), which is incorporated herein by reference in its entirety. In some embodiments, the PEG lipid may be PEG2K-DSA. In other embodiments, the PEG lipid may be PEG2K-C11. In some embodiments, the PEG lipid may be PEG2K-C14. In some embodiments, the PEG lipid may be PEG2K-C16. In some embodiments, the PEG lipid may be PEG2K-C18. In some embodiments, the PEG lipid may be DSG-PEG2K-C18.

[0192] In certain aspects, the structural PEG-lipid is selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSG), PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG- dipalmitoylglycamide, PEG-distearoylglycamide, 1-[8’-(cholest-5-en-3[beta]- oxy)carboxamido-3’,6’-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol) (PEG-cholesterol), 3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol)ether (PEG-DMB), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2K-DMPE), 1,2-dimyristoyl-rac-glycero-3-[methoxy(polyethylene glycol)-2000] (PEG2K-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (PEG2K-DSPE), 1,2-distearoyl-sn-glycerol- [methoxy(polyethylene glycol)-2000] (PEG2K-DSG), poly(ethylene glycol)-2000- dimethacrylate (PEG2K-DMA), 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2K-DSA), methoxy-PEG2000-carbamoyl-1,2-tetradecyoxypropylamine (C14 Ether), and methoxy-PEG2000-carbamoyl-1,2-tridecyoxypropylamine (C13 Ether), or a combination thereof. For example, the PEG lipid is selected from PEG2K-DMG, C13 ether and C14 ether. For example, the PEG lipid comprises dimyristoylglycerol (DMG). Structures for C14 Ether, C13 Ether, and PEG2K-DMG are shown below: berberNTLA-103PCT; 5640-104.PCT n is

[0193] Neutral lipids suitable for use in a lipid composition of the disclosure include, for example, a variety of neutral, uncharged or zwitterionic lipids. In certain aspects, the neutral lipid is selected from dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), 1- palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC), 1,2-diarachidoyl-sn- glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1- myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2- dibehenoyl-sn-glycero-3-phosphocholine (DBPC), 1- stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), and lysophosphatidylethanolamine, or a combination thereof.

[0194] “Helper lipids” include steroids, sterols, and alkyl resorcinols. Helper lipids suitable for use in the present disclosure include, but are not limited to, cholesterol, 5- heptadecylresorcinol, and cholesterol hemisuccinate. In certain embodiments, the helper lipid may be cholesterol or a derivative thereof, such as cholesterol hemisuccinate. In certain aspects, the helper lipid is cholesterol.

[0195] The LNPs and t-LNPs of the present disclosure comprise an “anchor PEG-lipid” that may act as a conjugation handle and anchor for attachment to a targeting ligand as detailed herein including in the Definitions above. In some aspects, the anchor PEG-lipid is selected from DSPE-PEG(1000), DSPE-PEG(2000), DSPE-PEG(3400), DSPE-PEG(5000)or a combination thereof. In some aspects, the “anchor PEG-lipid” and the “structural PEG-lipid” 86NTLA-103PCT; 5640-104.PCT may comprise the same PEG lipids, except for the difference of a conjugation handle. In some aspects, a structural PEG-lipid may be used as an anchor PEG-lipid.

[0196] In some aspects, the anchor PEG-lipid and the first coupling moiety comprise DSPE- PEG(1000) Maleimide, DSPE-PEG(2000) Maleimide, DSPE-PEG(3400) Maleimide, DSPE- PEG(5000) Maleimide, DSPE-PEG(1000) Azide, DSPE-PEG(2000) Azide, DSPE- PEG(3400) Azide, DSPE-PEG(5000) Azide, DSPE-PEG(1000) DBCO, DSPE-PEG(2000) DBCO, DSPE-PEG(3400) DBCO, DSPE-PEG(5000) DBCO, DSPE-PEG(1000) FITC, DSPE-PEG(2000) FITC, DSPE-PEG(3400) FITC, DSPE-PEG(5000) FITC, DSPE- PEG(1000) TCO (trans-cyclooctene), DSPE-PEG(2000) TCO, DSPE-PEG(3400) TCO, DSPE-PEG(5000) TCO, or a combination thereof.

[0197] In some aspects, the PEG lipid includes a PEG spacer (aka PEG linker) that is functionalized with a first coupling moiety. In some embodiments, the PEG spacer between the lipid and the first coupling moiety comprises at least about 5, 10, 20, 30, 50, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160170, 180, 190, or 200 ethylene glycol units. In some embodiments, the PEG spacer comprises about 10-120 ethylene glycol units. In some embodiments, the molecular weight of the pegylated lipid bonded to the first coupling moiety is from about 500 (i.e., PEG500) to about 5,000 (i.e., PEG5000).

[0198] In some aspects, the LNP composition, after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 freeze / thaw cycles has less than 5%, 4%, 3%, 2%, or 1% increase in impurities over the LNP composition before the freeze / thaw cycle(s). For example, impurities may include degradation products of the LNP or a lipid component or a cargo encapsulated by the LNP. In some aspects, the LNP composition, after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 freeze / thaw cycles has less than 10% 5%, 4%, 3%, 2%, or 1% increase in sub-visible particles over the LNP composition before the freeze / thaw cycle(s). Subvisible particles may be quantified using techniques known in the art, such as light scattering, light obscuration or other suitable techniques. Targeting ligand

[0199] A targeting ligand with a second coupling moiety may be introduced such that the targeting ligand may be attached to the first coupling moiety on the anchor PEG-lipid via the second coupling moiety. In some aspects, the anchor PEG-lipid can be functionalized to serve as an attachment site for a targeting ligand. For example, the first coupling moiety on the anchor PEG-lipid couples to the second coupling moiety on the targeting ligand resulting in attachment of the targeting ligand to the anchor PEG-lipid. Conjugating a cell- or tissue- 87NTLA-103PCT; 5640-104.PCT specific targeting ligand to the anchor PEG-lipid enables a targeted LNP (t-LNP) to bind to its target tissue or cell type.

[0200] In some aspects, the first coupling moiety and the second coupling moiety form a covalent bond. In some aspects, the first coupling moiety and the second coupling moiety form a non-covalent bond.

[0201] In some aspects, the first coupling moiety comprises dibenzocyclooctyne (DBCO), cysteine, a bioconjugation protein (e.g., a SpyCatcher protein of a SpyTag-SpyCatcher), streptavidin, protein G, protein G-derived peptide, or an immunoglobulin Fab domain. In some aspects, the second coupling moiety comprises azide, maleimide, a bioconjugation peptide (e.g., a SpyTag peptide of a SpyTag-SpyCatcher), biotin, an immunoglobulin Fc domain, or fluorescein isothiocyanate (FITC).

[0202] In some aspects, the first coupling moiety comprises azide, maleimide, a bioconjugation peptide, biotin, an immunoglobulin Fc domain, or FITC. In some aspects, the second coupling moiety comprises dibenzocyclooctyne (DBCO), cysteine, a bioconjugation protein, streptavidin, protein G, protein G-derived peptide, or an immunoglobulin Fab domain.

[0203] For example in some aspects, the first coupling moiety may be DBCO and the second coupling moiety may be azide or the second coupling moiety may be DBCO and the first coupling moiety may be azide. In some aspects, the first coupling moiety may be a cysteine side chain and the second coupling moiety may be a maleimide functional group or the second coupling moiety may be a cysteine side chain and the first coupling moiety may be a maleimide functional group. In some aspects, the first coupling moiety may be a bioconjugation protein (e.g., a SpyCatcher protein of a SpyTag-SpyCatcher) and the second coupling moiety may be a bioconjugation peptide (e.g., a SpyTag peptide of a SpyTag- SpyCatcher) or the second coupling moiety may be a bioconjugation protein (e.g., a SpyCatcher protein of a SpyTag-SpyCatcher) and the first coupling moiety may be a bioconjugation peptide (e.g., a SpyTag peptide of a SpyTag-SpyCatcher). In some aspects, the first coupling moiety may be streptavidin and the second coupling moiety may be biotin or the second coupling moiety may be streptavidin and the first coupling moiety may be biotin. In some aspects, the first coupling moiety may be a protein G or protein G-derived peptide and the second coupling moiety may be an immunoglobulin Fc domain or the second coupling moiety may be a protein G or protein G-derived peptide and the first coupling moiety may be an immunoglobulin Fc domain. In some aspects, the first coupling moiety 88NTLA-103PCT; 5640-104.PCT may be FITC and the second coupling moiety may be an amine functional group or the second coupling moiety may be FITC and the first coupling moiety may be an amine functional group.

[0204] In some aspects, the first coupling moiety comprises a peptide sequence selected from: AHIVMVDAYKPTK (SEQ ID NO: 10), VPTIVMVDAYKRYK (SEQ ID NO: 11), or RGVPHIVMVDAYKRYK (SEQ ID NO: 12). In some aspects, the second coupling moiety comprises a protein selected from: GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGK TISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKAT KGDAHI (SEQ ID NO: 13), VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIST WISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGD AHI (SEQ ID NO: 14), VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTIST WISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGEATKGD AHT (SEQ ID NO: 15), VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTIST WISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDA HT (SEQ ID NO: 16), or EDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFV ETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI (SEQ ID NO: 17).

[0205] In some aspects, the first coupling moiety comprises a protein selected from: GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGK TISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKAT KGDAHI (SEQ ID NO: 13), VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIST WISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGD AHI (SEQ ID NO: 14), VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTIST WISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGEATKGD AHT (SEQ ID NO: 15), 89NTLA-103PCT; 5640-104.PCT VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTIST WISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDA HT (SEQ ID NO: 16), or EDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFV ETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI (SEQ ID NO: 17).

[0206] In some aspects, the second coupling moiety comprises a peptide sequence selected from AHIVMVDAYKPTK (SEQ ID NO: 10), VPTIVMVDAYKRYK (SEQ ID NO: 11), or RGVPHIVMVDAYKRYK (SEQ ID NO: 12).

[0207] In some aspects, the targeting ligand is selected from an antibody, an antibody fragment, small molecule, or a peptide that is capable of specifically binding to a target or multiple targets.

[0208] In some aspects, the targeting ligand is an antibody. Exemplary antibodies, include, without limitation, immunoglobulins of the IgG subtype, such as IgG1, IgG2, IgG3, or IgG4, monoclonal antibodies, humanized antibodies, chimeric antibodies, bi- or multi-specific antibodies.

[0209] In some aspects, the targeting ligand is an antibody fragment. Exemplary antibody fragments, including without limitation, a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, or a single domain light chain antibody. Additional examples of the antigen-binding fragments include a VH domain, a VHH domain, a VNAR domain, and a single chain fragment variable (scFv), BiTE or a component thereof, a (scFv)2, a NANOBODY®, a nanobody-HSA, VHH-scAb, a VHH-Fab, a Dual scFab, a F(ab’)2, a diabody, a CROSSMAB®, a DAF (two-in-one), a DAE (four-in-one), a DUTAMAB®, a DT- IgG, a knobs-in-holes common light chain, a knobs-in-holes assembly, a charge pair, a Fab-arm exchange, a SEEDbody, a LUZ-Y, a FcAb, a kl-body, an orthogonal Fab, a DVD-IgG, a IgG(H)-scFv, a scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4- Ig, ZYBODY™, DVI-IgG, Diabody-CH3, a triple body, a miniantibody, a minibody, a TriBi minibody, scFv-CH3 KIH , Fab-scFv, a F(ab’)2-scFv2, a scFv-KIΉ, a Fab-scFv-Fc, a tetravaient HCAb, a scDiabody-Fc, a Diabody-Fc, a tandem scFv-Fc, a VHH-Fc, a tandem VHH-Fc, a L'ΉH-Fc KiH, a Fab- VHH-Fc, an Intrabody, a dock and lock, an ImmTAC® (immune-mobilizing monoclonal TCRs (T cell receptors) against cancer), an IgG-IgG conjugate, a Cov-X-Body, a scFvl- PEG-scFv2, an Adnectin, a DARPin®, a fibronectin, an 90NTLA-103PCT; 5640-104.PCT IgG, an IgM, an IgA, an IgE, an IgD, a DEP conjugate, TMEAbodyTM, SAFEbody®, TRITAC® , or SHIELD antibody.

[0210] In some aspects, the antibody fragment is selected from a Fab, a Fab', a F(ab')2, VHH- scAb, a VHH-Fab, a Dual scFab, a Fv fragment, a single chain variable fragment (scFv), a (scFv)2, a disulfide-linked Fv (sdFv), a Fd fragment consisting of VH and CH1 domains, a linear antibody, a nanobody, a diabody, a triple body, a miniantibody, a minibody, a TriBi minibody, a single domain antibody, or a VHH domain.

[0211] In some aspects, the targeting ligand is a small molecule or peptide, including without limitation, peptide analogs and derivatives thereof. In some embodiments, the amino acid range of the small molecule or peptide is about 5 to 10 amino acids, about 15 to 25 amino acids, or about 40 amino acids or longer.

[0212] In some aspects, the targeting ligand is added post LNP formulation. For example, the LNP comprising an anchor PEG-lipid having a first coupling moiety may be incubated in the presence of a targeting ligand having a second coupling moiety so that the first coupling moiety on the anchor PEG-lipid couples to the second coupling moiety on the targeting ligand resulting in attachment of the targeting ligand to the LNP containing the anchor PEG- lipid.

[0213] In some aspects, the targeting ligand targets an epitope on a target cell. In some aspects, the target cell is a bone marrow cell or bone marrow derived cell. In some aspects, the target cell is a hematopoetic stem cell (HSC). In some aspects, the target epitope may be CD34, CD38, CD45, CD117, CD123, or a CD135. In some aspects the target cell may be a T-cell. In some aspects, the LNP, t-LNP, or composition thereof, respectively, targets CD2, CD3, CD4, CD5, CD6, CD7, CD8, or CD45. In some aspects, the LNP, t-LNP, or composition thereof, respectively, targets CD7 or CD8. Lipid Nanoparticle (LNP) and Targeted LNP (t-LNP) Compositions

[0214] The lipid compositions may be provided as lipid nanoparticles (LNPs) or targeted lipid nanoparticles (t-LNPs) or compositions thereof. Lipid nanoparticles may be solid lipid nanoparticles.

[0215] In some aspects, the anchor PEG-lipid comprises a first coupling moiety that is conjugated with a second coupling moiety on a targeting ligand so that the targeting ligand is conjugated to the anchor PEG-lipid portion of the LNP. The conjugated targeting ligand can thus serve as a targeting moiety for the t-LNP. 91NTLA-103PCT; 5640-104.PCT

[0216] In some embodiments, LNP compositions are formed by mixing an aqueous RNA solution with an organic solvent-based lipid solution. Suitable solutions or solvents include or may contain: water, PBS, Tris buffer, NaCl, citrate buffer, acetate buffer, ethanol, chloroform, diethylether, cyclohexane, tetrahydrofuran, methanol, isopropanol. For example, the organic solvent may be 100% ethanol. A pharmaceutically acceptable buffer, e.g., for in vivo administration of LNP compositions, may be used. In certain embodiments, a buffer is used to maintain the pH of the composition comprising LNPs at or above pH 6.5. In certain embodiments, a buffer is used to maintain the pH of the composition comprising LNPs at or above pH 7.0. In certain embodiments, the composition has a pH ranging from about 7.2 to about 7.8. In additional embodiments, the composition has a pH ranging from about 7.3 to about 7.8 or ranging from about 7.4 to about 7.6. In further embodiments, the composition has a pH of about 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8. The pH of a composition may be measured with a micro pH probe. In certain embodiments, a cryoprotectant is included in the composition. Non-limiting examples of cryoprotectants include sucrose, trehalose, glycerol, DMSO, and ethylene glycol. Exemplary compositions may include up to 10% cryoprotectant, such as, for example, sucrose. In certain embodiments, the composition may comprise tris saline sucrose (TSS). In certain embodiments, the LNP composition may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% cryoprotectant. In certain embodiments, the LNP composition may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% sucrose. In some embodiments, the LNP composition may include a buffer. In some embodiments, the buffer may comprise a phosphate buffer (PBS), a Tris buffer, a citrate buffer, and mixtures thereof. In certain exemplary embodiments, the buffer comprises NaCl. In certain embodiments, the buffer lacks NaCl. Exemplary amounts of NaCl may range from about 20 mM to about 45 mM. Exemplary amounts of NaCl may range from about 40 mM to about 50 mM. In some embodiments, the amount of NaCl is about 45 mM. In some embodiments, the buffer is a Tris buffer. Exemplary amounts of Tris may range from about 20 mM to about 60 mM. Exemplary amounts of Tris may range from about 40 mM to about 60 mM. In some embodiments, the amount of Tris is about 50 mM. In some embodiments, the buffer comprises NaCl and Tris. Certain exemplary embodiments of the LNP compositions contain 5% sucrose and 45 mM NaCl in Tris buffer. In other exemplary embodiments, compositions contain sucrose in an amount of about 5% w / v, about 45 mM NaCl, and about 50 mM Tris at pH 7.5. The salt, buffer, and cryoprotectant amounts may be varied such that the osmolality of the overall composition is maintained. For example, the final osmolality may be 92NTLA-103PCT; 5640-104.PCT maintained at less than 450 mOsm / L. In further embodiments, the osmolality is between 350 and 250 mOsm / L. Certain embodiments have a final osmolality of 300 + / - 20 mOsm / L or 310 + / - 40 mOsm / L.

[0217] In some embodiments, microfluidic mixing, T-mixing, or cross-mixing of the aqueous RNA solution and the lipid solution in an organic solvent is used. In certain aspects, flow rates, junction size, junction geometry, junction shape, tube diameter, solutions, and / or RNA and lipid concentrations may be varied. LNPs or LNP compositions may be buffer exchanged, concentrated or purified, e.g., via dialysis, centrifugal filter, tangential flow filtration, chromatography, or gravity size exclusion chromatography. The LNP compositions may be stored as a suspension, an emulsion, or a lyophilized powder, for example. In some embodiments, an LNP composition is stored at 2-8° C, in certain aspects, the LNP compositions are stored at room temperature. In additional embodiments, an LNP composition is stored frozen, for example at -20° C or -80° C. In some embodiments, the LNP composition is stored frozen at about -70 to -80° C. In other embodiments, an LNP composition is stored at a temperature ranging from about 0° C to about -80° C. Frozen LNP compositions may be thawed before use, for example on ice, at room temperature, or at 25° C, preferably at room temperature.

[0218] Preferred lipid compositions, such as LNP compositions, are biodegradable, for example, in that they do not accumulate to cytotoxic levels in vivo at a therapeutically effective dose. In some embodiments, the compositions do not cause an innate immune response that leads to substantial adverse effects at a therapeutic dose level. In some embodiments, the compositions provided herein do not cause toxicity at a therapeutic dose level.

[0219] In some embodiments, the concentration of the LNPs in the LNP composition is about 1-10 µg / mL, about 2-10 µg / mL, about 2.5-10 µg / mL, about 1-5 µg / mL, about 2-5 µg / mL, about 2.5-5 µg / mL, about 0.04 µg / mL, about 0.08 µg / mL, about 0.16 µg / mL, about 0.25 µg / mL, about 0.63 µg / mL, about 1.25 µg / mL, about 2.5 µg / mL, or about 5 µg / mL.

[0220] In some embodiments, Dynamic Light Scattering (“DLS”) may be used to characterize the polydispersity index (PDI) and size of the LNPs of the present disclosure. DLS measures the scattering of light that results from subjecting a sample to a light source. PDI, as determined from DLS measurements, represents the distribution of particle size (around the mean particle size) in a population, with a perfectly uniform population having a PDI of zero. 93NTLA-103PCT; 5640-104.PCT

[0221] In some embodiments, the LNPs disclosed herein have a PDI from about 0.005 to about 0.75. In some embodiments, the LNPs disclosed herein have a PDI from about 0.005 to about 0.1. In some embodiments, the LNPs disclosed herein have a PDI from about 0.005 to about 0.09, about 0.005 to about 0.08, about 0.005 to about 0.07, or about 0.006 to about 0.05. In some embodiments, the LNP have a PDI from about 0.01 to about 0.5. In some embodiments, the LNP have a PDI from about zero to about 0.4. In some embodiments, the LNP have a PDI from about zero to about 0.35. In some embodiments, the LNP PDI may range from about zero to about 0.3. In some embodiments, the LNP have a PDI that may range from about zero to about 0.25. In some embodiments, the LNP PDI may range from about zero to about 0.2. In some embodiments, the LNP have a PDI from about zero to about 0.05. In some embodiments, the LNP have a PDI from about zero to about 0.01. In some embodiments, the LNP have a PDI less than about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.1, about 0.15, about 0.2, or about 0.4.

[0222] LNP size may be measured by various analytical methods known in the art. In some embodiments, LNP size may be measured using Asymmetric-Flow Field Flow Fractionation – Multi-Angle Light Scattering (AF4-MALS). In certain embodiments, LNP size may be measured by separating particles in the composition by hydrodynamic radius, followed by measuring the molecular weights, hydrodynamic radii and root mean square radii of the fractionated particles. In some embodiments, LNP size and particle concentration may be measured by nanoparticle tracking analysis (NTA, Malvern Nanosight). In certain embodiments, LNP samples are diluted appropriately and injected onto a microscope slide. A camera records the scattered light as the particles are slowly infused through field of view. After the movie is captured, the Nanoparticle Tracking Analysis processes the movie by tracking pixels and calculating a diffusion coefficient. This diffusion coefficient can be translated into the hydrodynamic radius of the particle. Such methods may also count the number of individual particles to give particle concentration. In some embodiments, LNP size, morphology, and structural characteristics may be determined by cryo-electron microscopy (“cryo-EM”).

[0223] The LNPs of the LNP compositions disclosed herein have a size (e.g. Z-average diameter or number-average diameter) of about 1 to about 250 nm. In some embodiments, the LNPs have a size of about 10 to about 200 nm. In further embodiments, the LNPs have a size of about 20 to about 150 nm. In some embodiments, the LNPs have a size of about 50 to about 150 nm or about 70 to 130 nm. In some embodiments, the LNPs have a size of about 94NTLA-103PCT; 5640-104.PCT 50 to about 100 nm. In some embodiments, the LNPs have a size of about 50 to about 120 nm. In some embodiments, the LNPs have a size of about 60 to about 100 nm. In some embodiments, the LNPs have a size of about 75 to about 150 nm. In some embodiments, the LNPs have a size of about 75 to about 120 nm. In some embodiments, the LNPs have a size of about 75 to about 100 nm. In some embodiments, the LNPs have a size of about 50 to about 145 nm, about 50 to about 120 nm, about 50 to about 120 nm, about 50 to about 115 nm, about 50 to about 100 nm, about 60 to about 145 nm, about 60 to about 120 nm, about 60 to about 115 nm, or about 60 to about 100 nm. In some embodiments, the LNPs have a size of less than about 145 nm, less than about 120 nm, less than about 115 nm, less than about 100 nm, or less than about 80 nm. In some embodiments, the LNPs have a size of greater than about 50 nm or greater than about 60 nm. In some embodiments, the particle size is a Z-average particle size. In some embodiments, the particle size is a number-average particle size. In some embodiments, the particle size is the size of an individual LNP. Unless indicated otherwise, all sizes referred to herein are the average sizes (diameters) of the fully formed nanoparticles, as measured by dynamic light scattering on a Malvern Zetasizer or Wyatt NanoStar. The nanoparticle sample is diluted in phosphate buffered saline (PBS) so that the count rate is approximately 200-400 kcps.

[0224] In some embodiments, the LNP compositions are formed with an average encapsulation efficiency ranging from about 50% to about 100%. In some embodiments, the LNP compositions are formed with an average encapsulation efficiency ranging from about 50% to about 95%. In some embodiments, the LNP compositions are formed with an average encapsulation efficiency ranging from about 70% to about 90%. In some embodiments, the LNP compositions are formed with an average encapsulation efficiency ranging from about 90% to about 100%. In some embodiments, the LNP compositions are formed with an average encapsulation efficiency ranging from about 75% to about 95%. In some embodiments, the LNP compositions are formed with an average encapsulation efficiency ranging from about 90% to about 100%. In some embodiments, the LNP compositions are formed with an average encapsulation efficiency ranging from about 95% to about 100%. In some embodiments, the LNP compositions are formed with an average encapsulation efficiency ranging from about 98% to about 100%. In some embodiments, the LNP compositions are formed with an average encapsulation efficiency ranging from about 99% to about 100%. 95NTLA-103PCT; 5640-104.PCT

[0225] In some aspects, any of the pharmaceutical compositions described herein can include one or more buffers (e.g., a neutral-buffered saline, a phosphate-buffered saline (PBS), amino acids (e.g., glycine), one or more preservatives, and / or a pharmaceutically acceptable carrier (e.g., bacteriostatic water, PBS, or saline).

[0226] In some aspects, the LNP composition is stable following one or more freeze / thaw cycles, for example after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 freeze / thaw cycle(s). In some aspects, the LNP in the LNP composition retains biological activity, gene editing activity, expression activity, and / or biophysical characteristics, as described above, after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 freeze / thaw cycle(s). LNP Cargo

[0227] The cargo delivered via an LNP composition described herein includes a biologically active agent. The biologically active agent may be a nucleic acid, such as an mRNA or gRNA. In certain embodiments, the cargo is or comprises one or more biologically active agent, such as mRNA, gRNA, expression vector, RNA-guided DNA-binding agent, antibody (e.g., monoclonal, chimeric, humanized, nanobody, and fragments thereof, etc.), cholesterol, hormone, peptide, protein, chemotherapeutic and other types of antineoplastic agent, low molecular weight drug, vitamin, co-factor, nucleoside, nucleotide, oligonucleotide, enzymatic nucleic acid, antisense nucleic acid, triplex forming oligonucleotide, antisense DNA or RNA composition, chimeric DNA:RNA composition, allozyme, aptamer, ribozyme, decoys and analogs thereof, plasmid and other types of vectors, and small nucleic acid molecule, RNAi agent, short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA) and “self-replicating RNA” (encoding a replicase enzyme activity and capable of directing its own replication or amplification in vivo) molecules, peptide nucleic acid (PNA), a locked nucleic acid ribonucleotide (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), sisiRNA (small internally segmented interfering RNA), and iRNA (asymmetrical interfering RNA). The above list of biologically active agents is exemplary only, and is not intended to be limiting. Such compounds may be purified or partially purified, and may be naturally occurring or synthetic, and may be chemically modified.

[0228] In certain aspects, the cargo is a coding polynucleotide, a non-coding polynucleotide, a polypeptide, or a combination thereof. In some aspects, the cargo comprises a guide RNA, a template nucleic acid, a shortmer, an antagomir, an antisense, a ribozyme, a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA 96NTLA-103PCT; 5640-104.PCT (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), a modified RNA, a self-amplifying mRNA, or a combination thereof.

[0229] In some aspects, the cargo may be an mRNA encoding a polypeptide. The polypeptide may be an enzyme or a therapeutic polypeptide or protein. In some aspects, the polypeptide may be a fluorescent protein or marker. In some aspects, the mRNA encodes a polypeptide component of a gene editing system. In some aspects, the polypeptide encoded by the mRNA cargo may be an endonuclease. In some aspects, the endonuclease may be a CRISPR-Cas nuclease, such as a Class II Cas nuclease, e.g., a Cas9 protein.

[0230] In some aspects, the cargo comprising an mRNA may encode a CAR. For example, the mRNA may encode an extracellular antigen binding domain, a transmembrane domain, and an intracellular domain comprising a functional signaling domain derived from a stimulatory molecule (e.g., an activation domain) and / or costimulatory molecule (e.g., a costimulatory domain).

[0231] In certain aspects, compositions comprising the LNPs according to any of the aspects described herein comprise a buffer, an excipient, a pharmaceutically acceptable carrier, or a combination thereof. In some aspects, the excipient comprises a cryoprotectant, a tonicity modifying agent, or a combination thereof. Parenteral formulations are typically aqueous or oily solutions or suspensions. Where the formulation is aqueous, excipients may be included such as sugars (including but not restricted to glucose, mannose, dextran, sucrose, mannitol, sorbitol, etc.), one or more carbohydrates, one or more antioxidants, one or more chelating agents (e.g., EDTA or glutathione). For some applications, they may be more suitably formulated with a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water (WFI).

[0232] The cargo delivered via LNP composition may be an RNA, such as an mRNA molecule encoding a protein of interest. For example, an mRNA for expressing a protein such as green fluorescent protein (GFP), an RNA-guided DNA-binding agent, or a Cas nuclease is included. LNP compositions that include a Cas nuclease mRNA, for example a Class 2 Cas nuclease mRNA that allows for expression in a cell of a Class 2 Cas nuclease such as a Cas9 or Cpf1 (also referred to as Cas12a) protein are provided. Further, the cargo may contain one or more gRNAs or nucleic acids encoding gRNAs. A template nucleic acid, e.g., for repair or recombination, may also be included with the compositions or a template nucleic acid may be used in the methods described herein. In a sub-embodiment, the cargo comprises an mRNA that encodes a Streptococcus pyogenes Cas9, optionally and an S. pyogenes gRNA. In a 97NTLA-103PCT; 5640-104.PCT further sub-embodiment, the cargo comprises an mRNA that encodes a Neisseria meningitidis Cas9, optionally and an Nme (Neisseria meningitidis) gRNA.

[0233] Various suitable gene editing systems comprising genome editing tools for delivery with the LNP compositions are described herein, including but not limited to the CRISPR / Cas system; zinc finger nuclease (ZFN) system; and the transcription activator-like effector nuclease (TALEN) system. Generally, the gene editing systems involve the use of engineered cleavage systems to induce a double strand break (DSB) or a nick (e.g., a single strand break, or SSB) in a target DNA sequence. Cleavage or nicking can occur through the use of specific nucleases such as engineered ZFN, TALENs, or using the CRISPR / Cas system with an engineered guide RNA to guide specific cleavage or nicking of a target DNA sequence. Further, targeted nucleases are being developed based on the Argonaute system (e.g., from T. thermophilus, known as ‘TtAgo’, see Swarts et al (2014) Nature 507(7491): 258-261), which also may have the potential for uses in genome editing and gene therapy.

[0234] In certain embodiments, the disclosed compositions comprise one or more DNA modifying agents, such as a DNA cutting agent. A variety of DNA modifying agents may be included in the LNP compositions described herein. For example, DNA modifying agents include nucleases (both sequence-specific and non-specific), topoisomerases, methylases, acetylases, chemicals, pharmaceuticals, and other agents. In some embodiments, proteins that bind to a given DNA sequence or set of sequences may be employed to induce DNA modification such as strand breakage. Proteins can either be modified by many means, such as incorporation of125I, the radioactive decay of which would cause strand breakage, or modifying cross- linking reagents such as 4-azidophenacylbromide which form a cross-link with DNA on exposure to UV-light. Such protein-DNA cross-links can subsequently be converted to a double-stranded DNA break by treatment with piperidine. Yet another approach to DNA modification involves antibodies raised against specific proteins bound at one or more DNA sites, such as transcription factors or architectural chromatin proteins, and used to isolate the DNA from nucleoprotein complexes.

[0235] In certain embodiments, the disclosed compositions comprise one or more DNA cutting agents. DNA cutting agents include technologies such as Zinc-Finger Nucleases (ZFN), Transcription Activator-Like Effector Nucleases (TALEN), mito-TALEN, and meganuclease systems. TALEN and ZFN technologies use a strategy of tethering endonuclease catalytic domains to modular DNA binding proteins for inducing targeted DNA double-stranded breaks (DSB) at specific genomic loci. Additional DNA cutting agents 98NTLA-103PCT; 5640-104.PCT include small interfering RNA, micro RNA, anti-microRNA, antagonist, small hairpin RNA, and aptamers (RNA, DNA or peptide based (including affimers)).

[0236] In some embodiments, the gene editing system is a TALEN system. Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands). Transcription activator-like effectors (TALEs) can be engineered to bind to a desired DNA sequence, to promote DNA cleavage at specific locations (see, e.g., Boch, 2011, Nature Biotech). The restriction enzymes can be introduced into cells, for use in gene editing or for genome editing in situ, a technique known as genome editing with engineered nucleases. Such methods and compositions for use therein are known in the art. See, e.g., WO2019147805, WO2014040370, WO2018073393, the contents of which are hereby incorporated in their entireties.

[0237] In some embodiments, the gene editing system is a zinc-finger system. Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA- binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences to enables zinc-finger nucleases to target unique sequences within complex genomes. The non-specific cleavage domain from the type IIs restriction endonuclease FokI is typically used as the cleavage domain in ZFNs. Cleavage is repaired by endogenous DNA repair machinery, allowing ZFN to precisely alter the genomes of higher organisms. Such methods and compositions for use therein are known in the art. See, e.g., WO2011091324, the contents of which are hereby incorporated in their entireties.

[0238] In preferred embodiments, the disclosed compositions comprise an mRNA encoding an RNA-guided DNA-binding agent, such as a Cas nuclease. In particular embodiments, the disclosed compositions comprise an mRNA encoding a Class 2 Cas nuclease, such as S. pyogenes Cas9.

[0239] Non-limiting exemplary species that the Cas nuclease can be derived from include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus 99NTLA-103PCT; 5640-104.PCT acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, Acidaminococcus sp., Lachnospiraceae bacterium ND2006, and Acaryochloris marina.

[0240] In some embodiments, the Cas nuclease is the Cas9 nuclease from Streptococcus pyogenes. In other embodiments, the Cas nuclease is the Cas9 nuclease from Streptococcus thermophilus. In still other embodiments, the Cas nuclease is the Cas9 nuclease from Neisseria meningitidis. In some embodiments, the Cas nuclease is the Cas9 nuclease is from Staphylococcus aureus. In some embodiments, the Cas nuclease is the Cpf1 nuclease from Francisella novicida. In other embodiments, the Cas nuclease is the Cpf1 nuclease from Acidaminococcus sp. In still other embodiments, the Cas nuclease is the Cpf1 nuclease from Lachnospiraceae bacterium ND2006. In further embodiments, the Cas nuclease is the Cpf1 nuclease from Francisella tularensis, Lachnospiraceae bacterium, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium, Parcubacteria bacterium, Smithella, Acidaminococcus, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi, Leptospira inadai, Porphyromonas crevioricanis, Prevotella disiens, or Porphyromonas macacae. In some embodiments, the Cas nuclease is a Cpf1 nuclease from an Acidaminococcus or Lachnospiraceae.

[0241] Wild type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, and the HNH domain cleaves the target strand of DNA. In some embodiments, the Cas9 nuclease comprises more than one RuvC domain and / or more than one HNH domain. In some embodiments, the Cas9 nuclease is a wild type Cas9. In 100NTLA-103PCT; 5640-104.PCT some embodiments, the Cas9 is capable of inducing a double strand break in target DNA. In other embodiments, the Cas nuclease may cleave dsDNA, it may cleave one strand of dsDNA, or it may not have DNA cleavase or nickase activity.

[0242] In some embodiments, chimeric Cas nucleases are used, where one domain or region of the protein is replaced by a portion of a different protein. In some embodiments, a Cas nuclease domain may be replaced with a domain from a different nuclease such as Fok1. In some embodiments, a Cas nuclease may be a modified nuclease.

[0243] In other embodiments, the Cas nuclease or Cas nickase may be from a Type-I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a component of the Cascade complex of a Type-I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a Cas3 protein. In some embodiments, the Cas nuclease may be from a Type-III CRISPR / Cas system. In some embodiments, the Cas nuclease may have an RNA cleavage activity.

[0244] In some embodiments, the RNA-guided DNA-binding agent has single-strand nickase activity, i.e., can cut one DNA strand to produce a single-strand break, also known as a “nick.” In some embodiments, the RNA-guided DNA-binding agent comprises a Cas nickase. A nickase is an enzyme that creates a nick in dsDNA, i.e., cuts one strand but not the other of the DNA double helix. In some embodiments, a Cas nickase is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which an endonucleolytic active site is inactivated, e.g., by one or more alterations (e.g., point mutations) in a catalytic domain. See, e.g., US Pat. No.8,889,356 for discussion of Cas nickases and exemplary catalytic domain alterations. In some embodiments, a Cas nickase such as a Cas9 nickase has an inactivated RuvC or HNH domain.

[0245] In some embodiments, the RNA-guided DNA-binding agent is modified to contain only one functional nuclease domain. For example, the agent protein may be modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, a nickase is used having a RuvC domain with reduced activity. In some embodiments, a nickase is used having an inactive RuvC domain. In some embodiments, a nickase is used having an HNH domain with reduced activity. In some embodiments, a nickase is used having an inactive HNH domain.

[0246] In some embodiments, a conserved amino acid within a Cas protein nuclease domain is substituted to reduce or alter nuclease activity. In some embodiments, a Cas nuclease may comprise an amino acid substitution in the RuvC or RuvC-like nuclease domain. Exemplary 101NTLA-103PCT; 5640-104.PCT amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015) Cell Oct 22:163(3): 759-771. In some embodiments, the Cas nuclease may comprise an amino acid substitution in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015). Further exemplary amino acid substitutions include D917A, E1006A, and D1255A (based on the Francisella novicida U112 Cpf1 (FnCpf1) sequence (UniProtKB - A0Q7Q2 (CPF1_FRATN)).

[0247] In some embodiments, an mRNA encoding a nickase is provided in combination with a pair of guide RNAs that are complementary to the sense and antisense strands of the target sequence, respectively. In this embodiment, the guide RNAs direct the nickase to a target sequence and introduce a DSB by generating a nick on opposite strands of the target sequence (i.e., double nicking). In some embodiments, use of double nicking may improve specificity and reduce off-target effects. In some embodiments, a nickase is used together with two separate guide RNAs targeting opposite strands of DNA to produce a double nick in the target DNA. In some embodiments, a nickase is used together with two separate guide RNAs that are selected to be in close proximity to produce a double nick in the target DNA.

[0248] In some embodiments, the RNA-guided DNA-binding agent lacks cleavase and nickase activity. In some embodiments, the RNA-guided DNA-binding agent comprises a dCas DNA-binding polypeptide. A dCas polypeptide has DNA-binding activity while essentially lacking catalytic (cleavase / nickase) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, the RNA-guided DNA-binding agent lacking cleavase and nickase activity or the dCas DNA-binding polypeptide is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which its endonucleolytic active sites are inactivated, e.g., by one or more alterations (e.g., point mutations) in its catalytic domains. See, e.g., US 2014 / 0186958 A1; US 2015 / 0166980 A1.

[0249] In some embodiments, the RNA-guided DNA binding agent comprises a APOBEC3 deaminase. In some embodiments, an APOBEC3 deaminase is an APOBEC3A (A3A). In some embodiments, the A3A is a human A3A. In some embodiments, the A3A is a wild-type A3A.

[0250] In some embodiments, the RNA-guided DNA binding agent comprises an editor. An exemplary editor comprises a human A3A fused to S. pyogenesD10A Cas9 nickase. In some embodiments the editor comprises a human A3A fused to a N. meningitidis D16A nickase. In 102NTLA-103PCT; 5640-104.PCT some embodiments, the editor is provided with a uracil glycosylase inhibitor (“UGI”). In some embodiments, the editor is fused to the UGI. In some embodiments the UGI is not fused to the editor. In some embodiments, the mRNA encoding the editor and an mRNA encoding the UGI are formulated together in an LNP. In other embodiments, the editor and UGI are provided in separate LNPs.

[0251] In some embodiments, the RNA-guided DNA-binding agent comprises one or more heterologous functional domains (e.g., is or comprises a fusion polypeptide).

[0252] In some embodiments, the heterologous functional domain may facilitate transport of the RNA-guided DNA-binding agent into the nucleus of a cell. For example, the heterologous functional domain may be a nuclear localization signal (NLS).

[0253] In some embodiments, the heterologous functional domain may be capable of modifying the intracellular half-life of the RNA-guided DNA binding agent. In some embodiments, the half-life of the RNA-guided DNA binding agent may be increased. In some embodiments, the half-life of the RNA-guided DNA-binding agent may be reduced. In some embodiments, the heterologous functional domain may be capable of increasing the stability of the RNA-guided DNA-binding agent. In some embodiments, the heterologous functional domain may be capable of reducing the stability of the RNA-guided DNA-binding agent. In some embodiments, the heterologous functional domain may act as a signal peptide for protein degradation. In some embodiments, the protein degradation may be mediated by proteolytic enzymes, such as, for example, proteasomes, lysosomal proteases, or calpain proteases. In some embodiments, the heterologous functional domain may comprise a PEST sequence. In some embodiments, the RNA-guided DNA-binding agent may be modified by addition of ubiquitin or a polyubiquitin chain. In some embodiments, the ubiquitin may be a ubiquitinlike protein (UBL). Non-limiting examples of ubiquitin-like proteins include small ubiquitinlike modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferonstimulated gene-15 (ISG15)), ubiquitin-related modifier-1 (URM1), neuronal- precursor-cellexpressed developmentally downregulated protein-8 (NEDD8, also called Rub1 in S. cerevisiae), human leukocyte antigen F-associated (FAT10), autophagy-8 (ATG8) and - 12 (ATG12), Fau ubiquitin-like protein (FUB1), membrane-anchored UBL (MUB), ubiquitin fold-modifier-1 (UFM1), and ubiquitin-like protein-5 (UBL5).

[0254] In some embodiments, the heterologous functional domain may be a marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, epitope tags, and reporter gene sequences. In some embodiments, the marker domain may be 103NTLA-103PCT; 5640-104.PCT a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1 ), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire,), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira- Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the marker domain may be a purification tag and / or an epitope tag. Non- limiting exemplary tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis, 8xHis, biotin carboxyl carrier protein (BCCP), poly-His, and calmodulin. Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent proteins.

[0255] In additional embodiments, the heterologous functional domain may target the RNA- guided DNA-binding agent to a specific organelle, cell type, tissue, or organ. In some embodiments, the heterologous functional domain may target the RNA-guided DNA-binding agent to mitochondria.

[0256] In further embodiments, the heterologous functional domain may be an effector domain such as an editor domain. When the RNA-guided DNA-binding agent is directed to its target sequence, e.g., when a Cas nuclease is directed to a target sequence by a gRNA, the effector domain such as an editor domain may modify or affect the target sequence. In some embodiments, the effector domain such as an editor domain may be chosen from a nucleic acid binding domain, a nuclease domain (e.g., a non-Cas nuclease domain), an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. In some embodiments, the heterologous functional domain is a nuclease, such as a FokI nuclease. See, e.g., US Pat. No.9,023,649. In some embodiments, the heterologous functional domain is a transcriptional activator or repressor. See, e.g., Qi et al., “Repurposing 104NTLA-103PCT; 5640-104.PCT CRISPR as an RNA-guided platform for sequence-specific control of gene expression,” Cell 152:1173-83 (2013); Perez-Pinera et al., “RNA-guided gene activation by CRISPR-Cas9- based transcription factors,” Nat. Methods 10:973-6 (2013); Mali et al., “CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering,” Nat. Biotechnol.31:833-8 (2013); Gilbert et al., “CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes,” Cell 154:442-51 (2013). As such, the RNA-guided DNA-binding agent essentially becomes a transcription factor that can be directed to bind a desired target sequence using a guide RNA. In some embodiments, the DNA modification domain is a methylation domain, such as a demethylation or methyltransferase domain. In some embodiments, the effector domain is a DNA modification domain, such as a base-editing domain. In particular embodiments, the DNA modification domain is a nucleic acid editing domain that introduces a specific modification into the DNA, such as a deaminase domain. See, e.g., WO 2015 / 089406; US 2016 / 0304846. The nucleic acid editing domains, deaminase domains, and Cas9 variants described in WO 2015 / 089406 and U.S.2016 / 0304846, each of which is hereby incorporated by reference in its entirety.

[0257] The nuclease may comprise at least one domain that interacts with a guide RNA (“gRNA”). Additionally, the nuclease may be directed to a target sequence by a gRNA. In Class 2 Cas nuclease systems, the gRNA interacts with the nuclease as well as the target sequence, such that it directs binding to the target sequence. In some embodiments, the gRNA provides the specificity for the targeted cleavage, and the nuclease may be universal and paired with different gRNAs to cleave different target sequences. Class 2 Cas nuclease may pair with a gRNA scaffold structure of the types, orthologs, and exemplary species listed above.

[0258] In some embodiments of the present disclosure, the cargo for the LNP composition includes at least one gRNA comprising guide sequences that direct an RNA-guided DNA- binding agent, which can be a nuclease (e.g., a Cas nuclease such as Cas9), to a target DNA. The gRNA may guide the Cas nuclease or Class 2 Cas nuclease to a target sequence on a target nucleic acid molecule. In some embodiments, a gRNA binds with and provides specificity of cleavage by a Class 2 Cas nuclease. In some embodiments, the gRNA and the Cas nuclease may form a ribonucleoprotein (RNP), e.g., a CRISPR / Cas complex such as a CRISPR / Cas9 complex. In some embodiments, the CRISPR / Cas complex may be a Type-II CRISPR / Cas9 complex. In some embodiments, the CRISPR / Cas complex may be a Type-V CRISPR / Cas complex, such as a Cpf1 / gRNA complex. Cas nucleases and cognate gRNAs 105NTLA-103PCT; 5640-104.PCT may be paired. The gRNA scaffold structures that pair with each Class 2 Cas nuclease vary with the specific CRISPR / Cas system.

[0259] In some embodiments, an mRNA encoding a RNA-guided DNA binding agent is formulated in a first LNP composition and a gRNA nucleic acid is formulated in a second LNP composition. In some embodiments, the first and second LNP compositions are administered simultaneously. In other embodiments, the first and second LNP compositions are administered sequentially. In some embodiments, the first and second LNP compositions are combined prior to the preincubation step. In other embodiments, the first and second LNP compositions are preincubated separately.

[0260] In some embodiments, the cargo may comprise a DNA molecule. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding a crRNA. In some embodiments, the nucleotide sequence encoding the crRNA comprises a targeting sequence flanked by all or a portion of a repeat sequence from a naturally occurring CRISPR / Cas system. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding a tracr RNA. In certain embodiments, the crRNA and the tracr RNA may be encoded by two separate nucleic acids. In other embodiments, the crRNA and the tracr RNA may be encoded by a single nucleic acid. In some embodiments, the crRNA and the tracr RNA may be encoded by opposite strands of a single nucleic acid. In other embodiments, the crRNA and the tracr RNA may be encoded by the same strand of a single nucleic acid. In some embodiments, the gRNA nucleic acid encodes an sgRNA. In some embodiments, the gRNA nucleic acid encodes a Cas9 nuclease sgRNA. In come embodiments, the gRNA nucleic acid encodes a Cpf1 nuclease sgRNA.

[0261] The nucleotide sequence encoding the guide RNA may be operably linked to at least one transcriptional or regulatory control sequence, such as a promoter, a 3' UTR, or a 5' UTR. In one example, the promoter may be a tRNA promoter, e.g., tRNALys3, or a tRNA chimera. See Mefferd et al., RNA.201521:1683-9; Scherer et al., Nucleic Acids Res.200735: 2620– 2628. In some embodiments, the promoter may be recognized by RNA polymerase III (Pol III). Non-limiting examples of Pol III promoters also include U6 and H1 promoters. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human U6 promoter. In some embodiments, the gRNA nucleic acid is a modified nucleic acid. In some embodiments, the gRNA nucleic acid includes a modified nucleoside or nucleotide. In some embodiments, the gRNA nucleic acid includes a 5' end modification, for example a modified nucleoside or nucleotide to stabilize and prevent integration of the 106NTLA-103PCT; 5640-104.PCT nucleic acid. In other embodiments, the gRNA nucleic acid comprises a double-stranded DNA having a 5' end modification on each strand. In some embodiments, the gRNA nucleic acid includes an inverted dideoxy-T or an inverted abasic nucleoside or nucleotide as the 5' end modification. In some embodiments, the gRNA nucleic acid includes a label such as biotin, desthiobiotin- TEG, digoxigenin, and fluorescent markers, including, for example, FAM, ROX, TAMRA, and AlexaFluor.

[0262] In certain embodiments, multiple LNP compositions may be used collaboratively and / or for separate purposes. In some embodiments, a cell may be contacted with first and second LNP compositions described herein. In some embodiments, the first and second LNP compositions each independently comprise one or more of an mRNA, a gRNA, and a guide RNA nucleic acid. In some embodiments, the first and second LNP compositions are administered simultaneously. In some embodiments, the first and second LNP compositions are administered sequentially.

[0263] In some embodiments, a method of producing multiple genome edits in a cell is provided (sometimes referred to herein and elsewhere as “multiplexing” or “multiplex gene editing” or “multiplex genome editing”). The ability to engineer multiple attributes into a single cell depends on the ability to perform edits in multiple targeted genes efficiently, including knockouts and in locus insertions, while retaining viability and the desired cell phenotype. In some embodiments, the method comprises culturing a cell in vitro, contacting the cell with two or more lipid nucleic acid assembly compositions, wherein each lipid nucleic acid assembly composition comprises a nucleic acid genome editing tool capable of editing a target site, and expanding the cell in vitro. The method results in a cell having more than one genome edit, wherein the genome edits differ. In certain embodiments, the first LNP composition comprises a first gRNA and the second LNP composition comprises a second gRNA, wherein the first and second gRNAs comprise different guide sequences that are complementary to different targets. In such embodiments, the LNP compositions may allow for multiplex gene editing. In some embodiments, the cell is contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 lipid nucleic acid assembly compositions. In some embodiments, the cell is contacted with at least 6 lipid nucleic acid assembly compositions.

[0264] Target sequences for RNA-guided DNA-binding proteins such as Cas proteins include both the positive and negative strands of genomic DNA (i.e., the sequence given and the sequence’s reverse complement), as a nucleic acid substrate for a Cas protein is a double stranded nucleic acid. Accordingly, where a guide sequence is said to be “complementary to a 107NTLA-103PCT; 5640-104.PCT target sequence”, it is to be understood that the guide sequence may direct a gRNA to bind to the reverse complement of a target sequence. Thus, in some embodiments, where the guide sequence binds the reverse complement of a target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., the target sequence not including the PAM) except for the substitution of U for T in the guide sequence.

[0265] In certain embodiments, at least one of the foregoing LNP compositions comprises a nucleic acid genome editing tool as described herein. In some embodiments, a further lipid composition comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9.

[0266] In some embodiments, LNPs or LNP compositions or pharmaceutical formulations comprising the LNPs are provided comprising at least one gRNA (e.g., sgRNA, short- sgRNA, dgRNA, or crRNA) described herein and a nuclease or a nucleic acid (e.g., an mRNA) encoding a nuclease. In some embodiments, the nuclease is an RNA-guided DNA binding agent, such as a Cas protein. In some embodiments, the short-sgRNA together with a Cas protein or nucleic acid (e.g., mRNA) encoding Cas protein is called a Cas RNP. In some embodiments, the RNA-guided DNA binding agent is one that functions with the short- sgRNA to direct a RNA-guided DNA binding agent to a target nucleic acid sequence. In some embodiments, the RNA-guided DNA binding agent is a Cas protein from the Type-II CRISPR / Cas system. In some embodiments, the Cas protein is Cas9. In some embodiments, the Cas9 protein is a wild type Cas9. In some embodiments, the Cas9 protein is derived from the Streptococcus pyogenes Cas9 protein, e.g., a S. pyogenes Cas9 (sypCas9). In some embodiments, compositions are provided comprising at least one short-sgRNA and a nuclease or an mRNA encoding a spyCas9. In some embodiments, the Cas9 protein is not derived from S. pyogenes, but functions in the same way as S. pyogenes Cas9 such that short- sgRNA that is specific to S. pyogenes Cas9 will direct the non-S. pyogenes Cas9 to its target site. In some embodiments, the Cas9 protein is derived from the Staphylococcus aureus Cas9 protein, e.g., a SaCas9. In some embodiments, compositions are provided comprising at least one short-sgRNA and a nuclease or an mRNA encoding a saCas9. In some embodiments, the Cas induces a double strand break in target DNA. Equivalents of spyCas9 and saCas9 protein are encompassed by the embodiments described herein.

[0267] In some embodiments, the RNA-guided DNA binding agent may be modified to contain only one functional nuclease domain. For example, the RNA-guided DNA binding agent may be modified such that one of the nuclease domains is mutated or fully or partially 108NTLA-103PCT; 5640-104.PCT deleted to reduce its nucleic acid cleavage activity. In some embodiments, a nickase Cas is used having a RuvC domain with reduced activity. In some embodiments, a nickase Cas is used having an inactive RuvC domain. In some embodiments, a nickase Cas is used having an HNH domain with reduced activity. In some embodiments, a nickase Cas is used having an inactive HNH domain.

[0268] In some embodiments, the RNP complex described herein comprises a nickase or an mRNA encoding a nickase and a pair of gRNAs (one or both of which may be sgRNAs and / or short-sgRNAs) that are complementary to the sense and antisense strands of the target sequence, respectively. In this embodiment, the gRNAs (e.g., sgRNAs and / or short-sgRNAs) direct the nickase to a target sequence and introduce a double stranded break (DSB) by generating a nick on opposite strands of the target sequence (i.e., double nicking). In some embodiments, use of double nicking may improve specificity and reduce off-target effects. In some embodiments, a nickase RNA-guided DNA binding agent is used together with two separate short-sgRNAs targeting opposite strands of DNA to produce a double nick in the target DNA. In some embodiments, a nickase RNA-guided DNA binding agent is used together with two separate gRNAs (e.g., sgRNAs or short-sgRNAs) that are selected to be in close proximity to produce a double nick in the target DNA.

[0269] In some embodiments, chimeric Cas proteins are used, where one domain or region of the protein is replaced by a portion of a different protein. In some embodiments, a Cas nuclease domain may be replaced with a domain from a different nuclease such as Fok1. In some embodiments, a Cas protein may be a modified nuclease. In some embodiments, the target sequence may be adjacent to a PAM. In some embodiments, the PAM may be adjacent to or within 1, 2, 3, or 4, nucleotides of the 3′ end of the target sequence. The length and the sequence of the PAM may depend on the Cas protein used. For example, the PAM may be selected from a consensus or a particular PAM sequence for a specific Cas9 protein or Cas9 ortholog, including those disclosed in FIG.1 of Ran et al., Nature 520:186-191 (2015). In some embodiments, the PAM may comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Non-limiting exemplary PAM sequences include NGG, NAG, NGA, NGAG, NGCG, NNGRRT, TTN, NGGNG, NG, NAAAAN, NNAAAAW, NNNNACA, GNNNCNNA, and NNNNGATT (wherein N is defined as any nucleotide, and W is defined as either A or T, and R is defined as either A or G). In some embodiments, the PAM sequence may be NGG. In some embodiments, the PAM sequence may be NGGNG. In some embodiments, the PAM sequence may be NNAAAAW. 109NTLA-103PCT; 5640-104.PCT

[0270] In some embodiments, an nucleic acid (e.g., mRNA) comprising an ORF encoding an RNA-guided DNA binding agent is used which has one or more of the following features. In some embodiments, the ORF encoding the RNA-guided DNA-binding agent, e.g. a Cas9 nuclease such as an S. pyogenes Cas9, has an adenine content ranging from its minimum adenine content to about 150% of its minimum adenine content. In some embodiments, the adenine content of the ORF is less than or equal to about 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of its minimum adenine content. In some embodiments, the ORF has an adenine content equal to its minimum adenine content. In some embodiments, the ORF has an adenine content less than or equal to about 150% of its minimum adenine content. In some embodiments, the ORF has an adenine content less than or equal to about 145% of its minimum adenine content. In some embodiments, the ORF has an adenine content less than or equal to about 140% of its minimum adenine content. In some embodiments, the ORF has an adenine content less than or equal to about 135% of its minimum adenine content. In some embodiments, the ORF has an adenine content less than or equal to about 130% of its minimum adenine content. In some embodiments, the ORF has an adenine content less than or equal to about 125% of its minimum adenine content. In some embodiments, the ORF has an adenine content less than or equal to about 120% of its minimum adenine content. In some embodiments, the ORF has an adenine content less than or equal to about 115% of its minimum adenine content. In some embodiments, the ORF has an adenine content less than or equal to about 110% of its minimum adenine content. In some embodiments, the ORF has an adenine content less than or equal to about 105% of its minimum adenine content. In some embodiments, the ORF has an adenine content less than or equal to about 104% of its minimum adenine content. In some embodiments, the ORF has an adenine content less than or equal to about 103% of its minimum adenine content. In some embodiments, the ORF has an adenine content less than or equal to about 102% of its minimum adenine content. In some embodiments, the ORF has an adenine content less than or equal to about 101% of its minimum adenine content. The disclosures of US20210087568A1 are incorporated herein by reference in their entirety.

[0271] In some embodiments, the Cas protein comprises a fusion protein comprising a catalytically inactive Cas (e.g., Cas9) linked to a heterologous functional domain (see, e.g., WO2014152432). In some embodiments, the catalytically inactive Cas9 is from S. pyogenes. In some embodiments, the catalytically inactive Cas comprises mutations that inactivate the Cas. In some embodiments, the heterologous functional domain is a domain 110NTLA-103PCT; 5640-104.PCT that modifies gene expression, histones, or DNA. In some embodiments, the heterologous functional domain is a transcriptional activation domain or a transcriptional repressor domain.

[0272] In some embodiments, a conserved amino acid within an RNA-guided DNA binding agent nuclease domain is substituted to reduce or alter nuclease activity. In some embodiments, a Cas protein may comprise an amino acid substitution in the RuvC or RuvC- like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). In some embodiments, the Cas protein may comprise an amino acid substitution in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the spyCas9 protein).

[0273] RNA-guided DNA binding agents, including Cas9, encompass modified and variants thereof. Modified versions having one catalytic domain, either RuvC or HNH, that is inactive are termed “nickases.” Nickases cut only one strand on the target DNA, thus creating a single-strand break. A single-strand break may also be known as a “nick.” In some embodiments, the compositions and methods comprise nickases. In some embodiments, the compositions and methods comprise a nickase RNA-guided DNA binding agent, such as a nickase Cas9, that induces a nick rather than a double strand break in the target DNA.

[0274] In some aspects, the present disclosure provides kits comprising one or more gRNAs (e.g., sgRNAs, short-sgRNAs, dgRNAs, or crRNAs), LNPs, compositions, or pharmaceutical formulations described herein. In some embodiments, a kit further comprises one or more of a solvent, solution, buffer, each separate from the composition or pharmaceutical formulation, instructions, or desiccant. In some aspects, compositions of the present disclosure include gRNAs (e.g., sgRNAs, short-sgRNAs, dgRNAs, or crRNAs) and a carrier, excipient, diluent, or the like. In some aspects, the excipient or diluent is inert. In some aspects, the excipient or diluent is not inert. In some aspects, a pharmaceutical formulation is provided comprising gRNAs (e.g., sgRNAs, short-sgRNAs, dgRNAs, or crRNAs) and a pharmaceutically acceptable carrier, excipient, diluent, or the like. In some embodiments, the pharmaceutical formulation further comprises a Cas9 protein or an mRNA encoding a Cas9 protein. In some embodiments, the pharmaceutical formulation comprises gRNAs (e.g., sgRNAs, short-sgRNAs, dgRNAs, or crRNAs), an LNP, and a Cas9 protein or mRNA encoding a Cas9 protein.

[0275] In some embodiments, the methods of the present disclosure further comprise contacting the cell with a donor nucleic acid. In some embodiments, a further lipid 111NTLA-103PCT; 5640-104.PCT composition comprises a donor nucleic acid. The donor nucleic acid may be inserted in a target sequence. In some embodiments, a donor nucleic acid sequence is provided as a vector. In some embodiments, the donor nucleic acid encodes a targeting receptor. In certain embodiments, the donor nucleic acid comprises regions having homology with corresponding regions of a T cell receptor sequence. A “targeting receptor” is a polypeptide present on the surface of a cell, e.g., a T cell, to permit binding of the cell to a target site, e.g., a specific cell or tissue in an organism. In some embodiments, the targeting receptor is a chimeric antigen receptor (CAR). In some embodiments, the targeting receptor is a universal CAR (UniCAR). In some embodiments, the targeting receptor is a TCR. In some embodiments, the targeting receptor is a T cell receptor fusion construct (TRuC). In some embodiments, the targeting receptor is a B cell receptor (BCR) (e.g., expressed on a B cell). In some embodiments, the targeting receptor is chemokine receptor.

[0276] In some embodiments, the targeting receptor is a cytokine receptor.

[0277] The length of the targeting sequence may depend on the CRISPR / Cas system and components used. For example, different Class 2 Cas nucleases from different bacterial species have varying optimal targeting sequence lengths. Accordingly, the targeting sequence may comprise 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, 35, 40, 45, 50, or more than 50 nucleotides in length. In some embodiments, the targeting sequence length is 0, 1, 2, 3, 4, or 5 nucleotides longer or shorter than the guide sequence of a naturally-occurring CRISPR / Cas system. In certain embodiments, the Cas nuclease and gRNA scaffold will be derived from the same CRISPR / Cas system. In some embodiments, the targeting sequence may comprise or consist of 18-24 nucleotides. In some embodiments, the targeting sequence may comprise or consist of 19-21 nucleotides. In some embodiments, the targeting sequence may comprise or consist of 20 nucleotides.

[0278] In some embodiments, the sgRNA is a “Cas9 sgRNA” capable of mediating RNA- guided DNA cleavage by a Cas9 protein. In some embodiments, the sgRNA is a “Cpf1 sgRNA” capable of mediating RNA-guided DNA cleavage by a Cpf1 protein. In certain embodiments, the gRNA comprises a crRNA and tracr RNA sufficient for forming an active complex with a Cas9 protein and mediating RNA-guided DNA cleavage. In certain embodiments, the gRNA comprises a crRNA sufficient for forming an active complex with a Cpf1 protein and mediating RNA-guided DNA cleavage. See Zetsche 2015.

[0279] In certain embodiments, the lipid compositions, such as LNP compositions include an RNA-guided DNA-binding agent, such as a Class 2 Cas mRNA and at least one gRNA. In 112NTLA-103PCT; 5640-104.PCT some embodiments, the gRNA is a sgRNA. In some embodiments, the RNA-guided DNA- binding agent is a Cas9 mRNA In certain embodiments, the LNP composition includes a molar ratio of gRNA to RNA-guided DNA-binding agent mRNA, such as Class 2 Cas nuclease mRNA of about 1:1 or about 1:2. In some embodiments, the ratio of by weight is from about 25:1 to about 1:25, about 10:1 to about 1:10, about 8:1 to about 1:8, about 4:1 to about 1:4, about 2:1 to about 1:2, about 2:1 to 1:4 by weight, or about 1:1 to about 1:2.

[0280] In certain aspects, a guide RNA nucleic acid and a Class 2 Cas nuclease mRNA, are provided in a molar ratio of the mRNA to the guide RNA nucleic acid from about 2:1 to about 1:4 by weight, or about 1:1 by weight.

[0281] In certain aspects, the gRNA is a modified gRNA, for example the modified gRNA comprises a modification at one or more of the first five nucleotides at a 5’ end, or the modified gRNA comprises a modification at one or more of the last five nucleotides at a 3’ end, or both.

[0282] In certain aspects, the gene editing system, for example a TtAgo system, may comprise one or more single stranded guide DNA molecules that are complimentary the DNA sequence to be cleaved or nicked. In some aspects, the guide DNA molecules are phosphorylated on the 5’-end. In some aspects, the guide DNA may be 10 to 20 nucleotides, 12 to 18 nucleotides, or 14 to 16 nucleotides in length.

[0283] Certain embodiments also provide nucleic acids, e.g., expression cassettes, encoding the gRNA described herein Modified RNAs

[0284] In certain embodiments, the lipid compositions, such as LNP compositions comprise modified nucleic acids, including modified RNAs. For example, modified RNAs may include gRNAs or mRNAs. Modified nucleosides or nucleotides can be present in an RNA, for example a gRNA or mRNA. A gRNA or mRNA comprising one or more modified nucleosides or nucleotides, for example, is called a “modified” RNA to describe the presence of one or more non-naturally and / or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues. In some embodiments, a modified RNA is synthesized with a non-canonical nucleoside or nucleotide, here called “modified.”

[0285] Modified nucleosides and nucleotides can include one or more of: (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and / or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification); (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, 113NTLA-103PCT; 5640-104.PCT e.g., of the 2’ hydroxyl on the ribose sugar (an exemplary sugar modification); (iii) wholesale replacement of the phosphate moiety with “dephospho” linkers (an exemplary backbone modification); (iv) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification); (v) replacement or modification of the ribose-phosphate backbone (an exemplary backbone modification); (vi) modification of the 3’ end or 5’ end of the polynucleotide, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, cap or linker (such 3’ or 5’ cap modifications may comprise a sugar and / or backbone modification); and (vii) modification or replacement of the sugar (an exemplary sugar modification). Certain embodiments comprise a 5’ end modification to an mRNA, gRNA, or nucleic acid. Certain embodiments comprise a modification to an mRNA, gRNA, or nucleic acid. Certain embodiments comprise a 3’ end modification to an mRNA, gRNA, or nucleic acid. A modified RNA can contain 5’ end and 3’ end modifications. A modified RNA can contain one or more modified residues at non-terminal locations. In certain embodiments, a gRNA includes at least one modified residue. In certain embodiments, an mRNA includes at least one modified residue. In certain embodiments, the modified gRNA comprises a modification at one or more of the first five nucleotides at a 5’ end. In certain embodiments, the modified gRNA comprises a modification at one or more of the first five nucleotides at a 5’ end. In certain embodiments, wherein the modified gRNA comprises a modification at one or more of the last five nucleotides at a 3’ end.

[0286] In some aspects, the cargo comprises a nucleic acid encoding a Cas protein, a guide RNA (gRNA), a nucleic acid of interest, or a combination thereof. In some aspects, the gRNA is a modified gRNA. In some aspects, the gRNA is modified at one or more of the first five nucleotides at a 5’ end, is modified at one or more of the last five nucleotides at a 3’ end, or both. In some aspects, the guide RNA is selected from a single-guide RNA (sgRNA) or a dual-guide RNA (dgRNA). In some aspects, the Cas protein comprises a Class 2 Cas nuclease or a Cas9 nuclease. In some aspects, the ratio of the nucleic acid encoding a Cas protein to the guide RNA is from about 2:1 to 1:4 by weight.

[0287] Unmodified nucleic acids can be prone to degradation by, e.g., intracellular nucleases or those found in serum. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Accordingly, in one aspect the RNAs (e.g. mRNAs, gRNAs) described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward intracellular or serum-based nucleases. In some embodiments, the modified RNA molecules 114NTLA-103PCT; 5640-104.PCT described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo. The term “innate immune response” includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, which involves the induction of cytokine expression and release, particularly the interferons, and cell death.

[0288] Accordingly, in some embodiments, an RNA or nucleic acid comprises at least one modification which confers increased or enhanced stability to the nucleic acid, including, for example, improved resistance to nuclease digestion in vivo. As used herein, the terms “modification” and “modified” as such terms relate to the nucleic acids provided herein, include at least one alteration which preferably enhances stability and renders the RNA or nucleic acid more stable (e.g., resistant to nuclease digestion) than the wild-type or naturally occurring version of the RNA or nucleic acid. As used herein, the terms “stable” and “stability” and such terms relate to the nucleic acids described herein, and particularly with respect to the RNA, refer to increased or enhanced resistance to degradation by, for example nucleases (i.e., endonucleases or exonucleases) which are normally capable of degrading such RNA. Increased stability can include, for example, less sensitivity to hydrolysis or other destruction by endogenous enzymes (e.g., endonucleases or exonucleases) or conditions within the target cell or tissue, thereby increasing or enhancing the residence of such RNA or nucleic acid in the target cell, tissue, subject and / or cytoplasm. The stabilized RNA or nucleic acid molecules provided herein demonstrate longer half-lives relative to their naturally occurring, unmodified counterparts (e.g. the wild-type version of the molecule). Also contemplated by the terms “modification” and “modified” as such terms related to the mRNA of the LNP compositions disclosed herein are alterations which improve or enhance translation of mRNA nucleic acids, including for example, the inclusion of sequences which function in the initiation of protein translation (e.g., the Kozak consensus sequence). (Kozak, M., Nucleic Acids Res 15 (20): 8125-48 (1987)).

[0289] In some embodiments, the RNA or nucleic acid has undergone a chemical or biological modification to render it more stable. Exemplary modifications to an RNA or nucleic acid include the depletion of a base (e.g., by deletion or by the substitution of one nucleotide for another) or modification of a base, for example, the chemical modification of a base. The phrase “chemical modifications” as used herein, includes modifications which introduce chemistries which differ from those seen in naturally occurring RNA or nucleic acids, for example, covalent modifications such as the introduction of modified nucleotides, 115NTLA-103PCT; 5640-104.PCT (e.g., nucleotide analogs, or the inclusion of pendant groups which are not naturally found in such RNA, such as a deoxynucleoside, or nucleic acid molecules).

[0290] In some embodiments of a backbone modification, the phosphate group of a modified residue can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified residue, e.g., modified residue present in a modified nucleic acid, can include the wholesale replacement of an unmodified phosphate moiety with a modified phosphate group as described herein. In some embodiments, the backbone modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.

[0291] Examples of modified phosphate groups include, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non- bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral. The stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp). The backbone can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens. The phosphate group can be replaced by non- phosphorus containing connectors in certain backbone modifications. In some embodiments, the charged phosphate group can be replaced by a neutral moiety. Examples of moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. mRNA

[0292] In some aspects, the t-LNPs described herein provide targeted delivery of cargo comprising an mRNA to a cell or tissue of interest. In some embodiments, a composition or formulation disclosed herein comprises an mRNA encoding a polypeptide sequence for expression. For example, the mRNA can be translated in a cell to express a therapeutic 116NTLA-103PCT; 5640-104.PCT protein, an enzyme, a polypeptide, a receptor, or other polypeptide having an amino acid sequence of interest.

[0293] In some embodiments, a composition or formulation disclosed herein comprises an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA-binding agent, such as a Cas nuclease, or Class 2 Cas nuclease as described herein. In some embodiments, an mRNA comprising an ORF encoding an RNA-guided DNA-binding agent, such as a Cas nuclease or Class 2 Cas nuclease, is provided, used, or administered. An mRNA may comprise one or more of a 5’ cap, a 5’ untranslated region (UTR), a 3’ UTRs, and a polyadenine tail. The mRNA may comprise a modified open reading frame, for example to encode a nuclear localization sequence or to use alternate codons to encode the protein.

[0294] The mRNA in the disclosed LNP compositions may encode a cell surface or intracellular polypeptide. The mRNA in the disclosed LNP compositions may encode, for example, a secreted hormone, enzyme, receptor, polypeptide, peptide or other protein of interest that is normally secreted. In some embodiments, the mRNA may optionally have chemical or biological modifications which, for example, improve the stability and / or half- life of such mRNA or which improve or otherwise facilitate protein production. In some aspects, mRNA in the disclosed LNP compositions may encode a CAR.

[0295] In addition, suitable modifications include alterations in one or more nucleotides of a codon such that the codon encodes the same amino acid but is more stable than the codon found in the wild-type version of the mRNA. For example, an inverse relationship between the stability of RNA and a higher number cytidines (C’s) and / or uridines (U’s) residues has been demonstrated, and RNA devoid of C and U residues have been found to be stable to most RNases (Heidenreich, et al. J Biol Chem 269, 2131-8 (1994)). In some embodiments, the number of C and / or U residues in an mRNA sequence is reduced. In another embodiment, the number of C and / or U residues is reduced by substitution of one codon encoding a particular amino acid for another codon encoding the same or a related amino acid. Contemplated modifications to the mRNA nucleic acids also include the incorporation of pseudouridines. In some embodiments, a modified uridine is a pseudouridine modified at the 1 position, e.g., with a halogen, methyl, or ethyl. The modified uridine can be, for example, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof. In some embodiments, the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified 117NTLA-103PCT; 5640-104.PCT uridine is pseudouridine. In some embodiments, the modified uridine is N1-methyl- pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methyl pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5- methoxyuridine. The incorporation of pseudouridines into the mRNA nucleic acids may enhance stability and translational capacity, as well as diminishing immunogenicity in vivo. See, e.g., Karikó, K., et al., Molecular Therapy 16 (11): 1833-1840 (2008). Substitutions and modifications to the mRNA may be performed by methods readily known to one or ordinary skill in the art.

[0296] The constraints on reducing the number of C and U residues in a sequence will likely be greater within the coding region of an mRNA, compared to an untranslated region, (i.e., it will likely not be possible to eliminate all of the C and U residues present in the message while still retaining the ability of the message to encode the desired amino acid sequence). The degeneracy of the genetic code, however presents an opportunity to allow the number of C and / or U residues that are present in the sequence to be reduced, while maintaining the same coding capacity (i.e., depending on which amino acid is encoded by a codon, several different possibilities for modification of RNA sequences may be possible). The term modification also includes, for example, the incorporation of non-nucleotide linkages or modified nucleotides into the mRNA sequences (e.g., modifications to one or both the 3′ and 5′ ends of an mRNA molecule encoding a functional secreted protein or enzyme). Such modifications include the addition of bases to an mRNA sequence (e.g., the inclusion of a poly A tail or a longer poly A tail), the alteration of the 3′ UTR or the 5′ UTR, complexing the mRNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and inclusion of elements which change the structure of an mRNA molecule (e.g., which form secondary structures).

[0297] The poly A tail is thought to stabilize natural messengers. Therefore, a long poly A tail may be added to an mRNA molecule thus rendering the mRNA more stable. Poly A tails can be added using a variety of art-recognized techniques. For example, long poly A tails can be added to synthetic or in vitro transcribed mRNA using poly A polymerase (Yokoe, et al. 118NTLA-103PCT; 5640-104.PCT Nature Biotechnology.1996; 14: 1252-1256). A transcription vector can also encode long poly A tails. In addition, poly A tails can be added by transcription directly from PCR products. In some embodiments, the length of the poly A tail is at least about 90, 200, 300, 400 at least 500 nucleotides. In certain embodiments, the length of the poly A tail is adjusted to control the stability of a modified mRNA molecule and, thus, the transcription of protein. For example, since the length of the poly A tail can influence the half-life of an mRNA molecule, the length of the poly A tail can be adjusted to modify the level of resistance of the mRNA to nucleases and thereby control the time course of protein expression in a cell. In some embodiments, the stabilized mRNA molecules are sufficiently resistant to in vivo degradation (e.g., by nucleases), such that they may be delivered to the target cell without a transfer vehicle.

[0298] In certain embodiments, an mRNA can be modified by the incorporation 3′ and / or 5′ untranslated (UTR) sequences which are not naturally found in the wild-type mRNA. In some embodiments, 3′ and / or 5′ flanking sequence which naturally flanks an mRNA and encodes a second, unrelated protein can be incorporated into the nucleotide sequence of an mRNA molecule encoding a therapeutic or functional protein in order to modify it. For example, 3′ or 5′ sequences from mRNA molecules which are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) can be incorporated into the 3′ and / or 5′ region of a sense mRNA nucleic acid molecule to increase the stability of the sense mRNA molecule. See, e.g., US2003 / 0083272.

[0299] More detailed descriptions of the mRNA modifications can be found in US2017 / 0210698A1, at pages 57-68, the contents of which are incorporated herein.

[0300] In some aspects, the biologically active agent comprises a gene editing system. In certain aspects, the gene editing system comprises a DNA nuclease or an RNA-guided DNA nuclease. In certain aspects the gene editing system may comprise a CRISPR / Cas system; Tth Argonaute (e.g., from T. thermophilus, known as ‘TtAgo’, see Swarts et al (2014) Nature 507(7491): 258-261); zinc finger nuclease (ZFN) system; ARCUS nuclease system; megaTALs; and the transcription activator-like effector nuclease (TALEN) system. Template Nucleic Acid

[0301] The methods disclosed herein may include using a template nucleic acid. The template may be used to alter or insert a nucleic acid sequence at or near a target site for an RNA-guided DNA-binding protein such as a Cas nuclease, e.g., a Class 2 Cas nuclease. In 119NTLA-103PCT; 5640-104.PCT some embodiments, the methods comprise introducing a template into the cell. In some embodiments, a single template may be provided. In other embodiments, two or more templates may be provided such that editing may occur at two or more target sites. For example, different templates may be provided to edit a single gene in a cell, or two different genes in a cell.

[0302] In some embodiments, the template may be used in homologous recombination. In some embodiments, the homologous recombination may result in the integration of the template sequence or a portion of the template sequence into the target nucleic acid molecule. In other embodiments, the template may be used in homology-directed repair, which involves DNA strand invasion at the site of the cleavage in the nucleic acid. In some embodiments, the homology-directed repair may result in including the template sequence in the edited target nucleic acid molecule. In yet other embodiments, the template may be used in gene editing mediated by non-homologous end joining. In some embodiments, the template sequence has no similarity to the nucleic acid sequence near the cleavage site. In some embodiments, the template or a portion of the template sequence is incorporated. In some embodiments, the template includes flanking inverted terminal repeat (ITR) sequences.

[0303] In some embodiments, the template sequence may correspond to, comprise, or consist of an endogenous sequence of a target cell. It may also or alternatively correspond to, comprise, or consist of an exogenous sequence of a target cell. As used herein, the term “endogenous sequence” refers to a sequence that is native to the cell. The term “exogenous sequence” refers to a sequence that is not native to a cell, or a sequence whose native location in the genome of the cell is in a different location. In some embodiments, the endogenous sequence may be a genomic sequence of the cell. In some embodiments, the endogenous sequence may be a chromosomal or extrachromosomal sequence. In some embodiments, the endogenous sequence may be a plasmid sequence of the cell.

[0304] In some embodiments, the template contains ssDNA or dsDNA containing flanking invert-terminal repeat (ITR) sequences. In some embodiments, the template is provided as a vector, plasmid, minicircle, nanocircle, or PCR product.

[0305] In some embodiments, the template nucleic acid is purified. In some embodiments, the nucleic acid is purified using a precipitation method (e.g., LiCl precipitation, alcohol precipitation, or an equivalent method, e.g., as described herein). In some embodiments, the template nucleic acid is purified using a chromatography-based method, such as an HPLC- based method or an equivalent method (e.g., as described herein). In some embodiments, the 120NTLA-103PCT; 5640-104.PCT template nucleic acid is purified using both a precipitation method (e.g., LiCl precipitation) and an HPLC-based method. In some embodiments, the template nucleic acid is purified by tangential flow filtration (TFF). Cell and Tissue Cargo Delivery

[0306] LNPs and targeted LNPs (t-LNPs) provide efficient delivery of encapsulated cargo to a cell. LNP cargo may include a variety of biologically active agents, as described above, so long as the agents may be stably encapsulated by the lipid component formulation. The LNPs are taken up by the cell through endocytosis, then escape the endosome and release the cargo into the cytosol. LNPs may be targeted or untargeted depending on desired specificity, route of administration, or risk of toxicity.

[0307] Targeted LNPs (t-LNPs) are particularly useful for in vivo administration because the t-LNPs are targeted to a desired tissue or cell type within a living organism, thereby minimizing off-target effects. t-LNPs are conjugated to ligands that bind to molecules on target cells and enhance the precision and efficacy of LNP-mediated treatments at specific tissues and cell populations when administered in vivo. In some aspects, t-LNPs provide targeted delivery of cargo to a cell or tissue of interest in vivo. In one embodiment, one or more LNP compositions described herein may be administered to a subject in need thereof. In one embodiment, a therapeutically effective amount of a composition described herein may contact a cell of a subject in need thereof. In one embodiment, a genetically engineered cell may be produced by contacting a cell with an LNP composition described herein.

[0308] The LNP or t-LNP according to any one or combination of aspects may also be used to deliver a cargo to an ex vivo cell. Ex vivo gene editing is a process where genetic material in cells is modified outside the body (in vitro) and then reintroduced into the body. In some embodiments, an ex vivo method of the present disclosure includes contacting a target cell from a patient or donor or other suitable source with an LNP or t-LNP. In some embodiments, the cell is maintained in culture. In some embodiments, the cell is transplanted into a patient. In some embodiments, the cell is removed from a subject, genetically modified ex vivo, and then administered back to the same patient. In some embodiments, the cell is removed from a subject, genetically modified ex vivo, and then administered to a subject other than the subject from which it was removed. In some embodiments, the cell is from a cell line. In some embodiments, the cell line is derived from a human subject. The cell may be cryopreserved and thawed. The cell may not have been previously cryopreserved. In some embodiments, the cell is from a cell bank. In some embodiments, the cell is genetically 121NTLA-103PCT; 5640-104.PCT modified and then transferred into a cell bank. In some embodiments the cell is removed from a subject, genetically modified ex vivo, and transferred into a cell bank. In some embodiments, a genetically modified population of cells is transferred into a cell bank. In some embodiments, a genetically modified population of immune cells is transferred into a cell bank. In some embodiments, a genetically modified population of immune cells comprising a first and second subpopulations, wherein the first and second sub-populations have at least one common genetic modification and at least one different genetic modification are transferred into a cell bank. Targeted delivery to specific cell types in ex vivo gene editing can ensure that the gene-editing machinery modifies only the intended cells and minimizes off-target effects. Cell Types Stem Cells

[0309] In some aspects, the targeted LNPs described herein can be targeted to a stem cell, such as a hematopoietic stem cell, an induced pluripotent stem cell, or another multipotent or pluripotent cell. In some aspects, the cell is a stem cell, for example, a mesenchymal stem cell that can develop into a bone, cartilage, muscle, or fat cell. In some aspects, the stem cells comprise ocular stem cells. In certain aspects, the cell is selected from mesenchymal stem cells, hematopoietic stem cells (HSCs) (for example CD45+ / Lin- / cKit+ / Sca1+ / Flk2- / CD34- cells), Epithelial Stem Cells, mononuclear cells, endothelial progenitor cells (EPCs), neural stem cells (NSCs), limbal stem cells (LSCs), tissue-specific primary cells or cells derived therefrom (TSCs), induced pluripotent stem cells (iPSCs), ocular stem cells, pluripotent stem cells (PSCs), embryonic stem cells (ESCs), and cells for organ or tissue transplantations. In some aspects, the stem cell is a cancer stem cell. HSCs divide throughout life, allowing them to give rise to the blood and immune system due to their self-renewal ability. Their multipotency enables the formation of myeloid (erythroid, megakaryocytic, and myeloid- immune) and lymphoid progenitors. HSCs are dependent on stromal-derived factors, including stem cell factor (SCF), which binds to CD117. Replacement of diseased HSCs with healthy HSCs can treat or cure various diseases and disorders. However, not all patients have a suitable immunologic match to minimize graft versus host disease (GVHD), a morbid, and potentially fatal, complication. Gene therapy can eliminate the risk of GVHD and cure NMHD by using autologous HSCs with the genetic defect corrected, either by gene addition 122NTLA-103PCT; 5640-104.PCT or editing. The present disclosure provides compositions and methods for targeted delivery of gene editing components to a target HSC. Bone Marrow and bone marrow derived cells

[0310] In some aspects, the targeted LNPs described herein can be targeted to a target tissue that is bone marrow, or a target cell that is a bone marrow cell or a bone marrow derived cell. Bone marrow or bone marrow derived cells may include Multipotent Progenitor Cell (MPP) (for example CD45+ / Lin- / cKit+ / CD34+ / Flk2+ / - cells), Common Myeloid Progenitor (CMP), Common Lymphoid Progenitor (CLP) (for example CD45+ / Lin- / Flk2+ / CD127+ cells), Granulocyte-Macrophage Progenitor (GMP), Megakaryocyte-Erythroid Progenitor (MEP), Erythroid Precursor Cells (Erythroblasts), Megakaryoblast, Granulocyte Progenitors (Myeloblasts), Monoblast, Promyelocyte, Myelocyte, Metamyelocyte, Band Cells (Immature Granulocytes), Mature Granulocytes (Neutrophils, Eosinophils, Basophils), Mature Monocytes, Mature Erythrocytes (Red Blood Cells), Mature Megakaryocytes (responsible for platelet formation), Plasma Cells (differentiated from B lymphocytes, responsible for antibody production), B Lymphocytes (immature B cells), or T Lymphocytes (immature T cells). In some aspects, the LNP may target CD34, CD38, CD45, CD117, CD123, or CD135. Immune cells

[0311] In some embodiments, the targeted LNPs described herein can be targeted to an immune cell. As used herein, “immune cell” refers to a cell of the immune system, including e.g., a lymphocyte (e.g., T cell (Helper T Cells (CD3+ T Cells), (CD4+ T cells), Cytotoxic T Cells (CD8+ T cells), Regulatory T Cells (Tregs), Memory T Cells, Gamma Delta (γδ) T Cells, Natural Killer T Cells (NKT Cells)), B cell, natural killer cell (“NK cell”, and NKT cell, or iNKT cell)), monocyte, macrophage, mast cell, dendritic cell, or granulocyte (e.g., neutrophil, eosinophil, and basophil), Follicular Dendritic Cells, Antigen-Presenting Cells (APCs).. In some embodiments, the cell is a primary immune cell. In some embodiments, the cell is an adaptive immune cell. In some embodiments, the target cell is selected from T cells, Macrophages, Neutrophils, Dendritic Cells, Natural Killer (NK) Cells, Eosinophils, Basophils, Mast Cells, B cells, Plasma Cells, Follicular Dendritic Cells, or Antigen- Presenting Cells (APCs). In some embodiments, the cell is a NK cell. In some aspects, the LNP may target CD2, CD3, CD4, CD5, CD6, CD7, CD8, or CD45.

[0312] The target cell may also include other cells, such as endothelial cells, epithelial cells, fibroblasts, mesenchymal cells, keratinocytes, melanocytes, monocytes, mononuclear cells, 123NTLA-103PCT; 5640-104.PCT adipocytes, preadipocytes, neurons, glial cells, hepatocytes, skeletal myoblasts, and smooth muscle cells. For example, primary cells can be derived from connective tissues, muscle tissues, nervous system tissues, or epithelial tissues. In some embodiments, the cell is a liver cell (hepatocyte). Methods of Delivering a Cargo

[0313] In certain aspects, methods are provided for delivering a cargo into a target cell, comprising contacting the target cell with the LNP or t-LNP, as described above. LNP and t- LNP formulations and biophysical properties may be altered in order to increase selectivity using a particular ligand type and for a particular target site within a subject. For example, the targeting ligand that is selected may influence parameters such as size, encapsulation efficiency, Z-average diameter, PDI, and mean diameter size. In some aspects, the method comprises contacting the target cell with an LNP and / or t-LNP carrying the cargo. In some aspects, the LNP or t-LNP, or the composition thereof, respectively, delivers an mRNA molecule encoding a CAR to the target cell. In some aspects, the target cell is a CD4 positive T cell, a CD8 positive T cell, or both.

[0314] In some aspects, the method comprises contacting the target cell with an LNP or LNP composition carrying a first cargo, e.g., a first nucleic acid, and an LNP or LNP composition carrying a second cargo, e.g., a second nucleic acid, wherein the second cargo is different from the first cargo. In some aspects, the method produces multiple genome edits. In some aspects, the method comprises contacting the target cell with one or more additional LNPs or LNP compositions, e.g., a third LNP or LNP composition, a fourth LNP or LNP composition, a fifth LNP or LNP composition, or a sixth LNP or LNP composition, etc, carrying one or more additional cargos, e.g., a third cargo, a fourth cargo, fifth cargo, sixth cargo, etc. In certain embodiments, at least two of the LNPs or LNP compositions are administered sequentially. In some embodiments, at least two of the LNPs or LNP compositions are administered simultaneously. In some aspects, the target cell is contacted with the LNP or LNP composition in vitro. In some aspects, the target cell is contacted with the LNP or LNP composition in vivo. In some aspects, the target cell is contacted with the LNP or LNP composition ex vivo. In some aspects, the method further comprises expanding the contacted cell in vitro. In some embodiments, the expanded cell exhibits increased survival.

[0315] Exemplary target cells include, but are not limited to, prokaryotic cells (e.g., a bacterial cell) or eukaryotic cells. As used herein, the term “eukaryotic cell” refers to a cell 124NTLA-103PCT; 5640-104.PCT having a distinct, membrane-bound nucleus. Such cells may include, for example, mammalian, insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryote, such as mammalian, avian, plant, or insect cells. Non-limiting examples of mammalian cells include a rodent cell (e.g., a mouse cell, a rat cell, a hamster cell, such as Chinese hamster ovary (CHO) cells, or a non-human primate cell, or a human cell, such as human embryonic kidney cells (e.g., HEK293 cells). In some aspects, the target tissue or cell may comprise spleen cells, liver cells, bone marrow cells, immune cells, kidney cells, endocrine cells, muscle cells, heart cells, lung cells, ocular cells, or cells in the central nervous system.

[0316] In some aspects, the cell is a type of cell useful in a therapy, for example, adoptive cell therapy (ACT). Examples of ACT include autologous and allogeneic cell therapies.

[0317] In certain aspects, methods are provided for expressing a cargo in a target cell comprising contacting the target cell with the LNP and / or t-LNP, as described above, or compositions thereof. In some aspects, the method comprises expressing the cargo in the target cell in greater than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the target cells contacted with the LNP or t-LNP composition. In some aspects, the target cell is in a living subject, wherein expression of the cargo in the target cell after contacting with the t-LNP or t-LNP composition is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5- fold or 5-fold higher relative to expression in the same cell-type contacted with 4- or 5- component LNP or LNP compositions without a targeting ligand.

[0318] In some aspects, cargo delivery and expression mediated by LNP, t-LNP, or a composition thereof, respectively, is enhanced by controlling the neutral lipid content. In some aspects, gene editing mediated by LNP, t-LNP, or composition thereof, respectively, is enhanced by controlling the neutral lipid content. In some aspects, hepatic cell delivery or gene editing by the LNP, t-LNP, or composition thereof, respectively, is reduced by controlling the neutral lipid content. In certain aspects, the lipid component comprises the neutral lipid in an amount from about 12 mol% to about 25 mol% or about 15 mol% to about 22.5 mol%. In certain aspects, expression of the cargo in the target cell after contacting with the LNP, t-LNP, or composition thereof, respectively, is at least 1.5-fold, 2-fold, 2.5-fold, 3- fold, 3.5-fold, 4-fold, 4.5-fold or 5-fold higher relative to expression after contacting with a LNP, t-LNP, or composition thereof, respectively, with a neutral lipid content outside the above-described ranges. In certain aspects, gene editing in the target cell after contacting 125NTLA-103PCT; 5640-104.PCT with the LNP, t-LNP, or composition thereof, respectively, is at least 1.5-fold, 2-fold, 2.5- fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold or 5-fold higher relative to gene editing after contacting with an LNP, t-LNP, or composition thereof, respectively, with a neutral lipid content outside an amount from about 12 mol% to about 25 mol% or about 15 mol% to about 22.5 mol%. In some aspects, hepatic cell delivery or gene editing by the LNP, t-LNP, or composition thereof, respectively, is reduced by about 10% to about 60%, about 15% to about 50%, or about 20% to about 40% relative to hepatic cell delivery or gene editing after contacting with an LNP, t-LNP, or composition thereof, respectively, with a neutral lipid content outside the above-described ranges.

[0319] In certain aspects, methods are provided for genetically engineering a target cell comprising contacting the target cell with the LNP, as described above, or compositions thereof. In some aspects, the cargo is selected from a CRISPR / Cas system, a Tth Argonaute (TtAgo) system, a zinc finger nuclease (ZFN) system, ARCUS nuclease system, megaTALs, or a transcription activator-like effector nuclease (TALEN) system. In some aspects, the cargo comprises a guide RNA and optionally an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the guide RNA comprises a sequence for targeting a genomic sequence in the target cell. In some aspects, the contacting step comprises introducing a single stranded DNA nick. In some aspects, the contacting step comprises introducing a double-stranded DNA break. In some aspects, the method further comprising introducing at least one template nucleic acid into the target cell. In some aspects, the method comprises administering the guide RNA and the RNA-guided DNA binding agent in a single LNP composition. In some aspects, the gene editing comprises introducing a gene knockout. In some aspects, the gene editing comprises introducing a gene modification. In some aspects, the method comprises contacting a tissue of an animal with the lipid composition. In some aspects, the animal is a human.

[0320] In certain aspects, methods are provided for administering a cargo to a subject in need thereof, comprising administering a composition comprising the LNP, as described above, or compositions thereof.

[0321] In some aspects, the method comprises administering the LNP(s) or LNP composition(s) in an amount sufficient to obtain at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% biological activity, e.g., gene editing, in a target cell of the subject. In some aspects, the subject has a genetic disease or disorder. In some aspects, the LNP comprises a composition comprising a buffer, an excipient, or a combination thereof. 126NTLA-103PCT; 5640-104.PCT

[0322] In some aspects, the excipient comprises a cryoprotectant, a tonicity modifying agent, or a combination thereof. In some aspects, the method comprises administering the LNP systemically, parenterally, or intratumorally. In some aspects, the method comprises administering the LNP via injection or infusion. In some aspects, the composition is stable following one or more freeze / thaw cycles. Bone Marrow Derived Cell Delivery

[0323] LNPs according to the present disclosure may be targeted in vivo or ex vivo to a bone marrow derived cell using a 5-component LNP composition according to the present disclosure. In some embodiments, the LNPs are used to target bone marrow derived cells in vivo. For example, an LNP may be delivered to a bone marrow derived cell by contacting the bone marrow derived cell with an LNP comprising: (i) a lipid component comprising: a) an anchor PEG-lipid; b) a helper lipid; c) a neutral lipid; d) a structural PEG-lipid; and e) an ionizable lipid, (ii) a first coupling moiety attached to the anchor PEG-lipid; and (iii) a cargo.

[0324] Various types of bone marrow derived cells may be targeted as described above. In some aspects, the bone marrow derived cell is a CD34 positive cell. In some aspects, the bone marrow derived cell is a hematopoietic stem cell (HSC).

[0325] The lipid composition, the targeting ligand, or both the lipid composition and the targeting ligand of the LNP may provide improved cargo delivery to a bone marrow derived cell. Suitable ionizable lipids, anchor PEG-lipids, helper lipids, neutral lipids, and structural PEG lipids for targeting an LNP to a bone marrow derived cell are provided herein. In some aspects, the LNP is a non-targeted LNP and the lipid composition of the LNP improves delivery of a cargo to a bone marrow derived cell. In some aspects, the LNP is a targeted LNP comprising a targeting ligand and the lipid composition of the LNP further improves delivery of a cargo to a bone marrow derived cell. In some aspects, the ionizable lipids used in the LNP formulation improve delivery of a cargo to a bone marrow derived cell. In some 127NTLA-103PCT; 5640-104.PCT aspects, the ionizable lipid is a lipid according to Compound 3 as described below in Examples 12-14.

[0326] Various cargos may be delivered to the bone marrow derived cell as described in any of the aspects according to the present disclosure. For example, the cargo may be a polynucleotide or a polypeptide. In some aspects, the polynucleotide is a vector or an mRNA comprising a gene of interest. In some aspects, the cargo is expressed in the bone marrow derived cell. In some aspects, the gene of interest is expressed in a specific type of cell of specific types of cells, for example in a bone marrow derived cell.

[0327] In some aspects, provided herein is method of delivering a cargo to a bone marrow derived cell, comprising contacting the bone marrow derived cell with the LNP, wherein the LNP comprises: (i) a lipid component comprising: f) an anchor PEG-lipid; g) a helper lipid; h) a neutral lipid; i) a structural PEG-lipid; and j) an ionizable lipid comprising:(iii) a cargo. In some aspects, the cargo comprises a gene editing system. In some aspects, the method comprises contacting the bone marrow derived cell with the LNP as a first LNP comprising a first cargo and contacting the bone marrow derived cell with a second LNP comprising a second cargo. In some aspects, the first cargo and the second cargo together comprise the gene editing system.

[0328] In some aspects, gene editing in the bone marrow derived cell after contacting with the LNP is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold or 5-fold higher 128NTLA-103PCT; 5640-104.PCT relative to gene editing after contacting with a LNP comprising a different ionizable lipid than compound 3.

[0329] In some aspects, the cargo expression in the bone marrow derived cell after contacting with the LNP is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold or 5-fold higher relative to cargo expression after contacting with a LNP comprising a different ionizable lipid than compound 3.

[0330] In some aspects, the content of the neutral lipid in the LNP formulation improves delivery of a cargo to a bone marrow derived cell. In some aspects, the lipid component comprises (a) the anchor PEG-lipid in an amount from about 0.001 to about 1.5 mol % of the lipid component; (b) the helper lipid in an amount from about 25 to about 50 mol % of the lipid component; (c) the neutral lipid in an amount from about 7.5 to about 25 mol % of the lipid component; (d) the structural PEG-lipid in an amount from about 0.5 to about 5 mol % of the lipid component; and (e) the ionizable lipid in an amount of from about 30 to about 55 mol % of the lipid component. In some aspects, the neutral lipid in an amount from about 10 to about 25 mol % of the lipid component, or about 15 to about 22.5 mol % of the lipid component.

[0331] In some aspects, the LNP comprises neutral lipid having a content that is about 1.1x to about 3.0x, for example 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2.0x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3.0x, relative to a reference LNP composition. In some aspects, the reference LNP composition comprises the neutral lipid in an amount of about 10 mol%.

[0332] In some aspects, the cargo expression in the bone marrow derived cell after contacting with the LNP is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold or 5-fold higher relative to cargo expression after contacting with a LNP comprising a different content of neutral lipid. In some aspects, gene editing in the bone marrow derived cell after contacting with the LNP is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold or 5-fold higher relative to gene editing after contacting with a LNP comprising a different content of neutral lipid. In some aspects, the different content of neutral lipid is above or below about 7.5 to about 25 mol %. In some aspects, the different content of neutral lipid is above or below about 10 to about 25 mol %. In some aspects, the different content of neutral lipid is below about 10 mol% or below about 15 mol%.

[0333] In some aspects, the method comprises contacting the bone marrow derived cell with the LNP comprising a targeting ligand attached to the first coupling moiety via a second 129NTLA-103PCT; 5640-104.PCT coupling moiety. In some aspects, the targeting ligand is selected from an antibody, an antibody fragment, a small molecule, or a peptide. In some aspects, the targeting ligand targets CD117. In some aspects, the targeting ligand is an anti-CD117 antibody or antibody fragment.

[0334] In some aspects, gene editing in the bone marrow derived cell after contacting with the LNP is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold or 5-fold higher relative to gene editing after contacting with a LNP lacking the targeting ligand.

[0335] In some aspects, the cargo expression in the bone marrow derived cell after contacting with the LNP is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold or 5-fold higher relative to cargo expression after contacting with a LNP lacking the targeting ligand.

[0336] Contacting the bone marrow derived cell may be conducted in vitro, in vivo, or ex vivo. In some aspects, the contacting comprises administering the LNP to a subject. In some aspects, the LNP is administered one or more times. In some aspects, the LNP is administered as a multi-dose regimen. In some aspects, the LNP is administered two or more times, up to three times, up to four times, or up to five times. CAR Delivery

[0337] LNP based delivery of mRNA-encoding CAR therapeutics in an ex vivo or in vivo setting provides several advantages including reducing the risk of target cell induced toxicities, because of their transient nature, as well as avoiding the risk of genomic integration. LNP mediated CAR delivery is applicable to many diseases that require short- term reduction in specific pathogenic cells, such as for the treatment of myocardial fibrosis, autoimmune diseases, cancer, and many other diseases. In some aspects, the LNP, t-LNP, or composition thereof, respectively, delivers an mRNA molecule encoding a CAR to a target cell, such as a T cell.

[0338] Targeted LNPs also provide a means to deliver an mRNA encoding a CAR in vivo by selectively targeting a cell surface antigen. In some aspects, a t-LNP, or composition thereof, respectively, targets a T cell epitope.

[0339] B cell depletion is an important mode of treatment for various conditions including conditions like rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), and pemphigus vulgaris, B cells contribute to inflammation and tissue damage. By depleting B cells, these therapies can reduce autoimmune responses and alleviate symptoms. In some cases, B cell depletion can be used to manage infections where B cells 130NTLA-103PCT; 5640-104.PCT play a role in exacerbating the disease. For example, some treatments aim to reduce B cell activity in cases of chronic viral infections like hepatitis B. In the context of organ transplants, B cell depletion is sometimes used to prevent organ rejection by reducing the immune system’s ability to mount an antibody-mediated attack on the transplanted organ. In some embodiments, the CAR can comprise an antigen-binding domain that specifically recognizes an antigen on the surface of an immune cell, e.g., a B cell. In some embodiments, the CAR can comprise an antigen-binding domain that specifically binds to CD19. In some embodiments, the CAR can comprise an antigen-binding domain that specifically binds to CD20.

[0340] In some embodiments, t-LNPs delivered in vivo may be used to deplete B cells in a subject, while minimizing off-target effects.

[0341] In some aspects, provided herein is a method of delivering a lipid nanoparticle (LNP) to an immune cell, e.g., a T cell, comprising contacting an immune cell with a lipid nanoparticle comprising (i) a lipid component comprising: a) an anchor PEG-lipid; b) a helper lipid; c) a neutral lipid; d) a structural PEG-lipid; and e) an ionizable lipid, (ii) a first coupling moiety attached to the anchor PEG-lipid; and (iii) a cargo, wherein the cargo is a mRNA molecule encoding a CAR encapsulated in the LNP.

[0342] In some embodiments, the step of contacting comprises administering the LNP to a subject. Suitable ionizable lipids, anchor PEG-lipids, helper lipids, neutral lipids, and structural PEG lipids are provided herein. In some embodiments, the CAR comprises an antigen-binding region that specifically binds to CD19 or CD20.

[0343] In some aspects, provided herein is a method of delivering a lipid nanoparticle (LNP) to a T cell, comprising: contacting a T cell with a lipid nanoparticle comprising a (i) lipid component comprising: a) an anchor PEG-lipid; b) a helper lipid; 131NTLA-103PCT; 5640-104.PCT c) a neutral lipid; d) a structural PEG-lipid; and e) an ionizable lipid comprising: (ii)(iii) a cargo, wherein the cargo is a mRNA molecule encoding a CAR encapsulated in the LNP.

[0344] In some embodiments, the step of contacting comprises administering the LNP to a subject. Suitable anchor PEG-lipids, helper lipids, neutral lipids, and structural PEG lipids are provided herein. In some embodiments, the CAR comprises an antigen-binding region that specifically binds to CD19 or CD20.

[0345] In some aspects, the LNP targets a T cell epitope. In some aspects, the LNP targets a cell selected from CD2+ cell, CD3+ cell, CD4+ cell, CD5+ cell, CD6+ cell, CD7+ cell, CD8+ cell, or a CD45+ cell. For example, the LNP can comprise an antigen-binding moiety coupled to the anchor PEG lipid, where the antigen-binding moiety specifically binds to CD2, CD3, CD4, CD5, CD6, CD7, CD8, or CD45. In some embodiments, the antigen-binding moiety is an antibody, or an antigen-binding fragment thereof, e.g., an scFv, a Fab, or a VHH. In some aspects, the target cell is a CD4 positive T cell, a CD8 positive T cell, or both. In some aspects, the contacting comprises administering the LNP to a subject. In some aspects, the subject has a depletion of B cells 48 hours after administration, 72 hours after administration, 1 week after administration, or 1 month after administration. In some aspects, the LNP is administered one or more times. In some aspects, the LNP is administered as a multi-dose regimen. In some aspects, the LNP is administered two or more times, up to three times, up to four times, or up to five times.

[0346] In some aspects, about 50% to about 99%, about 60% to about 95%, about 70% to about 90% of B cells are depleted in the subject relative to untreated control. In some aspects, the LNP is dosed one or more times, e.g., 2, 3, 4, 5, or more doses of the LNP are administered to the subject. In some aspects, redosing is performed within 1, 2, 3, 4, 5, 6, or 7 132NTLA-103PCT; 5640-104.PCT days, 1, 2, 3, 4, 5, or 6 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 months, or 1, 2, 3, 4, or 5 years between any 1, 2, 3, 4, 5, or more doses. In some aspects, the subject is dosed multiple times with the LNP and has a depletion of B cells after redosing with an LNP. In one aspect, the CAR stimulatory molecule is the zeta chain associated with the T-cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule. In one aspect, the costimulatory molecule is chosen from the costimulatory molecules 41BB (i.e., CD137), CD27 and / or CD28. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule.

[0347] While the inventions are described in conjunction with the illustrated embodiments, it is understood that they are not intended to limit the invention to those embodiments. On the contrary, the disclosure is intended to cover all alternatives, modifications, and equivalents, including equivalents of specific features, which may be included within the inventions as defined by the appended claims.

[0348] Both the foregoing general description and detailed description, as well as the following examples, are exemplary and explanatory only and are not restrictive of the teachings. The section headings used herein are for organizational purposes only and are not to be construed as limiting the desired subject matter in any way. In the event that any literature incorporated by reference contradicts any term defined in this specification, this 133NTLA-103PCT; 5640-104.PCT specification controls. All ranges given in the application encompass the endpoints unless stated otherwise. EXAMPLES

[0349] The following examples are intended to exemplify the present disclosure and are not limitations of the claimed invention. All molecules, compositions, methods, assays, and results disclosed in the examples form non-limiting parts of the present disclosure. Example 1: General Methods

[0350] 1.1 Preparation of LNPs

[0351] LNPs were prepared in a 4-component or 5-component formulation. The 4- component LNP formulations comprised an ionizable lipid (e.g., Compounds 1-8), distearoylphosphatidylcholine (DSPC), cholesterol, and a structural PEG-lipid (e.g., C13 Ether PEG(2000), C14 DMG PEG(2000), C14 Ether PEG(2000)). The 5-component LNP formulations comprised an ionizable lipid (e.g., Compounds 1-8), DSPC, cholesterol, a structural PEG-lipid (e.g., C13 Ether PEG(2000), C14 DMG PEG(2000), C14 Ether PEG(2000)), and an anchor PEG-lipid (e.g., DSPE-PEG(2000) or DSPE-PEG(1000)); the anchor PEG-lipid can be functionalized with a coupling / conjugation moiety, and incubated before or after the 5-component formulation is produced with a ligand in various amounts to allow for the ligand to attach to the anchor PEG, thereby producing a targeted LNP with a specific ligand density. Table 1 shows a list of compounds used for the formulation of 4- component and 5-component LNPs. Table 2 shows a list of targeting ligands used to produce targeted LNPs described in this disclosure. The lipids were combined to yield desired molar ratios of 50% ionizable lipid, 10% DSPC, 38% cholesterol, and 2% total PEG (structural PEG-lipid and anchor PEG-lipid) lipid, unless otherwise indicated (see for example, Table 3, below). In assays for in vitro editing in cell cultures or in vivo editing in mice, Cas9 mRNA and chemically modified sgRNA targeting a genomic sequence were formulated in LNPs as cargo encapsulated in the LNP. In some cases, LNPs were prepared with a single RNA species such as a mRNA or a gRNA. In some cases, LNPs were prepared with a mixture of mRNA and a guide RNA, at a 1:2 w / w sgRNA:Cas9 mRNA ratio, unless otherwise indicated. The LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 3 to 9.

[0352] In general, the LNP lipid components were dissolved in 100% ethanol. The RNA cargos (e.g., Cas9 mRNA and sgRNA) were dissolved in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in a RNA solution with a concentration of RNA cargo of approximately 0.45 134NTLA-103PCT; 5640-104.PCT mg / mL, unless otherwise indicated. The LNPs were formed by an impinging jet mixing of the lipid in ethanol with two volumes of RNA solution and one volume of water. First, the lipid in ethanol was mixed through a mixing cross with the two volumes of RNA solution. Then, a fourth stream of water was mixed with the outlet stream of the cross through an inline tee. (See, e.g., WO2016010840, Fig.2). The LNPs were held for 1 hour at room temperature, and further diluted with water (approximately 1:1 v / v). Diluted LNPs were buffer exchanged into 50 mM Tris, 45 mM NaCl, 5% (w / v) sucrose, pH 7.5 (TSS) and concentrated as needed by methods known in the art. The resulting LNP mixtures were then filtered using a 0.2 μm sterile filter. The final LNPs were characterized as described below and were stored at 4°C or -80°C until further use. 135NTLA-103PCT; 5640-104.PCT

[0353] Table 1. Structures of compounds used for the preparation of 4-component or 5-component LNP formulations Com D Chol Com Com136NTLA-103PCT; 5640-104.PCT Com Com Com Com Com137NTLA-103PCT; 5640-104.PCT Com Com Com138NTLA-103PCT; 5640-104.PCT

[0354] Table 2. List of targeting ligands used for the production in targeted LNPs. Ligand ID Target

[0355] 1.2 LNP Composition Analytics

[0356] Dynamic Light Scattering (“DLS”) can be used to characterize the polydispersity index (“pdi” or “PDI”) and size of the LNPs of the present disclosure. DLS measures the scattering of light that results from subjecting a sample to a light source. PDI, as determined from DLS measurements, represents the distribution of particle size (around the mean particle size) in a population, with a perfectly uniform population having a PDI of zero.

[0357] Electrophoretic light scattering can be used to characterize the surface charge of the LNP at a specified pH. The surface charge, or the zeta potential, is a measure of the magnitude of electrostatic repulsion / attraction between particles in the LNP suspension.

[0358] Asymmetric-Flow Field Flow Fractionation – Multi-Angle Light Scattering (AF4- MALS) can be used to separate particles in the composition by hydrodynamic radius and then measure the molecular weights, hydrodynamic radii and root mean square radii of the 139NTLA-103PCT; 5640-104.PCT fractionated particles. This allows for the ability to assess molecular weight and size distributions as well as secondary characteristics such as the Burchard-Stockmeyer Plot (ratio of root mean square (“rms”) radius to hydrodynamic radius over time suggesting the internal core density of a particle) and the rms conformation plot (log of rms radius vs log of molecular weight where the slope of the resulting linear fit gives a degree of compactness vs elongation).

[0359] Nanoparticle tracking analysis (NTA, Malvern Nanosight) can be used to determine particle size distribution as well as particle concentration. LNP samples are diluted appropriately and injected onto a microscope slide. A camera records a movie of the scattered light as the particles are slowly infused through field of view. After the movie is captured, the Nanoparticle Tracking Analysis processes the movie by tracking pixels and calculating a diffusion coefficient. This diffusion coefficient is translated into the hydrodynamic radius of the particle. The instrument also counts the number of individual particles counted in the analysis to give particle concentration.

[0360] Cryo-electron microscopy (“cryo-EM”) can be used to determine the particle size, morphology, and structural characteristics of an LNP.

[0361] Lipid compositional analysis of the LNPs can be determined from liquid chromatography followed by charged aerosol detection (LC-CAD). This analysis provides a comparison of the actual lipid content versus the theoretical lipid content.

[0362] LNP compositions were analyzed for average particle size, polydispersity index (pdi), total RNA content, encapsulation efficiency of RNA, and zeta potential. LNP compositions were further characterized by lipid analysis, AF4-MALS, NTA, and / or cryo-EM. Average particle size and polydispersity were measured by dynamic light scattering (DLS) using a Malvern Zetasizer DLS instrument. LNP samples were diluted with PBS buffer prior to being measured by DLS. Z-average diameter (“Z-avg”) which is a light intensity-weighted measurement of average particle size was reported, along with number average diameter (“number mean,” “number average,” or “num-avg”) which is a number-weighted measurement of average particle size, and PDI. A Malvern Zetasizer instrument was used to measure the zeta potential of the LNP. Samples were diluted 1:17 (50 µL into 800 µL) in 0.1X PBS, pH 7.4 prior to measurement.

[0363] Encapsulation efficiency (“%E”) was calculated as the percentage of (Total RNA - Free RNA) / Total RNA. A fluorescence-based assay (Ribogreen®, ThermoFisher Scientific) was used to determine total RNA concentration and free RNA concentration. LNP samples 140NTLA-103PCT; 5640-104.PCT were diluted appropriately with 1x TE buffer containing 0.2% Triton-X 100 to determine total RNA or 1x TE buffer to determine free RNA. Standard curves were prepared by utilizing the starting RNA solution used to make the compositions and diluted in 1x TE buffer + / - 0.2% Triton-X 100. Diluted RiboGreen® dye was then added (according to the manufacturer's instructions) to each of the standards and samples, and the solutions were allowed to incubate for approximately 10 minutes at room temperature, in the absence of light. A SpectraMax M5 Microplate Reader (Molecular Devices) was used to read the samples with excitation, auto cutoff and emission wavelengths set to 488 nm, 515 nm, and 525 nm respectively. Total RNA and free RNA concentrations were determined from the appropriate standard curves. Alternatively, the total RNA concentration was determined by a reverse-phase ion-pairing (RP-IP) HPLC method. Triton X-100 was used to disrupt the LNPs, releasing the RNA. The RNA was then separated from the lipid components chromatographically by RP-IP HPLC and quantified against a standard curve using UV absorbance at 260 nm.

[0364] The same procedure may be used for determining the encapsulation efficiency of a DNA-based cargo component, in which case encapsulation efficiency is calculated as the percentage of (Total DNA - Free DNA) / Total DNA. In a fluorescence-based assay, Oligreen Dye may be used for single-stranded DNA, and Picogreen Dye may be used for double- stranded DNA.

[0365] AF4-MALS was used to obtain molecular weight and size distributions as well as secondary statistics from those calculations. LNPs were diluted as appropriate and injected into a AF4 separation channel using an HPLC autosampler where they were focused and then eluted with an exponential gradient in cross flow across the channel. All fluid was driven by an HPLC pump and Wyatt Eclipse Instrument. Particles eluting from the AF4 channel flowed through a UV detector, multi-angle light scattering detector, quasi-elastic light scattering detector and differential refractive index detector. Raw data was processed by using a Debye model to determine molecular weight and rms radius from the detector signals.

[0366] Lipid components in LNPs were analyzed quantitatively by HPLC coupled to a charged aerosol detector (CAD) and / or a UV detector (UV). Chromatographic separation of lipid components was achieved by reverse phase HPLC. CAD is a destructive mass-based detector which detects all non-volatile compounds and the signal is consistent regardless of analyte structure. 141NTLA-103PCT; 5640-104.PCT

[0367] The pKa of each ionizable lipid was determined according to the method in Jayaraman, et al. (Angew Chem Int Ed Engl 51(34), 2012, 8529-8533) with the following adaptations. The pKa was determined for unformulated ionizable lipid in ethanol. Lipid stock solutions (2.94 mM) were diluted into Sodium Phosphate Buffers (0.1 M, Boston Bioproducts) of different pH (pH-range: 4.5-9.0) yielding a final lipid concentration of approx.100 µM. The test samples were supplemented with TNS {6-(p-Toluidino)-2- naphthalenesulfonic acid sodium salt}, incubated and the fluorescence intensity was measured using excitation and emission wavelengths of 321 nm and 448 nm, respectively. The recorded data were normalized and the respective pKa values were derived from sigmoidal fitting. The pKa values of ionizable lipids used in the present disclosure are shown in Table 3.

[0368] Table 3. pKa values of ionizable lipids used in the present disclosure Compound # Lipid pKa

[0369] 1.3 Cas9 mRNA and gRNA Cargos

[0370] Capped and polyadenylated mRNA containing N1-methyl pseudo-U was generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase. The linearized plasmid DNA containing a T7 promoter, and a sequence for transcription was linearized by restriction endonuclease digestion followed by heat inactivation of the reaction mixture and purified from enzyme and buffer salts. Messenger RNA was synthesized and purified using standard techniques known in the art.

[0371] Messenger RNA was generated from plasmid DNA encoding an open reading frame according to ORF sequences for mRNA encoding a GFP protein (SEQ ID NO: 6) and mRNA encoding a SpyCas9 protein (SEQ ID NO: 3) as shown in the Sequence Table below. When 142NTLA-103PCT; 5640-104.PCT the sequences cited in this paragraph are referred to below with respect to RNAs, it is understood that Ts should be replaced with Us (which can be modified nucleosides as described above). Messenger RNAs used in the Examples include a 5' cap and a 3' polyadenylation sequence e.g., up to 100 nts. Guide RNAs were chemically synthesized by commercial vendors or using standard in vitro synthesis techniques with modified nucleotides.

[0372] 1.4 Formulation Stability Assessment

[0373] Biophysical characteristics of LNP formulations before and after they were subjected to one or more freeze-thaw cycles were compared to assess stability through the freeze-thaw cycles. Biophysical characteristics such as encapsulation efficiency (“%E”), Z-average diameter (“Z-avg”), polydispersity index (“PDI”) and number average (“num-avg”) were measured, as described above, for LNPs stored at 4°C (pre-freeze-thaw) and at -80°C (post- freeze-thaw). Each freeze-thaw cycle consisted of freezing the LNP formulation (0.5 mL in 1.5 mL aliquot tube, or 7 mL in 10 mL vial) at -80°C for 15-18 hours, then thawing the frozen LNPs at room temperature for 0.5-2 hours. Thawed LNP formulations were mixed with gentle inversions for homogeneity. Freeze-thaw cycles were repeated one or more times prior to analysis to assess stability over multiple consecutive freeze-thaw cycles.

[0374] 1.5 LNP Delivery In Vivo

[0375] Wild-typeC57Bl / 6J female mice, or humanized NOG mice, ranging from 6-15 weeks of age, were used in each study. C57Bl / 6J animals were either wild type (WT), or had a ApoE or LDLR gene KO, as indicated in the corresponding experiments. Animals were weighed and dosed based on individual body weights at time of dosing. LNPs were dosed via the lateral tail vein in a volume of 10 uL per gram of animal body weight, at a dose of 2 mpk (mg per kg of animal body weight) of total RNA, unless otherwise indicated. The animals were periodically observed for adverse effects for at least 24 hours post dose.

[0376] Animals were euthanized by carbon dioxide asphyxiation, at days 4, 5 or 6 after dosing for editing readout, and at 6, 14 or 24 hours after dosing for protein expression readout. Liver tissue, lung tissue, spleen tissue, blood, and bones were collected. Cells from blood were obtained by red blood cell lysis and centrifugation. Cells from the spleen were obtained by dissociating the spleen tissue and passing the resulting cell suspension through a filter, followed by centrifugation. Whole bone marrow (WBM) material was obtained from bones, and hematopoietic stem and progenitor cell (HSPC) populations were obtained from flow cytometry sorting as described below. Protein expression (e.g., GFP, mScarlet) in cells 143NTLA-103PCT; 5640-104.PCT was measured using flow cytometry analysis as described below. For gene editing readout, genomic DNA was isolated from the liver tissue, the lung tissue, the whole bone marrow (WBM), and HSPC. Editing was measured using Next-Generation Sequencing (NGS).

[0377] 1.6 Flow Cytometry

[0378] Cells were obtained from mouse tissues as described above. A mix of relevant antibodies in FACS buffer was prepared to select for the cell population of interest (e.g., CD4+ T cells, CD8+ T cells, B cells, NK cells, Hematopoietic Stem Cells (HSCs), Multipotent Progenitors (MPPs), Myeloid Progenitors (MPs), Common Lymphoid Progenitors (CLPs), lin− / SCA-1+ / c-KIT+ cells (LSKs), lineage depleted cells), according to methods known in the art. HSCs were defined as CD45+ / Lin- / cKit+ / Sca1+ / Flk2- / CD34-; MPPs were defined as CD45+ / Lin- / cKit+ / CD34+ / Flk2+ / -; MPs were defined as CD45+ / Lin- / cKit+ / Sca1-; and CLPs were defined as CD45+ / Lin- / Flk2+ / CD127+. Pelleted cells were resuspended in the antibody mix and incubated at the appropriate temperature in the dark for 30 minutes to 1.5 hours depending on the antibody panel.

[0379] For protein expression analysis, the antibody mix was washed off after incubation, and the cells were run through a Beckman Coulter CytoFLEX LX. Protein expression was analyzed using FlowJo software.

[0380] For gene editing analysis, the antibody mix was washed off after incubation, and the cells were run through a BD FACSAria™ Fusion Flow Cytometer one sample at a time. Sorted material for the populations of interest was collected individually to be processed for NGS Sequencing.

[0381] 1.7 NGS Sequencing

[0382] In brief, to quantitatively determine the efficiency of editing at a target location in the genome, genomic DNA was isolated and deep sequencing was utilized to identify the presence of insertions and deletions (“indels”) introduced by gene editing.

[0383] PCR primers were designed around the target site (e.g., B2M), and the genomic area of interest was amplified. Additional PCR was performed according to the manufacturer's protocols (Illumina) to add the necessary chemistry for sequencing. The amplicons were sequenced on an Illumina MiSeq instrument. The reads were aligned to the human reference genome (e.g., hg38) after eliminating those having low quality scores. The resulting files containing the reads were mapped to the reference genome (BAM files), where reads that overlapped the target region of interest were selected and the number of wild type reads 144NTLA-103PCT; 5640-104.PCT versus the number of reads which contain an insertion, substitution, or deletion was calculated.

[0384] The editing percentage (e.g., the “indel efficiency” or “percent indels” or “% editing” or “% indel”) can be defined as the total number of sequence reads with insertions or deletions over the total number of sequence reads, including wild type. 145NTLA-103PCT; 5640-104.PCT Fab 8 Fab 3 Fab 2 Fab 7 Fab 10 VHH (CD7) VHH (CD8)(SEQ ID NO: 32) (SEQ ID NO: 34) (SEQ ID NO: 36) 146NTLA-103PCT; 5640-104.PCT Example 2 – Production of 5-Component LNP Formulations

[0385] To evaluate the biophysical profiles, stability, and efficacy of 5-component LNPs compared to 4-component LNPs, a range of LNP formulations comprising a fifth lipid component (an anchor PEG-lipid) were prepared and tested. The purpose was to convert conventional 4-component LNPs into targeted LNPs having a fifth lipid to serve as a conjugation handle and anchor to a targeting ligand. For LNP formulations lacking neutral lipid (e.g., DSPC), the anchor lipid serves as the fourth lipid component.

[0386] 4-component or 5-component LNP formulations were prepared as described in Example 1. Ionizable lipids according to Formula (I), (II), (III), (IV), and (V) were used in combination with DSPC, cholesterol, and various structural and anchorPEG-lipids.

[0387] For 5-component LNPs, the anchor PEG-lipid included the first coupling moiety as an unpaired conjugation moiety for attachment to a second coupling moiety on a corresponding targeting ligand. LNP formulations were prepared using anchor PEG-lipids including DSPE- PEG(1000)-Maleimide, DSPE-PEG(2000)-Maleimide, DSPE-PEG(2000)-Azide, and DSPE- PEG(2000)-FITC, as listed in Table 1. Table 4 describes the molar compositions and RNA cargo used for the formulation of 4-component and 5-component LNPs. 147NTLA-103PCT; 5640-104.PCT

[0388] Table 4. Compositions of 4-component or 5-component LNPs.LNP ILNP#1 LNP#2 LNP#3 LNP#4 LNP#5 LNP#6 LNP#7 LNP#8 LNP#9 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#2 LNP#2 LNP#2 LNP#2 LNP#2LNP#25 Compound 3 C13 Ether PEG(2000) DSPE-PEG(2000) (non functionalized) 50:9:38:2.925:0.075 gRNA:Cas9 mRNA, 1:2 mass ratio 148NTLA-103PCT; 5640-104.PCTLNP ILNP#2 LNP#2 LNP#2 LNP#2 LNP#3 LNP#3 LNP#3 LNP#3 LNP#3 LNP#3 LNP#3 LNP#3 LNP#3 LNP#3 LNP#4 LNP#4 LNP#4 LNP#4 LNP#4 LNP#4 LNP#4 LNP#4 LNP#4 LNP#4 LNP#5LNP#53 Compound 3 C14 DMG PEG(2000) DSPE-PEG(2000) (non functionalized) 50:10:37.8:1.98:0.22 gRNA:Cas9 mRNA, 1:2 mass ratio 149NTLA-103PCT; 5640-104.PCTLNP ILNP#5 LNP#5 LNP#5 LNP#5 LNP#5 LNP#5 LNP#6 LNP#6 LNP#6 LNP#6 LNP#6 LNP#6 LNP#6 LNP#6 LNP#6 LNP#6 LNP#7 LNP#7 LNP#7 LNP#7 LNP#7 LNP#7 LNP#7 LNP#7 LNP#7LNP#79 Compound 3 C13 Ether PEG(2000) DSPE-PEG(2000)-SpyCatcher 50:10:37.8:2.155:0.045 gRNA:Cas9 mRNA, 1:2 mass ratio 150NTLA-103PCT; 5640-104.PCTLNP ILNP#8 LNP#8 LNP#8 LNP#8 LNP#8 LNP#8 LNP#8 LNP#8 LNP#8 LNP#8 LNP#9 LNP#9 LNP#9 LNP#9 LNP#9 LNP#9 LNP#9 LNP#9 LNP#9 LNP#9 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1LNP#105 Compound 2 C13 Ether PEG(2000) DSPE-PEG(2000) (non functionalized) 50:10:38.5:1.425:0.075 gRNA:Cas9 mRNA, 1:2 mass ratio 151NTLA-103PCT; 5640-104.PCTLNP ILNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 ¥Molar ratio (ionizable lipid:DSPC:cholesterol:structural PEG:anchor PEG) 152NTLA-103PCT; 5640-104.PCT Example 3 – Characterization of targeted LNPs comprising antibodies or antibody fragments

[0389] To evaluate the biophysical characteristics and stability of targeted LNPs, 5- component LNP formulations were conjugated with various antibodies or antibody fragments targeting a specific epitope of CD117 (c-KIT) or MPL, as listed in Table 2, and characterized as described in Example 1.

[0390] The formulations described in Example 2, and listed in Table 4, were used to produce targeted LNPs comprising a targeting ligand conjugated to an anchor PEG-lipid, as described in Example 1, using different conjugation chemistries, many of which are known in the art. The ligands comprised an antibody or a fragment thereof, as well as a conjugation moiety for attachment to the anchor PEG-lipid component of a 5-component LNP. Comparative controls consisted of untargeted LNPs prepared, as described in Example 1, as 4-component formulations, or as 5-component formulations where the anchor PEG-lipid was not conjugated to a targeting ligand. Table 5 illustrates experiments that measured the biophysical characteristics, including encapsulation efficiency (%E), Z-average diameter (Z- avg) polydispersity index (PDI), and number average (num-avg), as described in Example 1, of LNP formulations stored at 4°C (“pre-freeze-thaw”, or “pre-FT”), and after subjecting them to one or more freeze-thaw cycles (“post-freeze-thaw”, or “post-FT”), as described in Example 1. Each analysis number corresponds to the characterization of a set of LNPs for which the anchor PEG-lipid density, conjugation chemistry, ligand type, or formulation were varied. The data in Table 5 show formulatability (e.g., desired particle size, encapsulation efficiency, Z-average diameter) and freeze-thaw stability for the various antibody or antibody fragment targeted LNPs. The LNPs with the most desirable formulation characteristics and stability were then selected for further analyses (e.g., GFP delivery and editing efficiency). The LNPs analyzed were produced either at small scale (1-5mL) or at large scale (25- 1000mL), as indicated in Table 5. 153NTLA-103PCT; 5640-104.PCT

[0391] Table 5. Biophysical characteristics of targeted LNPs comprising a targeting ligand comprising antibodies or antibody fragments. Unless otherwise indicated, ligand density is equal to anchor PEG-lipid density for all targeted LNPs listed. LNPLNP#8LNP#5LNP#5LNP#6LNP#6LNP#6LNP#6LNP#6LNP#6LNP#6LNP#6LNP#6LNP#6LNP#70 Fab 1Spy-tag / Spy-Catchersmall92% 86 0.026 72 92% 85 0.061 663154NTLA-103PCT; 5640-104.PCTLNP#7LNP#7LNP#7LNP#7LNP#6LNP#7LNP#7LNP#7LNP#7LNP#7LNP#7LNP#8LNP#8LNP#5LNP#5 LNP#6 LNP#6 LNP#6 LNP#8 LNP#8 LNP#8 LNP#8 LNP#8 LNP#8 LNP#8LNP#88 N / A N / Alarge93% 71 0.05 56 93% 71 0.06 568155NTLA-103PCT; 5640-104.PCT LNP#8LNP#9LNP#9 LNP#9LNP#9LNP#8 LNP#9LNP#1LNP#9 LNP#9 LNP#9 LNP#9 LNP#9 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#8 LNP#1 LNP#1 LNP#8 LNP#1 LNP#1LNP#110 Fab 3 Spy-tag / Spy-Catchersmall99% 78 0.061 60 99% 78 0.064 6113156NTLA-103PCT; 5640-104.PCT LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#8 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#1 LNP#3LNP#40 Fab 6 Spy-tag / Spy-Catchersmall97% 78 0.068 58 97% 78 0.016 6416157NTLA-103PCT; 5640-104.PCT LNP#4 LNP#4 LNP#4158NTLA-103PCT; 5640-104.PCT Example 4 – Characterization of targeted LNPs comprising peptides or small molecules as targeting ligands

[0392] To evaluate the biophysical characteristics and stability of targeted LNPs, 5- component LNP formulations were conjugated with various peptides targeting an epitope of MPL or CXCR4 or with small molecules targeting an epitope of VLA4, as listed in Table 2, and characterized as described in Example 1.

[0393] The formulations described in Example 2 were used to produce targeted LNPs comprising a targeting ligand conjugated to an anchor PEG-lipid, as described in Example 1, using different conjugation chemistries, many of which are known in the art. The targeting ligands comprised a peptide or a small molecule, as well as a second coupling moiety for attachment to the first coupling moiety attached to the anchor PEG-lipid component of a 5- component LNP. Comparative controls consisted of untargeted LNPs prepared, as described in Example 1, as 4-component formulations, or as 5-component formulations where the anchor PEG-lipid (without a first coupling moiety attached to the anchor PEG-lipid) was not conjugated to a targeting ligand. Table 6 compiles a list of experiments that measured the biophysical characteristics, including encapsulation efficiency (%E), Z-average diameter (Z- avg) polydispersity index (PDI) and number average (num-avg), as described in Example 1, of LNP formulations stored at 4°C (“pre-freeze-thaw”, or “pre-FT”), and after subjecting them to one or more freeze-thaw cycles (“post-freeze-thaw”, or “post-FT”), as described in Example 1. Each analysis number corresponds to the characterization of a set of LNPs for which the anchor PEG-lipid density, conjugation chemistry, ligand type, or formulation were varied. The data in Table 6 show formulatability and freeze-thaw stability for the various peptide or small molecule targeted LNPs. The LNPs with the most desirable formulation characteristics and stability were then selected for further analyses (e.g., GFP delivery and editing efficiency). The LNPs analyzed were produced either at small scale (1-5mL) or at large scale (25-1000mL), as indicated in Table 6. 159NTLA-103PCT; 5640-104.PCT

[0394] Table 6. Biophysical characteristics of targeted LNPs comprising a targeting ligand comprising peptides or small molecules. Unless otherwise indicated, ligand density is equal to anchor PEG-lipid density for all targeted LNPs listed. LNP I LNP#1LNP#2LNP#3LNP#4LNP#5LNP#6LNP#7LNP#8LNP#9LNP#1LNP#1LNP#1LNP#1LNP#1LNP#15Peptide 1 DBCO-Az small 95% 77 0.102 65 19 160NTLA-103PCT; 5640-104.PCTLNP#1LNP#1LNP#1LNP#1LNP#1LNP#1LNP#1LNP#1LNP#1LNP#1LNP#2LNP#2LNP#2LNP#2LNP#2LNP#2LNP#2LNP#8LNP#2LNP#2LNP#1LNP#8LNP#1LNP#1LNP#1LNP#19Peptide 8 Cys-Mal small 94% 82 0.128 65 95% 82 0.142 69 25 161NTLA-103PCT; 5640-104.PCTLNP#1LNP#2LNP#2LNP#2LNP#2LNP#2LNP#2LNP#3LNP#3LNP#3LNP#3LNP#3LNP#3LNP#3LNP#3LNP#3LNP#4LNP#1LNP#4LNP#3LNP#8LNP#1LNP#3LNP#3LNP#4LNP#46Peptide 1 Cys-Mal small 97% 83 0.016 76 97% 82 0.019 75 29 162NTLA-103PCT; 5640-104.PCTLNP#4LNP#4LNP#3LNP#5LNP#5LNP#3LNP#5LNP#5LNP#5LNP#5ep e ys- a arge . . 163NTLA-103PCT; 5640-104.PCT Example 5 – Evaluation of the delivery efficiency of targeted LNPs comprising antibodies or antibody fragments by measuring GFP protein expression in vivo

[0395] To evaluate the delivery efficiency of targeted LNPs in vivo, wild-type C57Bl / 6J mice were dosed with targeted LNPs conjugated with antibodies or antibody fragments and comprising a GFP-encoding mRNA. Upon takedown, various bone marrow cell types were collected from the mice and analyzed for GFP expression. Controls consisted of untreated mice or mice treated with untargeted LNPs. GFP protein expression indicates successful delivery of the mRNA using the targeted LNPs.

[0396] GFP protein expression was assessed in cells obtained from wild-type mouse WBM material, as described in Example 1, using LNPs comprising a mRNA encoding a GFP protein (SEQ ID NO: 5) in the Sequence Table below. Table 7 and FIGS.1A-1B (Experiment #1) and FIGS.2A-2B (Experiment #2) show the percentage of cells expressing GFP protein (%GFP+) and the Median Fluorescent Intensity (MFI) in GFP+ cells, at 6 or 14 hours after dosing. LNPs used in Experiment #1 were produced at large scale (25-1000mL). LNPs used in Experiment #2 were produced at small scale (1-5mL). 164NTLA-103PCT; 5640-104.PCT Table 7. Percentage of cells expressing GFP protein (%GFP+) and the Median Fluorescent Intensity (MFI) in GFP+ cells, at 6 or 14 hours after dosing. Cell Type LSK MP LSK MPP HSCLNP#89 Fab 3 1 6 36.15 7.43 4 5745.5 746.9 4 2 165NTLA-103PCT; 5640-104.PCT Example 6 – Evaluation of in vivo editing efficacy of targeted LNPs comprising antibodies or antibody fragments

[0397] To evaluate in vivo editing efficacy, various tissue types from wild-type mice treated with selected targeted LNPs conjugated with antibodies or antibody fragments and comprising a Cas9 mRNA and a guide RNA, and were analyzed for editing outcome.

[0398] Multiple in vivo experiments were conducted to evaluate editing, as described in Example 1, in liver tissue, lung tissue, whole bone marrow (WBM) tissue, or in specific cell types (e.g., lineage depleted cells, Hematopoietic Stem Cells (HSCs), Multipotent Progenitors (MPPs), Myeloid Progenitors (MPs), Common Lymphoid Progenitors (CLPs), lin− / SCA- 1+ / c-KIT+ cells (LSK cells)) of wild-type C57Bl / 6J mice. The animals were dosed with 2 mpk of total RNA, using the LNPs formulated as described in Example 3. The LNPs comprised a modified guide RNA-1 (SEQ ID NO: 1) which targets the mouse B2M gene and a Cas9 mRNA (SEQ ID NO: 2) at a gRNA:mRNA mass ratio of 1:2. Table 8 shows the editing results obtained from these experiments. Experiments # 4 (FIGS.4A-4D) and 6 (FIGS.6A-6E) were conducted with LNPs produced at a small scale (1-5mL), while Experiments # 3 (FIGS.3A-3G) and 5 (FIGS.5A-5H) were conducted with LNPs produced at large scale (25-1000mL).

[0399] Table 8. Editing results obtained with LNPs comprising an antibody or antibody fragment targeting ligand. LNP ID Ligand ID Cell or Tissue Type Mean %editing SD N Experiment #166NTLA-103PCT; 5640-104.PCT LNP ID Ligand ID Cell or Tissue Type Mean %editing SD N Experiment #167NTLA-103PCT; 5640-104.PCT LNP ID Ligand ID Cell or Tissue Type Mean %editing SD N Experiment #Example 7 – Evaluation of in vivo editing efficacy of targeted LNPs comprising peptides

[0400] To evaluate in vivo editing efficacy, various tissue types from mice treated with selected targeted LNPs conjugated with peptides and comprising a Cas9 mRNA and a guide RNA, and were analyzed for editing outcome.

[0401] Multiple in vivo experiments were conducted to evaluate editing, as described in Example 1, in liver tissue, lung tissue, whole bone marrow (WBM) tissue, or in specific cell types (e.g., lineage depleted cells, Hematopoietic Stem Cells (HSCs), Multipotent Progenitors 168NTLA-103PCT; 5640-104.PCT (MPPs), Myeloid Progenitors (MPs), Common Lymphoid Progenitors (CLPs), lin− / SCA- 1+ / c-KIT+ cells (LSK cells)) of C57Bl / 6J mice, using the LNPs formulated as described in Example 4. The LNPs comprised a modified guide RNA-1 (SEQ ID NO: 1) which targets the mouse B2M gene and a Cas9 mRNA referred to as mRNA encoding a SpyCas9 protein (SEQ ID NO: 2) at a gRNA:mRNA mass ratio of 1:2. Experiments # 7 (FIGS.7A-7J), 8 (FIGS. 8A-8F), 9 (FIGS.9A-9E), 11 (FIGS.11A-11F), 12 (FIGS.12A-12F), 13 (FIGS.13A-13F), 14 (FIGS.14A-14F), 15 (FIGS 15A-15E), and 16 (FIGS.16A-16D) were conducted with LNPs produced at a small scale (1-5mL), while Experiments # 10 (FIGS.10A-10E), and 18 (FIGS.18A-18E) were conducted with LNPs produced at large scale (25-1000mL). In Experiment #17 (FIGS.17A-17D), all LNPs used were produced at small scale (1-5mL), except for LNP#34 which was produced at large scale (25-1000mL). Table 9 shows the editing results obtained from these experiments.

[0402] Table 9. Editing results obtained with LNPs comprising a peptide targeting ligand. Cell or Dose Mean LNP ID Li d ID A i l t Ti SD N E i t #169NTLA-103PCT; 5640-104.PCT Cell or Dose Mean LNP ID Ligand ID Animal type Tissue SD N Experiment #170NTLA-103PCT; 5640-104.PCT Cell or Dose Mean LNP ID Ligand ID Animal type Tissue SD N Experiment #171NTLA-103PCT; 5640-104.PCT Cell or Dose Mean LNP ID Ligand ID Animal type Tissue SD N Experiment #172NTLA-103PCT; 5640-104.PCT Cell or Dose Mean LNP ID Ligand ID Animal type Tissue SD N Experiment #173NTLA-103PCT; 5640-104.PCT Cell or Dose Mean LNP ID Ligand ID Animal type Tissue SD N Experiment #174NTLA-103PCT; 5640-104.PCT Cell or Dose Mean LNP ID Ligand ID Animal type Tissue SD N Experiment #175NTLA-103PCT; 5640-104.PCT Cell or Dose Mean LNP ID Ligand ID Animal type Tissue SD N Experiment #LNP formulations comprising an antibody fragment

[0403] The delivery efficiency of high DSPC targeted LNPs was evaluated in wild-type C57Bl / 6J mice treated with targeted LNPs conjugated with an antibody fragment and formulated with various lipid molar compositions. The LNPs contained increased percentages of DSPC – 1.5X, 2.25X and 2.5X (14.3, 22.5, and 25 mol %) – relative to a control targeted LNP (10 mol % DSPC).

[0404] Wild-type mice (N=3 animals per group) were treated, as described in Example 1, with 0.125 mpk of total RNA delivered via an LNP selected from the LNPs listed in Table 10. The LNPs were produced as described in Example 1, at small scale (1-5mL), and comprised a mRNA encoding a GFP protein (SEQ ID NO: 5). 176NTLA-103PCT; 5640-104.PCT

[0405] Table 10. Compositions of exemplary targeted LNPs, and corresponding biophysical analysis post-freeze-thaw. LNP I LNP#1 LNP#1 LNP#1 (1.5X LNP#1 (1.5X LNP#1 (2.25X LNP#1 (2.25X LNP#1 (2.5X LNP#1 (2.5X (1)Mol (2)Tar (3)Conjugation chemistry 177NTLA-103PCT; 5640-104.PCT

[0406] Upon takedown, various bone marrow cell types were collected from the mice and analyzed for GFP expression, as described in Example 1. An untreated control group (N=2 untreated animals per control group) consisted of mice injected with TSS (buffer control). GFP protein expression indicates successful delivery of the mRNA using the targeted LNPs. TThe percentage of cells expressing GFP protein (%GFP+) and the Median Fluorescent Intensity (MFI) in GFP+ cells, at 6 hours after dosing is shown in Table 11 and FIGS.19A- 19H. FIGS.19A-19H demonstrate the LNPs containing increased percentages of DSPC (14.3, 22.5, and 25 mol %) result in higher GFP protein expression levels in bone marrow cell types relative to a control targeted LNP (10 mol % DSPC).

[0407] Table 11. Percentage of cells expressing GFP protein (%GFP+) and Median Fluorescent Intensity (MFI) in GFP+ cells, at 6 hours after dosing. Cell or tissue type LNP treatment %GFP+ SD MFI SD .8 .0.6.3 .5 .5.3 .5 .0.9.9 .2 .0.0 .1 .0 .8.1 .5 .2.4.7 .1 .2178NTLA-103PCT; 5640-104.PCT Cell or tissue type LNP treatment %GFP+ SD MFI SD .0 .0.2.5 .7.3.6 .0 .6.0.9 .5 ExaLNP formulations comprising an antibody fragment

[0408] The in vivo editing efficacy of selected high DSPC targeted LNPs conjugated with an antibody fragment targeting CD117 and comprising a Cas9 mRNA and a guide RNA, was evaluated in various tissues of wild-type C57Bl / 6J mice. The selected LNPs contained increased percentages of DSPC – 1.5X and 2.25X (14.3 and 22.5 mol %) – relative to a control targeted LNP (10 mol % DSPC).

[0409] Wild-type mice were treated, as described in Example 1, with 0.5 mpk of total RNA delivered via an LNP selected from the LNPs listed in Table 12. The LNPs were produced as described in Example 1, and conjugated with a Fab 3 (anti-CD117) ligand, with a ligand density of 0.045%, through a SpyTag-SpyCatcher conjugation chemistry. 179NTLA-103PCT; 5640-104.PCT

[0410] Table 12. Compositions of exemplary targeted LNPs, and corresponding biophysical analysis post-freeze-thaw. LNP I LNP#8 LNP#1LNP#1LNP#1Molarra o (onza e p : :c oesero: srucura :anc or ) 180NTLA-103PCT; 5640-104.PCT

[0411] The LNPs comprised a guide RNA-1(SEQ ID NO: 1) which targets the mouse B2M gene and a Cas9 mRNA (SEQ ID NO: 2) at a gRNA:mRNA mass ratio of 1:2. Editing was evaluated in liver tissue, whole bone marrow (WBM) tissue, or in specific cell types including HSCs, MPPs, MPs, and CLPs. The LNPs were produced at mid-scale (25- 1000mL). Table 13 and FIGs.20A-20F show the editing results at the B2M locus in the different cell and tissue types. Animals treated with LNP #137 (2.25X DSPC) showed a significant reduction in liver editing compared to the control LNP (LNP #82). 181NTLA-103PCT; 5640-104.PCT

[0412] Table 13. Editing results obtained with LNPs comprising a peptide targeting ligand. Cell o tissue type HSC MPP MP CLP WBM liver63.51 4.45 4 64.22 6.17 3 22.83 2.48 4 62.68 1.57 3 182NTLA-103PCT; 5640-104.PCT Example 10 – Evaluation of in vivo delivery efficiency of targeted-LNPs comprising an anti-CD7 or an anti-CD8 targeting ligand

[0413] The delivery efficiency of targeted LNPs compared to non-targeted LNPs in vivo was evaluated in CD34+ humanized NOG mice treated with the same 5-component LNP formulation, that was either (i) conjugated to a targeting ligand consisting of an anti-CD7 VHH or an anti-CD8 VHH, or (ii) not conjugated to a targeting ligand. Delivery efficiency was assessed by detection of mScarlet protein expression.

[0414] The LNPs were produced as described in Example 1, with the compositions described in Table 14, and comprised a mRNA encoding a mScarlet protein (SEQ ID NO: 8). The LNPs comprised a FITC moiety in their anchor PEG-lipid component. An anti-FITC-anti- CD7 bispecific construct was used to produce CD7-targeted LNPs. An anti-FITC-anti-CD8 bispecific construct was used to produce CD8-targeted LNPs. The biophysical analysis of the

[0415] LNPs used in this experiment is indicated in Table 15 for pre-freeze-thaw, and in Table 16 for post-freeze-thaw. The fluorescence resulting from FITC was measured as follows. A sample of 5 uL of LNP was diluted in 195 uL of Tris-EDTA (TE) buffer. A SpectraMax M5 Microplate Reader (Molecular Devices) was used to read the samples with excitation, auto cutoff and emission wavelengths set to 488 nm, 515 nm, and 525 nm respectively. The fluorescence measurements, normalized to the RNA cargo mass, are reported in Tables 15 and 16.

[0416] CD34+ humanized NOG mice (Taconic Biosciences) (N=5 animals per group) were treated, as described in Example 1, with 1 mpk of mRNA delivered via untargeted or targeted LNP formulations as listed in Table 14, with an injection volume of 150 uL per animal. An untreated control group consisted of mice injected with the injection media alone. 183NTLA-103PCT; 5640-104.PCT

[0417] Table 14. Compositions of exemplary LNPs. LNPLNP#1 LNP#1 LNP#1(1)Mol (2)Targetng gan an ensty ( ) 184NTLA-103PCT; 5640-104.PCT

[0418] Table 15. Biophysical analysis of exemplary LNPs pre-freeze-thaw. LNP ID Z-Ave [nm] PDI Num Ave [nm] Fluorescence [a.u. / µg RNA] [0LNP ID Z-Ave [nm] PDI Num Ave [nm] Fluorescence [a.u. / µg RNA] [harvested. Cells from blood and spleen tissue were analyzed using flow cytometry, as described in Example 1, to evaluate mScarlet protein expression. FIGs.21A-21B, and Table 17 show the percentage of CD4+ T cells, CD8+ T cells and B cells positive for mScarlet in the collected blood, as well as the Mean Fluorescent Intensity (MFI) measured in the mScarlet positive cells. FIGs.22A-22B, and Table 18 show the percentage of CD4+ T cells, CD8+ T cells, B cells, and NK cells positive for mScarlet in the collected spleen tissue, as well as the MFI measured in the mScarlet positive cells. These data, taken together, show that CD7- and CD8- targeted LNPs significantly increase uptake of mScarlet protein in specific T cell populations, such as CD4+ T cells and CD8+ T cells, compared to untargeted LNPs. 185NTLA-103PCT; 5640-104.PCT

[0421] Table 17. Percentage of CD4+ T cells, CD8+ T cells and B cells positive for mScarlet, and corresponding MFI in blood. Untr Unt CD7- CD8-MFI SD 367 2707 20 *N=4 186NTLA-103PCT; 5640-104.PCT

[0422] Table 18. Percentage of CD4+ T cells, CD8+ T cells and B cells positive for mScarlet, and corresponding MFI in spleen. Untre Unta Tar TarMFI SD 360 1989 43 40 187NTLA-103PCT; 5640-104.PCT Example 11 – Evaluation of in vivo B cell depletion using targeted-LNPs comprising an anti-CD7 targeting ligand by measuring B cell depletion

[0423] The ability of targeted LNPs to deplete B cells in humanized NOG mice was evaluated using varying amounts of CD7-targeted LNPs containing an mRNA encoding an anti-CD19 CAR, compared to vehicle (injection media) alone. CD7-targeted LNPs were produced as described in Example 1, and contained a mRNA encoding an anti-CD19 CAR (SEQ ID NO: 9. The LNPs comprised a FITC moiety in their anchor PEG-lipid component. An anti-FITC-anti-CD7 bispecific construct was used to produce CD7-targeted LNPs. The compositions and biophysical analysis of the LNPs are shown in Table 19. The fluorescence resulting from FITC was measured as follows. A sample of 5 uL of LNP was diluted in 195 uL of Tris-EDTA (TE) buffer. A SpectraMax M5 Microplate Reader (Molecular Devices) was used to read the samples with excitation, auto cutoff and emission wavelengths set to 488 nm, 515 nm, and 525 nm respectively. The fluorescence measurements, normalized to the RNA cargo mass, are reported in Table 19. 188NTLA-103PCT; 5640-104.PCT

[0424] Table 19. Composition and biophysical analysis of exemplary LNPs post-freeze-thaw. LNP I LNP#1 (untar LNP#1 (CD7-t (1) Mo (2)Target ng l gand ID and dens ty (%) 189NTLA-103PCT; 5640-104.PCT

[0425] CD34+ humanized NOG mice (Taconic Biosciences) (N=5 animals per group) were treated, as described in Example 1, with varying doses (0.04, 0.2, or 1 mpk) of mRNA encoding an anti-CD19 CAR, delivered via a CD7-targeted LNP, with an injection volume of 150 uL per animal. An untreated control group consisted of mice injected with the injection media alone.

[0426] Following treatment using the LNPs, blood from the mice was collected via the tail vein on Day 2, Day 7, and Day 14 and analyzed by flow cytometry, as described in Example 1. The baseline used for comparison was the percentage of B cells at Day -2 (i.e., two days before the injection on Day 0).

[0427] Percent depletion of B cells 48 hours after dosing (on Day 2), and the recovery of B cells one week and two weeks after administration of the LNPs (on Days 7 and 14), relative to the baseline of B cells at Day -2, are shown in FIG.23 and Table 20. The results demonstrate that CD7-targeted LNPs containing the anti-CD19 CAR mRNA significantly enhance B cell depletion, and that the effect is transient, lasting up to one week after administration.

[0428] Table 20. Percent of B cell depletion relative to the baseline on Days 2, 7, and 14 after the first LNP dose. % B cell % B cell % B cell depletion on depletion on depletion on 6 3 8 0

[0009] On ay 9 a ter t e rst ose, an mas were a m n stere a second dose of the same CD7-targeted LNPs at the same doses, as described above. The percent depletion of B cells 48 hours after the second dose (on Day 21), relative to the baseline of B cells at Day 14, is shown in FIG.24 and Table 21. The results demonstrate that redosing with CD7-targeted LNPs results in depletion of B cells after a recovery following the first dose.

[0430] Table 21. Percent of B cell depletion relative to the baseline 48 hours after the first LNP dose, and 48 hours after the second LNP dose. 190NTLA-103PCT; 5640-104.PCT % B cell depletion % B cell depletion on on Day 2 (48 hours Day 21 (48 hoursExample 12 – Evaluation of in vivo editing efficacy of non-targeted LNPs with various ionizable lipids in humanized mice

[0431] To evaluate in vivo editing efficacy of LNPs having high DSPC with various ionizable lipids, humanized mice were treated with a 5-component LNP having the same lipid molar ratios and comprising a Cas9 mRNA and a guide RNA (Table 22). The LNPs with the various ionizable lipids were analyzed for editing outcome, as described in Example 1.

[0432] Humanized mice (N=5 animals per group) were treated, as described in Example 1, with 2 mpk of total RNA delivered via an LNP selected from the LNPs listed in Table 22. The LNPs were produced as described in Example 1, at small scale (1-5mL). The LNPs comprised a modified guide RNA (SEQ ID NO: 18) which targets BCL11a and a SpyCas9 protein (SEQ ID NO: 2) at a gRNA:mRNA weight ratio of 1:2. The editing results in specific cell types (lineage depleted / CD34(+) cells and Hematopoietic Stem Cells (HSCs)) are shown in FIGs.25A-25B.

[0433] Table 22. Compositions of 5-component LNPs. Ionizable Molar ratio¥Z-AVE (nm) PDI Num Ave (nm) Lipid pKa191NTLA-103PCT; 5640-104.PCT

[0434] As shown in FIGs.25A-25B, LNPs comprising Compound 3 as the ionizable lipid showed greater editing efficacy relative to LNPs with the same lipid ratios, but comprising either Compound 4 or Compound 7 as the ionizable lipid.

[0435] To evaluate in vivo editing efficacy of LNPs having high DSPC with Compound 3, humanized mice were treated with a 5-component LNP with a lipid molar ratio of 47 / 22.5 / 28 / 2.365 / 0.135 comprising Compound 3 with high DSPC and comprising a Cas9 mRNA and a guide RNA. The LNPs with the various ionizable lipids were analyzed for editing outcome, as described in Example 1.

[0436] Humanized mice (N=5 animals per group) were treated, as described in Example 1, with 2 mpk of total RNA delivered via an LNP. The LNPs were produced as described in Example 1, at small scale (1-5mL). The LNPs comprised a modified guide RNA (SEQ ID NO: 18) which targets the BCL11a and a SpyCas9 protein (SEQ ID NO: 2) at a gRNA:mRNA weight ratio of 1:2. The editing results in specific cell types (lineage depleted / CD34(+) cells and Hematopoietic Stem Cells (HSCs)) are shown in Table 23.

[0437] Table 23. Compositions of 5-component LNP comprising high DSPC and Compound 3. HSC CD34+Example 13 – Evaluation of in vivo editing and delivery efficacy of non-targeted LNPs with varying DSPC amounts in humanized mice.

[0438] To evaluate in vivo editing efficacy of LNPs having varying DSPC with Compound 3 as the ionizable lipid, humanized mice were treated with a 5-component LNP (LNP-A, LNP- B, or LNP-C) comprising a Cas9 mRNA and a guide RNA (Table 24). The LNPs were analyzed for editing outcome, as described in Example 1.

[0439] Humanized mice (N=5 animals per group) were treated, as described in Example 1, with 2 mpk of total RNA delivered via an LNP selected from the LNPA, LNPB, or LNPC listed in Table 24. The LNPs were produced as described in Example 1, at small scale (1- 5mL). The LNPs comprised a modified guide RNA (SEQ ID NO: 18) which targets the BCL11A gene and a SpyCas9 protein (SEQ ID NO: 2) at a gRNA:mRNA weight ratio of 1:2. 192NTLA-103PCT; 5640-104.PCT The editing results in specific cell types (lineage depleted / CD34(+) cells and Hematopoietic Stem Cells (HSCs)) are shown in Table 25 and FIGs.26A-26B. 193NTLA-103PCT; 5640-104.PCT

[0440] Table 24. Compositions of 5-component LNPs. LNP LNP- LNP- LNP-¥Mola

[0441] Table 25. In vivo editing efficacy for 5-component LNPs. Mean Stand194NTLA-103PCT; 5640-104.PCT

[0442] The delivery efficiency of LNPs corresponding to LNP-A and LNP-B shown in Table 24 was evaluated in the humanized mice treated with the LNPs.

[0443] Humanized mice (N=5 animals per group) were treated, as described in Example 1, with 2 mpk of total RNA delivered via an LNP selected from the LNP-A or LNP-B listed in Table 24. The LNPs were produced as described in Example 1, at small scale (1-5mL). The LNPs comprised a mRNA encoding a GFP protein (SEQ ID NO: 5).

[0444] The percentage of cells expressing GFP protein (%GFP+) at 6 hours after dosing is shown in Table 26.

[0445] Table 26. Delivery efficacy for 5-component LNPs. HSC Editing [%] LNP-A LNP-B Example 14 – Evaluon-targeted LNP formulations compared to targeted LNP formulations comprising an antibody fragment in humanized mice

[0446] The in vivo editing efficacy of non-targeted LNP formulations compared to LNPs conjugated with an antibody fragment targeting CD117 and comprising a Cas9 mRNA and a guide RNA, was evaluated in humanized mice.

[0447] Humanized mice were treated, as described in Example 1, with 2 mpk of total RNA delivered via an LNP selected from the LNPs listed in Table 27. The LNPs were produced as described in Example 1, and conjugated with a Fab8 (anti-CD117) ligand, with a ligand density of 0.045%, through a cysteine-maleimide conjugation chemistry. The targeted and non-targeted LNPs comprised a modified guide RNA (SEQ ID NO: 18) which targets the BCL11a and a SpyCas9 protein (SEQ ID NO: 2) at a gRNA:mRNA weight ratio of 1:2.

[0448] The editing efficiency of the LNPs listed in Table 27 was evaluated in the humanized mice treated with the LNPs and the results are shown in FIG. 27A.

[0449] The in vivo delivery efficiency of the targeted and non-targeted LNPs listed in Table 27 was evaluated in the humanized mice treated with the LNPs.

[0450] Humanized mice (N=5 animals per group) were treated, as described in Example 1, with 2 mpk of total RNA delivered via a targeted LNP or a non-targeted LNP listed in Table 27. The 195NTLA-103PCT; 5640-104.PCT LNPs were produced as described in Example 1, at small scale (1-5mL). The LNPs comprised a mRNA encoding a GFP protein (SEQ ID NO: 5).

[0451] The percentage of cells expressing GFP protein (%GFP+) at 6 hours after dosing is shown in FIG. 27B. 196NTLA-103PCT; 5640-104.PCT

[0452] Table 27. Compositions of t-LNP and nt-LNP. LNP t-LN Nt-L ¥Mola197NTLA-103PCT; 5640-104.PCT

[0453] SEQUENCE TABLE Descri modifi B2M- modifi modifi phosp consec mRN (mRNUGUUCGGCAACCUGAUCGCCCUGUCCCUGGGCCUGACCCCCAACUUCA 198NTLA-103PCT; 5640-104.PCT DescriAGGUGAUGAAGCAGCUGAAGCGGCGGCGGUACACCGGCUGGGGCCGG 199NTLA-103PCT; 5640-104.PCT DescriACGGCGAGAUCCGGAAGCGGCCCCUGAUCGAGACCAACGGCGAGACC 200NTLA-103PCT; 5640-104.PCT DescriAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG 201NTLA-103PCT; 5640-104.PCT Descri mRN (ORF)ACCTTCGACAACGGCTCCATCCCCCACCAGATCCACCTGGGCGAGCTGC 202NTLA-103PCT; 5640-104.PCT DescriGTACGTGGACCAGGAGCTGGACATCAACCGGCTGTCCGACTACGACGT 203NTLA-103PCT; 5640-104.PCT DescriCACTACGAGAAGCTGAAGGGCTCCCCCGAGGACAACGAGCAGAAGCAG 204NTLA-103PCT; 5640-104.PCT Descri a SpyEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV 205NTLA-103PCT; 5640-104.PCT Descri mRNUACAAGUGACUAGCACCAGCCUCAAGAACACCCGAAUGGAGUCUCUA 206NTLA-103PCT; 5640-104.PCT Descri mRN (ORF) GFP pSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK 207NTLA-103PCT; 5640-104.PCT Descri mRN mRNUGUCCGCCUCCCUGGGCGACCGGGUGACCAUCUCCUGCCGGGCCUCCC 208NTLA-103PCT; 5640-104.PCT Descri coupli coupli coupli coupliQVTVNGKATKGDAHI 209NTLA-103PCT; 5640-104.PCT Descri coupli coupli coupli coupli modif BCL1 modif modif phospconsecutive nucleotides) 210NTLA-103PCT; 5640-104.PCT NUMBERED ITEMS

[0454] The following numbered items also form part of the instant disclosure. Any and all combinations of the following numbered items and subparts or elements thereof are encompassed by the present disclosure. 1. A lipid nanoparticle (LNP) comprising: (i) a lipid component comprising: a) an anchor PEG-lipid; b) a helper lipid; c) a neutral lipid; d) a structural PEG-lipid; and e) an ionizable lipid represented by any one of structural Formula I, or, wherein,t bond, X1is a C1-5 alkylene, R1and R2are each independently a C1-3alkyl, or R1taken together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X1form a 4-, 5-, or 6-membered ring, or R1taken together with R2and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, and R3is H or C1-3 alkyl, Z1and Z2are each independently a C1-5 alkylene, 211NTLA-103PCT; 5640-104.PCT Z3and Z4are each independently a -C(=O)O- in either direction, Z5and Z6are each independently a direct bond or a C1-3 alkylene, Y1is selected from H, a C1-10alkyl, C3-10alkenyl, and C3-10alkynyl, Y2, Y3, and Y4are each independently selected from a C3-10alkyl, C3-10alkenyl, or C3-10 alkynyl, and n is 0 or 1; and (ii) a first coupling moiety attached to the anchor PEG-lipid; and (iii) a cargo, wherein the cargo is encapsulated in the LNP. 2. The LNP of item 1, wherein the ionizable lipid is selected from: ,212NTLA-103PCT; 5640-104.PCT , , ,NTLA-103PCT; 5640-104.PCT , ,NTLA-103PCT; 5640-104.PCT , ,3. The LNP of item 2, wherein the ionizable lipid is: , or a salt t. 4. A lipid nanoparticle (LNP) comprising: (i) a lipid component comprising: a) an anchor PEG-lipid; b) a helper lipid; c) a neutral lipid; d) a structural PEG-lipid; and e) an ionizable lipid comprising: 215NTLA-103PCT; 5640-104.PCT(iii) a cargo, wherein the cargo is encapsulated in the LNP. 5. A lipid nanoparticle (LNP) comprising: (i) a lipid component comprising: a) an anchor PEG-lipid; b) a helper lipid; c) a neutral lipid; d) a structural PEG-lipid; and e) an ionizable lipid represented by any one of structural Formula II, [Formula II], wherein: Q is CH or A is O or NH, X1is a C1-5 alkylene, R1and R2are each independently a C1-3alkyl, or R1taken together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X1form a 4-, 5-, or 6-membered ring, or 216NTLA-103PCT; 5640-104.PCT R1taken together with R2and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, and Z1is a C2-9alkylene, Z2is a C1-3alkylene or a direct bond, and Y1and Y2are each independently selected from a C3-10 alkyl, C3-10 alkenyl, or C3-10alkynyl; (ii) a first coupling moiety attached to the anchor PEG-lipid; and (iii) a cargo, wherein the cargo is encapsulated in the LNP. 6. The LNP of item 5, wherein the ionizable lipid is selected from: , , , ,NTLA-103PCT; 5640-104.PCT , , , , ,NTLA-103PCT; 5640-104.PCT , , , ,7. A lipid nanoparticle (LNP) comprising: (i) a lipid component comprising: a) an anchor PEG-lipid; b) a helper lipid; c) a neutral lipid; d) a structural PEG-lipid; and e) an ionizable lipid represented by any one of structural Formula III, [Formula III] 219NTLA-103PCT; 5640-104.PCT O R2 X1 N A O OY3 4 ,wherein: A is O or NH, X1is a C1-5 alkylene, R1and R2is each independently a C1-3alkyl, or R1taken together with R2and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, and Z1is a C1-5alkylene, Y1and Y2is each independently a C3-10 alkoxyl or O(C3-10 alkynyl), Z2is a C1-5alkylene or a direct bond, and Y3and Y4is each independently a C3-10 alkoxyl or O(C3-10 alkynyl), or Y3and Y4is each independently a C3-10alkyl or C3-10alkynyl, provided that if Y1, Y2, Y3, and Y4is each independently a C3-10 alkoxy, then R1and R2are not C2alkyl, and R1taken together with R2and the nitrogen atom to which they are attached do not form a 6-membered ring; (ii) a first coupling moiety attached to the anchor PEG-lipid; and (iii) a cargo, wherein the cargo is encapsulated in the LNP. 8. The LNP of item 7, wherein the ionizable lipid is selected from: ,NTLA-103PCT; 5640-104.PCT , , ,9. A lipid nanoparticle (LNP) comprising: (i) a lipid component comprising: a) an anchor PEG-lipid; b) a helper lipid; c) a neutral lipid; d) a structural PEG-lipid; and e) an ionizable lipid represented by any one of structural Formula IV, [Formula IV] 221NTLA-103PCT; 5640-104.PCT wurrence, X1is C5-11alkylene, Y1is C3-11 alkylene, a bon1d to R , Z1is C2-4 alkylene, Z2is selected from -OH, -NH2, -OC(=O)R3, -OC(=O)NHR3, -NHC(=O)NHR3, and - NHS(=O)2R3, R1is C4-12 alkyl or C3-12 alkenyl, each R2is independently C4-12alkyl, and R3is C1-3 alkyl, or a salt thereof; (ii) a first coupling moiety attached to the anchor PEG-lipid; and (iii) a cargo, wherein the cargo is encapsulated in the LNP. 222NTLA-103PCT; 5640-104.PCT 10. The LNP of item 9, wherein the ionizable lipid is selected from:223NTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCT , or a saltthereof. 11. The LNP of item 10, wherein the ionizable lipid is selected from: ,NTLA-103PCT; 5640-104.PCT , or a salt thereof. 1(i) a lipid component comprising: a) an anchor PEG-lipid; b) a helper lipid; c) a neutral lipid; d) a structural PEG-lipid; and e) an ionizable lipid represented by any one of structural Formula V, [Formula V] wX1is O, NR1, or a direct bond, X2is C2-5 alkylene, X3is C(=O) or a direct bond, R1is H or Me, R3is C1-3alkyl, R2is C1-3 alkyl, or R2taken together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X2form a 4-, 5-, or 6-membered ring, or X1is NR1, R1and R2taken together with the nitrogen atoms to which they are attached form a 5- or 6-membered ring, or R2taken together with R3and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, 231NTLA-103PCT; 5640-104.PCT Y1is C2-12 alkylene, Y2is selected from ( ientation),on),tation), n is 0 to 3, R4is C1-15 alkyl, Z1is C2-6alkylene or a direct bond, on) or abse1 2nt, provided that if Z is a direct bond, Z is absent; R5is C5-9alkyl or C6-10alkoxy, R6is C5-9 alkyl or C6-10 alkoxy, W is methylene or a direct bond, and R7is H or Me, or a salt thereof, provided that if R3and R2are C2 alkyls, X1is O, X2is linear C3 alkylene, X3is C(=O), Y1is linear C6 alkylene, (Y2)n-R4is , R4is linear C5 alkyl, Z1is C2 alkylene, Z2is absent, W ismet y ene, an s H, then R5and R6are not C8alkoxy; (ii) a first coupling moiety attached to the anchor PEG-lipid; and (iii) a cargo, wherein the cargo is encapsulated in the LNP. 232NTLA-103PCT; 5640-104.PCT 13. The LNP of item 12, wherein the ionizable lipid is selected from:233NTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCTNTLA-103PCT; 5640-104.PCT 14. The LNP of item 13, wherein the ionizable lipid is:or a salt thereof. 15. The LNP of any one or combination of items, wherein the lipid component comprises: a) the anchor PEG-lipid in an amount from about 0.001 to about 1.5 mol % of the lipid component; b) the helper lipid in an amount from about 25 to about 65 mol % of the lipid component; c) the neutral lipid in an amount from about 5 to about 15 mol % of the lipid component; d) the structural PEG-lipid in an amount from about 0.5 to about 5 mol % of the lipid component; and e) the ionizable lipid in an amount of from about 40 to about 60 mol % of the lipid component. 16. The LNP of any one or combination of items, wherein the structural PEG-lipid comprises about 2.5 to about 3.5 mol % of the lipid component. 17. The LNP of any one or combination of items, wherein the structural PEG-lipid comprises about 1.2 to about 2.2 mol % of the lipid component. 18. The LNP of any one or combination of items, wherein the anchor PEG-lipid comprises about 0.05 to about 0.2 mol % of the lipid component. 19. The LNP of any one or combination of items, wherein the anchor PEG-lipid comprises about 0.03 to about 0.1 mol % of the lipid component. 20. The LNP of any one or combination of items, wherein the anchor PEG-lipid comprises about 0.25 to about 0.7 mol % of the lipid component. 21. The LNP of any one or combination of items, wherein a targeting ligand is attached to the first coupling moiety via a second coupling moiety. 254NTLA-103PCT; 5640-104.PCT 22. The LNP of any one or combination of items, wherein the amount of the anchor PEG- lipid is the same as an anchor PEG-lipid density. 23. The LNP of item 22, wherein the targeting ligand has a density that is the same or less than the anchor PEG-lipid density. 24. The LNP of any one or combination of items, wherein the targeting ligand is selected from an antibody, an antibody fragment, a small molecule, or a peptide. 25. The LNP of item 21, wherein the targeting ligand is an antibody. 26. The LNP of item 25, wherein the anchor PEG-lipid is in an amount from about 0.005 to about 0.045 mol% of the lipid component. 27. The LNP of item 21, wherein the targeting ligand is an antibody fragment. 28. The LNP of item 27, wherein the antibody fragment is selected from a Fab, a Fab', a F(ab')2, VHH-scAb, a VHH-Fab, a Dual scFab, a Fv fragment, a single chain variable fragment (scFv), a (scFv)2, a disulfide-linked Fv (sdFv), a Fd fragment consisting of VH and CH1 domains, a linear antibody, a nanobody, a diabody, a triple body, a miniantibody, a minibody, a TriBi minibody, a single domain antibody, or a VHH domain. 29. The LNP of any one of items 27 or 28, wherein the anchor PEG-lipid is in an amount from about 0.005 to about 0.075 mol% of the lipid component. 30. The LNP of item 21, wherein the targeting ligand is a peptide. 31. The LNP of item 30, wherein the anchor PEG-lipid is in an amount from about 0.1 to about 0.9 mol% of the lipid component. 32. The LNP of any one of items 22-31, wherein the anchor PEG-lipid is about 0.5 to about 30 mol % of a total of structural PEG-lipid and anchor PEG-lipids in the LNP. 33. The LNP of item 21, wherein first coupling moiety and the second coupling moiety form a covalent bond. 34. The LNP of item 21, wherein first coupling moiety and the second coupling moiety form a non-covalent bond. 35. The LNP of item 21, wherein the first coupling moiety comprises dibenzocyclooctyne (DBCO), cysteine, a bioconjugation protein, streptavidin, protein G, protein G- derived peptide, or an immunoglobulin Fab domain, and wherein the second coupling moiety comprises azide, maleimide, a bioconjugation peptide, biotin, an immunoglobulin Fc domain, or FITC. 255NTLA-103PCT; 5640-104.PCT 36. The LNP of item 21, wherein the first coupling moiety comprises azide, maleimide, a bioconjugation peptide, biotin, an immunoglobulin Fc domain, or FITC, and wherein the second coupling moiety comprises dibenzocyclooctyne (DBCO), cysteine, a bioconjugation protein, streptavidin, protein G, protein G-derived peptide, or an immunoglobulin Fab domain. 37. The LNP of item 21, wherein the first coupling moiety comprises a peptide sequence selected from AHIVMVDAYKPTK (SEQ ID NO: 10), VPTIVMVDAYKRYK (SEQ ID NO: 11), or RGVPHIVMVDAYKRYK (SEQ ID NO: 12), and wherein the second coupling moiety comprises a protein selected from: GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDS SGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVT VNGKATKGDAHI (SEQ ID NO: 13), VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKT ISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGK ATKGDAHI (SEQ ID NO: 14), VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGK TISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG EATKGDAHT (SEQ ID NO: 15), VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGK TISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGE ATEGDAHT (SEQ ID NO: 16), or EDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGK YTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI (SEQ ID NO: 17). 38. The LNP of item 21, wherein the first coupling moiety comprises a protein selected from: 256NTLA-103PCT; 5640-104.PCT GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDS SGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVT VNGKATKGDAHI (SEQ ID NO: 13), VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKT ISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGK ATKGDAHI (SEQ ID NO: 14), VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGK TISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG EATKGDAHT (SEQ ID NO: 15), VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGK TISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGE ATEGDAHT (SEQ ID NO: 16), or EDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGK YTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI (SEQ ID NO: 17), and wherein the second coupling moiety comprises a peptide sequence selected from AHIVMVDAYKPTK (SEQ ID NO: 10), VPTIVMVDAYKRYK (SEQ ID NO: 11), or RGVPHIVMVDAYKRYK (SEQ ID NO: 12). 39. The LNP of any one or combination of items, wherein the cargo is a coding polynucleotide, a non-coding polynucleotide, a polypeptide, or a combination thereof. 40. The LNP of any one or combination of items, wherein the cargo comprises a guide RNA, nucleic acid of interest, a shortmer, an antagomir, an antisense, a ribozyme, a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), a modified RNA, a self-amplifying mRNA, or a combination thereof. 41. The LNP of any one or combination of items, wherein the cargo comprises a nucleic acid encoding a Cas protein, a guide RNA (gRNA), a template nucleic acid, or a combination thereof. 257NTLA-103PCT; 5640-104.PCT 42. The LNP of item 41, wherein the nucleic acid of interest comprises a template nucleic acid. 43. The LNP of item 41, wherein the gRNA is a modified gRNA. 44. The LNP of item 43, wherein the gRNA is modified at one or more of the first five nucleotides at a 5’ end, is modified at one or more of the last five nucleotides at a 3’ end, or both. 45. The LNP of any one of items 41-44, wherein the guide RNA is selected from a single- guide RNA (sgRNA) or a dual-guide RNA (dgRNA). 46. The LNP of any one of items 41-45, wherein the Cas protein comprises a Class 2 Cas nuclease or a Cas9 nuclease. 47. The LNP of any one of items 41-46, wherein the ratio of the nucleic acid encoding a Cas protein to the guide RNA is from about 2:1 to 1:4 by weight. 48. The LNP of any one or combination of items, wherein the structural PEG-lipid is selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG- dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSG), PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-distearoylglycamide, 1- [8’-(cholest-5-en-3[beta]-oxy)carboxamido-3’,6’-dioxaoctanyl]carbamoyl-[omega]- methyl-poly(ethylene glycol) (PEG-cholesterol), 3,4-ditetradecoxylbenzyl-[omega]- methyl-poly(ethylene glycol)ether (PEG-DMB), 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2K-DMPE), 1,2- dimyristoyl-rac-glycero-3-[methoxy(polyethylene glycol)-2000] (PEG2K-DMG), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)- 2000] (PEG2K-DSPE), 1,2-distearoyl-sn-glycerol-[methoxy(polyethylene glycol)- 2000] (PEG2K-DSG), poly(ethylene glycol)-2000-dimethacrylate (PEG2K-DMA), 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2K- DSA), methoxy-PEG2000-carbamoyl-1,2-tetradecyoxypropylamine (C14 Ether), and methoxy-PEG2000-carbamoyl-1,2-tridecyoxypropylamine (C13 Ether), or a combination thereof. 49. The LNP of item 48, wherein the structural PEG-lipid is PEG2K-DMG, methoxy- PEG2000-carbamoyl-1,2-tetradecyoxypropylamine (C14 Ether), methoxy-PEG2000- carbamoyl-1,2-tridecyoxypropylamine (C13 Ether), or a combination thereof. 258NTLA-103PCT; 5640-104.PCT 50. The LNP of item 49, wherein the structural PEG-lipid comprises dimyristoyl glycerol (DMG). 51. The LNP of any one or combination of items, wherein the neutral lipid is selected from dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), 1- palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC), 1,2-diarachidoyl-sn- glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2-dibehenoyl-sn-glycero-3- phosphocholine (DBPC), 1- stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2- dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), and lysophosphatidylethanolamine, or a combination thereof. 52. The LNP of item 51, wherein the neutral lipid is DSPC or DMPE. 53. The LNP of item 52, wherein the neutral lipid is DSPC. 54. The LNP of any one or combination of items, wherein the helper lipid is cholesterol, 5-heptadecylresorcinol, cholesterol hemisuccinate, or a combination thereof. 55. The LNP of item 54, wherein the helper lipid is cholesterol. 56. The LNP of any one or combination of items, wherein the anchor PEG-lipid is selected from DSPE-PEG(1000), DSPE-PEG(2000), DSPE-PEG(3400), DSPE- PEG(5000), DSPE-PEG(1000), DSPE-PEG(2000), DSPE-PEG(3400), DSPE- PEG(5000), DSPE-PEG(1000), DSPE-PEG(2000), DSPE-PEG(3400), DSPE- PEG(5000), or a combination thereof. 57. The LNP of any one of items 1-32, wherein the anchor PEG-lipid and the first coupling moiety comprise DSPE-PEG(1000) Maleimide, DSPE-PEG(2000) Maleimide, DSPE-PEG(3400) Maleimide, DSPE-PEG(5000) Maleimide, DSPE- PEG(1000) Azide, DSPE-PEG(2000) Azide, DSPE-PEG(3400) Azide, DSPE- 259NTLA-103PCT; 5640-104.PCT PEG(5000) Azide, DSPE-PEG(1000) DBCO, DSPE-PEG(2000) DBCO, DSPE- PEG(3400) DBCO, DSPE-PEG(5000) DBCO, DSPE-PEG(1000) FITC, DSPE- PEG(2000) FITC, DSPE-PEG(3400) FITC, DSPE-PEG(5000) FITC or a combination thereof. 58. The LNP of any one or combination of items, wherein a molar ratio of the structural PEG-lipid to the anchor PEG-lipid is between about 2:1 to about 300:1. 59. The LNP of item 58, wherein the molar ratio of the structural PEG-lipid to the anchor PEG-lipid is about 20:1, about 19:1, about 18:1, about 17:1, about 16:1, about 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, or about 2:1. 60. A composition comprising the LNP of any one or combination of items, wherein the composition comprises a buffer, an excipient, a pharmaceutically acceptable carrier, or a combination thereof. 61. The composition of item 60, wherein the excipient comprises a cryoprotectant, a tonicity modifying agent, or a combination thereof. 62. A method of delivering a cargo to a target cell, comprising contacting the target cell with the LNP of any one of items 1-59 or the composition of any one of items 60-61. 63. A method of expressing a cargo in a target cell comprising contacting the target cell with the LNP of any one of items 1-59 or the composition of any one of items 60-61. 64. The method of item 63, comprising expressing the cargo in greater than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the target cells contacted with the LNP or the composition. 65. The method of any one of items 62-64, comprising contacting the target cell in a living subject, wherein expression of the cargo in the target cell is at least 1.5-fold, 2- fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold or 5-fold higher relative to expression in the same cell-type contacted with a non-targeted LNP. 66. A method for administering a cargo to a subject in need thereof, comprising administering a composition comprising the LNP of any one of item 1-59 or the composition of any one of items 60-61 to the subject. 67. The method of item 66, wherein the subject has a genetic disease or disorder. 68. The method of any one of item 66-67, comprising administering the LNP in an amount sufficient to obtain at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% gene editing in a target cell of the subject. 260NTLA-103PCT; 5640-104.PCT 69. The method of any one of item 66-68, comprising administering the LNP systemically, parenterally, or intratumorally. 70. The method of any one of items 66-69, comprising administering the LNP via injection or infusion. 71. A method for genetically engineering a target cell comprising contacting the target cell with the LNP of any one of items 1-59, wherein the cargo is selected from a CRISPR / Cas system, a Tth Argonaute (TtAgo) system, a zinc finger nuclease (ZFN) system, ARCUS nuclease system, megaTALs, or a transcription activator-like effector nuclease (TALEN) system. 72. The method of item 71, wherein the cargo comprises a guide RNA and optionally an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the guide RNA comprises a se...

Claims

1. NTLA-103PCT; 5640-104.PCT CLAIMS What is claimed is:

1. A lipid nanoparticle (LNP) comprising: (i) a lipid component comprising: a) an anchor PEG-lipid; b) a helper lipid; c) a neutral lipid; d) a structural PEG-lipid; and e) an ionizable lipid represented by any one of structural Formula I, or , wherein, bond, X1is a C1-5alkylene, R1and R2are each independently a C1-3 alkyl, or R1taken together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X1form a 4-, 5-, or 6-membered ring, or R1taken together with R2and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, and R3is H or C1-3 alkyl, Z1and Z2are each independently a C1-5alkylene, Z3and Z4are each independently a -C(=O)O- in either direction, 271 NTLA-103PCT; 5640-104.PCT Z5and Z6are each independently a direct bond or a C1-3alkylene, Y1is selected from H, a C1-10 alkyl, C3-10 alkenyl, and C3-10 alkynyl, Y2, Y3, and Y4are each independently selected from a C3-10alkyl, C3-10alkenyl, or C3-10alkynyl, and n is 0 or 1; and (ii) a first coupling moiety attached to the anchor PEG-lipid; and (iii) a cargo, wherein the cargo is encapsulated in the LNP.

2. The LNP of claim 1, wherein the ionizable lipid is selected from: , 272 NTLA-103PCT; 5640-104.PCT , , , 273 NTLA-103PCT; 5640-104.PCT , , NTLA-103PCT; 5640-104.PCT , , 3. The LNP of claim 2, wherein the ionizable lipid is: , or a salt t e eo .

4. A lipid nanoparticle (LNP) comprising: (i) a lipid component comprising: a) an anchor PEG-lipid; b) a helper lipid; c) a neutral lipid; d) a structural PEG-lipid; and e) an ionizable lipid comprising: 275 NTLA-103PCT; 5640-104.PCT (iii) a cargo, wherein the cargo is encapsulated in the LNP.

5. A lipid nanoparticle (LNP) comprising: (i) a lipid component comprising: a) an anchor PEG-lipid; b) a helper lipid; c) a neutral lipid; d) a structural PEG-lipid; and e) an ionizable lipid represented by any one of structural Formula II, [Formula II] , wherein: Q is CH o A is O or NH, X1is a C1-5alkylene, R1and R2are each independently a C1-3 alkyl, or 276 NTLA-103PCT; 5640-104.PCT R1taken together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X1form a 4-, 5-, or 6-membered ring, or R1taken together with R2and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, and Z1is a C2-9 alkylene, Z2is a C1-3alkylene or a direct bond, and Y1and Y2are each independently selected from a C3-10 alkyl, C3-10 alkenyl, or C3-10alkynyl; (ii) a first coupling moiety attached to the anchor PEG-lipid; and (iii) a cargo, wherein the cargo is encapsulated in the LNP.

6. The LNP of claim 5, wherein the ionizable lipid is selected from: , , , NTLA-103PCT; 5640-104.PCT , , , , , NTLA-103PCT; 5640-104.PCT , , , , , o r a sa ereo .

7. A lipid nanoparticle (LNP) comprising: (i) a lipid component comprising: a) an anchor PEG-lipid; 279 NTLA-103PCT; 5640-104.PCT b) a helper lipid; c) a neutral lipid; d) a structural PEG-lipid; and e) an ionizable lipid represented by any one of structural Formula III, [Formula III] O R2 X1 N A 3 4 , wherein: A is O or NH, X1is a C1-5 alkylene, R1and R2is each independently a C1-3 alkyl, or R1taken together with R2and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, and Z1is a C1-5alkylene, Y1and Y2is each independently a C3-10 alkoxyl or O(C3-10 alkynyl), Z2is a C1-5alkylene or a direct bond, and Y3and Y4is each independently a C3-10 alkoxyl or O(C3-10 alkynyl), or Y3and Y4is each independently a C3-10alkyl or C3-10alkynyl, provided that if Y1, Y2, Y3, and Y4is each independently a C3-10 alkoxy, then R1and R2are not C2 alkyl, and R1taken together with R2and the nitrogen atom to which they are attached do not form a 6-membered ring; (ii) a first coupling moiety attached to the anchor PEG-lipid; and (iii) a cargo, wherein the cargo is encapsulated in the LNP. 280 NTLA-103PCT; 5640-104.PCT 8. The LNP of claim 7, wherein the ionizable lipid is selected from: , , , , .

9. A lipid nanoparticle (LNP) comprising: (i) a lipid component comprising: a) an anchor PEG-lipid; b) a helper lipid; 281 NTLA-103PCT; 5640-104.PCT c) a neutral lipid; d) a structural PEG-lipid; and e) an ionizable lipid represented by any one of structural Formula IV, [Formula IV] w urrence, X1is C5-11alkylene, Y1is C3-11 alkylene, a bond t1 o R , Z1is C2-4alkylene, Z2is selected from -OH, -NH2, -OC(=O)R3, -OC(=O)NHR3, -NHC(=O)NHR3, and - NHS(=O)2R3, R1is C4-12 alkyl or C3-12 alkenyl, each R2is independently C4-12alkyl, and R3is C1-3 alkyl, or a salt thereof; (ii) a first coupling moiety attached to the anchor PEG-lipid; and (iii) a cargo, wherein the cargo is encapsulated in the LNP. 282 NTLA-103PCT; 5640-104.PCT 10. The LNP of claim 9, wherein the ionizable lipid is selected from: 283 NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT , or a salt thereof.

11. The LNP of claim 10, wherein the ionizable lipid is selected from: , NTLA-103PCT; 5640-104.PCT , or a salt thereof.

12. A lipid nanoparticle (LNP) comprising: (i) a lipid component comprising: a) an anchor PEG-lipid; b) a helper lipid; c) a neutral lipid; d) a structural PEG-lipid; and e) an ionizable lipid represented by any one of structural Formula V, [Formula V] X1is O, NR1, or a direct bond, X2is C2-5 alkylene, X3is C(=O) or a direct bond, R1is H or Me, R3is C1-3alkyl, R2is C1-3 alkyl, or R2taken together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X2form a 4-, 5-, or 6-membered ring, or X1is NR1, R1and R2taken together with the nitrogen atoms to which they are attached form a 5- or 6-membered ring, or 291 NTLA-103PCT; 5640-104.PCT R2taken together with R3and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y1is C2-12 alkylene, Y2is selected from ( ientation), on), tation), n is 0 to 3, R4is C1-15 alkyl, Z1is C2-6alkylene or a direct bond, ion) or absent, provided that if Z1is a direct bond, Z2is absent; R5is C5-9alkyl or C6-10alkoxy, R6is C5-9 alkyl or C6-10 alkoxy, W is methylene or a direct bond, and R7is H or Me, or a salt thereof, provided that if R3and R2are C2alkyls, X1is O, X2is linear C3alkylene, X3is C(=O), Y1is linear C6 alkylene, (Y2)n-R4is , R4is linear C5 alkyl, Z1is C2 alkylene, Z2is absent, W is methylene, and R is H, then R5and R6are not C8alkoxy; (ii) a first coupling moiety attached to the anchor PEG-lipid; and 292 NTLA-103PCT; 5640-104.PCT (iii) a cargo, wherein the cargo is encapsulated in the LNP.

13. The LNP of claim 12, wherein the ionizable lipid is selected from: 293 NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT NTLA-103PCT; 5640-104.PCT 14. The LNP of claim 13, wherein the ionizable lipid is: or a salt thereof.

15. The LNP of any preceding claim, wherein the lipid component comprises: a) the anchor PEG-lipid in an amount from about 0.001 to about 1.5 mol % of the lipid component; b) the helper lipid in an amount from about 25 to about 65 mol % of the lipid component; c) the neutral lipid in an amount from 0 to about 25 mol % of the lipid component; d) the structural PEG-lipid in an amount from about 0.5 to about 5 mol % of the lipid component; and e) the ionizable lipid in an amount of from about 40 to about 60 mol % of the lipid component.

16. The LNP of any preceding claim, wherein the lipid component comprises: a) the anchor PEG-lipid in an amount from about 0.001 to about 1.5 mol % of the lipid component; b) the helper lipid in an amount from about 25 to about 65 mol % of the lipid component; c) the neutral lipid in an amount from about 5 to about 15 mol % of the lipid component; d) the structural PEG-lipid in an amount from about 0.5 to about 5 mol % of the lipid component; and e) the ionizable lipid in an amount of from about 40 to about 60 mol % of the lipid component. 314 NTLA-103PCT; 5640-104.PCT 17. The LNP of any one of claims 1-14, wherein the lipid component comprises: a) the anchor PEG-lipid in an amount from about 0.001 to about 1.5 mol % of the lipid component; b) the helper lipid in an amount from about 25 to about 50 mol % of the lipid component; c) the neutral lipid in an amount from about 7.5 to about 25 mol % of the lipid component; d) the structural PEG-lipid in an amount from about 0.5 to about 5 mol % of the lipid component; and e) the ionizable lipid in an amount of from about 30 to about 55 mol % of the lipid component.

18. The LNP of any preceding claim, wherein the structural PEG-lipid comprises about 2.5 to about 3.5 mol % of the lipid component.

19. The LNP of any preceding claim, wherein the structural PEG-lipid comprises about 1.2 to about 2.2 mol % of the lipid component.

20. The LNP of any preceding claim, wherein the anchor PEG-lipid comprises about 0.05 to about 0.2 mol % of the lipid component.

21. The LNP of any preceding claim, wherein the anchor PEG-lipid comprises about 0.03 to about 0.1 mol % of the lipid component.

22. The LNP of any preceding claim, wherein the anchor PEG-lipid comprises about 0.25 to about 0.7 mol % of the lipid component.

23. The LNP of any one of claims 1-15 and 17-22, wherein the neutral lipid is present in an amount from about 10 to about 25 mol % of the lipid component.

24. The LNP of any one of claims 1-15 and 17-23, wherein the neutral lipid is present in an amount from about 15 to about 22.5 mol % of the lipid component. 315 NTLA-103PCT; 5640-104.PCT 25. The LNP of any preceding claim, wherein a targeting ligand is attached to the first coupling moiety via a second coupling moiety.

26. The LNP of any preceding claim, wherein the amount of the anchor PEG-lipid is the same as an anchor PEG-lipid density.

27. The LNP of claim 26, wherein the targeting ligand has a density that is the same or less than the anchor PEG-lipid density.

28. The LNP of any preceding claim, wherein the targeting ligand is selected from an antibody, an antibody fragment, a small molecule, or a peptide.

29. The LNP of claim 25, wherein the targeting ligand is an antibody.

30. The LNP of claim 23, wherein the anchor PEG-lipid is in an amount from about 0.005 to about 0.045 mol% of the lipid component.

31. The LNP of claim 25, wherein the targeting ligand is an antibody fragment.

32. The LNP of claim 31, wherein the antibody fragment is selected from a Fab, a Fab', a F(ab')2, VHH-scAb, a VHH-Fab, a Dual scFab, a Fv fragment, a single chain variable fragment (scFv), a (scFv)2, a disulfide-linked Fv (sdFv), a Fd fragment consisting of VH and CH1 domains, a linear antibody, a nanobody, a diabody, a triple body, a miniantibody, a minibody, a TriBi minibody, a single domain antibody, or a VHH domain.

33. The LNP of any one of claims 31 or 32, wherein the anchor PEG-lipid is in an amount from about 0.005 to about 0.075 mol% of the lipid component.

34. The LNP of claim 25, wherein the targeting ligand is a peptide.

35. The LNP of claim 34, wherein the anchor PEG-lipid is in an amount from about 0.1 to about 0.9 mol% of the lipid component.

36. The LNP of any one of claims 26-35, wherein the anchor PEG-lipid is about 0.5 to about 30 mol % of a total of structural PEG-lipid and anchor PEG-lipids in the LNP. 316 NTLA-103PCT; 5640-104.PCT 37. The LNP of claim 25, wherein first coupling moiety and the second coupling moiety form a covalent bond.

38. The LNP of claim 25, wherein first coupling moiety and the second coupling moiety form a non-covalent bond.

39. The LNP of claim 25, wherein the first coupling moiety comprises dibenzocyclooctyne (DBCO), cysteine, a bioconjugation protein, streptavidin, protein G, protein G-derived peptide, or an immunoglobulin Fab domain, and wherein the second coupling moiety comprises azide, maleimide, a bioconjugation peptide, biotin, an immunoglobulin Fc domain, or FITC.

40. The LNP of claim 25, wherein the first coupling moiety comprises azide, maleimide, a bioconjugation peptide, biotin, an immunoglobulin Fc domain, or FITC, and wherein the second coupling moiety comprises dibenzocyclooctyne (DBCO), cysteine, a bioconjugation protein, streptavidin, protein G, protein G-derived peptide, or an immunoglobulin Fab domain.

41. The LNP of claim 25, wherein the first coupling moiety comprises a peptide sequence selected from AHIVMVDAYKPTK (SEQ ID NO: 10), VPTIVMVDAYKRYK (SEQ ID NO: 11), or RGVPHIVMVDAYKRYK (SEQ ID NO: 12), and wherein the second coupling moiety comprises a protein selected from: GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDS SGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVT VNGKATKGDAHI (SEQ ID NO: 13), VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKT ISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGK ATKGDAHI (SEQ ID NO: 14), VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGK TISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG EATKGDAHT (SEQ ID NO: 15), 317 NTLA-103PCT; 5640-104.PCT VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGK TISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGE ATEGDAHT (SEQ ID NO: 16), or EDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGK YTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI (SEQ ID NO: 17).

42. The LNP of claim 25, wherein the first coupling moiety comprises a protein selected from: GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDS SGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVT VNGKATKGDAHI (SEQ ID NO: 13), VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKT ISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGK ATKGDAHI (SEQ ID NO: 14), VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGK TISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG EATKGDAHT (SEQ ID NO: 15), VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGK TISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGE ATEGDAHT (SEQ ID NO: 16), or EDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGK YTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI (SEQ ID NO: 17), and wherein the second coupling moiety comprises a peptide sequence selected from AHIVMVDAYKPTK (SEQ ID NO: 10), VPTIVMVDAYKRYK (SEQ ID NO: 11), or RGVPHIVMVDAYKRYK (SEQ ID NO: 12). 318 NTLA-103PCT; 5640-104.PCT 43. The LNP of any preceding claim, wherein the cargo is a coding polynucleotide, a non- coding polynucleotide, a polypeptide, or a combination thereof.

44. The LNP of any preceding claim, wherein the cargo comprises a guide RNA, a template nucleic acid, a shortmer, an antagomir, an antisense, a ribozyme, a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), a modified RNA, a self-amplifying mRNA, or a combination thereof.

45. The LNP of any preceding claim, wherein the cargo comprises a nucleic acid encoding a Cas protein, a guide RNA (gRNA), a nucleic acid of interest, or a combination thereof.

46. The LNP of claim 45, wherein the nucleic acid of interest comprises a template nucleic acid.

47. The LNP of claim 45, wherein the gRNA is a modified gRNA.

48. The LNP of claim 47, wherein the gRNA is modified at one or more of the first five nucleotides at a 5’ end, is modified at one or more of the last five nucleotides at a 3’ end, or both.

49. The LNP of any one of claims 45-48, wherein the guide RNA is selected from a single-guide RNA (sgRNA) or a dual-guide RNA (dgRNA).

50. The LNP of any one of claims 45-49, wherein the Cas protein comprises a Class 2 Cas nuclease or a Cas9 nuclease.

51. The LNP of any one of claims 45-50, wherein the ratio of the nucleic acid encoding a Cas protein to the guide RNA is from about 2:1 to 1:4 by weight.

52. The LNP of any preceding claim, wherein the structural PEG-lipid is selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG- 319 NTLA-103PCT; 5640-104.PCT dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSG), PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-distearoylglycamide, 1- [8’-(cholest-5-en-3[beta]-oxy)carboxamido-3’,6’-dioxaoctanyl]carbamoyl-[omega]- methyl-poly(ethylene glycol) (PEG-cholesterol), 3,4-ditetradecoxylbenzyl-[omega]- methyl-poly(ethylene glycol)ether (PEG-DMB), 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2K-DMPE), 1,2- dimyristoyl-rac-glycero-3-[methoxy(polyethylene glycol)-2000] (PEG2K-DMG), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)- 2000] (PEG2K-DSPE), 1,2-distearoyl-sn-glycerol-[methoxy(polyethylene glycol)- 2000] (PEG2K-DSG), poly(ethylene glycol)-2000-dimethacrylate (PEG2K-DMA), 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2K- DSA), 1,2,-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol (PEG2K-DPG), methoxy-PEG2000-carbamoyl-1,2-tetradecyoxypropylamine (C14 Ether), and methoxy-PEG2000-carbamoyl-1,2-tridecyoxypropylamine (C13 Ether), or a combination thereof.

53. The LNP of claim 52, wherein the structural PEG-lipid is PEG2K-DMG, methoxy- PEG2000-carbamoyl-1,2-tetradecyoxypropylamine (C14 Ether), methoxy-PEG2000- carbamoyl-1,2-tridecyoxypropylamine (C13 Ether), or a combination thereof.

54. The LNP of claim 53, wherein the structural PEG-lipid comprises dimyristoyl glycerol (DMG).

55. The LNP of any preceding claim, wherein the neutral lipid is selected from dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), 1- palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC), 1,2-diarachidoyl-sn- glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2-dibehenoyl-sn-glycero-3- phosphocholine (DBPC), 1- stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2- 320 NTLA-103PCT; 5640-104.PCT dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), and lysophosphatidylethanolamine, or a combination thereof.

56. The LNP of claim 55, wherein the neutral lipid is DSPC or DMPE.

57. The LNP of claim 56, wherein the neutral lipid is DSPC.

58. The LNP of any preceding claim, wherein the helper lipid is cholesterol, 5- heptadecylresorcinol, cholesterol hemisuccinate, or a combination thereof.

59. The LNP of claim 58, wherein the helper lipid is cholesterol.

60. The LNP of any preceding claim, wherein the anchor PEG-lipid is selected from DSPE-PEG(1000), DSPE-PEG(2000), DSPE-PEG(3400), DSPE-PEG(5000), or a combination thereof.

61. The LNP of any one of claims 1-36, wherein the anchor PEG-lipid and the first coupling moiety comprise DSPE-PEG(1000) Maleimide, DSPE-PEG(2000) Maleimide, DSPE-PEG(3400) Maleimide, DSPE-PEG(5000) Maleimide, DSPE- PEG(1000) Azide, DSPE-PEG(2000) Azide, DSPE-PEG(3400) Azide, DSPE- PEG(5000) Azide, DSPE-PEG(1000) DBCO, DSPE-PEG(2000) DBCO, DSPE- PEG(3400) DBCO, DSPE-PEG(5000) DBCO, DSPE-PEG(1000) FITC, DSPE- PEG(2000) FITC, DSPE-PEG(3400) FITC, DSPE-PEG(5000) FITC or a combination thereof.

62. The LNP of any preceding claim, wherein a molar ratio of the structural PEG-lipid to the anchor PEG-lipid is between about 2:1 to about 300:

1.

63. The LNP of claim 62, wherein the molar ratio of the structural PEG-lipid to the anchor PEG-lipid is about 20:1, about 19:1, about 18:1, about 17:1, about 16:1, about 321 NTLA-103PCT; 5640-104.PCT 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, or about 2:

1.

64. A composition comprising the LNP of any preceding claim, wherein the composition comprises a buffer, an excipient, a pharmaceutically acceptable carrier, or a combination thereof.

65. The composition of claim 64, wherein the excipient comprises a cryoprotectant, a tonicity modifying agent, or a combination thereof.

66. A method of delivering a cargo to a target cell, comprising contacting the target cell with the LNP of any one of claims 1-63 or the composition of any one of claims 64- 65.

67. The method of claim 66, wherein the LNP or the composition delivers a mRNA molecule encoding a CAR to the target cell.

68. A method of expressing a cargo in a target cell comprising contacting the target cell with the LNP of any one of claims 1-63 or the composition of any one of claims 64- 65.

69. The method of claim 68, comprising expressing the cargo in greater than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the target cells contacted with the LNP or the composition.

70. The method of any one of claims 66-69, comprising contacting the target cell in a living subject, wherein expression of the cargo in the target cell is at least 1.5-fold, 2- fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold or 5-fold higher relative to expression in the same cell-type contacted with a non-targeted LNP.

71. The method of any one of claims 66-70, wherein the target cell is selected from an immune cell, a T cell, a resident T cell, a B cell, a natural killer (NK) cell, a cancerous cell, a cell associated with a disease or disorder, or any combination thereof.

72. The method of claim 71, wherein the cell is a CD4+ T cell, a CD8+ T cell, or a CD4+CD8+ T cell. 322 NTLA-103PCT; 5640-104.PCT 73. A method for administering a cargo to a subject in need thereof, comprising administering a composition comprising the LNP of any one of claims 1-60 or the composition of any one of claims 64-65 to the subject.

74. The method of claim 73, wherein the cargo is a mRNA molecule encoding a CAR.

75. The method of claim 73 or claim 74, wherein the subject has a genetic disease or disorder.

76. The method of any one of claims 73-75, comprising administering the LNP in an amount sufficient to obtain at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% gene editing in a target cell of the subject.

77. The method of any one of claims 73-76, wherein the lipid component comprises the neutral lipid in an amount from about 12 mol% to about 25 mol%.

78. The method of claim 73, wherein expression of the cargo in the target cell after contacting with the LNP or LNP composition is at least 1.5-fold, 2-fold, 2.5-fold, 3- fold, 3.5-fold, 4-fold, 4.5-fold or 5-fold higher relative to expression after contacting with a t-LNP or t-LNP composition with a different neutral lipid content.

79. The method of claim 73, wherein hepatic cell cargo delivery after administration with the LNP or LNP composition is reduced by about 10% to about 60%, about 15% to about 50%, or about 20% to about 40% relative to hepatic cell cargo delivery after administration with a LNP or LNP composition with a different neutral lipid content.

80. The method of any one of claims 73-79, comprising administering the LNP systemically, parenterally, or intratumorally.

81. The method of any one of claims 73-80, comprising administering the LNP via injection or infusion. 323 NTLA-103PCT; 5640-104.PCT 82. The method of any one of claims 73-81, wherein the LNP targets an immune cell, a T cell, a resident T cell, a B cell, a natural killer (NK) cell, a cancerous cell, a cell associated with a disease or disorder, or any combination thereof.

83. The method of any one of claims 66-81, wherein the LNP targets a T cell epitope.

84. The method of claim 83, wherein the LNP targets CD7 or CD8.

85. The method of any one of claims 73-84, wherein the subject has a depletion of B cells 48 hours after administration, 72 hours after administration, 1 week after administration, or 1 month after administration.

86. A method for genetically engineering a target cell comprising contacting the target cell with the LNP of any one of claims 1-63, wherein the cargo is selected from a CRISPR / Cas system, a Tth Argonaute (TtAgo) system, a zinc finger nuclease (ZFN) system, ARCUS nuclease system, megaTALs, or a transcription activator-like effector nuclease (TALEN) system.

87. The method of claim 86, wherein the cargo comprises a guide RNA and optionally an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the guide RNA comprises a sequence for targeting a genomic sequence in the target cell.

88. The method of any one of claims 86-87, wherein the contacting step comprises introducing a single stranded DNA nick.

89. The method of any one of claims 86-87, wherein the contacting step comprises introducing a double-stranded DNA break.

90. The method of any one of claims 86-89, further comprising introducing at least one template nucleic acid into the target cell.

91. The method of any one of claims 86-90, wherein the LNP comprises a composition comprising a buffer, an excipient, or a combination thereof. 324 NTLA-103PCT; 5640-104.PCT 92. The method of claim 91, wherein the excipient comprises a cryoprotectant, a tonicity modifying agent, or a combination thereof.

93. The method of any one of claims 86-92, wherein the composition is stable following one or more freeze / thaw cycles.

94. The method of any one of claims 86-93, wherein the target cell is a eukaryotic cell.

95. The method of any one of claims 86-94, wherein the method comprises administering a first lipid nanoparticle (LNP) composition and a second LNP composition.

96. The method of claim 95, wherein the first and second LNP compositions are administered simultaneously.

97. The method of claim 95, wherein the first and second LNP compositions are administered sequentially.

98. The method of any one of claims 86-96, wherein the method comprises administering the guide RNA and the RNA-guided DNA binding agent in a single LNP composition.

99. The method of any one of claims 86-98, wherein the gene editing comprises introducing a gene knockout.

100. The method of any one of claims 86-98, wherein the gene editing comprises introducing a gene modification.

101. The method of any one of claims 86-100, wherein the cell is contacted with the lipid composition in vitro.

102. The method of any one of claims 86-100, wherein the cell is contacted with the lipid composition in vivo.

103. The method of any one of claims 86-100, wherein the cell is contacted with the lipid composition ex vivo. 325 NTLA-103PCT; 5640-104.PCT 104. The method of any one of claims 86-100, wherein the method comprises contacting a tissue of an animal with the lipid composition.

105. The method of claim 104, wherein the animal is a human.

106. The method of any one of claims 86-105, wherein the lipid component comprises the neutral lipid in an amount from about 12 mol% to about 25 mol%.

107. The method of claim 106, wherein gene editing in the target cell after contacting with the t-LNP or t-LNP composition is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold or 5-fold higher relative to gene editing after contacting with a t-LNP or t-LNP composition with a different neutral lipid content.

108. The method of claim 106, wherein hepatic cell gene editing after administration with the t-LNP or t-LNP composition is reduced by about 10% to about 60%, about 15% to about 50%, or about 20% to about 40% relative to hepatic cell gene editing after administration with a t-LNP or t-LNP composition with a different neutral lipid content.

109. A composition comprising the LNP of any one of claims 1-63, wherein the composition is stable when stored at room temperature for more than 2 hours or at 4° C overnight without precipitation.

110. A composition comprising the LNP of any one of claims 1-63, wherein the composition is stable following one or more freeze / thaw cycles.

111. A lipid nanoparticle (LNP) according to any one of claims 1-63, wherein the LNP is stable following storage at about -70 to -80° C.

112. The composition of claim 109, wherein the LNP, after a freeze / thaw cycle: (a) retains at least 80%, 85%, 90%, 95%, 98%, or 99% biological activity of the LNP before the freeze / thaw cycle; (b) retains at least 80%, 85%, 90%, 95%, 98%, or 99% encapsulation efficiency of the LNP before the freeze / thaw cycle; 326 NTLA-103PCT; 5640-104.PCT (c) retains at least 80%, 85%, 90%, 95%, 98%, or 99% LNP size distribution of the LNP before the freeze / thaw cycle; (d) retains at least 80%, 85%, 90%, 95%, 98%, or 99% LNP mean size of the LNP before the freeze / thaw cycle; (e) retains at least 80%, 85%, 90%, 95%, 98%, or 99% expression activity of the LNP before the freeze / thaw cycle; (f) retains at least 80%, 85%, 90%, 95%, 98%, or 99% gene editing activity of the LNP before the freeze / thaw cycle; (g) has less than 20%, 15%, 10%, 8%, 5%, or 3% immunogenicity increase over the LNP before the freeze / thaw cycle; (h) has less than 5%, 4%, 3%, 2%, or 1% increase in impurities over the LNP before the freeze / thaw cycle; and / or (i) has less than 10% 5%, 4%, 3%, 2%, or 1% increase in sub-visible particles over the LNP before the freeze / thaw cycle.

113. The composition of claim 112, wherein the LNP has any one or combination of (a)-(i) after 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 freeze / thaw cycles.

114. A kit comprising the composition of any one of claims 64-65 and 109 in a container and instructions and / or a desiccant, optionally containing a second container containing a solvent, solution, or buffer.

115. A method of delivering a lipid nanoparticle (LNP) to a T cell, comprising: contacting the T cell with the LNP, wherein the LNP comprises: (i) a lipid component comprising: a) an anchor PEG-lipid; b) a helper lipid; c) a neutral lipid; d) a structural PEG-lipid; and e) an ionizable lipid comprising: 327 NTLA-103PCT; 5640-104.PCT (iii) a cargo, wherein the cargo is a mRNA molecule encoding a CAR encapsulated in the LNP.

116. The method of claim 115, wherein the LNP targets a T cell epitope.

117. The method of any one of claims 115-116, wherein the LNP targets CD2, CD3, CD4, CD5, CD6, CD7, CD8, or CD45.

118. The method of any one of claims 115-117, wherein the target cell is a CD4 positive T cell, a CD8 positive T cell, or both.

119. The method of any one of claims 115-118, wherein the contacting comprises administering the LNP to a subject.

120. The method of any one of claims 115-119, wherein the subject has a depletion of B cells 48 hours after administration, 72 hours after administration, 1 week after administration, or 1 month after administration.

121. The method of any one of claims 115-120, wherein the LNP is administered one or more times.

122. The method of any one of claims 116-122, wherein the LNP is administered as a multi-dose regimen.

123. The method of claim 122, wherein the LNP is administered two or more times, up to three times, up to four times, or up to five times. 328 NTLA-103PCT; 5640-104.PCT 124. A method of depleting B cells in a subject, comprising administering the LNP of any one of claims 1-66 to the subject.

125. The method of claim 124, further comprising redosing the subject with the LNP after 1, 2, 3, or 4 weeks.

126. A method of delivering a cargo to a bone marrow derived cell, comprising contacting the bone marrow derived cell with the LNP, wherein the LNP comprises: (i) a lipid component comprising: a. an anchor PEG-lipid; b. a helper lipid; c. a neutral lipid; d. a structural PEG-lipid; and e. an ionizable lipid comprising: (ii) d (iii) a cargo.

127. The method of claim 126, wherein the cargo is expressed in the bone marrow derived cell.

128. The method of any one of claims 126-127, wherein the cargo comprises a gene editing system.

129. The method of any one of claims 126-128, wherein the method comprises contacting the bone marrow derived cell with the LNP as a first LNP comprising the cargo as 329 NTLA-103PCT; 5640-104.PCT a first cargo and contacting the bone marrow derived cell with a second LNP comprising a second cargo.

130. The method of claim 129, wherein the first cargo and the second cargo together comprise the gene editing system.

131. The method of any one of claims 128-130, wherein gene editing in the bone marrow derived cell after contacting with the LNP is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold or 5-fold higher relative to gene editing after contacting with a LNP comprising a different ionizable lipid.

132. The method of claim 127, wherein the cargo expression in the bone marrow derived cell after contacting with the LNP is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5- fold, 4-fold, 4.5-fold or 5-fold higher relative to cargo expression after contacting with a LNP comprising a different ionizable lipid.

133. The method of any one of claims 126-132, wherein the lipid component comprises: a) the anchor PEG-lipid in an amount from about 0.001 to about 1.5 mol % of the lipid component; b) the helper lipid in an amount from about 25 to about 50 mol % of the lipid component; c) the neutral lipid in an amount from about 7.5 to about 25 mol % of the lipid component; d) the structural PEG-lipid in an amount from about 0.5 to about 5 mol % of the lipid component; and e) the ionizable lipid in an amount of from about 30 to about 55 mol % of the lipid component.

134. The method of claim 127, wherein the lipid component comprises: a) the anchor PEG-lipid in an amount from about 0.001 to about 1.5 mol % of the lipid component; b) the helper lipid in an amount from about 25 to about 50 mol % of the lipid component; 330 NTLA-103PCT; 5640-104.PCT c) the neutral lipid in an amount from about 7.5 to about 25 mol % of the lipid component; d) the structural PEG-lipid in an amount from about 0.5 to about 5 mol % of the lipid component; and e) the ionizable lipid in an amount of from about 30 to about 55 mol % of the lipid component.

135. The method of claim 134, wherein the cargo expression in the bone marrow derived cell after contacting with the LNP is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5- fold, 4-fold, 4.5-fold or 5-fold higher relative to cargo expression after contacting with a LNP comprising a different content of neutral lipid.

136. The method of any one of claims 128-130, wherein the lipid component comprises: a) the anchor PEG-lipid in an amount from about 0.001 to about 1.5 mol % of the lipid component; b) the helper lipid in an amount from about 25 to about 50 mol % of the lipid component; c) the neutral lipid in an amount from about 7.5 to about 25 mol % of the lipid component; d) the structural PEG-lipid in an amount from about 0.5 to about 5 mol % of the lipid component; and e) the ionizable lipid in an amount of from about 30 to about 55 mol % of the lipid component.

137. The method of claim 136, wherein gene editing in the bone marrow derived cell after contacting with the LNP is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold or 5-fold higher relative to gene editing after contacting with a LNP comprising a different content of neutral lipid.

138. The method of any one of claims 133-137, comprising the neutral lipid in an amount from about 10 to about 25 mol % of the lipid component. 331 NTLA-103PCT; 5640-104.PCT 139. The method of any one of claims 133-138, comprising the neutral lipid in an amount from about 15 to about 22.5 mol % of the lipid component.

140. The method of any one of claims 126-139, wherein a targeting ligand is attached to the first coupling moiety via a second coupling moiety.

141. The method of claim 140, wherein the targeting ligand is selected from an antibody, an antibody fragment, a small molecule, or a peptide.

142. The method of any one of claims 126-141, wherein the bone marrow derived cell is a CD34 positive cell.

143. The method of any one of claims 126-141, wherein the bone marrow derived cell is a hematopoietic stem cell (HSC).

144. The method of any one of claims 126-143, wherein the contacting comprises administering the LNP to a subject.

145. The method of claim 144, wherein the LNP is administered one or more times.

146. The method of any one of claims 144-145, wherein the LNP is administered as a multi-dose regimen.

147. The method of claim 146, wherein the LNP is administered two or more times, up to three times, up to four times, or up to five times. 332

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