Lipid nanoparticles compositions with ribonucleoproteins

Abstract

Provided herein are lipid nanoparticles comprising a ribonucleoprotein complex (RNP) comprising a Type II Cas nuclease polypeptide (e.g., a Cas9 polypeptide) and a guide RNA (gRNA), e.g., a single guide RNA (sgRNA). Compositions and formulations comprising such lipid nanoparticles are also provided. Such lipid nanoparticles can be used for delivery of a RNP to a cell, for example, a cell that is present in a subject in vivo. Methods of producing such lipid nanoparticles, and methods of use for genome engineering, are also provided.

Description

NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01Lipid Nanoparticles Compositions with RibonucleoproteinsCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority to U. S. Provisional Application No. 63 / 726,067, which was filed on November 27, 2024, which is incorporated by reference in its entirety.INCORPORATION OF SEQUENCE LISTING

[0002] The patent application is filed with a sequence listing in electronic format. The Sequence Listing is provided as a file entitled “5640-106W01.xml,” which was created on November 26, 2025, and which is 77,824 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.FIELD

[0003] The instant disclosure relates generally to lipid nanoparticles (LNPs) and lipid nanoparticle compositions that facilitate the delivery of CRLSPR / Cas9 ribonucleoproteins (RNPs) into a cell, as well as methods of modifying a target nucleic acid using LNPs encapsulating RNPs.BACKGROUND

[0004] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas systems can comprise a CRISPR-associated (Cas) effector polypeptide and a guide nucleic acid. The CRISPR-Cas systems can bind to and modify a targeted nucleic acid. The programmable nature of these CRISPR-Cas effector systems has facilitated their use as a versatile technology for use in, e.g., gene editing.

[0005] Lipid nanoparticle (LNP) / mRNA complexes are currently used for delivering genome editors in vivo. Once delivered, the RNA relies on cellular machinery for translation or functional activity, which introduces a dependency on intracellular processes like ribosomal assembly for mRNA translation. In contrast, ribonucleoproteins (RNPs) are pre-assembled complexes of proteins (e.g., Cas enzymes) and RNA (e.g., gRNA), which are larger, more structurally complex, and less stable than the RNA equivalents alone. Due to their size and charge, RNPs do not readily cross cellular membranes unassisted. Traditionally, RNPs have been used in ex vivo CRISPR-Cas applications, where delivery to cells is possible using modalities such as electroporation or lipofection. Delivering RNPs in vivo is hindered by the challenges of preserving complex protein-RNA interactions and maintaining functional activity during transport and entry into the cell. RNP administration in vivo may result inNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01premature degradation of the RNP components including the Cas protein and also may cause immune responses, reducing efficient delivery of the RNP to the target cell or tissue and causing safety and tolerability issues.SUMMARY OF THE INVENTION

[0006] Efficient delivery of RNPs into target cells in vivo can be advantageous.Compositions and methods for efficient gene editing through the in vivo delivery of functional RNP containing a Cas enzyme and a guide RNA are provided herein. Methods for the efficient production of LNPs encapsulating RNPs are also provided. In exemplary embodiments, the RNPs contain a Cas polypeptide and a guide nucleic acid.

[0007] In some aspects, provided herein is a lipid nanoparticle (LNP) comprising (i) a lipid component comprising:a) an ionizable lipid having a pKa at or below about 7.0;b) a helper lipid;c) a neutral lipid; andd) a structural PEG-lipid; and(ii) a cargo comprising a ribonucleoprotein complex (RNP) comprising a Type II Cas nuclease polypeptide (Cas nuclease) and a guide RNA (gRNA). In some aspects, the ionizable lipid has a pKa of about 6.0 to about 7.0.

[0008] In some aspects, provided herein is a lipid nanoparticle (LNP) comprising:(i) a lipid component comprising:a) a structural PEG-lipid;b) a helper lipid;c) a neutral lipid;d) an anchor PEG-lipid comprising a coupling moiety; ande) an ionizable lipid having a pKa at or below 7.0; and(ii) a cargo comprising a ribonucleoprotein complex (RNP) comprising a Type II Cas nuclease polypeptide and a guide RNA (gRNA).

[0009] The LNP according to any one or combination of aspects disclosed herein can comprise a ratio of gRNA: Cas nuclease in a range of about 1:1 to about 16:1, and wherein the Type II Cas nuclease and the gRNA associate in the RNP complex.

[0010] In some aspects, provided herein is a method for genetically engineering a cell comprising contacting the cell with the LNP of any one or combination of aspects disclosed herein or the composition of any one or combination of aspects disclosed herein. In someNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01aspects, the method further comprises introducing a single stranded DNA nick or introducing a double-stranded DNA break.

[0011] In some aspects, provided herein is a composition comprising the LNP of any one or combination of aspects disclosed herein. In some aspects, the composition comprises a carrier. In some aspects, provided herein is a method for genetically engineering a cell in a subject comprising administering a therapeutically effective amount of the composition to a subject in need thereof.

[0012] In some aspects, provided herein is a method for genetically engineering a target cell in a subject comprising administering a therapeutically effective amount of a composition comprising the LNP of any one or combination of aspects disclosed herein. In some aspects, the lipid component of the LNP comprises an anchor PEG-lipid comprising a first coupling moiety. In some aspects, a targeting ligand is attached to the first coupling moiety via a second coupling moiety. 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. In some aspects, a targeting ligand targets a liver cell, a bone cell, a bone marrow cell, a cancerous cell, a cell associated with a disease or disorder, or any combination thereof.

[0013] In some aspects, provided herein is a container, vial, syringe, injector pen, or kit comprising at least one dose of the LNP of any one or combination of aspects disclosed herein or the composition of any one or combination of aspects disclosed herein.

[0014] In some aspects, provided herein is a method of preparing a lipid nanoparticle (LNP) encapsulating a cargo comprising a ribonucleoprotein (RNP), wherein the method comprises: (i) mixing a first solution comprising a RNP in a preformulation buffer, and a second solution comprising a lipid component dissolved in ethanol, to prepare an RNP-LNP solution;(ii) mixing the RNP-LNP solution downstream with a third solution; and(iii) diluting the solution in (ii) with a fourth solution to prepare a diluted RNP-LNP solution;wherein the preformulation buffer has a pH about 5 to about 7 when measured at 25 °C and wherein the lipid component comprises an ionizable lipid having a pKa at or below about 7.0.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0015] In some aspects, the ionizable lipid having a pKa of about 6.0 to about 7.0. In some aspects. In some aspects, the lipid component comprises an ionizable lipid having a pKa of about 6.0 to about 7.5.

[0016] In some aspects, provided herein is a method of preparing a lipid nanoparticle (LNP) encapsulating a cargo comprising a ribonucleoprotein (RNP), wherein the method comprises: (i) mixing a first solution comprising a RNP in a preformulation buffer, and a second solution comprising a lipid component dissolved in ethanol, to prepare an RNP- LNP solution; (ii) mixing the RNP-LNP solution downstream with a third solution; and(iii) diluting the solution in (ii) with a fourth solution to prepare a diluted RNP-LNP solution; wherein the preformulation buffer has a pH about 5.5 to about 7.0 when measured at 25 °C and wherein the lipid component comprises an ionizable lipid having a pKa of about 6.0 to about 7.5.

[0017] In some aspects, provided herein is a method of preparing a lipid nanoparticle (LNP) encapsulating a cargo comprising a ribonucleoprotein (RNP), wherein the method comprises: (i) mixing a first solution comprising a RNP in a preformulation buffer, and a second solution comprising a lipid component dissolved in ethanol, to prepare an RNP-LNP solution;(ii) mixing the RNP-LNP solution downstream with a third solution; and(iii) diluting the solution in (ii) with a fourth solution to prepare a diluted RNP-LNP solution; wherein the preformulation buffer has a pKa of about 5.5 to about 7.0.

[0018] In some aspects, the preformulation buffer has a pH of about 6.0 to about 7.0. In some aspects, the preformulation buffer has a pH of about 6.0 to about 6.5. In some aspects, the preformulation buffer has a pH of about 6.5 to about 7.0 when measured at 25 °C.

[0019] In some aspects, the preformulation buffer has a pH of about 5.5 to about 6.5 when measured at 25 °C.

[0020] In some aspects, the preformulation buffer has a pKa of about 6.0 to about 7.0. In some aspects, the preformulation buffer has a pKa of about 6.0 to about 6.5. In some aspects, the preformulation buffer has a pKa of about 6.5 to about 7.0.

[0021] In some aspects, the preformulation buffer has a concentration of about 40 mM to about 60 mM.

[0022] In some aspects, the preformulation buffer has a concentration of about 50 mM.

[0023] In some aspects, the ionizable lipid has a pKa of about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0024] In some aspects, the LNP has a particle hydrodynamic size Z-average of 120 nm or less. In some aspects, the LNP has a particle hydrodynamic size Z-average of about 100 nm or less. In some aspects, the LNP has a particle hydrodynamic size Z-average of about 90 nm or less. In some aspects, the LNP has a particle hydrodynamic size Z-average of about 80 nm or less. In some aspects, the LNP has a particle hydrodynamic size Z-average of about 70 nm or less. In some aspects, the LNP has a particle hydrodynamic size Z-average of about 60 nm. In some aspects, the LNP has a particle hydrodynamic size Z-average of 50 nm to 120 nm. In some aspects, the LNP has a particle hydrodynamic size Z-average of 55 nm to 100 nm. In some aspects, the LNP has a particle hydrodynamic size Z-average of 60 nm to 90 nm. In some aspects, the LNP has a particle hydrodynamic size Z-average of 50 nm to 60 nm. In some aspects, the LNP has a particle hydrodynamic size Z-average of 60 nm to 70 nm. In some aspects, the LNP has a particle hydrodynamic size Z-average of 70 nm to 80 nm. In some aspects, the LNP has a particle hydrodynamic size Z-average of 80 nm to 90 nm. In some aspects, the LNP has a particle hydrodynamic size Z-average of 90 nm to 100 nm.

[0025] In some aspects, the LNP has a polydispersity index (PDI) in a range of 0.001-0.2. In some aspects, the LNP has a polydispersity index (PDI) in a range of 0.001-0.15. In some aspects, the LNP has a polydispersity index (PDI) in a range of about 0.001-0.1. In some aspects, the LNP has a polydispersity index (PDI) in a range of about 0.03-0.7. In some aspects, the LNP has a polydispersity index (PDI) in a range of about 0.07-0.10. In some aspects, the LNP has a polydispersity index (PDI) in a range of about 0.11-0.15. In some aspects, the LNP has a polydispersity index (PDI) in a range of about 0.15-0.2. In some aspects, the LNP has a polydispersity index (PDI) in a range of about 0.003-0.009. In some aspects, the LNP has a polydispersity index (PDI) in a range of about 0.01-0.02. In some aspects, the LNP has a polydispersity index (PDI) in a range of about 0.001-0.05.

[0026] In some aspects, a population of LNPs comprising an RNP cargo described herein has a high encapsulation efficiency. In some aspects, the LNP has an encapsulation efficiency of about 80% to about 100%. In some aspects, the LNP has an encapsulation efficiency of about 85% to 100%. In some aspects, the LNP has an encapsulation efficiency of about 90% to 100%. In some aspects, the LNP has an encapsulation efficiency of about 95% to about 100%. In some aspects, the LNP has an encapsulation efficiency of about 96%, about 97%, about 98%. In some aspects, the LNP has an encapsulation efficiency of about 99%. In some aspects the LNP has an encapsulation efficiency at or above 80%, 81%, 82%, 83%, 84%,NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W0185%, 86%, 87%, 88%, or 89%. In some aspects the LNP has an encapsulation efficiency at or above 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0027] In some aspects, the LNP comprises:a) the helper lipid in an amount from about 25 to about 65 mol % of the lipid component; b) the neutral lipid in an amount from about 0 to about 25 mol % of the lipid component; c) the structural PEG-lipid in an amount from about 0.5 to about 5 mol % of the lipid component; andd) the ionizable lipid in an amount of from about 40 to about 60 mol % of the lipid component.

[0028] In some aspects, the LNP comprises the ionizable lipid, the neutral lipid, the helper lipid, and the structural PEG-lipid in a ratio of 32 to 40: 10 to 20: 40 to 50: 1.5 to 3.5.

[0029] In some aspects, the LNP comprises the ionizable lipid, the neutral lipid, the helper lipid, and the structural PEG-lipid in a ratio of about 35:15:47.5:2.5.

[0030] In some aspects, the LNP comprises the ionizable lipid, the neutral lipid, the helper lipid, and the structural PEGdipid in a ratio of about 50:9:38:3.

[0031] In some aspects, the LNP comprises the ionizable lipid, the neutral lipid, the helper lipid, and the structural PEGdipid in a ratio of about 45:7:45:3.

[0032] In some aspects, the LNP comprises the ionizable lipid, the neutral lipid, the helper lipid, and the structural PEGdipid in a ratio of about 50: 10:38.5: 1.5.

[0033] In some aspects, the LNP comprises the ionizable lipid, the neutral lipid, the helper lipid, and the structural PEGdipid in a ratio of about 50:10:37.8:2.2.

[0034] In some aspects, the LNP has a molar ratio of ionizable lipid to Type II Gas nuclease polypeptide of about 4,000: 1 to about 2,400: 1. In some aspects, the LNP has a molar ratio of ionizable lipid to Type II Gas nuclease polypeptide of about 3,800: 1. In some aspects, the LNP has a molar ratio of ionizable lipid to Type II Gas nuclease polypeptide of 3,792:1.

[0035] In some aspects, provided herein is a method for genetically engineering a cell, comprising contacting the cell with a LNP of the present disclosure and introducing a single stranded DNA nick or introducing a double-stranded DNA break in the genome of the cell.

[0036] 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 DRAWINGSNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0037] FIGS. 1A-1B show percent indels at the TTR locus in liver tissue of mice treated with 0.3 mpk of various RNP-LNPs formulated with ionizable lipids having different tail chemistries and pKa values at or below 7.0, as described in Table 3 (FIG. 1 A), and the TTR levels measured in the serum from the treated animals (FIG. IB; TSS = tris saline sucrose (negative control)).

[0038] FIG.2 shows percent indels as a function of lipid content (pg) per well at the TTR locus in primary mouse hepatocytes (PMH) treated with (i) a RNA-LNP comprising Compound 2 as the ionizable lipid, a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), (ii) a RNP-LNP comprising Compound 2 as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 8; Guide 1) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4), or (iii) an empty LNP comprising Compound 2 as the ionizable lipid, mixed with a free RNP complex prepared with a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4).

[0039] FIGS. 3A-3B show percent indels at the TTR locus in liver tissue (FIG. 3 A), and TTR levels in the serum (FIG. 3B), obtained from mice treated with (i) a RNA-LNP comprising Compound 2 as the ionizable lipid, a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), or (ii) a RNP-LNP comprising Compound 2 as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4) (TSS = tris saline sucrose (negative control)).

[0040] FIGS. 4A-4B show percent indels at the TTR locus in liver tissue (FIG. 4A), and TTR levels in the semm (FIG. 4B), obtained from mice treated with (i) a RNA-LNP comprising Compound 9 as the ionizable lipid, a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), or (ii) a RNP-LNP comprising Compound 9 as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4) (TSS = tris saline sucrose (negative control)).

[0041] FIG.5 shows percent indels as a function of lipid content (pg) per well at the TTR locus in PRH treated with (i) a RNA-LNP comprising Compound 2 as the ionizable lipid, a gRNA (SEQ ID NO: 9) targeting the TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), (ii) a RNP-LNP comprising Compound 2 as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 9) targeting the TTR locus, and a SpyCas9NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01protein (SEQ ID NO: 4), or (iii) an empty LNP comprising Compound 2 as the ionizable lipid, mixed with a free RNP complex prepared with a gRNA (SEQ ID NO: 9) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4).

[0042] FIGS. 6A-6B show percent indels at the TTR locus in liver tissue (FIG. 6A), and TTR levels in the serum (FIG. 6B), obtained from rats treated with (i) a RNA-LNP comprising Compound 2 as the ionizable lipid, a gRNA (SEQ ID NO: 9) targeting the TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), or (ii) a RNP-LNP comprising Compound 2 as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 9) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4) (TSS = tris saline sucrose (negative control)).

[0043] FIGS. 7A-7B show percent indels at the B2M locus in whole bone marrow (WBM) (FIG. 7A) and hematopoietic stem cells (HSCs) (FIG. 7B) of mice treated with 1 mpk of a CD117-targeted RNP-LNP or an untargeted RNP-LNP, wherein the RNP-LNPs comprised Compound 2 as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 1) targeting the B2M locus, and a SpyCas9 protein (SEQ ID NO: 4).

[0044] FIGS. 8A-8B show percent indels at the TTR locus in liver tissue (FIG. 8A), and TTR levels in the serum (FIG. 8B), obtained from mice treated with a RNP-LNP comprising Compound 2 as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 10) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4).

[0045] FIG.9 shows percent indels as a function of lipid content (pg) per well at the TTR locus in PMH treated with (i) a RNA-LNP comprising Compound 2 as the ionizable lipid, a 91-mer or a 100-mer gRNA targeting the TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), or (ii) a RNP-LNP comprising Compound 2 as the ionizable lipid, and a RNP cargo prepared with a 91-mer or a 100-mer gRNA targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4).

[0046] FIGS. 10A-10B show percent indels at the TTR locus in liver tissue (FIG. 10A), and TTR levels in the serum (FIG. 10B), obtained from mice treated with (i) a RNA-LNP comprising Compound 2 as the ionizable lipid, a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), or (ii) a RNP-LNP comprising Compound 8 (LNP#15) or Compound 2 (LNP#11 and LNP#16) as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4) (TSS = tris saline sucrose (negative control)).NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0047] FIGS. 11A-11B show percent indels at the TTR locus in liver tissue (FIG. 11 A), and TTR levels in the serum (FIG. 1 IB), obtained from mice treated with a RNP-LNP comprising a RNP cargo prepared with a gRNA (SEQ ID NO: 10) targeting the mouse TTR locus, and a SpyCas9 protein (SEQ ID NO: 4) (TSS = tris saline sucrose (negative control)). The RNP-LNPs were formulated with different ionizable lipids.

[0048] FIG. 12 shows the difference in editing efficiencies as a function of LNP dosage obtained at the TTR locus in mouse liver tissue using RNP-LNPs produced with different manufacturing processes described herein, and formulated with ionizable lipids having different pKa values.

[0049] FIGS. 13A-13E show RNP Stability using Dynamic Light Scattering (“DLS”) pH 6.0 Pre-Formulation Buffer Screen gRNA (SEQ ID NO: 10): Cas9 (2:1 mol:mol) particle size (FIG. 13A); Z-average size (FIG. 13B) and % encapsulation (FIG. 13C) for RNP-LNPs with Compound 2 as the ionizable lipid and using preformulation buffers; and formulation characteristics of RNP-LNPs with a ratio of ionizable lipid: neutral lipid: helper lipid: structural PEG-lipid of 47: 22.5: 28: 2.5 (FIG. 13D) and 50: 9: 38: 3 (FIG. 13E) with pH 6.0 pre-formulation buffers with pKas between 6.0-6.5.

[0050] FIGs. 14A-14C show LNP size and % encapsulation as a function of preformulation buffer Bis-Tris (FIG. 14A) and MES (FIG. 14B) buffer strength. FIG. 14C shows editing as a function of lipid content (pg) in PMH using RNP-LNPs prepared with 50 mM Bis-Tris and MES preformulation buffers at pH 6.0.

[0051] FIGS. 15A-15C show LNP Z-average particle size (FIG. 15A), % encapsulation (FIG. 15B), and editing (%indels) as a function of lipid content (pg) (FIG. 15C) for LNPs formulated with ionizable lipids: Compound 1, Compound 2, Compound 7, or Compound 8 using a final KC1 concentration of 21 mM in the 50mM MES pH 6.0 (Protocol 1) or 50mM Bis-Tris pH 6.0 (Protocol 4).

[0052] FIGS. 16A-16B show LNP particle size (FIG. 16A), % encapsulation (FIG. 16B) across different compositions using Protocol 1, Protocol 3, or Protocol 4.

[0053] FIGS. 17A-17E show the TTR levels in the serum (FIG. 17A) and editing efficiencies (FIGS. 17B-17E) obtained at the TTR locus in liver tissue of monkeys treated with TSS saline control or a dose of 0.5 mpk or 1.5 mpk of RNA-LNP or RNP-LNP formulated with Compound 7 as the ionizable lipid using Protocol 2 at TFF scale process.DETAILED DESCRIPTIONNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0054] The disclosure relates to lipid nanoparticles (LNPs) comprising a CRISPR / Cas polypeptide and a guide RNA (gRNA) ribonucleoprotein (RNP) complex, where the LNPs are made of a lipid component comprising an ionizable lipid, a helper lipid, a neutral lipid, a structural PEG-lipid, and, in some aspects, an anchor PEG-lipid. Targeted LNP compositions (t-LNPs) comprising RNP complexes are also disclosed herein, including LNP 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 LNP compositions, methods of preparing LNPs carrying a cargo comprising a RNP, and methods of administering lipid nanoparticles and lipid nanoparticle compositions comprising RNPs. The present disclosure includes methods for genetically engineering a target cell using CRISPR / Cas RNP complexes.

[0055] Described herein are formulation parameters and ionizable lipids that advantageously allow encapsulation of an RNP cargo (e.g., a Cas / sgRNA cargo) with high encapsulation efficiency, optimal size for in vivo administration, and / or low polydispersity index.

[0056] The disclosure also relates to RNP delivery using LNPs according to various aspects disclosed herein. Provided herein are LNPs for delivery of RNPs in vivo and methods of delivering RNPs in vivo. Delivery of the gene editing system as a preformed RNP complex encapsulated in an LNP overcomes the limitations of liposomal transfection-based delivery or electroporation, which are traditionally used for ex vivo RNP delivery to cells. In contrast, in vivo administration of RNPs using an LNP delivery system provides a complex that can stably reach a target cell or tissue and readily cross a cellular membrane to become active in a cell within a subject. As a result, LNP-based delivery of RNPs in vivo allows for efficient delivery of active Cas protein and guide RNA to a target tissue or cell, while reducing the potential for off-target effects, offering higher editing precision, and avoiding degradation of the encapsulated RNP. Further provided herein are methods of producing a stable LNP- RNP with high formulatability. Accordingly, the methods provided herein provide LNP compositions having desired particle size, encapsulation efficiency, and stability, thereby allowing for improved gene editing activity following in vivo administration compared to alternative methods of RNP delivery.

[0057] 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.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01Accordingly, the subject matter of this disclosure is not intended to be limited to the forms or aspects so described.

[0058] 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.

[0059] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0060] 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.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.

[0061] 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 theNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01presence 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.

[0062] 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.

[0063] 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 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.

[0064] 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.

[0065] 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 referenceNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01contradicts any term defined in this specification, this specification controls. All ranges given in the application encompass the endpoints unless stated otherwise.Definitions

[0066] The singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0067] 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.

[0068] 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”).

[0069] 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 limitation, a change in one or more biomarkers or physiological parameters relative to a control.

[0070] As used herein, a “buffer” refers to a solution that resists changes in pH when small amounts of acid or base are added to the solution.

[0071] ‘ ‘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 Cas 10, Csml, 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.

[0072] 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 Cas9 variants), which have RNA-guidedNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01DNA 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, Cpfl, C2cl, 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(l.l) (e.g., K848A, KI 003 A, R1060A variants) proteins and modifications thereof. Cpfl protein, Zetsche et al., Cell, 163: 1-13 (2015), is homologous to Cas9, and contains a RuvC-like nuclease domain. Cpfl 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., NmelCas9 or Nme2Cas9).

[0073] As used herein, the terms “editing efficiency”, “editing percentage”, “indel efficiency”, and “percent indels” 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 an intact gene or gene product (e.g., CD3) after treatment, relative to the number of the cells that initially contained that gene or gene product (e.g., CD3+ cells).

[0074] As used herein, “editing potency” means a dosage or amount of RNP-LNP producing a half-maximal gene editing response. In some aspects, editing potency is expressed as the LNP dosage (e.g., mg / kg) or LNP content (e.g., pg / well) that results in gene editing response that is half-way between baseline and maximum gene editing percent insertion / deletions (%indels) at a genomic locus in a target tissue or cell.

[0075] 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 usedNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01herein, “encapsulation” may refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement of cargo.

[0076] 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.

[0077] As used herein, “formulatability” or “LNP 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 about 80-120 nm, PDI less than about 0.1, free-thaw stability, or a combination thereof.

[0078] ‘ ‘Fold” change is a measure describing how much a quantity changes between a reference (e.g., control) and a comparator measurement. It is defined as the ratio between the two quantities; for quantities A and B the fold change of B with respect to A is B / A. In other words, a change from 30 to 60 is defined as a fold-change of 2. This can also be referred to as a "two fold increase" or “2- fold higher.” Similarly, a change from 30 to 15 is referred to as a "0.5-fold decrease".

[0079] 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).

[0080] 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. According to the present disclosure, a genome editing nuclease or nickase is delivered in the form of a RNP. 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.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0081] “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.

[0082] 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.

[0083] 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 byNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01one 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 pKa of the protonatable group, the concentration of acid form and conjugate base form of the protonatable group are equal.

[0084] As used herein, “knockdown” refers to a decrease in expression of a particular gene product (e.g., protein, mRNA, or both). Knockdown of a protein can be measured by detecting total cellular amount of the protein from a tissue or cell population of interest. Methods for measuring knockdown of mRNA are known and include sequencing of mRNA isolated from a tissue or cell population of interest. Flow cytometry analysis is a known method for measuring knockdown of protein expression. In some embodiments, “knockdown” may refer to some loss of expression of a particular gene product, for example a decrease in the amount of mRNA transcribed or a decrease in the amount of protein expressed by a population of cells. In some embodiments, “knockdown” may refer to some loss of expression of a particular gene product.

[0085] As used herein, “knockout” refers to a loss of expression of a particular protein in a cell. Knockout can be measured either by detecting total cellular amount of a protein in a cell, a tissue or a population of cells.

[0086] 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).

[0087] 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. InNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01some 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 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.

[0088] “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.

[0089] 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 arylNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01groups. 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.

[0090] 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.

[0091] 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 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.

[0092] 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.

[0093] In some embodiments, the “anchor PEG-lipid” and the “structural PEG-lipid” may comprise the same PEG-lipid.

[0094] 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 anyNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01conventional 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.

[0095] As used herein, the phrase “preformulation buffer’’ refers to a buffer solution containing cargo components (e.g., protein and / or nucleic acid components to be loaded into an LNP). In some embodiments, the preformulation buffer can be combined with a lipid-containing ethanol solution using controlled mixing (e.g., syringe pumps) during LNP formation.

[0096] 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.

[0097] As used herein, “ribonucleoprotein” (RNP) or “RNP complex” refers to a guide RNA (gRNA) together with an RNA-guided DNA-binding agent, such as a CRISPR / Cas nuclease, e.g., a Cas cleavase, Cas nickase, or dCas DNA-binding agent. In some embodiments, the RNP or RNP complex comprises a Type II Cas nuclease, e.g., a Cas9 nuclease. 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.

[0098] 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”).

[0099] “Stability,” “stabilized,” and “stable” refers to the increased or enhanced resistance of LNPs to changes in one or more physical properties, such as changes to LNP size or percent encapsulation (%E). Stability may be determined using methods described in the art, e.g., by using fluorescence-based assays.

[0100] 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.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0101] As used herein, a “subject” refers to any member of the animal kingdom. In some embodiments, “subject” refers to a human. In some embodiments, “subject” refers to a nonhuman animal. In some embodiments, “subject” refers to a primate. In some embodiments, a subject may be a transgenic animal, genetically engineered animal, or a clone. In certain embodiments of the present disclosure the subject is an adult, an adolescent, or an infant. In some embodiments, terms “individual” or “patient” are used and are intended to be interchangeable with “subject”.

[0102] As used herein, a “targeting ligand” refers to a moiety that is capable of specifically binding to a molecule on the surface of a “target cell,” such as a cell within a tissue of a subject. In some aspects, a targeting ligand specifically binds to a molecule on the surface of the target cell.Lipid Nanoparticles (LNPs)

[0103] Provided herein are LNP formulations for delivering ribonucleoprotein cargo, e.g., Cas9 / sgRNA RNP cargo, to cells of a subject in vivo. As further described herein, RNP-LNP formulatability is dependent on the pKa of the ionizable lipid (e.g., pKa at or below 7) in combination with features of the preformulation buffer and preformulation buffer pH for preparing the RNP-LNPs.

[0104] The LNP may contain (i) an ionizable lipid, (ii) an optional neutral lipid, (iii) a helper lipid, and (iv) a structural PEG-lipid. The lipid nucleic acid assembly, e.g., a LNP, may contain an ionizable lipid and one or more of a neutral lipid, a helper lipid, and structural PEG-lipid.

[0105] The lipid nanoparticle may contain (i) an ionizable lipid for encapsulation and for endosomal escape, (ii) a neutral lipid for stabilization, (iii) a helper lipid, also for stabilization, and (iv) a structural PEG lipid for reducing particle aggregation and controlling particle size. The lipid nanopaiticle may contain an ionizable lipid and one or more of a neutral lipid, a helper lipid, and a structural PEG lipid. Also provided herein are LNPs and LNP compositions comprising a fifth lipid component to serve as a conjugation handle and anchor for a targeting ligand (e.g., an anchor PEG-lipid comprising a coupling moiety). In some aspects, the LNP according to any one or combination of aspects disclosed herein is a targeted LNP (l-LNP).

[0106] In some aspects, provided herein is a lipid nanoparticle (LNP) comprising (i) a lipid component comprising:a) an ionizable lipid having a pKa at or below about 7.0;NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01b) a helper lipid;c) a neutral lipid; andd) a structural PEG-lipid; and(ii) a cargo comprising a ribonucleoprotein complex (RNP) comprising a Type II Cas nuclease polypeptide and a guide RNA (gRNA). In some aspects, the ionizable lipid has a pKa of about 6.0 to about 7.0.

[0107] In some aspects, provided herein is a lipid nanopaiticle (LNP) comprising (i) a lipid component comprising:a) a structural PEG-lipid;b) a helper lipid;c) a neutral lipid;d) an anchor PEG-lipid comprising a coupling moiety; ande) an ionizable lipid having a pKa at or below 7.0; and(ii) a cargo comprising a ribonucleoprotein complex (RNP) comprising a Type II Cas nuclease polypeptide and a guide RNA (gRNA). In some aspects, the ionizable lipid has a pKa of about 6.0 to about 7.0.

[0108] In some aspects, the LNP according to any one or combination of the foregoing aspects, comprises a ratio of gRNA: Cas nuclease protein is in a range of about 1:1 to about 16:1.

[0109] In some aspects, the LNP has a gene editing potency of 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100- fold higher than a control composition’s gene editing potency. In some embodiments, the control composition comprises a mixture of the Cas9 / gRNA RNP that is not encapsulated in an LNP and a corresponding LNP that lacks the cargo encapsulated therein. In other embodiments, the control composition comprises an LNP encapsulating mRNA (e.g., mRNA encoding the Cas nuclease protein) and gRNA. In some aspects, the LNP has a 20-fold to 100-fold higher gene editing potency than the control composition.Lipid componentIonizable Lipids

[0110] The disclosure provides ionizable lipids, also called amine lipids, that can be used in lipid nanoparticle (LNP) compositions. As described herein, ionizable lipids with pKa’s below 7.0 (particularly 6.0-7.0) are effective for delivering RNP cargo for gene editing in vivo.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0111] In some embodiments, the ionizable lipid is represented by Formula (I),whereinX1is 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, orR2taken together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X2form a 4-, 5-, or 6-membered ring, orX1is NR1, R1and R2taken together with the nitrogen atoms to which they are attached form a 5- or 6-membered ring, orR2taken 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(in either orientation),(in either orientation),(in either orientation),n is 0 to 3,NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01R4is Ci-15 alkyl,Z1is C2-6 alkylene or a direct bond.(in either orientation) or absent, provided that if Z1is a direct bond, Z2is absent;R5is C5-9 alkyl or Ce-io alkoxy,R6is C5-9 alkyl or Ce-io alkoxy,W is methylene or a direct bond, andR7is 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 C6alkylene, (Y2)n-R4isR4, R‘ is linear C5 alkyl, Z1is C2 alkylene, Z2is absent, W is methylene, and R7is H, then R5and R6are not Cs alkoxy,or a salt thereof.

[0112] In some embodiments, the ionizable lipid is Lipid A (also referred to as Compound #1 in Table 2), which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate. Lipid A can be depicted as:Nsalt thereof.

[0113] In some embodiments, the ionizable lipid is represented by structural Formula (II),NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01oor a salt thereof,wherein,A is O, NH, or a direct bond,X1is a Ci-5 alkylene,R1and R2are each independently a C1-3 alkyl, orR1taken together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X1form a 4-, 5-, or 6-membered ring, orR1taken together with R2and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, andR3is H or C1-3 alkyl,Z1and Z2are each independently a C1-5 alkylene,Z and Z4are each independently a -C(=O)O- in either direction,Z3and Z6are each independently a direct bond or a C1-3 alkylene,Y1is selected from H, a Ci-10 alkyl, C3-10 alkenyl, and C3 -10 alkynyl,Y2, Y3, and Y4are each independently selected from a C3-10 alkyl, C3-10 alkenyl, or C3- 10 alkynyl, andn is 0 or 1,or a salt thereof.

[0114] In some embodiments, the compound of Formula II is represented by structural formula Ila,NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01o

[0115] In some embodiments, n is 0. Alternatively, n is 1.

[0116] In some embodiments, A is O. Alternatively, A is NH. Alternatively yet, A is a direct bond.

[0117] In some embodiments, X1is 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, X1is a C2-3 alkylene, such as a C2 alkylene or a C alkylene. For example, X1is C2 alkylene.

[0118] In some embodiments, Z1is a C1-4 alkylene, C1-3 alkylene, C1-5 alkylene, C1-2 alkylene, C25 alkylene, C2-4 alkylene, or C2-3 alkylene. For example, Z1is a C2-3 alkylene, such as a C2 alkylene or a C3 alkylene.

[0119] In some embodiments, Z2is 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.

[0120] In some embodiments, Z1and Z2is each independently C3 alkylene. In some embodiments, Z1and Z2is each independently C5 alkylene.

[0121] In certain embodiments, Z1is C2 alkylene: and Z2is C3 alkylene.

[0122] In some embodiments, Z1and Z2is each independently C3 alkylene, A is NH, and X1is a C2 alkylene.O

[0123] In some embodiments, Z3and Z4is eacha, wherein a indicates the point of attachment to Z1and Z2, respectively.O

[0124] In some embodiments, Z3isby-0wherein b indicates the point of attachment to AZ1; and Z4isb°, wherein b indicates the point of attachment to Z2.

[0125] In some embodiments, Z and Z6is each independently a direct bond.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0126] In some embodiments, 7? is Ci alkylene; and Z6is a direct bond.

[0127] In some embodiment, Y1is H.

[0128] In some embodiments, Y1, Y2, Y3, and Y4is each independently a C3-9 alkyl. For example, Y1, Y2, Y3, and Y4is each independently a C4-9 alkyl, C5-9 alkyl., Ce-9 alkyl, C7-9 alkyl, Cs-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, Ce-8 alkyl, Ce-7 alkyl, or C7-8 alkyl. For example, Y1, Y2, Y3, and Y4is each independently a C7-9 alkyl.

[0129] In some embodiments, Y1and Y2is each independently a C5-7 alkyl and Y3and Y4is each independently a C3-5 alkyl.

[0130] In some embodiments, R1and R2is each independently a C1-3 alkyl. For example, R1and R2is each independently methyl ethyl, propyl, or isopropyl.

[0131] In some embodiments, 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.

[0132] In some embodiments, the compound of Formula II is represented by one of the following structural formulas:NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01or a salt thereof.

[0133] In some embodiments, the compound of Formula II is represented by one of the following structural formulas:NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01 oNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01or a salt thereof.

[0134] Lipid A may be synthesized according to WO2015 / 095340 (e.g., pp. 84-86), which is hereby incorporated by reference in its entirety. In some embodiments, the ionizable lipid is Lipid A, or an ionizable lipid provided in WO2020 / 219876, which is hereby incorporated by reference in its entirety.

[0135] In some embodiments, the ionizable lipid is Lipid B (also referred to as Compound #8 in Table 2), which is O, O'-(2-((((2-(diethylamino)ethyl)carbamoyl)oxy)methyl)propane-L3-diyl) di(heptadecan-9-yl) diglutarate. Lipid B can be depicted as:NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0136] Lipid B may be synthesized as follows: to a solution of di(heptadecan-9-yl) O, O'-(2-((((4-nitrophenoxy)carbonyl)oxy)methyl)propane-l,3-diyl) diglutarate in MeCN (0.05 - 0.25 M) was added N', N'-di ethylethane- 1,2-diamine (1.0 - 3.0 equiv.), pyridine (1.0 - 2.0 equiv.) and DMAP (0.1 - 1.0 equiv.). Then the mixture was stirred at 15-25 °C for at least 12 h under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was diluted with EtOAc and washed 2-5x with IN Nal ICO and 3x with H2O. The organic layer was dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography to afford product as a colorless oil.!H NMR (400 MHz, CDCI3) 85.35 (s, 1H), 4.85 (p, J = 6.3 Hz, 2H), 4.12 (t, J = 6.0 Hz, 6H), 3.22 (q, J = 5.8 Hz, 2H), 2.53 (d, J = 6.8 Hz, 6H), 2.36 (dt, J = 14.7, 7.4 Hz. 9H), 1.93 (p, J = 7.5 Hz, 4H), 1.49 (q, J = 5.9 Hz, 8H), 1.24 (s, 48H), 1.00 (t, J = 7.1 Hz, 6H), 0.87 (t, J = 6.7 Hz, 12H). MS: 953.6 m / z [M+H],

[0137] In some embodiments, the ionizable lipid is Lipid C (also referred to as Compound #2 in Table 2), which is 3-(((2-(azepan-l-yl)ethyl)carbamoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dicnoyl)oxy)mcthyl)propyl hcptadccan-9-yl glutarate. Lipid C can be depicted as:O

[0138] Lipid C may be synthesized from heptadecan-9-yl (3-(((4-nitrophenoxy)carbonyl)oxy)-2-((((9Z, 12Z)-octadeca-9, 12-dienoyl)oxy)methyl)propyl) glutarate and 2-(azepan-l-yl)ethan-l -amine using the same method employed for Lipid B. ’ll NMR (400 MHz. CDCL) 85.44 - 5.26 (m, 5H), 4.86 (p, J = 6.3 Hz, 1H), 4.13 (t, J = 5.8 Hz, 6H), 3.21 (t, J = 6.0 Hz, 2H), 2.81 - 2.73 (m, 2H), 2.66 - 2.56 (m, 5H), 2.42 - 2.26 (m, 7H), 2.05 (q,.1 = 6.8 Hz, 4H), 1.94 (p, J = 7.5 Hz, 2H), 1.62 (d, J = 16.8 Hz, 10H), 1.50 (d,.1 = 6.2NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01Hz, 4H), 1.41 - 1.26 (m, 24H), 1.25 (s, 16H), 0.88 (td, J = 6.9, 5.0 Hz, 9H). MS: 890.5 m / z [M+H],

[0139] In some embodiments, the ionizable lipid is Lipid E (also referred to as Compound #18 in Table 2), which is O, O'-(2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propane- 1,3 -diyl) di(heptadecan-9-yl) diglutarate. Lipid E can be depicted as:O 0J, or a salt thereof. II NMR (400 MHz, CDC13) 54.79 (tt, J = 6.5, 3.2 Hz, 2H), 4.18 - 4.02 (m, 8H), 2.44 (qd, J = 7.0, 2.5 Hz, 6H), 2.31 (tdd, J = 15.2, 6.3, 2.0 Hz, 9H), 1.88 (pd, J = 7.4, 2.0 Hz, 4H), 1.75 (qt, J = 9.0, 4.6 Hz, 3H), 1.44 (dt, J = 11.4, 5.5 Hz, 8H), 1.29 - 1.12 (m, 46H), 0.94 (td, J = 7.1, 2.4 Hz, 6H), 0.81 (td, J = 6.8, 2.3 Hz, 12H). MS: 968.7 m / z [M+H],

[0140] In some embodiments, the ionizable lipid is represented by structural Formula (III),OR2wherein, independently for each occurrence,X1is Ci-3 alkylene orX2is selected from O, NH, NMe, and a bond, provided that when X2is O, R2taken together with the nitrogen atom and either R1or a carbon atom of X3form a 4- membered, 5-membered, or 6-membered ring;NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01X3is C2-4 alkylene,X4is Ci alkylene or a bond,X5is Ci alkylene or a bond,R1is C1-3 alkyl,R2is C1-3 alkyl, orR2taken together with the nitrogen atom and either R1or a carbon atom of X3form a 4- membered, 5 -membered, or 6-membered ring,Y1is selected from a bond, -CH=CH-, -(C=O)O-, and -O(C=O)-,Y2is selected from -CH2-CH=CH- and C3-C4 alkylene,R3is selected from H, C5-7 cycloalkyl, Cs-Cio alkenyl, and C3-18 alkyl, andR4is C4-8 alkyl,or a salt thereof.

[0141] In some embodiments, the compound of Formula III is represented by structural Formula Illa,O (Illa).

[0142] In some embodiments, A is O. Alternatively, A is NH.

[0143] In some embodiments, X1is 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, X1is a C2-3 alkylene, such as a C2 alkylene or a C3 alkylene. For example, X1is a C3 alkylene.

[0144] In some embodiments, Z1is a C3-9 alkylene. For example, Z1is a C4-9 alkylene, C5-9 alkylene, C6-9 alkylene, C7-9 alkylene., Cs-9 alkylene, C3-8 alkylene, C3-7 alkylene, C3-6 alkylene, C3-5 alkylene, C3-4 alkylene, C4-8 alkylene, C4-7 alkylene, C4-6 alkylene, C4-5 alkylene, C5-8 alkylene, C5-7 alkylene, C5-6 alkylene, Ce-s alkyleneiCe-7 alkylene, or C7-8 alkoxy. For example, Z1is a C3-5 alkylene, C5-7 alkylene, or C7-9 alkylene.

[0145] In some embodiment, Z2is a direct bond. In some embodiment, Z2is a C1-3 alkylene.

[0146] In some embodiments, Y1and Y2is each independently a C3-9 alkyl. For example, Y1and Y2is each independently a C4-9 alkyl, C5-9 alkyl, Ce-9 alkyl, C7-9 alkyl, Cs-9 alkyl, C3-8NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01alkyl, 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, Ce-s alkyl, Ce-7 alkyl, or C7-8 alkyl. In some embodiments, Y1and Y2is each independently a C3-5 alkyl, C5-7 alkyl, or C7-9 alkyl.

[0147] In some embodiments, the compound of Formula III is represented by one of the following structural formulas:NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01andor a salt thereof.

[0148] In some embodiments, the ionizable lipid is Lipid D, which is 2-((4-(((3-(pyrrolidin- 1 -yl)propoxy)carbonyl)oxy)decanoyl)oxy)propane- 1,3 -diyl (9Z,9'Z, 12Z, 12'Z)-bis(octadeca-9,12-dienoate). Lipid D can be depicted as:or a salt thereof.

[0149] Lipid D may be synthesized according to WO 2020 / 118041, which is incorporated by reference in its entirety. In some embodiments, the ionizable lipid is Lipid D, or an ionizable lipid provided in WO 2020 / 118041.

[0150] Other suitable ionizable lipids can include, for example, the ionizable lipids of WO 2020 / 219876 (e.g., at pp. 13-33, 66-87), WO 2020 / 118041, WO 2019 / 067992,NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01WO 2017 / 173054, WO 2015 / 095340, and WO 2014 / 136086, which are hereby incorporated by reference.

[0151] Ionizable lipids and other “biodegradable lipids” suitable for use in the lipid nucleic acid assemblies described herein are biodegradable in vivo or ex vivo. The ionizable lipids have low toxicity (e.g., are tolerated in animal models without adverse effect in amounts of greater than or equal to 10 mg / kg). In some embodiments, lipid nucleic acid assemblies comprising an ionizable lipid include those where at least 75% of the ionizable lipid is cleared from the plasma or the engineered cell within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days. In some embodiments, lipid nucleic acid assemblies comprising an ionizable lipid include those where at least 50% of the lipid nucleic acid assembly is cleared from the plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days, for example by measuring a lipid (e.g., an ionizable lipid) or RNP. In some embodiments, lipid-encapsulated versus free lipid, RNP is measured.

[0152] Lipid clearance may be measured as described in literature. See Maier, M. A., et al. Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics. Mol. Ther. 2013, 21(8), 1570-78 (“Maier”).

[0153] Ionizable lipids for LNP delivery of RNPs may be selected with a pKa above the preformulation buffer pH to achieve a formulatable LNP-RNP. Specifically, the pKa of the ionizable lipids of the present disclosure (i.e., at or below 7) is tied to the formulatability of RNP-LNPs and demonstrating gene editing activity in vivo. In some aspects, effective RNP-LNP formulation may be achieved using a pre- formulation buffer pH and ionizable lipid pKa that are offset by about 0.5 pH units. In certain aspects, a lipid component including an ionizable lipid having pKa that is offset from the preformulation buffer pH, for example by about 0.5 pH units, may provide one or more of encapsulation efficiency greater than 50%, a PDI less than 0.1, particle size of about 80-120 nm, and stability. As described herein, the pKa is important for formulating RNP-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 formulatability and delivery of RNP cargo in vivo. In some embodiments, the ionizable lipids are positively charged at an acidic pH but neutral in the blood.

[0154] The pH at which the ionizable lipids is predominantly protonated is related to its intrinsic pKa. Provided herein is a lipid nanoparticle (LNP) including a lipid component with an ionizable lipid having a pKa at or below about 7.0. As described above, an ionizable lipid having a pKa at or below about 7.0 has been found to achieve desired formulatability withNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01RNP complexes and effective in vivo delivery of RNPs. In some embodiments, an ionizable lipid of the present disclosure has a pKa in the range of from about 5.0 to about 7.0, about 5.5 to 7.0 preferably from about 6.0 to about 7.0, even more preferably from about 6.5 to about 7.0. In some embodiments, an ionizable lipid of the present disclosure has a pKa in the range of from about 5.7 to about 7, or any increment in between. For example, an ionizable lipid of the present disclosure has a pKa of about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, or about 6.9. In some aspects, the ionizable lipid has a pKa of about 6.0 to about 7.0, a pKa of about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or about 7.0Additional LipidsNeutral Lipids

[0155] ‘ ‘Neutral lipids” suitable for use in a lipid composition of the disclosure include, for example, a variety of neutral, uncharged, or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include, but are not limited to, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), l,2-distearoyl-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), l-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), l,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), l,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), lysophosphatidyl ethanolamine and combinations thereof. In certain embodiments, the neutral phospholipid may be selected from distearoylphosphatidylcholine (DSPC) and dimyristoyl phosphatidyl ethanolamine (DMPE), preferably distearoylphosphatidylcholine (DSPC).

[0156] In certain aspects, the neutral lipid is DSPC or DMPE or preferably DSPC. In some aspects, the LNP does not include l,2-dioleoyl-3-trimethylammonium-propane (DOTAP).NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01Helper Lipids

[0157] “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.PEG-lipids

[0158] PEG-lipids are “stealth 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 lipid nucleic acid assembly or aid in stability of the nanoparticle ex vivo. Stealth lipids suitable for use in a lipid composition of the disclosure include, but are not limited to, stealth lipids having a hydrophilic head group linked to a lipid moiety. Stealth lipids suitable for use in a lipid composition of the present disclosure and information about the biochemistry of such lipids can be found in Romberg et al., Pharmaceutical Research, Vol. 25, No. 1, 2008, pg. 55-71 and Hoekstra et al., Biochimica et Biophysica Acta 1660 (2004) 41-52. Additional suitable PEG-lipids are disclosed, e.g., in WO 2006 / 007712. PEG-lipids may be structural PEG-lipids or anchor PEG-lipids, depending on the presence or absence of a conjugation handle. For example PEG- lipid may refer to both a structural PEG- lipid and an anchor PEG-lipid and the anchor PEG-lipid and the structural PEG-lipid may comprise the same PEG-lipid.

[0159] In one embodiment, the hydrophilic head group of stealth lipid comprises a polymer moiety selected from polymers based on PEG. Stealth lipids may comprise a lipid moiety. In some embodiments, the stealth lipid is a PEG-lipid.

[0160] In one embodiment, a stealth lipid comprises a polymer moiety selected from polymers based on PEG (sometimes referred to as poly(ethylene oxide)), poly(oxazoline), poly( vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), polyaminoacids and poly[N-(2-hydroxypropyl)methacrylamide],

[0161] Structural PEG-lipids and anchor PEG-lipids each comprise a PEG-lipid including a polymer moiety based on PEG (sometimes referred to as poly(ethylene oxide)).

[0162] The PEG-lipid further comprises a lipid moiety. In some embodiments, the lipid moiety may be derived from di acylglycerol or diacylglycamide, including those comprising a dialkylglycerol or dialkylglycamide group having alkyl chain length independentlyNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01comprising from about 04 to about C40 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups such as, for example, an amide or ester. In some embodiments, the alkyl chain length comprises about CIO to C20. The dialkylglycerol or dialky Iglycamide group can further comprise one or more substituted alkyl groups. The chain lengths may be symmetrical or asymmetrical.

[0163] In some embodiments, the PEG (e.g., conjugated to a lipid moiety or lipid, such as a stealth lipid), is a “PEG-2K,” also termed “PEG 2000,” which has an average molecular weight of about 2,000 Daltons. PEG-2K is represented herein by the following formula, wherein n is 45, meaning that the number averaged degree of polymerization comprises about45 subunitsn. However, other PEG embodiments known in the art may be used, including, e.g., those where the number-averaged degree of polymerization comprises about 23 subunits (n=23), or 68 subunits (n=68). In some embodiments, R may be selected from H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R may be unsubstituted alkyl. In some embodiments, R may be methyl.

[0164] In any of the embodiments described herein, the PEG-lipid may be selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG catalog # GM-020 from NOF, Tokyo, Japan), such as e.g., l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (PEG2k-DMG, also referred to herein as C14 DMG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSG), PEG- l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE) (catalog # DSPE-020CN, NOF, Tokyo, Japan), PEG-dilauiylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, and PEG-distearoylglycamide, PEG-cholesterol (l-[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) (cat. #880150P from Avanti Polar Lipids, Alabaster, Alabama, USA), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE) (cat. #880120C from Avanti Polar Lipids, Alabaster, Alabama, USA), 1,2-distearoyLsn-glycerol, methoxypolyethylene glycol (PEG2k-DSG; GS-020, NOF Tokyo, Japan), 1,2,-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol (PEG2K-DPG; GP-020, NOF Tokyo, Japan), polyethylene glycol)-2000-dimethacrylate (PEG2k-DMA), 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA), methoxy-PEG2000-carbamoy 1-1,2-NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01tetradecyoxypropylamine (Cl 4 Ether), methoxy-PEG2000-carbamoyl-l,2-tridecyoxypropylamine (Cl 3 Ether), or methoxy-PEG2000-carbamoyl-l,2-didecyoxypropylamine (Cl 2 Ether). In one embodiment, the PEG-lipid may be 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (PEG2k-DMG, also referred to herein as C14 DMG). In one embodiment, the PEG-lipid may be PEG2k-DMG (i.e., C14 DMG). In some embodiments, the PEG-lipid may be PEG2k-DSG. In one embodiment, the PEG-lipid may be PEG2k-DSPE. In one embodiment, the PEG-lipid may be PEG2k-DMA. In one embodiment, the PEG-lipid may be PEG2k-C-DMA. In one embodiment, the PEG-lipid may be compound S027, disclosed in WO2016 / 010840 (paragraphs [00240J to

[0244] ). In one embodiment, the PEG-lipid may be PEG2k-DSA. In one embodiment, the PEG-lipid may be PEG2k-Cll. 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.

[0165] In some embodiments, the PEG-lipid includes a glycerol group. In some embodiments, the PEG-lipid includes a dimyristoylglycerol (DMG) group. In some embodiments, the PEG-lipid comprises PEG- 2k. In some embodiments, the PEG-lipid is a PEG-DMG. In some embodiments, the PEG-lipid is a PEG-2k-DMG. In some embodiments, the PEG-lipid is l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol2000. In some embodiments, the PEG-2k-DMG is l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

[0166] In some 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), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2K-DMPE), l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (PEG2k-DMG, also referred to herein as C14 DMG), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (poly ethylene glycol)-2000] (PEG2K-DSPE), 1,2-distearoyl-sn-glycerol-[methoxy(polyethylene glycol)-2000] (PEG2K-DSG), polyethylene glycol)-2000-dimethacrylate (PEG2K-DMA), 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2K-DSA), methoxy-PEG2000-carbamoyl-l,2-NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01tetradecyoxypropylamine (Cl 4 Ether), methoxy-PEG2000-carbamoyl-l,2-tridecyoxypropylamine (Cl 3 Ether), methoxy-PEG2000-carbamoyl-l,2-didecyoxypropylamine (Cl 2 Ether), or a combination thereof.

[0167] In certain aspects, the structural PEG-lipid is l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (PEG2k-DMG, also referred to herein as Cl 4 DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2K-DSPE), methoxy-PEG2000-carbamoyl-l,2-didecyoxypropylamine (Cl 2 Ether), methoxy-PEG2000-carbamoyl-l,2-tetradecyoxypropylamine (C14 Ether), methoxy-PEG2000-carbamoyl-l,2-tridecyoxypropylamine (Cl 3 Ether), or a combination thereof.

[0168] In some aspects, the lipid component further comprises an anchor PEG-lipid comprising a first coupling moiety. 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.

[0169] In some aspects, the anchor PEG-lipid and the 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.

[0170] 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, 160 170, 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).LNP Formulations

[0171] Described herein are lipid compositions comprising at least one ionizable lipid of Formula (I)-(III), a helper lipid, a neutral lipid, and a structural PEG-lipid. In some aspects, the lipid component further comprises an anchor PEG-lipid. In some aspects, the lipid component includes a molar ratio of the ionizable lipid: neutral lipid: helper lipid: structuralNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01PEG-lipid of 30 to 60: 0 to 25: 25 to 65: 0.5 to 5. In some aspects, the lipid component includes a molar 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. In some aspects, the lipid component includes a molar ratio of the ionizable lipid: neutral lipid: helper lipid: structural PEG-lipid of 30 to 50: 0 to 25: 25 to 65: 0.5 to 5. In some aspects, the lipid component includes a molar ratio of the ionizable lipid, the neutral lipid, the helper lipid, and the structural PEG-lipid are in a ratio of 32 to 40: 10 to 20: 40 to 50: 1.5 to 3.5. In some aspects, the lipid component includes a molar ratio of the ionizable lipid: neutral lipid: helper lipid: structural PEG-lipid of 40 to 60: 5 to 15: 25 to 65: 0.5 to 5. In some aspects, the lipid component includes a molar ratio of the ionizable lipid: neutral lipid: helper lipid: structural PEG-lipid of 40 to 60: 5 to 25: 25 to 65: 0.5 to 5. Any of the foregoing compositions may further include 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 for delivering RNP cargo to a cell, for example a liver cell. In some aspects, the lipid component includes a molar ratio of the ionizable lipid: neutral lipid: helper lipid: structural PEG-lipid of 30 to 60: 0 to 25: 25 to 65: 0.5 to 5. In some aspects, the lipid component includes a molar 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. In some aspects, the lipid component includes a molar ratio of the ionizable lipid: neutral lipid: helper lipid: structural PEG-lipid of 30 to 50: 0 to 25: 25 to 65: 0.5 to 5. In some aspects, the lipid component includes a molar ratio of the ionizable lipid, the neutral lipid, the helper lipid, and the structural PEG-lipid are in a ratio of 32 to 40: 10 to 20: 40 to 50: 1.5 to 3.5. In some aspects, the lipid component includes a ratio of the ionizable lipid (e.g., referring to Table 2, below, compound 2, compound 8, compound 5, or compound 7): neutral lipid (e.g., DSPC): helper lipid (e.g., cholesterol): structural PEG-lipid (e.g., compound 11 in Table 2) of 40 to 60: 0 to 25: 25 to 65: 0.5 to 5, a ratio of 42 to 58: 2 to 23: 27 to 62: 0.7 to 5, a ratio of 44 to 56: 4 to 20: 30 to 60: 0.8 to 5, a ratio of 46 to 54: 6 to 18: 32 to 58: 1 to 5, a ratio of 48 to 52: 8 to 16: 34 to 56: 2 to 5, a ratio of 49 to 51: 8 to 10: 36 to 54: 2 to 4, or any subranges according to any of the aspects described below. In certain aspects, the lipid component includes a ratio of the ionizable lipid (e.g., compound 2, compound 8, compound 5, or compound 7 in Table 2): neutral lipid (e.g., DSPC): helper lipid (e.g., cholesterol): structural PEG-lipid (e.g., compound 11 in Table 2) in a ratio of 50:9:38:3. 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 a ratioNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01of 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. In some aspects, the lipid component includes a molar ratio of the ionizable lipid: neutral lipid: helper lipid: structural PEG-lipid in a ratio of 50:9:38:3, a ratio of 45:7:45:3, a ratio of 47:22.5:28:2.5, a ratio of 35:15:47.5:2.5, a ratio of 50:10:38.5:1.5, or a ratio of 50:10:37.8:2.2. In some aspects, the lipid component includes a molar ratio of the ionizable lipid: neutral lipid: helper lipid: structural PEG-lipid in a ratio of 50:9:38:3, a ratio of 35:15:47.5:2.5, or a ratio of 47:22.5:28:2.5.

[0173] LNPs may also include a five component LNP formulation, including an anchor PEG-lipid, which can be conjugated to a targeting moiety capable of targeting the LNP to certain tissues, e.g., bone marrow, and / or certain cell types. In some aspects, the lipid component includes a molar ratio of the ionizable lipid (e.g., referring to Table 2, below, compound 2, compound 8, compound 5, or compound 7): neutral lipid (e.g., DSPC): helper lipid (e.g., cholesterol): structural PEG-lipid (e.g., compound 11 in Table 2) of 40 to 60: 0 to 25: 25 to 65: 0.5 to 5, a ratio of 42 to 58: 2 to 23: 27 to 62: 0.7 to 5, a ratio of 44 to 56: 4 to 20: 30 to 60: 0.8 to 5, a ratio of 46 to 54: 6 to 18: 32 to 58: 1 to 5, a ratio of 48 to 52: 8 to 16: 34 to 56: 2 to 5, or a ratio of 49 to 51: 8 to 10: 36 to 54: 2 to 4, 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 0.01 to 1.5, 0.015 to 1, 0.02 to 0.9, 0.025 to 0.8, 0.03 to 0.7, 0.035 to 0.6, 0.04 to 0.5, 0.042 to 0.48, or about 0.045, 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 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. In certain aspects, the lipid component includes a ratio of the ionizable lipid (e.g., compound 2, compound 8, compound 5, or compound 7 in Table 2): neutral lipid (e.g., DSPC): helper lipid (e.g., cholesterol): structural PEG-lipid (e.g., compound 11 in Table 2): anchor PEG-lipid (e.g., DSPE-PEG-SpyTag) in a ratio of about 50:9:38:2.955:0.045.

[0174] In some aspects, the four component lipid formulation comprises 3-(((2-(azepan-I-yl)ethyl)carbamoyl)oxy)-2-((((9Z, I2Z)-octadeca-9, I2-dienoyl)oxy)methyl)propyl heptadecan-9-yl glutarate as the ionizable lipid (lipid C); DSPC as the neutral lipid; cholesterol as the helper lipid; and Cl 3 Ether (compound 11 in Table 2) as the slrucluralNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01PEG-lipid. In some aspects, the four component lipid formulation comprises O, O'-(2-((((2-(diethylamino)ethyl)carbamoyl)oxy)methyl)propane-l,3-diyl) di(heptadecan-9-yl) diglutarate as the ionizable lipid (lipid B); DSPC; cholesterol; and Cl 3 Ether structural PEG-lipid. In some aspects, the four component lipid formulation comprises O, O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane- 1,3 -diyl) di(heptadecan-9-yl) diglutarate (lipid E) as the ionizable lipid (Compound 7 in Table 2); DSPC as the neutral lipid; cholesterol as the helper lipid; and Cl 3 Ether (compound 11 in Table 2) as the structural PEG-lipid.

[0175] In some aspects, the five component lipid formulation comprises 3-(((2-(azepan-l-yl)ethyl)carbamoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl heptadecan-9-yl glutarate as the ionizable lipid (lipid C); DSPC as the neutral lipid; cholesterol as the helper lipid; C13 Ether (compound 11 in Table 2) as the structural PEG-lipid; and DSPE-PEG-SpyTag as the anchor PEG-lipid.

[0176] As used herein, the molar ratio (“mol %”) of the anchor PEG-lipid may also be referred to as the “anchor PEG-lipid density” or “anchor PEG-lipid density,” as both reflect the total anchor PEG-lipid component of the lipid composition. In some embodiments, the anchor PEG-lipid density is the same as the ligand density, i.e., every anchor PEG-lipid of a 5-component LNP is conjugated to a targeting ligand (100% conjugation efficiency). In some embodiments, the anchor PEG-lipid density is not the same as the ligand density, i.e., some anchor PEG-lipids of a 5-component LNP are not conjugated to a targeting ligand, thus creating a ligand density that is lower than the anchor PEG-lipid density. In some embodiments, only a subset of the anchor PEG-lipids are conjugated to a targeting ligand, e.g., 5%, 10%, 1 %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% conjugation efficiency.

[0177] 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 aboutNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W011.5 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 %, or 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 %, or 1.5 mol % of the lipid component.

[0178] In some aspects, 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%, or 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.

[0179] 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%, or 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.

[0180] 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%, or about 0.03 to about 0.075 mol% of the lipid component. In some aspects, the anchor PEG-lipid is in an amount fromNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01about 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.

[0181] 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%, or 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.

[0182] 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%, or 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.

[0183] 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 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 %,NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01about 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 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 molNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01%, 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 %, 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 22-2.2 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 %,NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01about 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.

[0184] 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.

[0185] 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.

[0186] 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 stmctural PEG-lipid and anchor PEG- lipids together in the lipid component. In some 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.

[0187] 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 isNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01included 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 %, 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 %,NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01about 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.

[0188] 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.

[0189] 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 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,NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01nominal, 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.

[0190] 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.

[0191] 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 %. 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.Targeting ligand

[0192] Conjugating a cell- or tissue-specific targeting ligand to the anchor PEG-lipid facilitates a targeted LNP (t-LNP) to bind to its target tissue or cell type.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0193] 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, a targeting ligand is attached to the first coupling moiety via a second coupling moiety. 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. 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.

[0194] 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). 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. 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 couplingNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01moiety may be an immunoglobulin Fc domain. In some aspects, the first coupling moiety 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.

[0195] In some aspects, the first coupling moiety comprises a peptide sequence selected from AHIVMVDAYKPTK (SEQ ID NO: 26), VPTIVMVDAYKRYK (SEQ ID NO: 27), or RGVPHIVMVDAYKRYK (SEQ ID NO: 28). In some aspects, the second coupling moiety comprises a protein selected from:GAMVDTLSGLSSEQGQSGDMT1EEDSATHIKFSKRDEDGKELAGATMELRDSSG KTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKAT KGDAHI (SEQ ID NO: 29), VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIST WISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDA HI (SEQ ID NO: 30), VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTIS TWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGEATKGDA HT (SEQ ID NO: 31), VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTIS TWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDA HT (SEQ ID NO: 32), or EDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTF VETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI (SEQ ID NO: 33).

[0196] In some aspects, the first coupling moiety comprises a protein selected from:GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSG KTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKAT KGDAHI (SEQ ID NO: 29), VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIST WISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDA HI (SEQ ID NO: 30), VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTIS TWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGEATKGDA HT (SEQ ID NO: 31),NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01VTTKSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTIS TWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDA HT (SEQ ID NO: 32), or EDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTF VETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI (SEQ ID NO: 33). In some aspects, the second coupling moiety comprises a peptide sequence selected from AHIVMVDAYKPTK (SEQ ID NO: 26), VPTIVMVDAYKRYK (SEQ ID NO: 27), or RGVPHIVMVDAYKRYK (SEQ ID NO: 28).

[0197] In some aspects, the targeting ligand is selected from an antibody, an antibody fragment, small molecule, or a peptide. In some aspects, the targeting ligand is capable of specifically binding to a target or multiple targets.

[0198] In some aspects, the targeting ligand is an antibody. Exemplary antibodies, include, without limitation, immunoglobulins of the IgG subtype, such as IgGl, IgG2, IgG3, or IgG4, monoclonal antibodies, humanized antibodies, chimeric antibodies, bi- or multi-specific antibodies.

[0199] 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-KIH, 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-HH-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, anNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01IgG, an IgM, an IgA, an IgE, an IgD, a DEP conjugate, TMEAbodyTM, SAFEbody®, TRITAC®, or SHIELD antibody.

[0200] 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, a disulfide-linked Fv (sdFv), a Fd fragment consisting of VH and CHI 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.

[0201] 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.

[0202] In some aspects, the targeting ligand is added after the LNP is formed. 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.

[0203] In some aspects, the amount of the anchor PEG-lipid is the same as an anchor PEG-lipid density. In some aspects, the targeting ligand has a density that is the same or less than the anchor PEG-lipid density.

[0204] 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 may be CD34, CD38, CD45, GDI 17, CD123, or CD135. In some aspects the target cell may be a T-cell. In some aspects, the target may be CD2, CD3, CD5, CD6, CD7, CD8, or CD45.LNP CargoRNP Complexes

[0205] Ribonucleoprotein (RNP) complexes are structures that facilitate RNA-guided gene editing in a cell. The complex comprises a guide RNA (gRNA) together with an RNA-guided DNA-binding agent, such as a CRISPR / Cas nuclease, wherein the Gas nuclease and the gRNA associate in a ribonucleoprotein (RNP) complex. In some embodiments, the RNP complex comprises a Type II Gas nuclease, e.g., a Cas9 nuclease. In some aspects, the gRNA guides the RNA-guided DNA-binding agent such as Cas9 to a target sequence, and the gRNANTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01hybridizes with the target sequence. LNPs according to any one or combination of aspects disclosed herein encapsulate a cargo comprising a ribonucleoprotein complex (RNP) comprising a Cas nuclease polypeptide and a guide RNA (gRNA). The endonuclease in the RNP can be modified, e.g., the endonuclease may comprise a gene fusion with a peptide tag or another protein, or the endonuclease in the RNP may be unmodified. Likewise, the gRNA, crRNA, tracrRNA, dgRNA, or sgRNA can be modified or unmodified.

[0206] Described herein are formulation parameters and ionizable lipids that advantageously allow encapsulation of an RNP cargo (e.g., a Cas / sgRNA cargo) with high encapsulation efficiency, optimal size for in vivo administration, and / or low polydispersity index. It has been found that effective RNP-LNP formulation, and in vivo gene editing activity by the LNP encapsulated RNP, can be achieved by selecting ionizable lipids having a pKa at or below 7.0, molar ratios of gRNA: Cas nuclease protein, molar ratios of ionizable lipid to Cas nuclease polypeptide, guide RNA length, and combinations thereof.

[0207] The gRNA may be included at various ratios with a Cas nuclease to provide a stable gRNA / Cas nuclease complex with high encapsulation efficiency in an LNP. In some aspects, a molar ratio of gRNA: Cas nuclease is in a range of about 1:1 to about 16:1. In some aspects, the gRNA: Cas nuclease molar ratio is about 1.1:1 to about 12:1, about 1.2:1 to about 10:1, about 1.4:1 to about 8:1, about 1.6:1 to about 6:1, about 1.8:1 to about 5:1, or about 2:1 to 4:1. In some aspects, the gRNA: Cas nuclease ratio is about 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, or 12: 1. In certain aspects, the gRNA: Cas nuclease ratio is about 2:1. In other aspects, the gRNA: Cas nuclease ratio is about 1:1 to about 3:1.

[0208] The LNPs may also be formulated with various ionizable lipid to Type II Cas nuclease polypeptide ratios to provide a stable LNP with high cargo encapsulation efficiency. For example, a molar ratio of ionizable lipid to Type II Cas nuclease polypeptide may be 11,000:1 to 1,600:1. In some aspects, a ratio of ionizable lipid to Type II Cas nuclease polypeptide may be 10,000:1 to 1,800:1, 8,000:1 to 2,000:1, 6,000:1 to 2,200:1, or 4,000:1 to 2,400:1. In some aspects, a ratio of ionizable lipid to Type II Cas nuclease polypeptide may be 11,000:1, 10,800:1, 10,600:1, 10,400:1, 10,200:1, 10,000:1, 9,800:1, 9,600:1, 9,400:1, 9,200:1, 9,000:1, 8,800:1, 8,600:1, 8,400:1, 8,200:1, 8,000:1, 7,800:1, 7,600:1, 7,400:1, 7,200:1, 7,000:1, 6,800:1, 6,600:1, 6,400:1, 6,200:1, 6,000:1, 5,800:1, 5,600:1. 5,400:1, 5,200:1, 5,000:1, 4,800:1, 4,600:1, 4,400:1, 4,200:1, 4,000:1, 3,950:1, 3,900:1, 3,850:1, 3,800:1, 3,750:1, 3,700:1, 3,650:1, 3,600:1, 3,550:1, 3,500:1, 3,450:1, 3,400:1, 3,350:1,NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W013,300:1, 3,250:1, 3,200:1, 3,150:1, 3,100:1, 3,050:1, 3,000:1, 2,950:1, 2,900:1, 2,850:1, 2,800:1, 2,750:1, 2,700:1, 2,650:1, 2,600:1, 2,550:1, 2,500:1, 2,450:1, 2,400:1, 2,350:1, 2,300:1, 2,250:1, 2,200:1, 2,150:1, 2,100:1, 2,050:1, 2,000:1, 1,950:1, 1,900:1, 1.850:1, 1,800:1, 1,750:1, 1,700:1, 1,650:1, or 1,600:1. In certain aspects, a molar ratio of ionizable lipid to Type II Cas nuclease polypeptide may be 2,800: 1 to 4,000:1, or about 2,850:1 or about 3,800:1. In certain aspects, a molar ratio of ionizable lipid to Type II Cas nuclease polypeptide is 4,000:1 to 2,400:1, or about 3,800:1, or 3,792:1. Methods for preparing such formulations are known in the art and may include measuring the concentration of the ionizable lipid and Cas nuclease and converting to molarity.

[0209] The nucleotide length of the gRNA associated with the Cas nuclease in the RNP complex may be selected to enhance formulatability of the LNP-RNP complex and in vivo gene editing potency. For example, the gRNA nucleotide length may be 200 nucleotides in length or less, for example 60 to 200 nucleotides. In some aspects, the gRNA nucleotide length is less than 200 nucleotides in length, for example, 150 nucleotides in length or less. In some aspects, the LNP according to any one or combination of the foregoing aspects, the gRNA is 60 to 150 nucleotides, 70 to 130 nucleotides, 70 to 120 nucleotides, 70 to 110 nucleotides, 70 to 100 nucleotides in length, or about 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82. 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93. 94, 95. 96, 97. 98, 99, 100 nucleotides in length. In certain aspects, an LNP-RNP according to aspects disclosed herein comprises a gRNA that is less than 100 nucleotides in length, and wherein the LNP has 2x to 5x, or about 3x, or about 4x higher gene editing potency relative to an LNP-RNP complex comprising a gRNA that is 100 nucleotides in length or more than 100 nucleotides in length. For example, gene editing potency of an LNP-RNP may be determined as the total LNP delivered (e.g., pg of LNP delivered) versus the percentage of identified insertions or deletions (“indels”) introduced by gene editing.EnzymesCRISPR / Cas system

[0210] The Cas nuclease polypeptide component of an RNP may comprise Class 1 or Class 2 system components. Cas proteins of Types II, V, and VI may be single-protein, RNA-guided endonucleases, herein called “Class 2 Cas nucleases.” Class 2 Cas nucleases include, for example, Cas9, Cpfl, C2cl, C2c2, and C2c3 proteins.

[0211] In some aspects, the RNP comprises a Type II Cas nuclease polypeptide. In some embodiments, the Cas protein may be from a Type-II CRISPR / Cas system, for example, aNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01Cas9 protein from a CRISPR / Cas9 system, or a Type-V CRISPR / Cas system, e.g., a Cpfl protein. In some embodiments, the Cas protein may be from a Cdass 2 CRISPR / Cas system, i.e.. a single-protein Cas nuclease such as a Cas9 protein or a Cpfl protein.

[0212] The Class 2 Cas nuclease families of proteins are enzymes with DNA endonuclease activity, and they can be directed to cleave a desired nucleic acid target by designing an appropriate guide RNA, as described further herein.

[0213] A Class 2 CRISPR / Cas system component may be from a Type-IIA, Type-IIB, Type-IIC, Type V, or Type VI system. Cas9 and its orthologs are encompassed. Non-limiting exemplary species that the Cas nuclease can be derived from include Streptococcus pyogenes (SpyCas9), Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis (“NmeCas9”, e.g., NmelCas9 or Nme2Cas9), Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus 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.

[0214] In some embodiments, the Cas nuclease is the Cas9 nuclease from Streptococcus pyogenes. In other embodiments, the Cas nuclease is the Cas9 nuclease from StreptococcusNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01thermophilus. 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 Cpf 1 nuclease from Francisella novicida. In other embodiments, the Cas nuclease is the Cpfl nuclease from Acidaminococcus sp. In still other embodiments, the Cas nuclease is the Cpfl nuclease from Lachnospiraceae bacterium ND2006. In further embodiments, the Cas nuclease is the Cpfl nuclease from Francisella tularensis, Lachnospiraceae bacterium, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium, Parcubacteria bacterium, Smithella, Acidaminococcus, Candidatus Methanoplasma termitum, Fubacterium eligens, Moraxella bovoculi, Leptospira inadai, Porphyromonas crevioricanis, Prevotella disiens, or Porphyromonas macacae. In some embodiments, the Cas nuclease is a Cpfl nuclease from an Acidaminococcus or Lachnospiraceae.

[0215] 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 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.

[0216] In some embodiments, the Cas polypeptide in the RNP can be SpyCas9 cleavase, nickase, or dead Cas9 (“dCas9”), and include the polypeptide sequences of these 4 sequences (cleavase, 2 nickase sequences, and dCas9). For example, in some embodiments, the RNP contains a SpyCas9 cleavase sequence comprising SEQ ID NO: 16, a SpyCas9 nickase sequence comprising SEQ ID NO: 17 or SEQ ID NO: 18, or a dead Cas9 with a sequence comprising (SEQ ID NO: 19) (see Table 30).

[0217] In some embodiments, the Cas polypeptide in the RNP can be Nme2Cas9. For example, in some embodiments, the RNP contains a Nme2Cas9 cleavase sequence comprising SEQ ID NO: 20, a Nme2Cas9 nickase sequence comprising SEQ ID NO: 21 or SEQ ID NO: 22, or a dead Nme2Cas9 sequence comprising SEQ ID NO: 23 (see Table 30).

[0218] 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 Fokl. In some embodiments, a Cas nuclease may be a modified nuclease.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0219] 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-Ill CRISPR / Cas system. In some embodiments, the Cas nuclease may have an RNA cleavage activity.

[0220] 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.

[0221] 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.

[0222] 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 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 acidNTEA-0118WO1-RNP-ENP; RFEM: 5640-106W01substitutions include D917A, E1006A, and D1255A (based on the Francisella novicida IJ112 Cpfl (FnCpfl) sequence (UniProtKB - A0Q7Q2 (CPF1_FRATN)).

[0223] In some aspects, the Type II Cas nuclease polypeptide is a fusion protein. In some aspects, the Cas nuclease is fused to a polymerase. In some aspects, the Cas nuclease is fused to a DNA-directed DNA polyermase. In some aspects, the Cas nuclease is fused to a RNA-directed DNA polyermase.

[0224] In some aspects, a LNP cargo comprises a RNP complex (RNP-LNP). For example, a Cas nuclease and the gRNA associated in a ribonucleoprotein (RNP) complex. For example, in some embodiments, an LNP may comprise the RNP component, an ionizable lipid, a helper lipid, a neutral lipid, and a structural PEG-lipid. In some compositions, the ionizable lipid is Eipid A-E, as described above, or an acetal analog thereof; the helper lipid is cholesterol; the neutral lipid is DSPC; and the stealth lipid is PEG2k-DMG (Cl 4 DMG). As used herein, if a FNP cargo comprises one or more RNA species (RNA-LNP), the LNP may be combined with an ionizable lipid; a helper lipid; a structural PEG-lipid.Guide RNA (gRNA)

[0225] The RNPs described herein can include a guide RNA, e.g., sgRNA, associated with an enzyme set forth herein, e.g., a Cas nuclease, forming a RNP complex. In some embodiments, a CRISPR / Cas system of the present disclosure may be directed to and cleave a target sequence on a target nucleic acid molecule. For example, the target sequence may be recognized and cleaved by the Cas nuclease. In some embodiments, a Class 2 Cas nuclease may be directed by the guide RNA to a target sequence of a target nucleic acid molecule, where the guide RNA hybridizes with the target sequence facilitating Cas protein-mediated cleavage of the target sequence. In some embodiments, the guide RNA complexed with the Cas protein cleaves the target DNA sequence upstream of a protospacer adjacent motif (PAM). In some embodiments, the target sequence may be complementary to the targeting sequence of the guide RNA. In some embodiments, the degree of complementarity between a targeting sequence of a guide RNA and the portion of the corresponding target sequence that hybridizes to the guide RNA may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments the homology region of the target is adjacent to a cognate PAM sequence. In some embodiments, the target sequence may comprise a sequence 100% complementary with the targeting sequence of the guide RNA. In other embodiments, the target sequence may comprise at least one mismatch, deletion, or insertion, as compared to the targeting sequence of the guide RNA. For example,NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01the target sequence and the targeting sequence of the guide RNA may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches, optionally in a portion of the target sequence adjacent to the PAM. In some embodiments, the target sequence and the targeting sequence of the guide RNA may contain 1-9 mismatches. In some embodiments, the target sequence and the targeting sequence of the guide RNA may contain 3-6 mismatches. In some embodiments, the target sequence and the targeting sequence of the guide RNA may contain 5 or 6 mismatches.

[0226] The length of the target sequence may depend on the nuclease system used. For example, the targeting sequence of a guide RNA for a CRISPR / Cas system 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 and the target sequence is a corresponding length, optionally adjacent to a PAM sequence. In some embodiments, the target sequence may comprise 15-24 nucleotides in length. In some embodiments, the target sequence may comprise 17-21 nucleotides in length. In some embodiments, the target sequence may comprise 20 nucleotides in length. The RuvC domain of Cas9 cleaves the non-target DNA strand, and the HNH domain of Cas9 cleaves the target strand of DNA. In certain aspects, the nickase may cleave the target strand. In certain aspects, the nickase may cleave the non-target strand. When nickases are used, the target sequence may comprise a pair of target sequences recognized by a pair of nickases that cleave opposite strands of the DNA molecule. In some embodiments, the target sequence may comprise a pair of target sequences recognized by a pair of nickases that cleave the same strands of the DNA molecule. In some embodiments, the target sequence may comprise a part of target sequences recognized by one or more Cas nucleases.

[0227] In embodiments involving a Cas nuclease, such as a Class 2 Cas nuclease, 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 Figure 1 of Ran et al., Nature, 520: 186-191 (2015), and Figure S5 of Zetsche 2015, the relevant disclosure of each of which is incorporated herein by reference. In some embodiments, the PAM may be 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Non-limiting exemplary PAM sequences include NGG, NGGNG, NG, NAAAAN, NNAAAAW, NNNNACA, GNNNCNNA, TTN, and NNNNGATT (wherein N is defined as any nucleotide, and W is defined as either A or T). In some embodiments, theNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01PAM sequence may be NGG. In some embodiments, the PAM sequence may be NGGNG. In some embodiments, the PAM sequence may be TTN. In some embodiments, the PAM sequence may be NNAAAAW.

[0228] In some aspects, the LNP according to any one or combination of the foregoing aspects the gRNA is 60 to 200 nucleotides, 60 to 150 nucleotides in length or 70 to 130, 70 to 110 nucleotides, or 70 to 100 nucleotides in length.

[0229] In some aspects, the guide RNA may comprise a single RNA molecule and is referred to herein as a “single guide RNA” or “sgRNA”. In some embodiments, the sgRNA may comprise a crRNA covalently linked to a tracr RNA. In some embodiments, the crRNA and the tracr RNA may be covalently linked via a linker. In some embodiments, the singlemolecule guide RNA may comprise a stem- loop structure via the base pairing between the flagpole on the crRNA and the tracr RNA. 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 “Cpfl sgRNA” capable of mediating RNA-guided DNA cleavage by a Cpfl protein. In certain embodiments, the guide RNA 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 guide RNA comprises a crRNA sufficient for forming an active complex with a Cpfl protein and mediating RNA-guided DNA cleavage.

[0230] Exemplary gRNAs that can be (i) complexed with a Cas9 polypeptide to form a RNP cargo for a RNP-LNP, or (ii) paired with a niRNA encoding a Cas9 polypeptide to form a RNA cargo for a RNA-LNP, are shown in Table 1 below.

[0231] Table 1. Exemplary gRNA sequences showing various scaffold modification patterns. The gRNA 20-mer spacer sequence is represented by “(N)20.” The gRNA 17-mer spacer sequence is represented by “(N)17”. “m” represents a 2’O-methyl modification; “*” represents a phospho rothioate bond; and “f” represents a 2’ -fluoro modification.Number of Sequence SEQ ID NO nucleotidesin gRNA100 (N)20- 11GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGcuuuuNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01Number of Sequence SEQ ID NO nucleotidesin gRNA100 mN*mN*mN*(N)17- 12GUUUUAGAmGmCmUmAmGniAmAmAmUmAniGmCAAGU UAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAm AmAmAmAmGmUmGniGmCmAmCmCmGmAmGmUmCmGm GmUmGmCmU*mU*mU*mU91 (N)20- 13GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCACGAAAGGGCACCGAGUCGGUGCU91 mN*mN*mN*(N)17- 14GUUUUAGAmGmCmUmAmGmAmAmAmUmAniGmCAAGU UAAAAUAAGGCUAGUCCGUUAUCACGAAAGGGCACCGA GUCGGmU*mG*mC*mU91mN*mN*mN*mNNNNNfNfNfNNfNfNNNfNfNNNmGUUUUA 15GmAmGmCmU mAmGmAm AmAmUmAmGmCmA AGUU mA A mAAmUmAmAGmGCUmAGUCmCmGUUAUmCAmCGmAm AmAmGGmGmCmAmCmCmGmAGUCmGmGmU*mG*mC*m U

[0232] In some aspects, a guide RNA may be a sgRNA or a dgRNA.

[0233] In some aspects, the gRNA is a modified gRNA. In some aspects, the gRNA comprises a 3’ end modification. In some aspects, the gRNA comprises a 3’ end extension. In some aspects, the gRNA comprises a modification in the hairpin region and / or a 5’ end modification. 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. For example, the 3’ and / or 5’ end modification comprises a protective end modification, such as a modified nucleotide selected from 2’-O-methyl (2’-OMe) modified nucleotide, 2’-O-(2-methoxyethyl) (2’- 0-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, an inverted abasic modified nucleotide, or combinations thereof. In certain aspects, the modification in the hairpin region comprises a modified nucleotide selected from 2’-O-methyl (2’-0Me) modified nucleotide, a 2’ -fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, or combinations thereof.Additional Cargo

[0234] In some aspects, provided herein are compositions comprising a plurality of LNPs encapsulating a ribonucleoprotein complex (RNP). In some embodiments, the plurality of LNPs can include an RNP, as described herein, and an additional cargo. In some aspects, theNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01additional cargo is in the same LNP. Tn some aspects, the additional cargo is encapsulated in a different LNP. For example, an LNP composition can comprise a first type of LNP carrying an RNP comprising a Type II Cas nuclease polypeptide and a guide RNA (gRNA) and a second LNP, different from the first LNP, carrying a different cargo.

[0235] The additional cargo delivered via an LNP or LNP composition described herein can include a biologically active agent. The biologically active agent may be an active pharmaceutical ingredient, a gene editing system, a nucleic acid, such as an mRNA or a gRNA, such as a sgRNA or a dsgRNA. In certain embodiments, the additional cargo is or comprises one or more biologically active agent, such as mRNA, 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.

[0236] 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 (miRNA), a Dicersubstrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), a modified RNA, a self-amplifying mRNA, or a combination thereof.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0237] The additional 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), or an RNA-guided DNA-binding agent.

[0238] Various suitable gene editing systems comprising genome editing tools for delivery with the LNP compositions are described herein (for example, LNPs including an RNP, as described herein, and an additional cargo encapsulated in the same LNP, or a first type of LNP carrying an RNP, as described herein, and a second type of LNP, different from the first type of LNP, carrying a different cargo), including but not limited to the 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. 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. When a first type of LNP carrying an RNP, as described herein, and a second type of LNP, different from the first type of LNP, carrying an additional cargo, are used, the first type of LNP and the second type of LNP may be delivered to a cell or to a subject together or separately.Nucleic Acids of Interest and Template Sequences

[0239] The methods disclosed herein may include using a template nucleic acid. For example, a template nucleic acid sequence may be encapsulated with a RNP according to any of the aspects disclosed here. The template may be used to alter or insert a nucleic acid sequence at or near a target site for an RNP comprising an RNA-guided DNA-binding protein such as a Cas nuclease, e.g., a Class 2 Cas nuclease and a guide RNA. In some embodiments, the methods comprise introducing a template to the cell. In some aspects, the template nucleic acid encodes a gene or a gene variant, e.g., comprising one or more mutations relative to a corresponding wild-type gene. 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.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0240] In some embodiments, the template sequence may correspond to an endogenous 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 bea 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. In some embodiments, the repair of the cleaved target nucleic acid molecule with the template may result in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of the target nucleic acid molecule.

[0241] In other embodiments, the template sequence may comprise an exogenous sequence. In some embodiments, the exogenous sequence may be a chromosomal or extrachromosomal sequence. In some embodiments, the exogenous sequence may provide a cDNA sequence encoding a protein or a portion of the protein. In some embodiments, the integration of the exogenous sequence may result in restored gene function. In some embodiments, the integration of the exogenous sequence may result in a gene knock-in. In some embodiments, the integration of the exogenous sequence may result in a gene knock-out.

[0242] The template may be of any suitable length. In some embodiments, the template may comprise 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, or more nucleotides in length. The template may be a singlestranded nucleic acid. The template can be double- stranded or partially double-stranded nucleic acid. In certain embodiments, the single stranded template is 20, 30, 40, 50, 75, 100, 125, 150, 175, or 200 nucleotides in length. In some embodiments, the template may comprise a nucleotide sequence that is complementary to a portion of the target nucleic acid molecule comprising the target sequence (i.e., a “homology arm”).

[0243] In some aspects, the template nucleic acid comprises a nucleic acid sequence that is complementary to at least a portion of a target nucleic acid. In some aspects, the template nucleic acid comprises a ssDNA or dsDNA containing flanking invert-terminal repeat (ITR) sequences, a plasmid, minicircle, nanocircle, or a PCR product.Lipid Nanoparticle (LNP) and Targeted LNP (t-LNP) Compositions

[0244] The RNP-containing lipid compositions described herein may be provided as lipid nanoparticles (LNPs) or targeted lipid nanopaiticles (t-LNPs) or compositions thereof. InNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01some aspects, a t- I NI’ includes an anchor PEG-lipid comprising 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.

[0245] Provided herein is a composition comprising the LNP or t-LNP of any one or combination of aspects disclosed herein, wherein the composition comprises a carrier.Suitable carrier solutions or solvents for an LNP or t-LNP composition include or may contain: water, PBS, Tris buffer, NaCl, citrate buffer, MES (2-(A-morpholino)ethanesulfonic acid) buffer, 3-(N-Morpholino)propanesulfonic acid, 4-Morpholinepropanesulfonic acid (MOPS), or acetate buffer. 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 for delivery to a cell or in vivo delivery at or above pH 6.5. In certain embodiments, a buffer is used to maintain the pH of the composition comprising LNPs for delivery to a cell or in vivo delivery at or above pH 7.0. In some aspects, the composition comprises a buffer having a pH of about 6.5 to about 8.0 or about 7.0 to about 7.8 at 25 °C. 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, 10, 11, or 12% cryoprotectant. In certain embodiments, the LNP composition may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% sucrose. In certain aspects, the cryoprotectant is from 5-12% (v / v) of the composition. 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, a MOPs 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 TrisNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01buffer. 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 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.

[0246] 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.

[0247] 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.

[0248] In some embodiments, the concentration of the LNPs in the LNP composition is about 1-10 pg / mL, about 2-10 pg / mL, about 2.5-10 pg / mL, about 1-5 pg / mL, about 2-5 pg / mL,NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01about 2.5-5 pg / mL, about 0.04 pg / mL, about 0.08 pg / mL, about 0.16 pg / mL, about 0.25 pg / mL, about 0.63 pg / mL, about 1.25 pg / mL, about 2.5 pg / mL, or about 5 pg / mL.

[0249] 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.

[0250] 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 0.05, about 0.08, about 0.1, about 0.15, about 0.2, or about 0.4. In some aspects, the LNP has a PDI in a range of 0.02-0.1, 0.02-0.09, 0.02-0.08, or 0.02-0.07. In some aspects, the LNP has a PDI of 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.010, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.020, 0.021, 0.022, 0.023, 0.024, 0.025, 0.026, 0.027, 0.028, 0.029, 0.030, 0.031, 0.032, 0.033, 0.034, 0.035, 0.036, 0.037, 0.038, 0.039, 0.040, 0.041, 0.042. 0.043, 0.044, 0.045, 0.046, 0.047, 0.048, 0.049, 0.050, 0.051, 0.052, 0.053. 0.054, 0.055, 0.056, 0.057, 0.058, 0.059, 0.060, 0.061, 0.062, 0.063, 0.064, 0.065, 0.066, 0.067, 0.068, 0.069, 0.070, 0.071, 0.072, 0.073, 0.074, 0.075, 0.076, 0.077, 0.078, 0.079, 0.080, 0.081, 0.082, 0.083, 0.084, 0.085, 0.086, 0.087, 0.088, 0.089, 0.090, 0.091, 0.092, 0.093, 0.094, 0.095, 0.096. 0.097, 0.098, 0.099, 0.100, 0.101, 0.102, 0.103, 0.104, 0.105, 0.106, 0.107, 0.108, 0.109, 0.110, 0.111, 0.112, 0.113, 0.114, 0.115, 0.116, 0.117, 0.118, 0.119, 0.120, 0.121, 0.122, 0.123, 0.124, 0.125, 0.126, 0.127, 0.128, 0.129, 0.130, 0.131, 0.132, 0.133, 0.134, 0.135, 0.136, 0.137, 0.138, 0.139, 0.140, 0.141, 0.142, 0.143, 0.144,NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W010.145, 0.146, 0.147, 0.148, 0.149, 0.150, 0.151, 0.152, 0.153, 0.154, 0.155, 0.156, 0.157, 0.158, 0.159, 0.160, 0.161, 0.162, 0.163, 0.164, 0.165, 0.166, 0.167, 0.168, 0.169, 0.170, 0.171, 0.172, 0.173, 0.174, 0.175, 0.176, 0.177, 0.178, 0.179, 0.180, 0.181, 0.182, 0.183, 0.184, 0.185. 0.186, 0.187, 0.188, 0.189, 0.190, 0.191, 0.192, 0.193, 0.194, 0.195, 0.196, 0.197, 0.198, 0.199, or 0.200.

[0251] 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”).

[0252] 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 50 to about 100 nm. In some embodiments, the LNPs have a size of about 50 to about 120 nm. In some embodiments, the LNP has a particle hydrodynamic size Z-average of 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, or about 60 nm. In some embodiments, the LNP has a particle hydrodynamic size Z-average of 60-80 nm or 80-100 nm. In some embodiments, the LNPs have a size of about 60 to about 100 nm. In some embodiments, the LNP has a particle hydrodynamic size Z-average of 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nm.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0253] 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 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 aspects, the LNP has a particle hydrodynamic size Z-average of 80-100 nm. 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.

[0254] In some embodiments, the LNP compositions are formed with an average encapsulation efficiency ranging from about 50% to about 100%, about 50% to 99%, or about 60% to 98%. 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%.

[0255] In certain aspects, compositions comprising the LNPs according to any of the aspects described herein comprises a buffer, an excipient, a pharmaceutically acceptable carrier, or aNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01combination 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).

[0256] In some aspects, the LNP composition is a pharmaceutical composition for delivery to a patient in need thereof. 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).

[0257] 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).

[0258] Embodiments of the present disclosure also provide lipid compositions described according to the molar ratio between the positively charged amine groups of the ionizable lipid (N) and the negatively charged phosphate groups (P) of the nucleic acid to be encapsulated. This may be mathematically represented by the equation N / P. In some embodiments, a LNP may comprise a lipid component that comprises an ionizable lipid, a helper lipid, a neutral lipid, and a PEG-lipid (i.e., a structural and / or PEG lipid); and a nucleic acid component, wherein the N / P ratio is about 3 to 10. For example, the N / P ratio may be about 4-7, about 5-7, or about 6 to 7. In some embodiments, the N / P ratio may about 6, e.g., 6 ±1, or 6 ± 0.5. In some embodiments, the N / P ratio may about 7, e.g., 7 ±1, or 7 ± 0.5. In some aspects, the LNP has a nitrogen to phosphorus ratio (N: P) ratio of about 3: 1 to about 9:1, about 4:1 to about 7:1, or about 6:1 to about 8:1.Cellular Engineering and Genomic Editing

[0259] Provided herein is a method for genetically engineering a cell comprising contacting the cell with the LNP of any one or combination of aspects disclosed herein or the composition of any one or combination of aspects disclosed herein and introducing a singleNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01stranded DNA nick or introducing a double-stranded DNA break. In some aspects, the genetic engineering comprises introducing an insertion or deletion into a genome (indel formation). For example, introducing an indel may comprise introducing a frameshift mutation, a change to an encoded amino acid (missense mutation), or introducing an a premature stop codon. In some aspects, indel formation leads to a change in gene expression. For example, indel formation may result in a gene expression that is reduced by 20 to 100-fold, 25 to 75-fold, or 30 to 50-fold compared to a control (for example buffer control).

[0260] In some aspects, the genetic engineering comprises inserting a nucleic acid of interest into the genome of the cell by non-homologous end joining (NHEJ) repair or homology directed (HD) repair in the cell.

[0261] In some aspects, the method for genetically engineering a cell comprises contacting the cell with the LNP of any one or combination of aspects disclosed herein, and introducing a single stranded DNA nick. In some aspects, the genetic engineering comprises inserting the template sequence into the genome of the cell by template-directed genomic editing, for example using a SpyCas9 nickase polypeptide that is operably linked to a DNA-dependent DNA polymerase polypeptide and a template guide RNA according to any one or combination of aspects disclosed herein.

[0262] In some aspects, the genetic engineering comprises a DNA substitution or a deletion. In some aspects, the genetic engineering comprises an insertion of a nucleotide in the genomic sequence. In some aspects, the genetic engineering comprises a change in an amino acid sequence encoded by a locus in which the genomic sequence is present. In some aspects, the genetic engineering comprises the genetic engineering does not result in a change in an amino acid sequence encoded by a locus in which the genomic sequence is present.Methods of Administration

[0263] The LNP or targeted LNP (t-LNP) compositions disclosed herein may be used for gene editing ex vivo, in vivo, and in vitro. According to any of the foregoing methods for genetically engineering a cell, the contacting is in vitro, in vivo, or ex vivo. Methods of administering LNP-RNPs in vivo to a target tissue or cell according to aspects disclosed herein, may avoid degradation of the encapsulated RNP in a subject, while allowing an RNP to efficiently enter a target cell.

[0264] In one embodiment, one or more LNP compositions described herein may be administered to a subject in need thereof. In one embodiment, one or more LNP compositionsNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01described herein may contact a cell. 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. In various embodiments, the methods comprise introducing a template nucleic acid to a cell or subject, as set forth above. In some embodiments, the cell is in vivo.

[0265] In some aspects, the LNP or LNP compositions are administered at a concentration of about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 5 mg / kg, or about 0.3 mg / kg to about 3 mg / kg to a subject. In some aspects, the LNP or LNP compositions are administered systemically, parenterally, or intratumorally to a subject. The LNP compositions can be administered parenterally. The LNP compositions may be administered directly into the blood stream, into tissue, into muscle, or into an internal organ. Administration may be systemic, e.g., to injection or infusion. Administration may be local. Suitable means for administration include intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, subretinal, intravitreal, intra-anterior chamber, intramuscular, intrasynovial, intradermal, and subcutaneous. Suitable devices for administration include needle (including microneedle) injectors, needle-free injectors, osmotic pumps, and infusion techniques. In some aspects, the LNP or LNP compositions are administered via injection or infusion to a subject. In some aspects, the LNP or LNP compositions are administered in an amount sufficient to obtain at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% gene editing, for example indel formation compared to control (for example a buffer control). In some aspects, the genetic engineering comprises introducing a gene modification.

[0266] In some aspects, the gene modification cuts, edits, blocks, marks or labels the gene. In some aspects, the gene modification inserts, deletes, or substitutes a base in the gene. In some aspects, the gene modification comprises an insertion or deletion of more than one base in the gene.

[0267] In some aspects, the method comprises altering expression of a gene, altering a gene, or deleting a gene in a target cell, e.g., a liver cell, a bone marrow cell, an immune cell, a tumor cell, or a cell associated with a disease or disorder.

[0268] In some aspects, the genetic engineering targets a transthyretin (TTR) gene. In some aspects, serum TTR is reduced by 20 to 100-fold, 25 to 75-fold, or 30 to 50-fold.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0269] In some aspects, the method comprises administering a first lipid nanoparticle (LNP) composition and a second LNP composition. In some aspects, the first LNP composition and the second LNP composition comprise different cargo. In some aspects, the first and second LNP compositions are administered simultaneously. In some aspects, the first and second LNP compositions are administered sequentially.

[0270] Provide herein is a container, vial, syringe, injector pen, or kit comprising at least one dose of the LNP of any one or combination of aspects disclosed herein or the composition of any one or combination of aspects disclosed herein.Cell Types

[0271] The LNPs according to the aspects described herein are suitable to deliver cargo to various cell types. The LNPs may target a specific cell type, for example using a targeting moiety, or the LNPs may not include a targeting moiety. In other aspects, the lipid component formulation may provide improved delivery to a certain cell or tissue with or without an anchor lipid conjugated to a targeting moiety. In some embodiments, the target tissue is the liver, or the target cell is a liver cell (hepatocyte).

[0272] In some aspects, the target tissue is bone marrow or the target cell is a bone marrow cell or bone marrow derived cell. For example, the cell may be a Hematopoietic Stem Cell (HSC), Multipotent Progenitor Cell (MPP), Common Myeloid Progenitor (CMP), Common Lymphoid Progenitor (CLP), 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 bone marrow cell may include the following markers: CD34, CD38, CD45, GDI 17, CD123, or CD135.

[0273] In some embodiments, the cell is 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, 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). In some embodiments, the cell is a primary immune cell. In some embodiments, the immune systemNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01cell may be selected from CD3+, CD4+ and CD8+ T cells, regulatory T cells (Tregs), B cells, NK cells, and dendritic cells (DC). In some embodiments, the immune cell is allogeneic. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is an adaptive immune cell. In some embodiments, the cell is a T cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is a NK cell. In some aspects, the T cell may be a CD2+ cell, CD3+ cell, CD4+ cell, CD5+ cell, CD6+ cell, CD7+ cell, CD8+ cell, or a CD45+ cell.

[0274] 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. In some aspects, the method comprises contacting the target cell with an LNP and / or t-LNP carrying the cargo.

[0275] In some aspects, the method comprises contacting the target cell with an LNP or LNP composition carrying a first cargo, e.g., a first RNP, and an LNP or LNP composition carrying a second cargo, e.g., a RNP, 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.

[0276] Exemplary cells for genetic engineering or 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 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. Nonlimiting examples of mammalian cells include a rodent cell (e.g., a mouse cell, a rat cell, aNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01hamster 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.

[0277] 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. In some aspects, the cell is 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), 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.Methods of preparing RNP-LNPs

[0278] Provided herein are methods of preparing a lipid nanoparticle (LNP) encapsulating a cargo comprising a ribonucleoprotein (RNP). In some embodiments, the RNP contains a Cas protein and a sgRNA. The lipid nanoparticle (LNP) encapsulating a cargo comprising a ribonucleoprotein complex (RNP) according to any of the aspects disclosed herein may be prepared by mixing a preformulation buffer solution containing an RNP cargo and potassium chloride or sodium chloride with an ethanol solution containing a lipid component mixture (e.g., a four or five lipid component formulation as described above). As described herein, methods that combine a preformulation buffer and preparation conditions for an LNP lipid component including an ionizable lipid with a pKa at or below 7.0 result in LNPs comprising an RNP cargo with optimal size and increased encapsulation efficiency (e.g., percent encapsulation, %E) compared to prior art methods. Specifically, methods of preparing a LNP as described herein can achieve encapsulation efficiency of more than 50%, particle size of about 80- 120 nm, PDI less than 0.1, free-thaw stability, or a combination thereof.

[0279] As described below, each of the first to fourth aspects for preparing an LNP encapsulating a cargo comprising an RNP may begin by preparing an RNP solution by first denaturing gRNA (for example using a heat denaturation step by heating the gRNA 95 °C forNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W011 minutes). The gRNA is then cooled and diluted in an aqueous solution (for example water) followed by mixing the aqueous solution of gRNA with a buffered solution of Cas nuclease polypeptide.

[0280] In a first aspect, a method of preparing an LNP encapsulating a cargo comprising an RNP may include mixing an RNP (e.g., Cas9-gRNA) in a solution to prepare an RNP solution in a preformulation buffer for example mixing gRNA and Cas nuclease protein in a molar ratio of gRNA: Cas nuclease protein of about 1:1 to about 16:1. The preformulation buffer may be prepared with a 2-(N-morpholino)ethanesulfonic acid (MBS) at a concentration of about 25 mM to about 100 mM, preferably at about 50 mM MES, at a pH of about 5.5 to about 7, preferably a pH of about 6.0, and a KC1 concentration of about 20 mM to 25 mM, preferably about 21 mM. The RNP solution may have a concentration of RNP cargo of about 0.025 mg / ml to about 0.5 mg / ml, about 0.1 mg / ml to about 0.5 mg / ml, or preferably about 0.2 mg / mL. The lipid component formulation may be prepared in an ethanol solution prior to mixing with the RNP solution in the preformulation buffer. In some embodiments, a method of preparing an RNP-LNP may include a first mixing step comprising mixing two volumes of the RNP solution in the preformulation buffer with one volume of the lipid component formulation in ethanol, followed by a second mixing step comprising mixing the RNP-LNP solution with a solution of MES buffer. The combined LNP-RNP solution after the second mixing step may be incubated, for example incubating for one hour at room temperature (e.g., 20 to 25 °C). Subsequently, a third mixing step may be conducted comprising mixing the combined LNP-RNP solution with water at a ratio of water to RNP-LNP solution of 1:1 v / v (See, e.g., W02016010840, Fig. 2).

[0281] In a second aspect, a method of preparing an LNP encapsulating a cargo comprising an RNP may include mixing an RNP (e.g., Cas9-gRNA) in a solution to prepare an RNP solution in a preformulation buffer for example mixing gRNA and Cas nuclease protein in a molar ratio of gRNA: Cas nuclease protein of about 1:1 to about 16:1. The preformulation buffer may be prepared with a bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (BisTris) at a concentration of about 25 mM to about 100 mM, preferably at about 50 mM BisTris, at a pH of about 5.5 to about 7, preferably a pH of about 6.0, and a KC1 concentration of about 20 mM to 25 mM, preferably about 21 mM. The RNP solution may have a concentration of RNP cargo of about 0.025 mg / ml to about 0.5 mg / ml, about 0.1 mg / ml to about 0.5 mg / ml, or preferably about 0.2 mg / mL. The lipid component formulation may be prepared in an ethanol solution prior to mixing with the RNP solution in the preformulationNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01buffer. In some embodiments, a method of preparing an RNP-LNP may include a first mixing step comprising mixing two volumes of the RNP solution in the preformulation buffer with one volume of the lipid component formulation in ethanol, followed by a second mixing step comprising mixing the RNP-LNP solution with a solution of Bis-Tris buffer. The combined LNP-RNP solution after the second mixing step may be incubated, for example incubating for one hour at room temperature (e.g., 20 to 25 °C). Subsequently, a third mixing step may be conducted comprising mixing the combined LNP-RNP solution with water at a ratio of water to RNP-LNP solution of 1:1 v / v (See, e.g., W02016010840, Fig. 2).

[0282] In a third aspect, a method of preparing an LNP encapsulating a cargo comprising an RNP may include mixing an RNP (e.g., Cas9-gRNA) in a solution to prepare an RNP solution in a preformulation buffer. The preformulation buffer may be prepared, as described above, with a 2-(N-morpholino)ethanesulfonic acid (MES) at a concentration of about 25 mM to about 100 mM, preferably at about 50 mM MES, at a pH of about 5.5 to about 7, preferably a pH of about 6.0, and a KC1 concentration of about 20 mM to 25 mM, preferably about 21 mM. The preformulation is then supplemented with KC1, for example supplementing the preformulation buffer with about 30 to about 35 mM KC1, preferably about 33 mM KC1, to reach final a KC1 concentration in the preformulation buffer of about 45 mM to 55 mM, preferably about 50 mM. The RNP solution may have a concentration of RNP cargo of about 0.025 mg / ml to about 0.5 mg / ml, about 0.1 mg / ml to about 0.5 mg / ml, or preferably about 0.2 mg / mL. The lipid component formulation may be prepared in an ethanol solution prior to mixing with the RNP solution in the preformulation buffer. The method of preparing an RNP-LNP may include a first mixing step comprising mixing two volumes of the RNP solution in the preformulation buffer with one volume of the lipid component formulation in ethanol, followed by a second mixing step comprising mixing the RNP-LNP solution with a solution of MES buffer. The combined LNP-RNP solution after the second mixing step may be incubated, for example incubating for one hour at room temperature (e.g., 20 to 25 °C). Subsequently, a third mixing step may be conducted comprising mixing the combined LNP-RNP solution with water at a ratio of water to RNP-LNP solution of 1: 1 v / v (See, e.g., WO2016010840, Fig. 2).

[0283] In a fourth aspect, a method of preparing an LNP encapsulating a cargo comprising an RNP may include mixing an RNP (e.g., Cas9-gRNA) in a solution to prepare an RNP solution in a preformulation buffer. The preformulation buffer may be prepared, as described above, with a 2-(N-morpholino)ethanesulfonic acid (MES) at a concentration of about 25NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01mM to about 100 mM, preferably at about 50 mM MES, at a pH of about 5.5 to about 7, preferably a pH of about 6.0, and a KC1 concentration of about 20 mM to 25 mM, preferably about 21 mM. The preformulation may be supplemented with KC1, for example supplementing the preformulation buffer with about 25 mM to about 40 mM KC1, or about 33 mM KC1, to reach final a KC1 concentration in the preformulation buffer of about 45 mM to 55 mM, preferably about 50 mM. The RNP solution may have a concentration of RNP cargo of about 0.025 mg / ml to about 0.5 mg / ml, about 0.1 mg / ml to about 0.5 mg / ml, or preferably about 0.2 mg / mL. The lipid component formulation may be prepared in an ethanol solution prior to mixing with the RNP solution in the preformulation buffer. The method of preparing an RNP-LNP may include a first mixing step comprising mixing two volumes of the RNP solution in the preformulation buffer with one volume of the lipid component formulation in ethanol, followed by a second mixing step comprising mixing the RNP-LNP solution with a solution of MES buffer. The combined LNP-RNP solution after the second mixing step may be incubated, for example incubating for one hour at room temperature (e.g., 20 to 25 °C). Subsequently, a third mixing step may be conducted comprising mixing the combined LNP-RNP solution with a MES / KC1 buffer (for example 30-40 mM MES, preferably about 35.6 mM MES and 20 mM to 30 mM KC1, preferably about 25 mM KC1) at a ratio of water to RNP-LNP solution of 1:1 v / v (See, e.g., W02016010840, Fig. 2).

[0284] Mixing, as described above, may be conducted using an impinging jet mixing of the solutions. For example, formation of the LNP-RNP solution may comprise mixing through a mixing cross a lipid component formulation dissolved in ethanol with two volumes of the foregoing RNP solution, as described above.

[0285] The LNP-RNP solutions may be diluted by undergoing a step of buffer exchanging the solution into a formulation buffer. For example, a formulation buffer may comprise 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 RNA-LNP or RNP-LNP mixtures may be then filtered using a 0.2 pm sterile filter and optionally stored at 4 °C or -80 °C until further use.

[0286] In some aspects, provided herein is a method of preparing a lipid nanoparticle (LNP) encapsulating a cargo comprising a ribonucleoprotein (RNP), wherein the method comprises:(i) mixing a first solution comprising a RNP in a preformulation buffer, and a second solution comprising a lipid component dissolved in ethanol, to prepare an RNP-LNP solution;(ii) mixing the RNP-LNP solution downstream with a third solution; andNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01(iii) diluting the solution in (ii) with a fourth solution to prepare a diluted RNP- LNP solution;wherein the preformulation buffer has a pH about 5 to about 7 when measured at 25 °C and wherein the lipid component comprises an ionizable lipid having a pKa at or below about 7.0.

[0287] In some aspects, the ionizable lipid has a pKa of about 5.0 to about 7.0, about 6.0 to about 6.9, about 6.1 to about 6.8, about 6.2 to about 6.7, or about 6.5. In some aspects, the ionizable lipid has a pKa of about 6.0 to about 7.0. In some aspects, the ionizable lipid has a pKa of about 0.5 units above the preformulation buffer pH.

[0288] In some aspects, provided herein is a method of preparing a lipid nanoparticle (LNP) encapsulating a cargo comprising a ribonucleoprotein (RNP), wherein the method comprises: (i) mixing a first solution comprising a RNP in a preformulation buffer, and a second solution comprising a lipid component dissolved in ethanol, to prepare an RNP-LNP solution;(ii) mixing the RNP-LNP solution downstream with a third solution; and(iii) diluting the solution in (ii) with a fourth solution to prepare a diluted RNP-LNP solution; wherein the preformulation buffer has a pH about 5.5 to about 7.0 when measured at 25 °C and wherein the lipid component comprises an ionizable lipid having a pKa of about 6.0 to about 7.5.

[0289] In some aspects, provided herein is a method of preparing a lipid nanoparticle (LNP) encapsulating a cargo comprising a ribonucleoprotein (RNP), wherein the method comprises: (i) mixing a first solution comprising a RNP in a preformulation buffer, and a second solution comprising a lipid component dissolved in ethanol, to prepare an RNP-LNP solution;(ii) mixing the RNP-LNP solution downstream with a third solution; and

[0290] diluting the solution in (ii) with a fourth solution to prepare a diluted RNP-LNP solution; wherein the preformulation buffer has a pKa of about 5.5 to about 7.0.

[0291] In some aspects, the ionizable lipid has a pKa of about 6.0 to about 7.0, about 6.0 to about 6.5, about 6.5 to about 7.0, about 7.0 to about 7.5. In some aspects, the ionizable lipid has apKa of about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5.

[0292] The lipid component may be a four-component lipid formulation, for example including an ionizable lipid, a neutral lipid, a helper lipid, and a structural PEG-lipid. InNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01some aspects, the four component lipid formulation may comprise the ionizable lipid, the helper lipid, the structural PEG-lipid, and an anchor PEG-lipid. The lipid component for preparing the LNP may be a five-component lipid formulation, for example including an ionizable lipid, a neutral lipid, a helper lipid, a structural PEG-lipid, and an anchor PEG-lipid.

[0293] In some aspects, the method further comprises incubating the RNP-LNP solution before step (iii) at room temperature for about one hour. In some aspects, the method further comprises a step (iv) of buffer exchanging the diluted RNP-LNP solution with a formulation buffer.

[0294] In some aspects, the preformulation buffer has a pH about 5 to about 7 when measured at 25 °C. In some aspects, the preformulation buffer has a pH of about 5.5 to about 6.5 when measured at 25 °C. In some aspects, the preformulation buffer has a pH of about 5.9 to about 6.3 when measured at 25 °C. In some aspects, the preformulation buffer has a pH of about 6.0 when measured at 25 °C. In some aspects, the preformulation buffer has a pKa of about 6.0 to about 7.0, about 6.0 to about 6.5, or about 6.5 to about 7.0 when measured at 25 °C. In some aspects, the preformulation buffer has a pKa of about 6.0 to about 6.5 when measured at 25 °C. In some aspects, the preformulation buffer has a pH of about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 when measured at 25 °C.

[0295] In some aspects, the preformulation buffer comprises a buffer agent at a concentration of 25 to 200 mM or 30 to 150 mM. In some aspects, the preformulation buffer comprises a buffer agent at a concentration of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55. 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 mM. In some aspects, the preformulation buffer comprises a buffer agent selected from an agent listed below, or a combination thereof.Buffer agent pKA2-(N-morpholino)ethanesulfonic acid (MES) 6.15N-(2-Acetamido)iminodiacetic acid (ADA) 6.6Piperazine-N, N'-bis(2-ethanesulfonic acid (PIPES) 6.76N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES) 6.9N, N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid 7.1(BES)3-(N-morpholino) propanesulfonic acid (MOPS) 7.2NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane 6.46(Bis-Tris)Sodium Phosphate 7.2Citrate (Avg. pKa: 4.6)(multiple pKa values (3.0-6.2)) Histidine 6.04Succinate 5.61

[0296] In some aspects, the preformulation buffer comprises potassium chloride (KCI) or sodium chloride (NaCl). In some aspects, the preformulation buffer comprises KCI. In some aspects, the preformulation buffer comprises a KCI or NaCl concentration of about 10 mM to about 100 ntM. about 12 mM to about 80 mM, about 15 mM to about 70 mM, about 18 mM to about 65 M. about 20 mM to about 55 M, In some aspects, the KCi or NaCl concentration is about 20 mM to about 25 mM, preferably about 21 mM, or about 45 mM to about 55 mM, preferably about 50 mM. In some aspects, the preform ulation buffer comprises about 5, 6, 7, 8. 9. 10, 11, 12, 13, 14. 15. 16, 17, 18, 19, 20. 21, 22, 23, 24, 25. 26, 27, 28, 29, 30. 31, 32, 33, 34, 35. 36. 37, 38, 39, 40. 41, 42, 43, 44, 45. 46. 47, 48, 49, 50, 51. 52. 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65. 66. 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100 mM: KCI or NaCl. In some aspects, the medium containing the LNP does not contain stabilizing counterions. In some aspects, the medium containing the LNP or the LNP composition is substantially free of negative counter-ions (i.e., anions). For example, the presence of negative counter-ions in an LNP formulation at least partially neutralizes the positively charged surface of the LNPs, thereby eliminating the aggregation reducing effect of charge repulsion.

[0297] In some aspects, the first solution comprising the RNP in the preformulation buffer is mixed with the second solution comprising the lipid component dissolved in ethanol at a ratio of about 4:1 to about 1:1, about 3:1 to about 1:1, or about 2:1 (v / v).

[0298] In some aspects, the third solution comprises histidine, BES, MOPS, succinate, MES, Bis-Tris, ADA, ACES, PIPES, citrate acetate, sodium citrate, phosphate buffer, or a combination thereof. In some aspects, the third solution comprises the same composition as the preformulation buffer. In some aspects, the third solution comprises a different composition from the preformulation buffer. For example, the preformulation buffer comprises Bis-Tris at a concentration of 25 to 200 mM, 30 to 150 mM, 40 mM to 60 mM, orNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01about 50 mM. For example, the preformulation buffer comprises Bis-Tris at a concentration of 50 mM.

[0299] In some aspects, the formulation buffer has a pH of about 5 to about 7, about 5.5 to about 6.5, or about 6.0. For example, the preformulation buffer comprises Bis-Tris at a pH of about 5.5. For example, the preformulation buffer comprises Bis-Tris at a pH of about 6.0. For example, the preformulation buffer comprises Bis-Tris at a pH of about 6.5. For example, the preformulation buffer comprises Bis-Tris at a pH of about 7.0.

[0300] In some aspects, the first solution and second solution are mixed perpendicular in a mixing cross. In some aspects, the solution in (ii) is mixed with a fourth solution at a ratio of about 4:1 to about 1:1, about 3:1 to about 1:1, about 2:1 to about 1:1, or about 1:1 (v / v). In some aspects, the fourth solution comprises water. In some aspects, the fourth solution comprises KC1 at a concentration of about 10 mM to about 100 mM, about 12 mM to about 80 mM, about 15 mM to about 70 mM, about 18 rnM to about 65 M, about 20 mM to about 55 mM. In some aspects, the fourth solution comprises KC1 at a concentration of about 20 mM to about 30 mM KC1, or about 25 mM KC1. In some aspects, the fourth solution comprises KC1 at a concentration of about 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 mM.

[0301] In some aspects, the fourth solution comprises a buffer agent at a concentration of about 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 rnM. In some aspects, the fourth solution comprises a buffer agent at a concentration of about 30 mM to about 40 mM, or about 35.6 mM. In some aspects, the fourth solution comprises histidine, BES, MOPS, succinate, MES, Bis-Tris, ADA, ACES, PIPES, citrate acetate, sodium citrate, phosphate buffer, or a combination thereof.

[0302] In some aspects, the formulation buffer comprises a buffer concentration of about 20 mM to about 100 mM, about 25 mM to about 75 mM, about 30 mM to 70 mM, about 35 mM to 65 mM, about 40 mM to 60 mM, about 43 mM to 57 mM, about 45 mM to 55 mM, about 48 mM to 52 mM, or about 50 mM.

[0303] In some aspects, the formulation buffer comprises one or more excipients. The excipients may be a pharmaceutically acceptable excipient, for example: fillers, diluents, binders, stabilizers, lubricants, disintegrants, wetting / solubilizing / emulsifying agents, or combinations thereof. Pharmaceutically acceptable excipients are well known in the art andNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01are described in Remington, the Science and Practice of Pharmacy, 21 (2006), pages 1058-1092 and Handbook of Pharmaceutical Excipients, 6 (2009). In certain aspects, the one or more excipients are selected from a salt, a sugar, or a combination thereof.

[0304] The salt may be for sodium chloride. In some aspects, the sodium chloride is at a concentration of about 10 mM to about 150 mM, about 15 mM to about 100 mM, about 20 mM to about 75 mM, about 30 mM to about 50 mM, or about 45 mM.

[0305] In some aspects, the sugar is at a concentration of about 1% to about 20%, about 2% to about 15%, about 3% to about 10%, or about 5% (w / v). The sugar may be sucrose.

[0306] In some aspects, a ratio of gRNA: Cas nuclease protein is in a range of about 1: 1 to about 16:1. In some aspects, the gRNA: Cas nuclease protein molar ratio is about 1.1:1 to about 12:1, about 1.2:1 to about 10:1, about 1.4:1 to about 8:1, about 1.6:1 to about 6:1, about 1.8:1 to about 5:1, or about 2:1 to 4:1. In some aspects, the gRNA: Cas nuclease protein ratio is about 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, or 12: 1. In certain aspects, the gRNA: Cas nuclease protein ratio is about 2:1.

[0307] In some aspects, at least 70% of the cargo that was present in the RNP solution prior to mixing with the lipid component in ethanol is encapsulated in the LNP. In some aspects, about 75% to about 99%, about 80% to 95% or about 90% of the cargo that was present in the preformulation buffer is encapsulated in the LNP. In some aspects, about 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100% of the cargo that was present in the preformulation buffer is encapsulated in the LNP.

[0308] 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.

[0309] 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 specification controls. All ranges given in the application encompass the endpoints unless stated otherwise.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01EXAMPLES

[0310] 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

[0311] 1.1 Preparation of cargo to be encapsulated in LNPs

[0312] 1.1.a. In vitro transcription (" IVT") of nuclease mRNA and synthesis of guide RNAs

[0313] Capped and poly adenylated mRNA containing N1 -methyl pseudo-U was generated by in vitro transcription using a linearized plasmid DNA template and 17 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.

[0314] Messenger RNA was generated from plasmid DNA encoding an open reading frame (“ORF”) according to ORF sequences as shown in the Table 30 below. When 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.

[0315] Guide RNAs (gRNAs) were chemically synthesized by commercial vendors or using standard in vitro synthesis techniques with modified nucleotides, unless otherwise indicated.

[0316] 1.1. b. Cas9-gRNA ribonucleoprotein (RNP) cargo preparation.

[0317] Cas9-gRNA RNP cargos were prepared as follows, using a Cas9 polypeptide (e.g., SpyCas9) and a chemically modified gRNA targeting a genomic sequence. First, the gRNA was denatured at 95 °C for 2 minutes, followed by cooling on ice for 10 minutes. The gRNA was then diluted to a working solution of 3 mg / mL in deionized water. The Cas9 polypeptide was diluted to a working solution of 5 mg / mL in a protein buffer composed of 20 mM HEPES, 300 mM KC1, 5% glycerol, 1 mM MgCl, and 1 mM dithiothreitol (DTT). The Cas9 polypeptide solution and the gRNA solution were mixed at a gRNA: Cas9 polypeptide molar ratio of 2:1, unless otherwise indicated. The mixture was stored at room temperature for 10 minutes to one hour. Finally, the mixture was diluted in preformulation buffer (e.g., 50 mM MES buffer) prior to formulation in the LNPs.

[0318] 1.2 Preparation of LNPsNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0319] LNPs were prepared as a 4-component or 5-component formulation. The 4-component LNP formulations comprised (i) an ionizable lipid (e.g., Compounds 1-9) and / or a cationic lipid (e.g., Compound 17), (ii) di stearoylphosphatidylcholine (DSPC), (iii) cholesterol, and (iv) a structural PEG-lipid (e.g., Compounds 10-16). The 5-component LNP formulations comprised (i) an ionizable lipid (e.g., Compounds 1-9) or a cationic lipid (e.g.. Compound 17), (ii) DSPC, (iii) cholesterol, (iv) a structural PEG-lipid (e.g., Compounds 10-16), and (v) an anchor PEG-lipid (e.g., a SpyTag-functionalized DSPE-PEG-lipid); 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 2 shows a list of compounds used for the formulation of 4-component and 5-component LNPs. 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 4, below).

[0320] In assays for in vitro editing in cell cultures or in vivo editing in mice, a cargo was encapsulated in the LNP.

[0321] In some cases, “RNA-LNPs” were prepared as a standard control, in which the cargo encapsulated in the LNP comprised a mRNA encoding a Cas9 protein and a chemically modified gRNA targeting a genomic sequence. In some cases, RNA-LNPs were prepared with a single RNA species such as a mRNA or a gRNA. In some cases, RNA-LNPs were prepared with a mixture of mRNA and a guide RNA, at a 1:2 w / w gRNA: Cas9 mRNA ratio, unless otherwise indicated. The RNA-LNPs were formulated with a lipid amine to RNA phosphate (N: P) molar ratio of about 3 to 9.

[0322] In other cases, “RNP-LNPs” were prepared, in which the cargo encapsulated in the LNP comprised a Cas9-gRNA RNP complex prepared as described in Section 1.1,b of this Example 1. Cas9-gRNA RNP cargos were prepared with a gRNA: Cas9 polypeptide molar ratio of 2:1, unless otherwise indicated. The RNP-LNPs were formulated with an ionizable lipid: Cas9 polypeptide molar ratio of 3,800, unless otherwise indicated.

[0323] RNA-LNPs and RNP-LNPs were produced as follows. In general, the LNP lipid components were dissolved in 100% ethanol.

[0324] To produce RNA-LNPs, the RNA cargos (e.g., Cas9 mRNA and gRNA) were dissolved in 25 mM citrate, 100 rnM NaCl, pH 5.0, resulting in a RNA solution with a concentration of RNA cargo of approximately 0.45 rng / mL, unless otherwise indicated. TheNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01RNA-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., W02016010840, Fig. 2). The LNPs were held for 1 hour at room temperature, and further diluted with water (approximately 1:1 v / v).

[0325] To produce RNP-LNPs, the RNP cargo (e.g., Cas9-gRNA) was dissolved in 50 mM 2-(N-morpholino)ethanesulfonic acid (MES buffer). pH 6.0, resulting in an RNP solution with a concentration of RNP cargo of approximately 0.5 mg / mL, unless otherwise indicated, and a final KC1 concentration of 21 mM. In some cases, the MES buffer was supplemented with 33 mM KC1, resulting in a final KC1 concentration of 50 mM. The RNP-LNPs were formed by an impinging jet mixing of the lipid in ethanol with two volumes of RNP solution and one volume of water. First, the lipid in ethanol was mixed through a mixing cross with the two volumes of RNP solution. Then, a fourth stream of MES was mixed with the outlet stream of the cross through an inline tee. The LNPs were held for 1 hour at room temperature, and further diluted with water (approximately 1:1 v / v) (See, e.g., W02016010840, Fig. 2), or with 35.6 mM MES buffer, 25 mM KC1 (approximately 1:1 v / v).

[0326] 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 RNA-LNP or RNP-LNP mixtures were then filtered using a 0.2 pm sterile filter. The final LNPs were characterized as described below and were stored at 4 °C or -80 °C until further use.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0327] Table 2. Structures of compounds used for the preparation of 4-component or 5-component LNP formulations.Compound Lipid Type Description StructureDSPC helper lipidCholesterol sterol lipidi fhI / 'X T1"1J HCompound 1 ionizable lipid Lipid A 0%Compound 2 ionizable lipid Lipid CH 1 o 11 — "" — — "" — % O\°= / o o / o z —_NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01Compound Lipid Type Description Structure Compound 3 ionizable lipidoOH 1 K% 0"Compound 4 ionizable lipid o1< Y oCompound 5 ionizable lipid0HZL_oCompound 6 ionizable lipidNN- 0v °%NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01Compound Lipid Type Description StructureCompound 7 ionizable lipid Lipid ECompound 8 ionizable lipid Lipid BCompound 9 ionizable lipid / V 9\ N. •hl. Y. -0- 1 v.. v n -O-.1 V.., 'o oCompound 10 structural PEG- C14DMG olipid PEG |1 J45 oCompound 11 structural PEG- Cl 3 Ether G' z —lipid PEGIZ°> >°°o / — \) V / o° ° \ \° )^ / / °= / Z) Oo= / / / oI o)°^ — \ / ) ° — \ ° / \ / ozz° 1 \ / z O cn / NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01Compound Lipid Type Description StructureCompound 12 structural PEG- C12 Ether o'o' y O' 'N' 'O\v-' o\ fa. lipid PEGCompound 13 structural PEG- C 14 Etherlipid PEGCompound 14 structural PEG- Cl 4 Amide 0o\ lipid PEG HJLoCompound 15 structural PEG- Cl 5 Amide 0.0? lipid PEG HN.oCompound 16 slruclural PEG- DSPE-PEG 9 II0ii n0Y lipid ^X X X H O- H I0%Compound 17 cationic lipid DOTAP 0XxX''Xxx''XxX''X^X'=XxxX'x^Xx^ / ^^x-'Yx-''XYXxx / xx==^^ H1 cr0o1NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0328] 1.3 LNP Composition Analytics

[0329] 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.

[0330] 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.

[0331] 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 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).

[0332] Nanoparticle tracking analysis (NT A, 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.

[0333] Cryo-electron microscopy (“cryo-EM”) can be used to determine the particle size, morphology, and structural characteristics of an LNP.

[0334] 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.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0335] 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” or “Z-ave”) 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 pL into 800 pL) in 0.1X PBS, pH 7.4 prior to measurement.

[0336] Encapsulation efficiency (“%E”) was calculated as the percentage of (Total RNA -Free RNA) / Total RNA. In the case of RNA-LNPs, Free RNA corresponds to RNA (e.g., gRNA or mRNA cargo) that is not encapsulated in the LNP lipid components. In the case of RNP-LNPs, the gRNA from the RNP cargo, whether bound or not bound to the Cas9 polypeptide, counts towards the Total RNA; and the Free RNA corresponds to gRNA, whether bound or not bound to the Cas9 polypeptide, that is not encapsulated in the LNP lipid components. A fluorescence-based assay (Ribogreen®, ThermoFisher Scientific) was used to determine total RNA concentration and free RNA concentration. LNP samples were diluted appropriately with lx TE buffer containing 0.2% Triton-X 100 to determine total RNA or lx TE buffer to determine free RNA. Standard curves were prepared by utilizing the starting RNA solution (for RNA-LNPs) or RNP solutions prepared at the appropriate gRNA: Cas9 polypeptide ratio (for RNP-LNPs) used to make the compositions and diluted in lx 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 componentsNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01chromatographically by RP-IP HPLC and quantified against a standard curve using UV absorbance at 260 nm.

[0337] 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 doublestranded DNA.

[0338] 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.

[0339] 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.

[0340] 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 pM. 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.

[0341] Table 3. pKa values of exemplary ionizable lipids.Compound # Lipid pKaNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01Compound 1 6.1Compound 2 6.4Compound 3 6.9Compound 4 6.1Compound 5 6.2Compound 6 6.9Compound 7 6.0Compound 8 6.6Compound 9 7.0

[0342] 1.4 Formulation Stability Assessment

[0343] 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 (postfreeze-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.

[0344] 1.5 LNP Delivery In Vivo

[0345] Female Sprague Dawley rats, or female C57B1 / 6J mice, ranging from 6-15 weeks of age, were used in each study. 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 LNP cargo (e.g., RNA or RNP cargo), unless otherwise indicated. The animals were periodically observed for adverse effects for at least 24 hours post dose.

[0346] Animals were euthanized by carbon dioxide asphyxiation, at days 4, 5, 6 or 7 after dosing for editing readout. Liver tissue, blood, and bones were collected. 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. For gene editing readout, genomic DNA was isolated from the liver tissue, the whole boneNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01marrow (WBM), and HSPC populations. Editing was measured using Next-Generation Sequencing (NGS).

[0347] 1.6 Flow Cytometry

[0348] 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-l+ / c-KIT+ cells (LSKs), lineage depleted cells), according to methods known in the art. 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.

[0349] For gene editing analysis, the antibody mix was washed off after incubation, and the cells were run through a BD FACS Aria™ Fusion Flow Cytometer one sample at a time. Sorted material for the populations of interest was collected individually to be processed for NGS Sequencing.

[0350] 1.7 Preparation of Cells for In Vitro Experiments

[0351] Primary rat hepatocytes (e.g., In Vitro ADMET Laboratories, Lot# 031-220), or primary mouse hepatocytes (e.g., Gibco, Lot MC980) were thawed and resuspended in Cryopreserved Hepatocyte Recovery Medium (Invitrogen, Cat. CM7000) followed by centrifugation. The supernatant was discarded, and the pelleted cells were resuspended in hepatocyte plating medium (Gibco Williams’ Medium E, Cat. A12176-01) plus supplement pack (Gibco, Cat. A15563 CM3000) and 5% FBS (Gibco, A13450). Cells were counted and plated on Bio-coat collagen I coated 96-well plates (e.g., Coming, Cat. 354407) at a density of 20,000 cells / well, unless otherwise indicated. Plated cells were allowed to settle and adhere for 4-6 hours in a tissue culture incubator at 37 °C and 5% CO2 atmosphere. After incubation, cells were checked for monolayer formation and washed once with hepatocyte maintenance medium (Gibco Williams’ Medium E, Cat. A12176-01) plus cell maintenance supplements (Gibco, Cat. A13448) and 3% FBS.

[0352] 1.8 Next-Generation Sequencing (NGS) Analysis

[0353] 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.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0354] 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 versus the number of reads which contain an insertion, substitution, or deletion was calculated.

[0355] 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.

[0356] 1.9. Mouse Serum TTR Level Analysis

[0357] Serum was isolated from blood using methods known in the art. The total TTR serum levels were determined using a Mouse Prealbumin (TTR) ELISA Kit (Aviva Systems Biology, OKIAOOl 11). Kit reagents and standards were prepared according to the manufacturer's protocol. Mouse serum was diluted between 2,500 to 10,000-fold. Both standard curve dilutions (100 L each) and diluted serum samples were added to each well of the ELISA plate pre-coated with capture antibody. The plate was incubated at room temperature for 30 minutes before washing. Enzyme- antibody conjugate (100 pL per well) was added for a 20-minute incubation. Unbound antibody conjugate was removed, and the plate was washed again before the addition of the chromogenic substrate solution. The plate was incubated for 10 minutes before adding 100 pL of the stop solution, e.g., sulfuric acid (approximately 0.3 M). The plate was read on a SpectraMax M5 or Clariostar plate reader at an absorbance of 450 nm. Serum TTR levels were calculated by SoftMax Pro software ver.6.4.2 or Mars software ver. 3.31 using a four-parameter logistic curve fit off the standard curve. Final serum values were adjusted for the assay dilution.

[0358] 1.10 Cynomolgus serum TTR analysis

[0359] The total TTR levels in cynomolgus serum were determined by surrogate-pep tide quantitative LC-MS / MS analysis. Briefly, serum proteins were denatured and subjected to proteolysis with trypsin. Three proteotypic peptides of TTR were selected for quantification based on a calibration curve prepared in matrix using a cynomolgus TTR protein standard (produced in-house). Since TTR is endogenously expressed in normal cynomolgus serum,NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01human serum was used as a surrogate matrix to prepare calibration standards and quality controls. Notably, none of the targeted tryptic TTR peptides are homologous between human and cynomolgus monkey. Stable-isotope-labeled versions of each quantitative peptide were as used as internal standards (IS). Assay signal (area-under-the-curve ratio of standard or QCs / intemal standard) was used to interpolate unknown samples from calibration curves (signal vs concentration). The nominal concentration dynamic range used was of 5.00 to 750 pg / mL of TTR in serum, and a 12-point curve was used. LC-MS measurements were performed on a Sciex 6500+ Triple Quad. Data integration and final Serum TTR concentration calculations were performed in Sciex Analyst v 1.7.2 using a quadratic fit to the calibration curve.Example 2 - Biophysical characterization and in vivo editing efficiency of RNP-LNPs formulated with ionizable lipids having various tail chemistries and pKa values at or below 7.0

[0360] An in vivo experiment was conducted in wild-type C57 / B16 female mice to assess editing efficiencies at the TTR locus, using RNP-LNPs formulated with ionizable lipids having various tail chemistries (as shown in Table 2) and pKa values (ranging from 6-7). The compositions and biophysical characteristics of the RNP-LNPs used in this experiment are shown in Table 4.RNP-LNPs were produced as described in Example 1, with a molar ratio of 50% ionizable lipid (e.g., Compounds 1-9), 38% cholesterol, 9% DSPC, and 3% PEG-lipid (e.g., Compounds 10-11). The RNP-LNPs were formulated with a final KC1 concentration of 50 mM in the preformulation buffer (50mM MES, pH 6.0), and diluted with 35.6 mM MES buffer, 25 mM KC1 prior to the buffer exchange step. The RNP-LNPs were formulated with a RNP cargo prepared with a SpyCas9 protein (SEQ ID NO: 4), which corresponds to an mRNA sequence according to SEQ ID NO: 2 and an amino acid sequence according to SEQ ID NO: 4, unless otherwise specified, and a gRNA (SEQ ID NO: 10) targeting the mouse TTR locus, in a gRNA: Cas9 molar ratio of 2:1. The pKas of the ionizable lipids ranged from 6.0 (Compound 7) to 7.0 (Compound 9).NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0361] Table 4. Compositions and biophysical characteristics of exemplary RNP-LNPs formulated with ionizable lipids having various tail chemistries and pKa values ranging from 6.0 to 7.0.LNPID Ionizable lipid Ionizable lipid PEG-lipid ¥ Z-Ave [nm] PDI Num Ave [nm] RNA %E pKa Molar ratioCompound 1 Compound 10LNP#1 6.1 50:9:38:3 89 0.03 81 90% Compound 2 Compound 11LNP#2 6.4 50:9:38:3 91 0.01 82 89% Compound 3 Compound 10LNP#3 6.9 50:9:38:3 106 0.02 97 89% Compound 4 Compound 10LNP#4 6.1 50:10:38.5:1.5 116 0.06 103 95% Compound 5 Compound 10LNP#5 6.2 50:10:38.5:1.5 103 0.04 94 95% Compound 6 Compound 10LNP#6 6.9 50:9:38:3 100 0.07 87 89% Compound 7 Compound 11LNP#7 6.0 50:9:38:3 93 0.00 89 91% Compound 8 Compound 11LNP#8 6.6 50:9:38:3 106 0.04 90 92% Compound 9 Compound 11LNP#9 7.0 50:9:38:3 113 0.05 99 93% ¥Molar ratio of ionizable lipid: DSPC:cholesterol: PEG-lipidNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0362] Animals (N=4 per group) were injected with a dose of 0.3 mpk of RNP cargo. A negative control group consisted of animals injected with TSS (vehicle solution). Animals were euthanized 5 days post-injection. Liver tissue was collected for NGS analysis, and serum from blood was collected for serum TTR level analysis, as described in Example 1.

[0363] FIGS. 1A-1B and Table 5 show editing efficiencies obtained at the TTR locus in liver tissue of mice treated with 0.3 mpk of the RNP-LNPs formulated as described in Table 4, and the TTR levels measured in the serum from the treated animals.

[0364] Table 5. Percent indels at the TTR locus in liver tissue of mice treated with 0.3 mpk of the RNP-LNPs formulated as described in Table 4.% Indels Serum 1 fTR level (ug / ml)Mean SD Mean SDTSS 0.01 0.01 772.67 56.48LNP#1 37.26 4.40 421.44 127.11LNP#2 63.80 5.25 138.16* 78.68*LNP#3 28.05 4.93 533.17 22.99LNP#4 24.55 3.74 593.92 73.17LNP#5 58.51 5.76 185.83 73.08LNP#6 48.18 7.63 321.32 89.09LNP#7 67.20* 1.23* 57.61* 15.94*LNP#8 61.01 4.06 167.46 63.79LNP#9 22.66 3.41 702.64 70.28Example 3 - Editing at the mouse TTR locus in primary mouse hepatocytes (in vitro) and in mouse liver (in vivo) using RNP-LNPs

[0365] The editing efficiency of RNP-LNPs was compared to that of RNA-LNPs in vitro in primary mouse hepatocytes (PMH), and in vivo in mouse liver.

[0366] PMH (Gibco, Lot MC980) were prepared as described in Example 1, and plated at a density of 20,000 cells / well. Cells were incubated at 37 °C, 5% CO2 for 24 hours prior to treatment with LNPs.

[0367] LNPs were produced as described in Example 1, with a molar ratio of 50% ionizable lipid (Compound 2), 38% cholesterol, 9% DSPC, and 3% PEG-lipid (Compound 11). The RNA-LNPs were formulated with a lipid amine to RNA phosphate (N: P) molar ratio of about 6, and a RNA cargo comprising a gRNA targeting the mouse TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), in a gRNA:mRNA mass ratio of 1:2. The RNP-NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01LNPs were formulated with a final KC1 concentration of 21 mM in the preformulation 50mM MES buffer, pH 6.0, and diluted with 35.6 mM MES buffer, 25 mM KC1 prior to the buffer exchange step. The RNP-LNPs were formulated with a RNP cargo prepared with a SpyCas9 protein (SEQ ID NO: 4), and gRNA SEQ ID NO: 8, in a gRNA: Cas9 molar ratio of 2:1. The compositions and biophysical characteristics of the RNA-LNP and the RNP-LNP used in this experiment are shown in Table 6.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0368] Table 6. Biophysical characteristics of exemplary RNA-LNP and RNP-LNP used in this experiment.V LNP ID Ionizable lipid PEG-lipid Cargo Molar ratio Z-Ave [nm] PDI Num Ave [nm] RNA %ECompound 2 CompoundLNP#10 11 RNA 50:9:38:3 70 0.02 66 99%Compound 2 CompoundLNP#11 11 RNP 50:9:38:3 93 0.07 80 79% VMolar ratio of ionizable lipid: DSPC:cholesterol: PEG-lipidNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0369] Empty LNPs (i.e., that did not comprise a RNA or a RNP cargo) were prepared and added in a tube with a free RNP complex, wherein the ratio of ionizable lipid in the empty LNP to the Cas9 protein in the RNP complex was about 3,800, and mixed by inversion for 30 seconds, then left at room temperature for 30 minutes to an hour; the free RNP complex was prepared as described in Section 1.1. b of Example 1, with a SpyCas9 protein (SEQ ID NO: 4) and gRNA (SEQ ID NO: 8), using a gRNA: Cas9 molar ratio of 2: 1.

[0370] Cells were treated with a RNA- LNP, a RNP-LNP, or an empty LNP mixed with a free RNP complex, in a 7-point, 3-fold dilution series starting at 13.8 ug of total LNP lipid / well. The cells were lysed 72 hours post-treatment for NGS analysis as described in Example 1. Samples were run in duplicates, unless otherwise indicated.

[0371] FIG.2 and Table 7 show editing efficiencies obtained at the TTR locus in PMH treated with (i) a RNA-LNP comprising Compound 2 as the ionizable lipid, a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), (ii) a RNP-LNP comprising Compound 2 as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4), or (iii) an empty LNP comprising Compound 2 as the ionizable lipid, mixed with a free RNP complex prepared with a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4). FIG.2 shows high editing in primary mouse hepatocytes treated with RNP-LNPs, comparable to the editing obtained with RNA-LNPs.

[0372] Table 7. Percent indels at the TTR locus in PMH treated with (i) a RNA-LNP comprising Compound 2 as the ionizable lipid, a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), (ii) a RNP-LNP comprising Compound 2 as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4), or (iii) an empty LNP comprising Compound 2 as the ionizable lipid, mixed with a free RNP complex prepared with a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4).RNA-LNP RNP-LNP Empty LNP + Free RNP % Indels % Indels % Indels Total Lipid / well[ug / well] Mean SD Mean SD Mean SD 13.8 95.41 0.42 89.01 5.40 40.72 10.31 4.6 96.02 0.41 88.22 7.38 16.34* N / A1.53 87.14 2.77 75.60 1.97 11.88* N / ANTLA-0118WO1-RNP-LNP; RFEM: 5640-106W010.51 69.61 1.34 69.76 1.84 2.27* N / A 0.17 27.93 1.71 35.02* N / A 0.53* N / A 0.06 7.50 1.01 19.65* N / A 0.14 0.20 0.02 0.00 0.00 8.82* N / A 0.00 0.000 0.00 0.00 0.31 0.44 0.00 0.00 *N=1

[0373] An in vivo experiment was conducted in wild-type C57 / B16 female mice to assess editing efficiencies at the TTR locus, using RNA-LNPs or RNP-LNPs.

[0374] LNPs were produced as described in Example 1, with a molar ratio of 50% ionizable lipid (Compound 2), 38% cholesterol, 9% DSPC, and 3% PEG-lipid (Compound 11). The RNA-LNPs were formulated with a lipid amine to RNA phosphate (N: P) molar ratio of about 6, and a RNA cargo comprising a gRNA targeting the mouse TTR locus (SEQ ID NO: 8), and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), in a gRNA:mRNA mass ratio of 1:2. The RNP-LNPs were formulated with a final KC1 concentration of 21 mM in the preformulation 50mM MES buffer, pH 6.0, and diluted with water prior to the buffer exchange step. The RNP-LNPs were formulated with a RNP cargo prepared with a SpyCas9 protein (SEQ ID NO: 4), and gRNA SEQ ID NO: 8, in a gRNA: Cas9 molar ratio of 2:1.

[0375] The compositions and biophysical characteristics of the RNA-LNP and the RNP-LNP used in this experiment are shown in Table 8.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0376] Table 8. Biophysical characteristics of exemplary RNA-LNP and RNP-LNP used in this experiment.LNPID Ionizable lipid PEG-lipid Cargo ¥Molar ratio Z-Ave [nm] PDI Num Ave [nm] RNA %ECompound 2 Compound 11LNP#10 RNA 50:9:38:3 74 0.02 66 98%Compound 2 Compound 11LNP#12 RNP 50:9:38:3 86 0.05 73 82%yMolar ratio of ionizable lipid: DSPC:cholesterol: PEG-lipidNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0377] Animals (N=4 per group) were injected with a dose of 0.03, 0.1, or 0.3 mpk of RNA, or with a dose of 0.1. 0.3, or 1 mpk of RNP. A negative control group consisted of animals injected with TSS (vehicle solution). Animals were euthanized 5 days post-injection. Liver tissue was collected for NGS analysis, and serum from blood was collected for serum TTR level analysis, as described in Example 1.

[0378] FIGS.3A-3B and Table 9 show the TTR levels in the serum, and the editing efficiencies at the TTR locus in the liver tissue, obtained from mice treated with (i) a RNA- LNP comprising Compound 2 as the ionizable lipid, a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO; 2), or (ii) a RNP-LNP comprising Compound 2 as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4).

[0379] Table 9. Percent indels at the TTR locus in liver tissue, and TTR levels in the serum, obtained from mice treated with (i) a RNA-LNP comprising Compound 2 as the ionizable lipid, a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), or (ii) a RNP-LNP comprising Compound 2 as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4).RNA-LNP RNP-LNP% Indels Serum TTR (ug / ml) % Indels Serum TTR (ug / ml) Dose (mpk) Mean SD Mean SD Mean SD Mean SD TSS(negative - - - - 0.02 0.02 1087.65 275.96 control)0.03 11.17 2.99 906.96 224.61 N / A N / A 697.91 24.83 0.1 51.67 7.95 287.96 33.87 14.28 2.20 394.64 90.66 0.3 59.67 12.91 99.14 57.47 40.53 6.71 83.75 35.75 1 N / A N / A 1087.65 275.96 68.04 3.58 697.91 24.83EC50 0.07 N / A 0.26 N / AExample 4 - In vivo editing at the TTR locus in mouse liver using RNP-LNPs

[0380] An in vivo experiment was conducted in wild-type C57 / B16 female mice to assess editing efficiencies at the TTR locus, using RNP-LNPs at different doses (0.03 mpk and 0.1 mpk).NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0381] LNPs were produced with a molar ratio of 50% ionizable lipid (Compound 9), 38% cholesterol, 9% DSPC, and 3% PEG-lipid (Compound 11). The RNP-LNPs were formulated by dissolving a RNP cargo in 50 mM 3- (N-morpholino) propanesulfonic acid (MOPS), pH 6.5. resulting in an RNP solution with a concentration of RNP cargo of approximately 0.5 mg / niL, and a final KC1 concentration of 21 mM. The RNP-LNPs were formed by an impinging jet mixing of the lipid in ethanol with two volumes of RNP solution and one volume of water. First, the lipid in ethanol was mixed through a mixing cross with the two volumes of RNP solution. Then, a fourth stream of MOPS was mixed with the outlet stream of the cross through an inline tee. The LNPs were held for 1 hour at room temperature, and further diluted with water (approximately 1: 1 v / v). The RNP-LNPs were formulated with a RNP cargo prepared with a SpyCas9 protein (SEQ ID NO: 4), and a gRNA (SEQ ID NO: 8) targeting the mouse TTR locus, in a gRNA: Cas9 molar ratio of 1:1. RNA-LNPs were used as a control, and were formulated, as described in Example 1, with a RNA cargo comprising a gRNA (SEQ ID NO: 8) targeting the mouse TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), in a gRNA:mRNA mass ratio of 1:2. The compositions and biophysical characteristics of the RNA-LNP and the RNP-LNP used in this experiment are shown in Table 10.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0382] Table 10. Biophysical characteristics of exemplary RNA-LNP and RNP-LNP used in this experiment.LNPID Ionizable lipid PEG-lipid Cargo ¥Molar ratio Z-Ave [nm] PDI Num Ave [nm] RNA %ECompound 9 Compound 11LNP#13 RNP 50:9:38:3 76 0.08 61 81%Compound 9 Compound 11LNP#14 RNA 50:9:38:3 67 0.02 55 99% ¥Molar ratio of ionizable lipid: DSPC:cholesterol: PEG-lipidNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0383] Animals (N=5 per group) were injected with a dose of 0.3, 1, or 3 mpk of RNP cargo, or with a dose of 0.3 mpk of RNA cargo. A negative control group consisted of animals injected with TSS (vehicle solution). Animals were euthanized 7 days post-injection. Liver tissue was collected for NGS analysis, and serum from blood was collected for serum TTR level analysis, as described in Example 1.

[0384] FIGS.4A-4B and Table 11 show the TTR levels in the serum, and the editing efficiencies at the TTR locus in the liver tissue, obtained from mice treated with (i) 0.3 mpk of a RNA-LNP comprising Compound 9 as the ionizable lipid, a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), or (ii) 0.3, 1 or 3 mpk of a RNP-LNP comprising Compound 9 as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4).

[0385] Table 11. Percent indels at the TTR locus in liver tissue, and TTR levels in the serum, obtained from mice treated with (i) a RNA-LNP comprising Compound 9 as the ionizable lipid, a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), or (ii) a RNP-LNP comprising a Compound 9 as the ionizable lipid, and RNP cargo prepared with a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4).% Indels Serum TTR (ug / ml) LNP cargo Dose (mpk) Mean SD Mean SDN / A TSS 0.11 0.12 1328.82 275.670.3 2.86 0.86 1197.73 126.63 RNP 1 9.18 3.53 829.90 78.863 39.98* 10.71* 413.04* 193.37*RNA 0.3 40.84 8.27 304.80 47.65Example 5 - Editing at the rat TTR locus in primary rat hepatocytes (in vitro) and in rat liver (in vivo) using RNP-LNPs

[0386] The editing efficiency of RNP-LNPs was compared to that of RNA-LNPs in vitro in primary rat hepatocytes (PRH), and in vivo in rat liver.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0387] PRH (Tn Vitro ADMET Laboratories, Lol# 031-220) were prepared as described in Example 1, and plated at a density of 30,000 cells / well. Cells were incubated at 37 °C, 5% CO2 for 24 hours prior to treatment with LNPs.

[0388] LNPs were produced as described in Example 1, with a molar ratio of 50% ionizable lipid (Compound 2), 38% cholesterol, 9% DSPC, and 3% PEG-lipid (Compound 11). The RNA-LNPs were formulated with a lipid amine to RNA phosphate (N: P) molar ratio of about 6, and a RNA cargo comprising a gRNA (SEQ ID NO: 9) targeting the rat TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), in a gRNA:mRNA mass ratio of 1:2. The RNP-LNPs were formulated with a final KC1 concentration of 21 mM in the preformulation 50mM MES buffer, pH 6.0, and diluted with water prior to the buffer exchange step. The RNP-LNPs were formulated with a RNP cargo prepared with a SpyCas9 protein (SEQ ID NO: 4), and gRNA SEQ ID NO: 9, in a gRNA: Cas9 molar ratio of 2: 1. The compositions and biophysical characteristics of the RNA-LNP and the RNP-LNP used in this experiment are shown in Table 12.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0389] Table 12. Biophysical characteristics of exemplary RNA-LNP and RNP-LNP used in this experiment.LNPID Ionizable lipid PEG-lipid Cargo ¥Molar ratio Z-Ave [nm] PDI Num Ave [nm] RNA %ECompound 2 Compound 11LNP#17 RNP 50:9:38:3 84 0.08 72 82%Compound 2 Compound 11LNP#18 RNA 50:9:38:3 67 0.02 56 98% ¥Molar ratio of ionizable lipid: DSPC:cholesterol: PEG-lipidNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0390] Empty LNPs (i.e., that did not comprise a RNA or a RNP cargo) were prepared and added in a tube with a free RNP complex, wherein the ratio of ionizable lipid in the empty LNP to the Cas9 protein in the RNP complex was about 3,800, and mixed by inversion for 30 seconds, then left at room temperature for 30 minutes to an hour; the free RNP complex was prepared as described in Section 1.1. b of Example 1, with a SpyCas9 protein (SEQ ID NO: 4) and gRNA (SEQ ID NO: 9), using a gRNA: Cas9 molar ratio of 2:1.

[0391] Cells were treated with a RNA-LNP or a RNP-LNP in a 7-point, 3-fold dilution series starting at 4.33 ug total LNP lipid / well. The cells were lysed 72 hours post-treatment for NGS analysis as described in Example 1. Samples were run in duplicates, unless otherwise indicated.

[0392] FIG.5 and Table 13 show editing efficiencies obtained at the TTR locus in PRH treated with (i) a RNA-LNP comprising Compound 2 as the ionizable lipid, a gRNA (SEQ ID NO: 9) targeting the rat TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), (ii) a RNP-LNP comprising Compound 2 as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 9) targeting the rat TTR locus, and a SpyCas9 protein (SEQ ID NO: 4), or (iii) an empty LNP comprising Compound 2 as the ionizable lipid, mixed with a free RNP complex prepared with a gRNA (SEQ ID NO: 9) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4).

[0393] Table 13. Percent indels at the TTR locus in PRH treated with (i) a RNA-LNP comprising Compound 2 as the ionizable lipid, a gRNA (SEQ ID NO: 9) targeting the TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), (ii) a RNP-LNP comprising Compound 2 as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 9) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4), or (iii) an empty LNP comprising Compound 2 as the ionizable lipid, mixed with a free RNP complex prepared with a gRNA (SEQ ID NO: 9) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4).RNA-LNP RNP-LNP Empty LNP + Free RNP % Indels % Indels % Indels Total LNP lipid [ug / well] Mean SD Mean SD Mean SD 4.33 97.72 2.24 97.13 0.14 88.29 0.43 1.44 96.48 4.30 94.58 1.84 85.17 4.59NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W010.48 97.36 1.51 83.50 0.35 60.56 0.07 0.16 93.76 1.72 73.58 6.64 22.41 1.57 0.05 92.76 4.99 38.76* N / A 10.52 1.09 0.018 72.51 8.90 53.88 0.93 4.98 0.37 0.006 37.62 5.87 20.41 5.11 1.33 1.15 0 0.87 1.22 0.09 0.13 0.00 0.00*N=1

[0394] An in vivo experiment was conducted in Sprague Dawley female rats to assess editing efficiencies at the TTR locus, using RNA-LNPs or RNP-LNPs.

[0395] LNP#17 and LNP#18 as described above were produced as described at the beginning of this Example 6. Animals (N=5 per group) were injected with a dose of 0.03, 0.1, or 0.3 mpk of RNA (LNP#18), or with a dose of 0.1, 0.3, or 1 mpk of RNP (LNP#17). A negative control group consisted of animals injected with TSS (vehicle solution). Animals were euthanized 5 days post-injection. Liver tissue was collected for NGS analysis, and serum from blood was collected for serum TTR level analysis, as described in Example 1.

[0396] FIGS.6A-6B and Table 14 show editing efficiencies obtained at the 1TR locus in liver tissue of rats treated with (i) a RNA-LNP comprising Compound 2 as the ionizable lipid, a gRNA (SEQ ID NO: 9) targeting the rat TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), or (ii) a RNP-LNP comprising Compound 2 as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 9) targeting the rat TTR locus, and a SpyCas9 protein (SEQ ID NO: 4).

[0397] Table 14. Percent indels at the TTR locus in liver tissue, and TTR levels in the serum, obtained from rats treated with (i) a RNA-LNP comprising Compound 2 as the ionizable lipid, a gRNA (SEQ ID NO: 9) targeting the TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), or (ii) a RNP-LNP comprising Compound 2 as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 9) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4).RNA-LNP RNP-LNP% Indels Serum TTR (ug / ml) % Indels Serum TTR (ug / ml) Dose (mpk) Mean SD Mean SD Mean SD Mean SD TSS - - - - 0.00 0.00 950.07 66.24NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W010.03 11.33 3.93 871.52 127.52 N / A N / A N / A N / A 0.1 45.64 7.30 427.45 169.62 52.66 3.67 373.88 106.99 0.3 68.29 2.16 67.80 19.40 69.54 3.10 70.26 43.67 1 N / A N / A N / A N / A 72.22 1.11 13.64 3.72Example -In vivo editing efficiency of targeted RNP-LNPs at the B2M locus in mouse bone marrow tissue and cells

[0398] An in vivo experiment was conducted in wild-type C57 / B16 female mice to assess editing efficiencies at the B2M locus, using targeted RNP-LNPs conjugated with an anti- CD117 Fab.

[0399] RNP-LNPs were produced as described in Example 1, as a 5-component LNP formulation, with a molar ratio of 50% ionizable lipid (Compound 2), 38% cholesterol, 9% DSPC, 2.955% structural PEG-lipid (Compound 11), and 0.045% anchor PEG-lipid (DSPE- PEG-SpyTag). The RNP-LNPs were formulated with a final KC1 concentration of 21 mM in the preformulation 50mM MES buffer, pH 6.0, and diluted with water prior to the buffer exchange step. The RNP-LNPs were formulated with a RNP cargo prepared with a SpyCas9 protein (SEQ ID NO: 4), and a gRNA (SEQ ID NO: 1) targeting the B2M locus, in a gRNA: Cas9 molar ratio of 1:1. In some cases, after being formulated, the RNP-LNPs were conjugated, via a SpyTag-SpyCatcher conjugation chemistry, with an anti-CD117 Fab (0.045%). The compositions and biophysical characteristics of the RNP-LNPs used in this experiment are shown in Table 15.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0400] Table 15. Compositions and biophysical characteristics of exemplary RNP-LNPs used in this experiment.LNPID Ionizable lipid PEG-lipid Targeting ligand ¥Molar ratio Z-Ave [nm] PDI Num Ave [nm] RNA %ELNP#19 Compound 2 Compound 11 anti-CDl 17 Fab (0.045%) 50:9:38:2.955:0.045 89 0.03 81 90% LNP#20 Compound 2 Compound 11 N / A 50:9:38:2.955:0.045 91 0.01 82 89% ¥Molar ratio of ionizable lipid: DSPC:cholesterol:structural PEG-lipid: anchor PEG-lipidNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0401] Animals (N=4 per group) were injected with a dose of 1 mpk of RNP, with or without an anti-CD117 Fab. Animals were euthanized 6 days post-injection. Whole bone marrow tissue, and HSCs were collected, as described in Example 1, for NGS analysis.

[0402] FIGS.7A-7B and Table 16 show editing efficiencies obtained at the B2M locus in liver, whole bone marrow (WBM) and HSCs of mice treated with 1 mpk of a CD117-targeted RNP-LNP or an untargeted RNP-LNP, wherein the RNP-LNPs comprised Compound 2 as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 1) targeting the B2M locus, and a SpyCas9 protein (SEQ ID NO: 4).

[0403] Table 16. Percent indels at the B2M locus in whole bone marrow (WBM) and HSCs of mice treated with 1 mpk of a CD117-targeted RNP-LNP or an untargeted RNP-LNP, wherein the RNP-LNPs comprised Compound 2 as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 1) targeting the B2M locus, and a SpyCas9 protein (SEQ ID NO: 4).CD117-targeted RNP-LNP Untargeted RNP-LNP% Indels % IndelsTissue / Cell Type Mean SD Mean SDWBM 3.84* 1.30* 1.27 0.26HSC 8.66* 3.41* 1.49 0.68*N=3Example 7 - In vivo editing at the mouse TTR locus using RNP-LNP formulations with different PEG- lipids

[0404] An in vivo experiment was conducted in wild-type C57 / B16 female mice to assess editing efficiencies at the TTR locus, using RNP-LNP formulations with different PEG-lipids.

[0405] RNP-LNPs produced as 4-component LNP formulations, with a molar ratio of 50% ionizable lipid (Compound 2), 38% cholesterol, 9% DSPC, and 3% PEG-lipid, wherein different PEG-lipids were used (e.g., Compounds 1-16). The RNP-LNPs were formulated with a final KC1 concentration of 21 mM in the preformulation 50mM MES buffer, pH 6.0, and diluted with water prior to the buffer exchange step, and a RNP cargo prepared with a gRNA targeting a TTR locus (SEQ ID NO: 10) and a SpyCas9 protein (SEQ ID NO: 4), in a gRNA: Cas9 molar ratio of 2:1.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0406] The RNP-LNPs were assessed for their biophysical characteristics, as described in Example 1. Table 17 shows the biophysical analysis for exemplary formulations.

[0407] Table 17. Biophysical characteristics of exemplary RNP-LNPs formulated with a molar ratio of 50% ionizable lipid (Compound 2), 38% cholesterol, 9% DSPC, and 3% PEG-lipid, wherein different PEG-lipids were used (e.g., Compounds 10-16).LNPID PEG-Lipid Z-Ave [nmj PDI Num Ave [nm] RNA %ELNP#2 Compound 11 82 0.06 71 84% LNP#21 Compound 10 83 0.05 70 84% LNP#22 Compound 12 84 0.04 73 82% LNP#23 Compound 13 86 0.05 74 84% LNP#24 Compound 14 86 0.06 69 84% LNP#25 Compound 15 91 0.06 76 85% LNP#26 Compound 16 80 0.04 67 83%

[0408] Animals (N=4 per group) were injected with a dose of 0.3 mpk of RNP. Animals were euthanized 6 days post-injection. A negative control group consisted of animals injected with TSS (vehicle solution). Liver tissue was collected for NGS analysis, and serum from blood was collected for serum TTR level analysis, as described in Example 1.

[0409] FIGS.8A-8B and Table 18 show the TTR levels in the serum, and the editing efficiencies at the TTR locus in the liver tissue, obtained from mice treated with 0.3 mpk of a RNP-LNP comprising Compound 2 as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 10) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4).

[0410] Table 18. Percent indels at the TTR locus in liver tissue, and TTR levels in the serum, obtained from mice treated with a RNP-LNP comprising Compound 2 as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 10) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4).NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01% Indels Serum TTR (ug / ml)LNPID Mean SD Mean SDTSS 0.01 0.00 849.50* 90.18*Compound 11 29.19 4.94 311.50* 84.96*Compound 10 36.40 4.27 390.90 17.52Compound 12 28.05 6.93 627.90 129.50Compound 13 33.12 1.62 482.00 94.92Compound 14 36.10 7.12 514.30 76.73Compound 15 45.01 2.36 348.00 21.15Compound 16 7.61 0.35 791.50 48.19*N=3Example 8 - Editing at the mouse TTR locus in primary mouse hepatocytes using RNP-LNPs with a RNP cargo comprising gRNAs of different lengths

[0411] The editing efficiency of RNP-LNPs was compared to that of RNA-LNPs in vitro in primary mouse hepatocytes (PMH), wherein the RNA-LNP and RNP-LNP cargos comprised gRNAs with different lengths.

[0412] PMH (Gibco, Lot MC980) were prepared as described in Example 1, and plated at a density of 20,000 cells / well. Cells were incubated at 37°C, 5% CO2 for 24 hours prior to treatment with LNPs.

[0413] LNPs were produced as described in Example 1, with a molar ratio of 50% ionizable lipid (Compound 2), 38% cholesterol, 9% DSPC, and 3% PEG-lipid (Compound 11). The RNA-LNPs were formulated with a RNA cargo comprising a gRNA targeting the mouse TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), in a gRNA:mRNA mass ratio of 1:2, wherein the gRNA was either 91 nucleotides long (SEQ ID NO: 10) or 100 nucleotides long (SEQ ID NO: 8). The RNP-LNPs were formulated with a final KC1 concentration of 21 mM in the preformulation 50mM MES buffer, pH 6.0, and diluted with water prior to the buffer exchange step, and comprised a RNP cargo prepared with a SpyCas9 protein (SEQ ID NO: 4), and a gRNA (SEQ ID NO: 8 or SEQ ID NO: 10) targeting the mouse TTR locus, in a gRNA: Cas9 molar ratio of 2:1.

[0414] The compositions and biophysical characteristics of the LNPs used in this experiment are shown in Table 19.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0415] Table 19. Compositions and biophysical characteristics of exemplary RNA-LNPs and RNP-LNPs comprising a gRNA with a length of 91 nucleotides (SEQ ID NO: 10) or 100 nucleotides (SEQ ID NO: 8).y LNPID Ionizable lipid PEG-lipid Cargo gRNA used in cargo Z-Ave [nm] PDI Num Ave [nm] RNA %E Molar ratioLNP#12 Compound 2 Compound 11 RNP Guide 1 82%(SEQ ID NO: 8) 50:9:38:3 86 0.05 73Guide 3LNP#2 RNP 0.04 Compound 2 Compound 11 50:9:38:3 87 73 83% (SEQ ID NO: 10)Guide 1 RNA 0.02 99% LNP#10 Compound 2 Compound 11 50:9:38:3 70 66(SEQ ID NO: 8)Guide 3 LNP#27 Compound 2 RNA Compound 11 50:9:38:3 70 0.05 61 98% (SEQ ID NO: 10)¥Molar ratio of ionizable lipid: DSPC:cholesterol:structural PEG-lipid: anchor PEG-lipidNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0416] Cells were treated with a RNA-LNP or a RNP-LNP in a 11 -point, 2- fold dilution series starting at 13 ug of total LNP lipid / well. The cells were lysed 72 hours post-treatment for NGS analysis as described in Example 1. Samples were run in duplicates.

[0417] FIG.9 and Table 20 show editing efficiencies obtained at the TTR locus in PMH treated with (i) a RNA-LNP comprising Compound 2 as the ionizable lipid, a 91-mer or a 100-mer gRNA targeting the TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), or (ii) a RNP-LNP comprising Compound 2 as the ionizable lipid, and a RNP cargo prepared with a 91-mer or a 100-mer gRNA targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4).NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0418] Table 20. Percent indels at the TTR locus in PMH treated with (i) a RNA-LNP comprising Compound 2 as the ionizable lipid, a 91-mer or a 100-mer gRNA targeting the TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), or (ii) a RNP-LNP comprising Compound 2 as the ionizable lipid, and a RNP cargo prepared with a 91-mer or a 100-mer gRNA targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4).RNP-LNP (100-mer gRNA) RNA-LNP (100-mer gRNA) RNP-LNP (91-mer gRNA) RNA-LNP (91-mer gRNA) % Indels % Indels % Indels % Indels Total LNP lipid [ug / well] Mean SD Mean SD Mean SD Mean SD 96.37 0 1.2113.38 93.56 95.46 0.67 94.20 0.60 94.72 1.74 95.04 0.28 95.70 0.10 95.14 1.09 6.593.70 2.46 93.96 1.61 95.43 0 94.49 3.25.25 1.13 94.19 0.03 92.52 0. 94.74 1.625 47 0.43 95.11 0.3388.62 90.39 5.0.813 91.77 1.96 0.11 90.53 4.03 5984.44 4.54 74.85 1.98 87.83 0.67 91.06 0.97 0.40669.85 2.81 53.79 4.24 74.83 0.61 80.68 1.56 0.20345.85 5.58 29.13 3.23 55.00 3.83 59.32 1.54 0.10224.67 6.23 13.95 1.25 31.68 0.33 31.20 2.92 0.0510.025 11.93 3.28 3.70 0.11 12.06 1.65 10.90 0.00 0 4.16 0.00 1.00 0.20 5.19 0.12.013 3.36 0.510.020 0.01 0.02 0.00 0.01 0.00 0.03 0.00NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01Example 9 -In vivo editing at the TTR locus in mouse liver using RNP-LNPs produced with different prefonnulation buffer concentrations

[0419] An in vivo experiment was conducted in wild-type C57 / B16 female mice to assess editing efficiencies at the TTR locus, using RNP-LNPs produced with different preformulation buffer concentrations.

[0420] LNPs were produced as described in Example 1, with a molar ratio of 50% ionizable lipid (e.g., Compound 2, or Compound 8), 38% cholesterol, 9% DSPC, and 3% PEG-lipid (Compound 11). The RNP-LNPs were formulated with a final KC1 concentration of 21 mM in the preformulation 50 mM MES buffer, pH 6.0, and diluted with water prior to the buffer exchange step. Alternatively, the preformulation buffer comprised 150 mM MES buffer, pH 6.0, and a final KC1 concentration of 21 mM. The RNP-LNPs were formulated with a RNP cargo prepared with a SpyCas9 protein (SEQ ID NO: 4), and a gRNA (SEQ ID NO: 8) targeting the mouse TTR locus, in a gRNA: Cas9 molar ratio of 2:1, and an ionizable lipid: Cas9 polypeptide molar ratio of about 2,850 or 3,800. In order to compare the RNP-LNP manufacturing processes, three lots of RNP-LNPs were produced with the 50mM MES preformulation buffer concentration, and two lots of RNP-LNPs were produced with the 150mM MES preformulation buffer concentration. RNA-LNPs were used as a control and were formulated with a RNA cargo comprising a gRNA (SEQ ID NO: 8) targeting the mouse TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), in a gRNA:mRNA mass ratio of 1:2, and a N: P molar ratio of about 6. The compositions and biophysical characteristics of the RNA-LNP and the RNP-LNPs used in this experiment are shown in Table 21.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0421] Table 21. Biophysical characteristics of exemplary RNA-LNP and RNP-LNPs used in this experiment.Buffer Z-Ave Num Ave RNA LNP ID Ionizable lipid PEG-lipid Cargo Molar ratio® Molar ratio® PDIconcent.ra *ti•on (1) [nm] [nm] %ELNP#11-lot#1 Compound 2 Compound 11 RNP 50 mM 2,850 50:9:38:3 93 0.07 80 79%LNP#15 Compound 8 Compound 11 RNP 50 mM 2,850 50:9:38:3 96 0.06 83 86%LNP#11-lot#2 50 mM 0.02 79 Compound 2 Compound 11 RNP 2,850 50:9:38:3 87 73%150 mM 84 0.04 79 74% LNP#16-lot#l Compound 2 Compound 11 RNP 2,850 50:9:38:350 mM LNP#ll-lot#3 Compound 2 Compound 11 RNP 3,800 50:9:38:3 93 0.05 83 73%LNP#16-lot#2 Compound 2 Compound 11 RNP 150 mM 3,800 50:9:38:3 88 0.04 77 73%NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01LNP#10 Compound 2 Compound 11 RNA N / A N / A 50:9:38:3 70 0.02 66 99% ®RNP-LNP preformulation MES Buffer concentration®Molar ratio RNP-LNP ionizable lipid: Cas9®Molar ratio of ionizable lipid: DSPC:cholesterol: PEG-lipidNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0422] Animals (N=5 per group) were injected with a dose of 3 mpk of RNP, or 0.3 mpk of RNA. A negative control group consisted of animals injected with TSS (vehicle solution). Animals were euthanized 5 days post-injection. Liver tissue was collected for NGS analysis, and serum from blood was collected for serum TTR level analysis, as described in Example 1.

[0423] FIGS. 10A-10B and Table 22 show the TTR levels in the serum, and the editing efficiencies at the TTR locus in the liver tissue, obtained from mice treated with (i) 3 mpk of a RNA-LNP comprising Compound 2 as the ionizable lipid, and a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), or (ii) 0.3 mpk of a RNP-LNP comprising Compound 8 (LNP#15) or Compound 2 (LNP#11 and LNP#16) as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4).

[0424] Table 22. Percent indels at the TTR locus in liver tissue, and TTR levels in the serum, obtained from mice treated with (i) a RNA-LNP comprising Compound 2 as the ionizable lipid, and a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), or (ii) a RNP-LNP comprising Compound 8 (LNP#15) or Compound 2 (LNP#11 and LNP#16) as the ionizable lipid, and a RNP cargo prepared with a gRNA (SEQ ID NO: 8) targeting the TTR locus, and a SpyCas9 protein (SEQ ID NO: 4).% Indels Serum TTR (ug / ml)LNP ID Mean SD Mean SDTSS 0.01 0.00 1283.74* 399.39*LNP#11 - Lot#1 79.26 4.64 53.56* 67.40*LNP# 15 79.32 1.51 24.86 3.56LNP#11 - Lot#2 79.23 1.89 20.73 3.18LNP#16 - Lot#l 77.26 0.70 21.46 2.36LNP#11 - Lot#3 82.25 1.87 21.55 2.93LNP#16 - Lot#2 80.32* 0.61* 14.70** 2.75**LNP# 10 69.53 2.75 35.81 10.79*N=4; **N=3NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01Example 10 - In vivo editing efficiency of RNP-LNPs having different ionizable lipids and produced using different formulation processes

[0425] An in vivo experiment was conducted in wild-type C57 / B16 female mice to assess the editing efficiency (at the TTR locus) of RNP-LNPs produced with different ionizable lipids, and using different formulation processes, as described in Example 1.RNP-LNPs were produced according to one of the following three protocols, as described in Example 1, with a molar ratio of 50% ionizable lipid (e.g., Compound 2, Compound 8, Compound 5, or Compound 7), 38% cholesterol, 9% DSPC, and 3% PEG-lipid (Compound 11).• In Protocol 1, the preformulation buffer comprised 50 mM MES buffer, pH 6.0, with a final KC1 concentration of 21 mM, the RNP-LNPs were further mixed with 50 mM MES. pH 6.0, and the RNP-LNPs were diluted in water prior to the buffer exchange step.• In Protocol 2, the preformulation buffer comprised 50 mM MES buffer, pH 6.0, and was supplemented with 33 mM KC1, resulting in a final KC1 concentration of 50 mM, the RNP-LNPs were further mixed with 50 mM MES, pH 6.0, and the RNP-LNPs were diluted in water prior to the buffer exchange step.• In Protocol 3, the preformulation buffer comprised 50 mM MES buffer, pH 6.0, and was supplemented with 33 mM KC1, resulting in a final KC1 concentration of 50 mM, the RNP-LNPs were further mixed with 50 mM MES, pH 6.0, and the RNP-LNPs were diluted in 35.6 mM MES buffer, 25 mM KC1 prior to the buffer exchange step.

[0426] The RNP-LNPs comprised a RNP cargo prepared with a SpyCas9 protein (SEQ ID NO: 4), and a gRNA (SEQ ID NO: 10) targeting the mouse TTR locus, in a gRNA: Cas9 molar ratio of 2:1. The compositions and biophysical characteristics of the LNPs used in this experiment are shown in Table 23.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0427] Table 23. Compositions and biophysical characteristics of exemplary RNP-LNPs having different ionizable lipids and formulated with one of Protocols 1, 2, or 3.¥LNPID Ionizable lipid PEG-lipid Formulation Process Molar ratio Z-Ave [nm] PDI Num Ave [nm] RNA %ELNP#2 Compound 2 Compound 11 Protocol 1 50:9:38:3 77 0.09 67 82%Protocol 2LNP#2 Compound 2 Compound 11 50:9:38:3 75 0.09 62 86%Protocol 3LNP#2 Compound 2 Compound 11 50:9:38:3 85 0.04 70 90%Compound 8 Compound 11LNP#8 Protocol 1 50:9:38:3 80 0.07 70 85%Compound 8 Compound 11 Protocol 2LNP#8 50:9:38:3 95 0.05 82 92%Compound 8 Compound 11 Protocol 3LNP#8 50:9:38:3 101 0.01 96 95%Compound 5 Compound 11LNP#28 Protocol 1 50:9:38:3 82 0.08 61 79%Compound 5 Compound 11 Protocol 2LNP#28 50:9:38:3 75 0.06 61 90%Compound 5 Compound 11 Protocol 3LNP#28 50:9:38:3 81 0.08 65 91%NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01Compound 7 Compound 11LNP#7 Protocol 1 50:9:38:3 78 0.06 69 83%Compound 7 Compound 11 Protocol 2LNP#7 50:9:38:3 83 0.06 71 87%Compound 7 Compound 11 Protocol 3LNP#7 50:9:38:3 94 0.05 79 88%¥Molar ratio of ionizable lipid: DSPC:cholesterol:structural PEG-lipid: anchor PEG-lipidNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0428] Animals (N=4 per group) were injected with a dose of 0.3 mpk of RNP. A negative control group consisted of animals injected with TSS (vehicle solution). Animals were euthanized 7 days post-injection. Liver tissue was collected for NGS analysis, and serum from blood was collected for serum TTR level analysis, as described in Example 1.

[0429] FIGS. 11A-11B and Table 24 show editing efficiencies obtained at the TTR locus in liver tissue of mice treated with a RNP-LNP comprising a RNP cargo prepared with a gRNA (SEQ ID NO: 10) targeting the mouse TTR locus, and a SpyCas9 protein (SEQ ID NO: 4).

[0430] Table 24. Percent indels at the TTR locus in liver tissue, and TTR levels in the serum, obtained from mice treated with a RNP-LNP comprising a RNP cargo prepared with a gRNA (SEQ ID NO: 10) targeting the mouse TTR locus, and a SpyCas9 protein (SEQ ID NO: 4). The RNP-LNPs were formulated with different ionizable lipids.% Indels Serum TTR levels (ug / ml)LNP ID Mean SD Mean SDTSS (negative control) 0.00 0.01 960.59 151.77LNP#2 - Protocol 1 30.65 8.48 490.99 113.63LNP#2 - Protocol 2 53.63 4.24 158.21 47.64LNP#2 - Protocol 3 63.47 4.91 126.63 21.91LNP#8 - Protocol 1 50.43 6.02 268.91 119.84LNP#8 - Protocol 2 63.46 3.68 129.94 24.81LNP#8 - Protocol 3 59.41 4.51 90.55 29.02LNP#30 - Protocol 1 35.95 5.74 353.18 45.44LNP#30 - Protocol 2 46.78 2.79 262.70 38.62LNP#30 - Protocol 3 57.37 4.15 140.37 49.72LNP#7 - Protocol 1 41.68 4.25 300.77 14.25LNP#7 - Protocol 2 58.82 2.13 111.92 22.30LNP#7 - Protocol 3 66.11 3.05 N / A* N / A**Note: TTR levels for this group are below the detection threshold of the assay.Example 11 - Comparison of the in vivo editing efficiency of RNP-LNPs made with different ionizable lipids and produced using different formulation processesNTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01

[0431] The in vivo editing efficiency of RNP-LNPs prepared with different ionizable lipids having different pKa values and produced using different formulation processes were evaluated. RNP-LNPs were produced according to the following protocols:• Protocol A: RNP-LNPs were produced with a molar ratio of 45% ionizable lipid (Compound 2), 9% cationic lipid (DOTAP; Compound 17), 35% cholesterol, 8% DSPC, and 3% PEG-lipid (Compound 11). The RNP-LNPs were formulated by dissolving a RNP cargo in IX PBS, pH 7.4, resulting in an RNP solution with a concentration of RNP cargo of approximately 0.5 mg / mL, and a final KC1 concentration of 21 mM. The RNP-LNPs were formed by an impinging jet mixing of the lipids (ionizable lipid, DOTAP, cholesterol, DSPC, and PEG lipid) in ethanol with two volumes of RNP solution and one volume of water. First, the lipids in ethanol were mixed through a mixing cross with the two volumes of RNP solution. Then, a fourth stream of PBS was mixed with the outlet stream of the cross through an inline tee. The LNPs were held for 1 hour at room temperature, and further diluted with water (approximately 1:1 v / v). The RNP-LNPs were formulated with a RNP cargo prepared with a SpyCas9 protein (SEQ ID NO: 4). and a gRNA (SEQ ID NO: 8) targeting the mouse TTR locus, in a gRNA: Cas9 molar ratio of 1:1.• Protocol B: RNP-LNPs were produced with a molar ratio of 50% ionizable lipid (Compound 9), 38% cholesterol, 9% DSPC, and 3% PEG-lipid (Compound 11), as described in Example 4. The RNP-LNPs comprised a RNP cargo prepared with a SpyCas9 protein (SEQ ID NO: 4), and a gRNA (SEQ ID NO: 8) targeting the mouse TTR locus, in a gRNA: Cas9 molar ratio of 1: 1.• Protocol 1: RNP-LNPs were produced according to Protocol 1, as described in Example 10, with a molar ratio of 50% ionizable lipid (Compound 2), 38% cholesterol, 9% DSPC, and 3% PEG-lipid (Compound 11), and a RNP cargo prepared with a SpyCas9 protein (SEQ ID NO: 4), and gRNA SEQ ID NO: 8, in a gRNA: Cas9 molar ratio of 2: 1.• Protocol 2: RNP-LNPs were produced according to Protocol 3, as described in Example 10, with a molar ratio of 50% ionizable lipid (Compound 2), 38% cholesterol, 9% DSPC, and 3% PEG-lipid (Compound 11), and a RNP cargo prepared with a SpyCas9 protein (SEQ ID NO: 4), and a gRNA (SEQ ID NO: 10) targeting the mouse TTR locus, in a gRNA: Cas9 molar ratio of 2:1.NTLA-0118WO1-RNP-LNP; RFEM: 5640-106W01• Protocol 3: RNP-LNPs were produced according to Protocol 3, as described in Example 10, with a molar ratio of 50% ionizable lipid (Compound 7), 38% cholesterol, 9% DSPC, and 3% PEG-lipid (Compound 11), and a RNP cargo prepared with a SpyCas9 protein (SEQ ID NO: 4), and a gRNA (SEQ ID NO: 10) targeting the mouse TTR locus, in a gRNA: Cas9 molar ratio of 2: 1.

[0432] To compare the editing efficiencies of the RNP-LNPs prepared according to the protocols listed above, data from different experiments were observed. In each experiment, animals were injected with RNP-LNPs or RNA-LNPs (wherein RNA-LNPs served as a positive control), at a dose of 0.03, 0.1, 0.3, 1, or 3 mpk of RNP or RNA, as shown in Table 25. Animals were euthanized 5- or 7-days post-injection, and liver tissue was collected for NGS analysis, as described in Example 1.

[0433] FIG. 12 and Table 25 show the difference in editing efficiencies obtained at the TTR locus in mouse liver tissue using RNP-LNPs produced with different manufacturing processes described herein, and formulated with ionizable lipids having different pKa values. Compound 9, with a pKa of 7.0, was considered a “high pKa lipid”, Compound 2, with a pKa of 6.4, was considered an “intermediate pKa lipid”, and Compound 7, with a pKa of 6.0, was considered a “low pKa lipid”.NTLA-0118USP01-RNP-LNP RFEM: 5640-106

[0434] Table 25. Percent indels obtained at the TTR locus in mouse liver tissue using RNP-LNPs produced with different manufacturing processes described herein, and formulated with ionizable lipids having different pKa values.Injection dose (mpk)RNP-LNPLNP injected manufacturing Ionizable lipid 0.03 0.1 0.3 1 3process%Indels 11.17 51.67 59.67 N / A N / ARNA-LNP Control Compound 2N / A SD 2.99 7.95 12.91 N / A N / A(data from Example 3) pKa = 6.4N 4 4 4 N / A N / ACompound 2, pKa = %Indels N / A N / A N / A N / A 20.36RNP-LNP 6.4Protocol A SD N / A N / A N / A N / A 7.41with Compound 17 with cationic lipidCompound 17 N N / A N / A N / A N / A 5RNP-LNP, %Indels N / A N / A 2.86 9.18 39.98Compound 9High pKa Lipid Protocol B SD N / A N / A 0.86 3.53 10.71pKa = 7.0(data from Example 4) N N / A N / A 5 5 4RNP-LNP, %Indels N / A 14.28 40.53 68.04 N / ACompound 2Intermediate pKa Lipid Protocol 1 SD N / A 2.20 6.71 3.58 N / ApKa = 6.4(data from Example 3) N N / A 4 4 4 N / ARNP-LNP, %Indels N / A N / A 53.63 N / A N / ACompound 2Intermediate pKa Lipid Protocol 2 SD N / A N / A 4.24 N / A N / ApKa = 6.4(data from Example 10) N N / A N / A 4 N / A N / ARNP-LNP, %Indels N / A N / A 66.11 N / A N / ACompound 7Low pKa Lipid Protocol 3 SD N / A N / A 3.05 N / A N / ApKa = 6.0(data from Example 10) N N / A N / A 4 N / A N / ANTLA-0118USP01-RNP-LNP RFEM: 5640-106Example 12: Comparison of different preformulation buffers for particle size and encapsulation properties.

[0435] The particle sizes, encapsulation efficiencies, and other biophysical properties of RNP-LNPs produced using various preformulation buffers were evaluated. RNP-LNPs were produced and tested using the following protocols:

[0436] RNP Cargo Stability in Different pH 6.0 preformulation buffers: Cas9-gRNA RNP cargos were prepared as follows, using a Cas9 polypeptide (e.g., SpyCas9) and a chemically modified gRNA targeting a genomic sequence (in this case, SEQ ID NO: 10, cyno 1TR). First, the gRNA was denatured at 95°C for 2 minutes, followed by cooling on ice for 10 minutes. The gRNA was then diluted to a working solution of 3 mg / mL in deionized water. The Cas9 polypeptide was diluted to a working solution of 5 mg / mL in a protein buffer composed of 20 mM HEPES, 300 mM KC1, 5% glycerol, 1 mM MgCl, and 1 mM dithiothreitol (DTT). The Cas9 polypeptide solution and the gRNA solution were mixed at a gRNA: Cas9 polypeptide molar ratio of 2:1, unless otherwise indicated. The mixture was stored at room temperature for 10 minutes to one hour. Finally, the mixture was diluted in different pH 6.0 preformulation buffer (50 mM) for stability assessment. FIG. 13A describes RNP cargo stability in different preformulation buffers. Stability assessment was performed using Dynamic Light Scattering. RNP cargo is found to be stable in all the tested pH 6.0 preformulation buffers.

[0437] Evaluation of RNP-LNP formulation using different pH 6.0 preformulation buffers. LNPs were produced as described in Example 1, with a molar ratio of 50% ionizable lipid (Compound 2), 38% cholesterol, 9% DSPC, and 3% PEG-lipid (Compound 11). The RNP-LNPs were formulated with a final KC1 concentration of 21 mM in the different 50mM preformulation buffer, pH 6.0 (MES, ADA, BES, Bis-Tris, Citrate, MOPS, Phosphate, PIPES), and diluted with water prior to the buffer exchange step. The RNP-LNPs were formulated with a RNP cargo prepared with a SpyCas9 protein (SEQ ID NO: 4), and gRNA (SEQ ID NO: 10), in a gRNA: Cas9 molar ratio of 2:1. The biophysical characteristics for each formulation are shown in Table 26.

[0438] Table 26. Formulation biophysical characteristics.LNP ID Buffer PH Z-Ave PDI Num Ave RNA[nm] [nm] %ELNP-A MES 6.0 82 0.05 68 86%NTLA-0118USP01-RNP-LNP RFEM: 5640-106LNP-B ADA 6.1 246 0.08 230 68%LNP-C BES 6.1 142 0.11 127 62%LNP-D Bis-Tris 6.0 84 0.05 72 91%LNP-E Citrate 6.1 314 0.09 316 53%LNP-F MOPS 6.0 108 0.07 100 67%LNP-G Phosphate 6.1 267 0.20 277 54%LNP-H PIPES 5.9 123 0.09 95 87%

[0439] FIGS. 13B and 13C show the trend in Post Freeze-Thaw particle size (Z-Average) and % Encapsulation for RNP-LNPs formulated using different pH 6.0 preformulation buffers. Each pH 6.0 preformulation buffer tested had a different pKa, and that data indicated that pH 6.0 preformulation buffers with a pKa range of 6.0-6.5 show the highest % encapsulation (>85%) and desired size in a range of about 80-85 nm.

[0440] LNPs (FIG. 13D) were produced as described in Example 1, with a molar ratio of 47% ionizable lipid (Compound 2), 28% cholesterol, 22.5% DSPC, and 2.5% PEG-lipid (Compound 11 ) and LNPs (FIG. 13E) were produced as described in Example 1, with a molar ratio of 50% ionizable lipid (Compound 8), 38% cholesterol, 9% DSPC, and 3% PEG-lipid (Compound 11). The RNP-LNPs were formulated with a final KC1 concentration of 21 mM in the different 50mM preformulation buffer, pH 6.0 (MES, Bis-Tris and Histidine), and diluted with water prior to the buffer exchange step. The RNP-LNPs were formulated with a RNP cargo prepared with a SpyCas9 protein (SEQ ID NO: 4), and gRNA (SEQ ID NO: 10), in a gRNA: Cas9 molar ratio of 2:1. FIGS. 13D and 13E shows plots of Particle size (Z-Average) and % Encapsulation across different preformulation buffers for both Compound 2 and Compound 8 RNP-LNPs respectively.Example 13: Comparison of different buffer concentrations for particle sizes and encapsulation properties

[0441] The particle sizes, encapsulation efficiencies, and gene editing properties of RNP-LNPs produced using various preformulation buffer concentrations were evaluated. RNP-LNPs were produced and tested using the following protocols. LNPs were produced as described in Example 1, with a molar ratio of 50% ionizable lipid (Compound 2), 38% cholesterol, 9% DSPC. and 3% PEG-lipid (Compound 11). The RNP-LNPs were formulatedNTLA-0118USP01-RNP-LNP RFEM: 5640-106with a final KC1 concentration of 21 mM in the MES preformulation buffer, pH 6.0 (Buffer Strength: 50, 150, 300mM) and Bis-Tris preformulation buffer, pH6.0 (Buffer Strength: 25, 50, 75, 100, 150, 200 mM), and diluted with water prior to the buffer exchange step. The RNP-LNPs were formulated with a RNP cargo prepared with a SpyCas9 protein (SEQ ID NO: 4), and gRNA (SEQ ID NO: 10), in a gRNA: Cas9 molar ratio of 2:1. FIG. 14A shows the particle size and % Encapsulation trend across buffer strength range of Bis-Tris, pH 6.0 preformulation buffer. FIG. 14B shows the particle size and % Encapsulation trend across buffer strength range of MES, pH 6.0 preformulation buffer. 50 mM buffer strength demonstrated superior particle size and % encapsulation properties. When comparing the potency of the two formulations - one formulated using 50mM MES and another formulated using 50 mM Bis-Tris, comparable potency was seen (FIG. 14C). Cells (PMH) were treated with RNP-LNPs in a 7-point, 3 -fold dilution series starting at 1.44 ug of total LNP lipid / well. The cells were lysed 72 hours post-treatment for NGS analysis. Samples were run in duplicates.Example 14: Comparison of different protocols for producing RNP-LNPs for particle sizes, encapsulation properties, and editing potency

[0442] The particle sizes and encapsulation efficiencies of RNP-LNPs produced using different formulation processes were evaluated. In a Protocol 4, the preformulation buffer comprised 50 mM Bis-Tris buffer, pH 6.0. with a final KC1 concentration of 21 mM, the RNP-LNPs were further mixed with 50 mM Bis-Tris, pH 6.0, and the RNP-LNPs were diluted in water prior to the buffer exchange step. RNP-LNPs were produced and tested using the following protocols. LNPs were produced as described in Example 1, with a molar ratio of 50% ionizable lipid (Compound 2, Compound 7, Compound 8 and Compound 1), 38% cholesterol, 9% DSPC, and 3% PEG-lipid (Compound 11). The RNP-LNPs were formulated with a final KC1 concentration of 21 mM in the 50mM MES pH 6.0 (Protocol 1) and 50mM Bis-Tris pH6.0 (Protocol 4) preformulation buffer, and diluted with water prior to the buffer exchange step. The RNP-LNPs were formulated with a RNP cargo prepared with a SpyCas9 protein (SEQ ID NO: 4), and gRNA (SEQ ID NO: 10), in a gRNA: Cas9 molar ratio of 2:1. FIG. 15A shows particle size trend for RNP-LNPs made with Protocol 1 and 4 for different ionizable lipids. Similar particle sizes were observed with both protocols for Compounds I, 2 and 7, however protocol 4 generated particles with smaller particle sizes compared to protocol 1 for Compound 8 RNP-LNPs. FIG. 15B shows % Encapsulation trend for RNP-LNPs made with Protocol 1 and 4 for different ionizable lipids. SignificantNTLA-0118USP01-RNP-LNP RFEM: 5640-106improvement in encapsulation was observed with Protocol 4. Comparable potency was seen between Protocol 1 and Protocol 4 across different compounds (FIG. 15C). Cells (PMH) were treated with RNP-LNPs in a 7-point, 3-fold dilution series starting at 1.44 ug of total LNP lipid / well. The cells were lysed 72 hours post-treatment for NGS analysis. Samples were run in duplicates.Example 15: Comparison of different LNP component ratios for particle sizes and encapsulation properties

[0443] LNPs were produced as described in Example 1 at three different molar ratios: 1) 50% ionizable lipid (Compound 2), 38% cholesterol, 9% DSPC, and 3% PEG-lipid (Compound 11); 2) 35% ionizable lipid (Compound 2), 47.5% cholesterol, 15% DSPC, and 2.5% PEG-lipid (Compound 11); and 3) 47% ionizable lipid (Compound 2), 28% cholesterol, 22.5% DSPC, and 2.5% PEG-lipid (Compound 11). The RNP-LNPs were formulated using Protocol 1, Protocol 3 and Protocol 4. The RNP-LNPs were formulated with a RNP cargo prepared with a SpyCas9 protein (SEQ ID NO: 4), and gRNA (SEQ ID NO: 10), in a gRNA: Cas9 molar ratio of 2:1. FIG. 16A show the particle size trend, RNP-LNPs across different compositions for a given protocol method. Protocol 4 generates desired particle size consistent across 3 compositions. FIG. 16B show % encapsulation trend for RNP-LNPs across different compositions for a given protocol method. Protocol 4 generated overall higher % encapsulation, with the 35 / 15 / 47.5 / 2.5 composition showing maximum encapsulation efficiency in general across all protocols.

[0444] RNP-LNPs formulated with Compound 2 as the ionizable lipid were produced as described in Example 1 at three different molar ratios using Protocol 1. The RNP-LNPs were formulated with a RNP cargo prepared with a SpyCas9 protein (SEQ ID NO: 4), and gRNA G000508 (which is SEQ ID NO: 120 in WO2020 / 198697, which is incorporated herein by reference), in a gRNA: Cas9 molar ratio of 2:1. The 35 / 15 / 47.5 / 2.5 composition showed improved % encapsulation while maintaining desired particle size. The biophysical characteristics for each formulation are shown in Table 27.NTLA-0118USP01-RNP-LNP RFEM: 5640-106

[0445] Table 27. Formulation biophysical characteristics.LNPID LNP Composition* gRNA: Cas9 Ratio Z-Ave [nm] PDI Num Ave [nm] RNA %ELNP-I 50 / 9 / 38 / 3 2:1 82 0.08 67 82% LNP-J Control 0:0 (Blank) 45 0.14 27 n / a LNP-K 45 / 7 / 45 / 3 2:1 81 0.07 71 86% LNP-L 0:0 (Blank) 45 0.13 34 n / a LNP-M 35 / 15 / 47.5 / 2.5 2d 85 0.03 76 93% LNP-N 0:0 (Blank) 52 0.01 49 n / a LNP-O 50 / 10 / 38.5 / 1.5 2:1 107 0.07 91 86% LNP-P 0:0 (Blank) 53 0.09 38 n / a LNP-Q 50 / 10 / 37.8 / 2.2 2:1 92 0.07 81 85% LNP-R 0:0 (Blank) 48 0.12 39 n / a ^Compound 2 / DSPC / Chol / C13 EthNTLA-0118USP01-RNP-LNP RFEM: 5640-106Example 16: In vivo editing in Non-Human Primates (NHP) using RNP-LNPs and RNA-LNPs

[0446] An in vivo experiment was conducted in Cynomolgus Macaque monkeys to assess editing efficiencies at the TTR locus, using RNP-LNP and RNA-LNP. The data show that the RNP-LNPs of the present disclosure surprisingly are able to efficiently deliver the cargo to target tissues and achieved editing efficiency results comparable to RNA-LNP.

[0447] LNPs were produced as described in Example 1 at a molar ratio of 50% ionizable lipid (Compound 7), 38% cholesterol, 9% DSPC, and 3% PEG-lipid (Compound 11). The RNP-LNPs with Compound 7 as the ionizable lipid were formulated using Protocol 2 at TFF scale process.

[0448] The RNP-LNPs were formulated with a RNP cargo prepared with a SpyCas9 protein (SEQ ID NO: 4), and gRNA (SEQ ID NO: 10), in a gRNA: Cas9 molar ratio of 2: 1.

[0449] The RNA-LNPs were formulated with a lipid amine to RNA phosphate (N: P) molar ratio of about 6, and a RNA cargo comprising a gRNA targeting the cyno TTR locus (SEQ ID NO: 10), and a mRNA encoding a SpyCas9 protein (SEQ ID NO: 2), in a gRNA:mRNA mass ratio of 1:2.

[0450] Animals (N=3 per group) were injected with a dose of 0.5 mpk or 1.5 mpk of RNA-LNP or RNP-LNP (Table 28). Animals were euthanized 93 days post-injection. A negative control group consisted of animals injected with TSS (vehicle solution). Liver tissue was collected for NGS analysis at necropsy. Serum from blood was collected for serum TTR level analysis across the study via a LC-MS method.

[0451] TTR levels in the semm were monitored over 92 days and quantified as a percent of baseline serum TTR for monkeys treated with TSS saline control or a dose of 0.5 mpk or 1.5 mpk of RNA-LNP or RNP-LNP (FIG. 17A, Table 29). Editing efficiencies obtained at the TTR locus in liver tissue of monkeys treated with TSS saline control or a dose of 0.5 mpk or 1.5 mpk of RNA-LNP or RNP-LNP are shown in FIGS. 17B- 17E.

[0452] Table 28: In vivo Study Design.Group Internal LNP ID Dose (mpk) Number of Animals1 Vehicle - 3 (IM, 2F)2 Compound 7 RNA LNP 0.5 3 (IM, 2F)3 Compound 7 RNA LNP 1.5 3 (IM, 2F)NTLA-0118USP01-RNP-LNP RFEM: 5640-1064 Compound 7 RNP LNP 0.5 3 (IM, 2F)5 Compound 7 RNP LNP 1.5 3 (IM, 2F)

[0453] Table 29. Knockdown from Baseline - Group AveragesGroup 20.5 mpk Group 3 Group 4 Group 5 Timepoint Group 1 mRNA- il.5 mpk 0.5 mpk 1.5 mpk(Days) |TSS LNP niRNA-I. NP RNP-LNP jRNP-LNP8 |-1% 49% |79% 14% ]65%15 |-1% 69% |94% 31% |85%29,4% 81% |95% 53% |88%64 |31% 72% |95% 35% ^88%92 |37% 76% |95% 36% |89%NTLA-0118USP01-RNP-LNP RFEM: 5640-106

[0454] Table 30 - SEQUENCESDescription Sequence SEQ ID NO. modified G000650 sgRNA targeting human mG*mA*mC*AAGCACCAGAAAGACCAGUUUUAGAmGmCmUmAmGmAmAmAmUmA 1B2M- mGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mUmodified sequence (m: 2'OMe modificationof a nucleotide; *: phosphorithioate bondbetween consecutive nucleotides)mRNA-A (mRNA) GGGAAGCUCAGAAUAAACGCUCAACUUUGGCCGGAUCUGCCACCAUGGACAAGA 2AGUACUCCAUCGGCCUGGACAUCGGCACCAACUCCGUGGGCUGGGCCGUGAUCAC CGACGAGUACAAGGUGCCCUCCAAGAAGUUCAAGGUGCUGGGCAACACCGACCG GCACUCCAUCAAGAAGAACCUGAUCGGCGCCCUGCUGUUCGACUCCGGCGAGACC GCCGAGGCCACCCGGCUGAAGCGGACCGCCCGGCGGCGGUACACCCGGCGGAAGA ACCGGAUCUGCUACCUGCAGGAGAUCUUCUCCAACGAGAUGGCCAAGGUGGACG ACUCCUUCUUCCACCGGCUGGAGGAGUCCUUCCUGGUGGAGGAGGACAAGAAGC ACGAGCGGCACCCCAUCUUCGGCAACAUCGUGGACGAGGUGGCCUACCACGAGA AGUACCCCACCAUCUACCACCUGCGGAAGAAGCUGGUGGACUCCACCGACAAGGC CGACCUGCGGCUGAUCUACCUGGCCCUGGCCCACAUGAUCAAGUUCCGGGGCCAC UUCCUGAUCGAGGGCGACCUGAACCCCGACAACUCCGACGUGGACAAGCUGUUC AUCCAGCUGGUGCAGACCUACAACCAGCUGUUCGAGGAGAACCCCAUCAACGCCU CCGGCGUGGACGCCAAGGCCAUCCUGUCCGCCCGGCUGUCCAAGUCCCGGCGGCU GGAGAACCUGAUCGCCCAGCUGCCCGGCGAGAAGAAGAACGGCCUGUUCGGCAA CCUGAUCGCCCUGUCCCUGGGCCUGACCCCCAACUUCAAGUCCAACUUCGACCUG GCCGAGGACGCCAAGCUGCAGCUGUCCAAGGACACCUACGACGACGACCUGGACA ACCUGCUGGCCCAGAUCGGCGACCAGUACGCCGACCUGUUCCUGGCCGCCAAGAA CCUGUCCGACGCCAUCCUGCUGUCCGACAUCCUGCGGGUGAACACCGAGAUCACC AAGGCCCCCCUGUCCGCCUCCAUGAUCAAGCGGUACGACGAGCACCACCAGGACC UGACCCUGCUGAAGGCCCUGGUGCGGCAGCAGCUGCCCGAGAAGUACAAGGAGA UCUUCUUCGACCAGUCCAAGAACGGCUACGCCGGCUACAUCGACGGCGGCGCCUCCCAGGAGGAGUUCUACAAGUUCAUCAAGCCCAUCCUGGAGAAGAUGGACGGCACNTLA-0118USP01-RNP-LNP RFEM: 5640-106Description Sequence SEQ ID NO.CGAGGAGCUGCUGGUGAAGCUGAACCGGGAGGACCUGCUGCGGAAGCAGCGGAC CUUCGACAACGGCUCCAUCCCCCACCAGAUCCACCUGGGCGAGCUGCACGCCAUC CUGCGGCGGCAGGAGGACUUCUACCCCUUCCUGAAGGACAACCGGGAGAAGAUC GAGAAGAUCCUGACCUUCCGGAUCCCCUACUACGUGGGCCCCCUGGCCCGGGGCA ACUCCCGGUUCGCCUGGAUGACCCGGAAGUCCGAGGAGACCAUCACCCCCUGGAA CUUCGAGGAGGUGGUGGACAAGGGCGCCUCCGCCCAGUCCUUCAUCGAGCGGAU GACCAACUUCGACAAGAACCUGCCCAACGAGAAGGUGCUGCCCAAGCACUCCCUG CUGUACGAGUACUUCACCGUGUACAACGAGCUGACCAAGGUGAAGUACGUGACC GAGGGCAUGCGGAAGCCCGCCUUCCUGUCCGGCGAGCAGAAGAAGGCCAUCGUG GACCUGCUGUUCAAGACCAACCGGAAGGUGACCGUGAAGCAGCUGAAGGAGGAC UACUUCAAGAAGAUCGAGUGCUUCGACUCCGUGGAGAUCUCCGGCGUGGAGGAC CGGUUCAACGCCUCCCUGGGCACCUACCACGACCUGCUGAAGAUCAUCAAGGACA AGGACUUCCUGGACAACGAGGAGAACGAGGACAUCCUGGAGGACAUCGUGCUGA CCCUGACCCUGUUCGAGGACCGGGAGAUGAUCGAGGAGCGGCUGAAGACCUACG CCCACCUGUUCGACGACAAGGUGAUGAAGCAGCUGAAGCGGCGGCGGUACACCG GCUGGGGCCGGCUGUCCCGGAAGCUGAUCAACGGCAUCCGGGACAAGCAGUCCG GCAAGACCAUCCUGGACUUCCUGAAGUCCGACGGCUUCGCCAACCGGAACUUCAU GCAGCUGAUCCACGACGACUCCCUGACCUUCAAGGAGGACAUCCAGAAGGCCCAG GUGUCCGGCCAGGGCGACUCCCUGCACGAGCACAUCGCCAACCUGGCCGGCUCCC CCGCCAUCAAGAAGGGCAUCCUGCAGACCGUGAAGGUGGUGGACGAGCUGGUGA AGGUGAUGGGCCGGCACAAGCCCGAGAACAUCGUGAUCGAGAUGGCCCGGGAGA ACCAGACCACCCAGAAGGGCCAGAAGAACUCCCGGGAGCGGAUGAAGCGGAUCG AGGAGGGCAUCAAGGAGCUGGGCUCCCAGAUCCUGAAGGAGCACCCCGUGGAGA ACACCCAGCUGCAGAACGAGAAGCUGUACCUGUACUACCUGCAGAACGGCCGGG ACAUGUACGUGGACCAGGAGCUGGACAUCAACCGGCUGUCCGACUACGACGUGG ACCACAUCGUGCCCCAGUCCUUCCUGAAGGACGACUCCAUCGACAACAAGGUGCU GACCCGGUCCGACAAGAACCGGGGCAAGUCCGACAACGUGCCCUCCGAGGAGGU GGUGAAGAAGAUGAAGAACUACUGGCGGCAGCUGCUGAACGCCAAGCUGAUCACCCAGCGGAAGUUCGACAACCUGACCAAGGCCGAGCGGGGCGGCCUGUCCGAGCUNTLA-0118USP01-RNP-LNP RFEM: 5640-106Description Sequence SEQ ID NO.GGACAAGGCCGGCUUCAUCAAGCGGCAGCUGGUGGAGACCCGGCAGAUCACCAA GCACGUGGCCCAGAUCCUGGACUCCCGGAUGAACACCAAGUACGACGAGAACGA CAAGCUGAUCCGGGAGGUGAAGGUGAUCACCCUGAAGUCCAAGCUGGUGUCCGA CUUCCGGAAGGACUUCCAGUUCUACAAGGUGCGGGAGAUCAACAACUACCACCA CGCCCACGACGCCUACCUGAACGCCGUGGUGGGCACCGCCCUGAUCAAGAAGUAC CCCAAGCUGGAGUCCGAGUUCGUGUACGGCGACUACAAGGUGUACGACGUGCGG AAGAUGAUCGCCAAGUCCGAGCAGGAGAUCGGCAAGGCCACCGCCAAGUACUUC UUCUACUCCAACAUCAUGAACUUCUUCAAGACCGAGAUCACCCUGGCCAACGGCG AGAUCCGGAAGCGGCCCCUGAUCGAGACCAACGGCGAGACCGGCGAGAUCGUGU GGGACAAGGGCCGGGACUUCGCCACCGUGCGGAAGGUGCUGUCCAUGCCCCAGG UGAACAUCGUGAAGAAGACCGAGGUGCAGACCGGCGGCUUCUCCAAGGAGUCCA UCCUGCCCAAGCGGAACUCCGACAAGCUGAUCGCCCGGAAGAAGGACUGGGACCC CAAGAAGUACGGCGGCUUCGACUCCCCCACCGUGGCCUACUCCGUGCUGGUGGUG GCCAAGGUGGAGAAGGGCAAGUCCAAGAAGCUGAAGUCCGUGAAGGAGCUGCUG GGCAUCACCAUCAUGGAGCGGUCCUCCUUCGAGAAGAACCCCAUCGACUUCCUGG AGGCCAAGGGCUACAAGGAGGUGAAGAAGGACCUGAUCAUCAAGCUGCCCAAGU ACUCCCUGUUCGAGCUGGAGAACGGCCGGAAGCGGAUGCUGGCCUCCGCCGGCG AGCUGCAGAAGGGCAACGAGCUGGCCCUGCCCUCCAAGUACGUGAACUUCCUGU ACCUGGCCUCCCACUACGAGAAGCUGAAGGGCUCCCCCGAGGACAACGAGCAGAA GCAGCUGUUCGUGGAGCAGCACAAGCACUACCUGGACGAGAUCAUCGAGCAGAU CUCCGAGUUCUCCAAGCGGGUGAUCCUGGCCGACGCCAACCUGGACAAGGUGCU GUCCGCCUACAACAAGCACCGGGACAAGCCCAUCCGGGAGCAGGCCGAGAACAUC AUCCACCUGUUCACCCUGACCAACCUGGGCGCCCCCGCCGCCUUCAAGUACUUCG ACACCACCAUCGACCGGAAGCGGUACACCUCCACCAAGGAGGUGCUGGACGCCAC CCUGAUCCACCAGUCCAUCACCGGCCUGUACGAGACCCGGAUCGACCUGUCCCAG CUGGGCGGCGACGGCGGCGGCUCCCCCAAGAAGAAGCGGAAGGUGUGACUAGCA CCAGCCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUUACA CUUUACAAAAUGUUGUCCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUUUCUUCACAUUCUCUCGAGAAAAAAAAAAAAAAAAAAAAAAAAAAANTLA-0118USP01-RNP-LNP RFEM: 5640-106Description Sequence SEQ ID NO.AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAUCUAGmRNA-A (ORF) AUGGACAAGAAGUACUCCAUCGGCCUGGACAUCGGCACCAACUCCGUGGGCUGG 3GCCGUGAUCACCGACGAGUACAAGGUGCCCUCCAAGAAGUUCAAGGUGCUGGGC AACACCGACCGGCACUCCAUCAAGAAGAACCUGAUCGGCGCCCUGCUGUUCGACU CCGGCGAGACCGCCGAGGCCACCCGGCUGAAGCGGACCGCCCGGCGGCGGUACAC CCGGCGGAAGAACCGGAUCUGCUACCUGCAGGAGAUCUUCUCCAACGAGAUGGC CAAGGUGGACGACUCCUUCUUCCACCGGCUGGAGGAGUCCUUCCUGGUGGAGGA GGACAAGAAGCACGAGCGGCACCCCAUCUUCGGCAACAUCGUGGACGAGGUGGC CUACCACGAGAAGUACCCCACCAUCUACCACCUGCGGAAGAAGCUGGUGGACUCC ACCGACAAGGCCGACCUGCGGCUGAUCUACCUGGCCCUGGCCCACAUGAUCAAGU UCCGGGGCCACUUCCUGAUCGAGGGCGACCUGAACCCCGACAACUCCGACGUGGA CAAGCUGUUCAUCCAGCUGGUGCAGACCUACAACCAGCUGUUCGAGGAGAACCC CAUCAACGCCUCCGGCGUGGACGCCAAGGCCAUCCUGUCCGCCCGGCUGUCCAAG UCCCGGCGGCUGGAGAACCUGAUCGCCCAGCUGCCCGGCGAGAAGAAGAACGGCC UGUUCGGCAACCUGAUCGCCCUGUCCCUGGGCCUGACCCCCAACUUCAAGUCCAA CUUCGACCUGGCCGAGGACGCCAAGCUGCAGCUGUCCAAGGACACCUACGACGAC GACCUGGACAACCUGCUGGCCCAGAUCGGCGACCAGUACGCCGACCUGUUCCUGG CCGCCAAGAACCUGUCCGACGCCAUCCUGCUGUCCGACAUCCUGCGGGUGAACAC CGAGAUCACCAAGGCCCCCCUGUCCGCCUCCAUGAUCAAGCGGUACGACGAGCAC CACCAGGACCUGACCCUGCUGAAGGCCCUGGUGCGGCAGCAGCUGCCCGAGAAGU ACAAGGAGAUCUUCUUCGACCAGUCCAAGAACGGCUACGCCGGCUACAUCGACG GCGGCGCCUCCCAGGAGGAGUUCUACAAGUUCAUCAAGCCCAUCCUGGAGAAGA UGGACGGCACCGAGGAGCUGCUGGUGAAGCUGAACCGGGAGGACCUGCUGCGGA AGCAGCGGACCUUCGACAACGGCUCCAUCCCCCACCAGAUCCACCUGGGCGAGCU GCACGCCAUCCUGCGGCGGCAGGAGGACUUCUACCCCUUCCUGAAGGACAACCGG GAGAAGAUCGAGAAGAUCCUGACCUUCCGGAUCCCCUACUACGUGGGCCCCCUG GCCCGGGGCAACUCCCGGUUCGCCUGGAUGACCCGGAAGUCCGAGGAGACCAUCA CCCCCUGGAACUUCGAGGAGGUGGUGGACAAGGGCGCCUCCGCCCAGUCCUUCAU CGAGCGGAUGACCAACUUCGACAAGAACCUGCCCAACGAGAAGGUGCUGCCCAA GCACUCCCUGCUGUACGAGUACUUCACCGUGUACAACGAGCUGACCAA...

Claims

NTLA-0118USP01-RNP-LNP RFEM: 5640-106CLAIMSWhat is claimed is:

1. A lipid nanoparticle (LNP) comprising:(i) a lipid component comprising:a) an ionizable lipid having a pKa of about 6.0 to about 7.0;b) a helper lipid;c) a neutral lipid; andd) a structural PEG-lipid; and(ii) a cargo comprising a ribonucleoprotein (RNP) complex comprising a Type II Cas nuclease polypeptide (Cas nuclease) and a guide RNA (gRNA), wherein a molar ratio of gRNA: Cas nuclease is in a range of about 1:1 to about 16:1, and wherein the Type II Cas nuclease and the gRNA associate in the RNP complex.

2. A lipid nanoparticle (LNP) comprising:(i) a lipid component comprising:a) a structural PEG-lipid;b) a helper lipid;c) a neutral lipid;d) an anchor PEG-lipid comprising a coupling moiety; ande) an ionizable lipid having a pKa of about 6.0 to 7.0; and(ii) a cargo comprising a ribonucleoprotein (RNP) complex comprising a Type II Cas nuclease polypeptide (Cas nuclease) and a guide RNA (gRNA), wherein a molar ratio of gRNA: Cas nuclease is in a range of about 1: 1 to about 16:1, and wherein the Type II Cas nuclease and the gRNA associate in the RNP complex.

3. The LNP of claim 1 or claim 2, wherein the ionizable lipid has a pKa of about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0.

4. The LNP of any preceding claim, wherein the LNP has a particle hydrodynamic size Z- average of 120 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, or about 60 nm.

5. The LNP of any preceding claim, wherein the LNP has a particle hydrodynamic size Z- average of 60-80 nm, 80-100 nm or 100-120 nm.NTLA-0118USP01-RNP-LNP RFEM: 5640-1066. The LNP of any preceding claim, wherein the LNP has a polydispersity index (PDI) in a range of 0.001-0.2, 0.001-0.15, 0.001-0.1, 0.03-0.7, 0.07-0.10, 0.11-0.15, 0.15-0.2, 0.003- 0.009, 0.01-0.02, or 0.001-0.05.

7. The LNP of any preceding claim, wherein the lipid component comprises:a) the helper lipid in an amount from about 30 to about 50 mol % of the lipid component; b) the neutral lipid in an amount from about 0 to about 25 mol % of the lipid component; c) the structural PEG-lipid in an amount from about 0.5 to about 5 mol % of the lipid component; andd) the ionizable lipid in an amount of from about 30 to about 60 mol % of the lipid component.

8. The LNP of any preceding claim, wherein the ionizable lipid is present in an amount from about 32 to about 40 mol % of the lipid component.

9. The LNP of any preceding claim, wherein the neutral lipid is present in an amount from about 5 to about 20 mol % of the lipid component.

10. The LNP of any preceding claim, wherein the ionizable lipid, the neutral lipid, the helper lipid, and the structural PEG-lipid are in a ratio of 32 to 40: 10 to 20: 40 to 50: 1.5 to 3.5.

11. The LNP of any preceding claim, wherein the ionizable lipid, the neutral lipid, the helper lipid, and the structural PEG-lipid are in a ratio of 35:15:47.5:2.5.

12. The LNP of any one of claims 1-7, wherein the ionizable lipid, the neutral lipid, the helper lipid, and the structural PEG-lipid are in a ratio of 50:9:38:3, a ratio of 45:7:45:3, a ratio of 50:10:38.5:1.5, or a ratio of 50:10:37.8:2.2.

13. The LNP of any one of claims 1-3, wherein the lipid component comprises:a) the helper lipid in an amount from about 25 to about 65 mol % of the lipid component; b) the neutral lipid in an amount from about 0 to about 25 mol % of the lipid component; c) the structural PEG-lipid in an amount from about 0.5 to about 5 mol % of the lipid component; andd) the ionizable lipid in an amount of from about 40 to about 60 mol % of the lipidcomponent.NTLA-0118USP01-RNP-LNP RFEM: 5640-10614. The LNP of any one of claims 1 -6 and 13, wherein the ionizable lipid, the neutral lipid, the helper lipid, and the structural PEG-lipid are in a ratio of 40 to 50: 20 to 25: 25 to 35: 1.5 to 3.5.

15. The LNP of any one of claims 1-6 and 13-14, wherein the ionizable lipid, the neutral lipid, the helper lipid, and the structural PEG-lipid are in a ratio of 47:22.5:28:2.5.

16. The LNP of any one of claims 1-3, wherein lipid component comprises:a) the helper lipid in an amount from about 27 to about 62 mol % or about 30 to about 60 mol% of the lipid component;b) the neutral lipid in an amount from about 2 to about 23 mol % or about 4 to about 20 mol% of the lipid component;c) the structural PEG-lipid in an amount from about 0.7 to about 5 mol % or about 0.8 to about 5 mol% of the lipid component; andd) the ionizable lipid in an amount of from about 42 to about 58 mol % or about 44 to about 56 mol% of the lipid component.

17. The LNP of any preceding claim, wherein the ratio of gRNA: Cas nuclease is in a range of about 1.1:1 to about 12:1, about 1.2:1 to about 10:1, about 1.4:1 to about 8:1, about 1.6:1 to about 6:1, about 1.8:1 to about 5:1, about 2:1 to 4:1, or about 2:1.

18. The LNP of any preceding claim, wherein the LNP has a molar ratio of ionizable lipid to Type II Gas nuclease polypeptide of 4,000:1 to 2,400:1, or about 3,800:1, or 3,792:1.

19. The LNP of claim 18, wherein the LNP has a molar ratio of ionizable lipid to Type II Cas nuclease polypeptide of 4,000:1 to 2,400:1.

20. The LNP of claim 19, wherein the LNP has a molar ratio of ionizable lipid to Type II Cas nuclease polypeptide of about 3,800:1 or about 3792:1.

21. The LNP of any one of claims 1-3, wherein of the LNP has a number average particle size of about 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, or about 60 nm.

22. The LNP of any one of claims 1-3, wherein the LNP has a number average particle size of 60-80 nm, 80-100 nm, or 100-120 nm.NTLA-0118USP01-RNP-LNP RFEM: 5640-10623. The LNP of any preceding claim, wherein the Type II Cas nuclease polypeptide is a CRISPR-Cas9 polypeptide.

24. The LNP of any preceding claim, wherein the Type II Cas nuclease polypeptide is genetically fused to a peptide tag, a protein, or both.

25. The LNP of any preceding claim, wherein the Type II Cas nuclease polypeptide is modified.

26. The LNP of claim 25, wherein the modification comprises a chemical modification.

27. The LNP of any preceding claim, wherein the ionizable lipid is a compound or a salt thereof represented by a structure selected from the group consisting of:O, O'-(2-((((2-(diethylamino)ethyl)carbamoyl)oxy)methyl)propane-l,3-diyl) di(heptadecan-9-yl) diglutarate,O, O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-l,3-diyl) di(heptadecan-9-yl) diglutarate,NTLA-0118USP01-RNP-LNP RFEM: 5640-1063-(((2-(azepan-1-yl)ethyl)carbamoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl heptadecan-9-yl glutarate, and(9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate.

28. The LNP of any preceding claim, 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, l-[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), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (PEG2K-DMPE), l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol 2000 (PEG2K-DMG), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2K-DSPE), 1,2- distearoyl-sn-glycerol-[methoxy(polyethylene glycol)-2000] (PEG2K-DSG), polyethylene glycol)-2000-dimethacrylate (PEG2K-DMA), l,2-distearyloxypropyl-3- amine-N-[methoxy(poly ethylene glycol)-2000] (PEG2K-DSA), methoxy-PEG2000- carbamoyl-l,2-tetradecyoxypropylamine (C14 Ether), methoxy-PEG2000-carbamoyl-l,2-NTLA-0118USP01-RNP-LNP RFEM: 5640-106tridecyoxypropylamine (C13 Ether), methoxy-PEG2000-carbamoyl-l,2- didecyoxypropylamine (Cl 2 Ether), or a combination thereof.

29. The LNP of claim 28, wherein the structural PEG-lipid is L2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol 2000 (PEG2K-DMG), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2K-DSPE), methoxy-PEG2000-carbamoyl-l,2-didecyoxypropylamine (Cl 2 Ether), methoxy - PEG2000-carbamoyl-l,2-tetradecyoxypropylamine (C14 Ether), methoxy-PEG2000- carbamoyl-l,2-tridecyoxypropylamine (Cl 3 Ether), or a combination thereof.

30. 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), l,2-diarachidoyl-sn-glycero-3- phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1 -myristoyl-2-palmitoyl phosphatidylcholine (MPPC), l-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), lysophosphatidylethanolamine, or a combination thereof.

31. The LNP of claim 30, wherein the neutral lipid is DSPC or DMPE.

32. The LNP of claim 31, wherein the neutral lipid is DSPC.

33. The LNP of any preceding claim, wherein the LNP does not include l,2-dioleoyl-3- trimethylammonium-propane (DOTAP).NTLA-0118USP01-RNP-LNP RFEM: 5640-10634. The LNP of any preceding claim, wherein the helper lipid is cholesterol, 5- heptadecylresorcinol, or cholesterol hemisuccinate.

35. The LNP of claim 34, wherein the helper lipid is cholesterol.

36. The LNP of any one of claims 1-35, wherein the lipid component comprises:(i) 3-(((2-(azepan- 1 -yl)ethyl)carbamoyl)oxy)-2-((((9Z, 12Z)-octadeca-9, 12- dienoyl)oxy)methyl)propyl heptadecan-9-yl glutarate; DSPC; cholesterol; and C13 Ether structural PEG-lipid;(ii) O, O'-(2-((((2-(diethylamino)ethyl)carbamoyl)oxy)methyl)propane- 1,3 -diyl) di(heptadecan-9-yl) diglutarate; DSPC; cholesterol; and C13 Ether structural PEG-lipid;(iii) 2-((4-(((2- (ethyl(methyl)amino)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propane-l,3-diyl (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate); DSPC; cholesterol; and C13 Ether stmctural PEG-lipid; or(iv) O, O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane- 1, 3 -diyl) di(heptadecan-9-yl) diglutarate; DSPC; cholesterol; and Cl 3 Ether stmctural PEG-lipid.

37. The LNP of claim 36, wherein the ionizable lipid, the neutral lipid, the helper lipid, and the stmctural PEG-lipid are in a ratio of about 50:9:38:3.

38. The LNP of claim 36, wherein the ionizable lipid, the neutral lipid, the helper lipid, and the stmctural PEG-lipid are in a ratio of about 35: 15:47.5:2.

539. The LNP of claim 34 or claim 35, wherein the ratio of gRNA: Cas nuclease is about 2:1.

40. The LNP of any one of claims 36-39, wherein the LNP has a molar ratio of ionizable lipid to Type II Cas nuclease polypeptide of 4,000:1 to 2,400:1, or about 3,800:1.

41. The LNP of any one of claims 36-39, wherein the LNP has a molar ratio of ionizable lipid to Type II Cas nuclease polypeptide of 3792:1.NTLA-0118USP01-RNP-LNP RFEM: 5640-10642. The LNP of any one of claims 1 and 3-41, wherein the lipid component further comprises an anchor PEG-lipid comprising a first coupling moiety.

43. The LNP of claim 42, wherein the anchor PEG-lipid is selected from DSPE-PEG(1000), DSPE-PEG(2000), DSPE-PEG(3400), DSPE-PEG(5000), or a combination thereof.

44. The LNP of claim 43, wherein the anchor PEG-lipid and the 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(IOOO) 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.

45. The LNP of any one of claims 42-43, wherein the lipid component comprises:a) the helper lipid in an amount from about 25 to about 65 mol % of the lipid component:b) the neutral lipid in an amount from about 0 to about 25 mol % of the lipid component;c) the structural PEG-lipid in an amount from about 0.5 to about 5 mol % of the lipid component:d) the ionizable lipid in an amount of from about 40 to about 60 mol % of the lipid component; ande) the anchor PEG-lipid in an amount from about 0.001 to about 1.5 mol % of the lipid component.

46. The LNP of any one of claims 42-45, wherein a targeting ligand is attached to the first coupling moiety via a second coupling moiety.

47. The LNP of any one of claims 42-46, wherein the amount of the anchor PEG-lipid is the same as an anchor PEG-lipid density.

48. The LNP of any one of claims 42-46, wherein the targeting ligand has a density that is the same or less than the anchor PEG-lipid density.NTLA-0118USP01-RNP-LNP RFEM: 5640-10649. The LNP of claim 48, wherein the targeting ligand is selected from an antibody, an antibody fragment, a small molecule, or a peptide.

50. The LNP of claim 49, wherein the targeting ligand is an antibody.

51. The LNP of claim 50, wherein the anchor PEG- lipid is in an amount from about 0.005 to about 0.045 mol% of the lipid component.

52. The LNP of claim 49, wherein the targeting ligand is an antibody fragment.

53. The LNP of claim 52, 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, a disulfide-linked Fv (sdFv), a Fd fragment consisting of VH and CHI 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.

54. The LNP of any one of claims 52 or 53, wherein the anchor PEG-lipid is in an amount from about 0.005 to about 0.075 mol% of the lipid component.

55. The LNP of claim 49, wherein the targeting ligand is a peptide.

56. The LNP of claim 55, wherein the anchor PEG-lipid is in an amount from about 0.1 to about 0.9 mol% of the lipid component.

57. The LNP of any one of claims 42-49, wherein lipid component comprises:a) the helper lipid in an amount from about 27 to about 62 mol % or about 30 to about 60 mol% of the lipid component;b) the neutral lipid in an amount from about 2 to about 23 mol % or about 4 to about 20 mol% of the lipid component;c) the structural PEG-lipid in an amount from about 0.7 to about 5 mol % or about 0.8 to about 5 mol% of the lipid component;d) the ionizable lipid in an amount of from about 42 to about 58 mol % or about 44 to about 56 mol% of the lipid component; andNTLA-0118USP01-RNP-LNP RFEM: 5640-106e) the anchor PEG-lipid in an amount from about 0.015 to about 1 mol% or about 0.02 to about 0.9 mol % of the lipid component.

58. The LNP of claim 57, wherein the anchor PEG-lipid comprises about 0.05 to about 0.2 mol % of the lipid component.

59. The LNP of claim 58, wherein the anchor PEG-lipid comprises about 0.03 to about 0.1 mol % of the lipid component.

60. The LNP of claim 59, wherein the anchor PEG-lipid comprises about 0.25 to about 0.7 mol % of the lipid component.

61. The LNP of claim 60, wherein the anchor PEG-lipid comprises about 0.042 to 0.48 mol% or about 0.045 mol%.

62. The LNP of any one of claims 42-61, wherein the lipid component comprises: 3-(((2- (azepan- 1 -yl)ethyl)carbamoyl)oxy)-2-((((9Z, 12Z)-octadeca-9, 12- dienoyl)oxy)methyl)propyl heptadecan-9-yl glutarate; DSPC; cholesterol; and C13 Ether structural PEG-lipid and DSPE-PEG-SpyTag anchor PEG-lipid.

63. The LNP of any one of claims 42-62, wherein the ionizable lipid, the neutral lipid, the helper lipid, the structural PEG-lipid, and the anchor PEG-lipid are in a ratio of 50:9:38:2.955:0.045.

64. The LNP of any one of claims 42-63, wherein the ratio of gRNA: Cas nuclease is about 2:1.

65. The LNP of any one of claims 42-64, wherein the LNP has a molar ratio of ionizable lipid to Type II Cas nuclease polypeptide of 4,000:1 to 2,400:1, or about 3,800:1, or 3,792:1.

66. The LNP of any one of claims 42-65, 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.

67. The LNP of any one of claims 46-66, wherein first coupling moiety and the second coupling moiety form a covalent bond.NTLA-0118USP01-RNP-LNP RFEM: 5640-10668. The LNP of any one of claims 46-61 and 63-66, wherein first coupling moiety and the second coupling moiety form a non-covalent bond.

69. The LNP of any one of claims 44-61 and 63-66, 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.

70. The LNP of any one of claims 44-61 and 63-66, 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.

71. The LNP of any one of claims 42-70, wherein a molar ratio of the structural PEG-lipid to the anchor PEG-lipid is between about 2:1 to about 300:1.

72. The LNP of claim 71, wherein a molar ratio of the structural PEG-lipid to the anchor PEG-lipid is between about 2:1 to about 300:1, about 3:1 to about 100:1, or about 5:1 to about 50:1.

73. The LNP of any preceding claim, wherein the LNP has a gene editing potency of 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold higher than a control composition’s gene editing potency, wherein the control composition comprises (i) a mixture of the Cas9 / gRNA RNP that is not encapsulated in an LNP and (ii) a corresponding LNP that lacks the cargo encapsulated therein.

74. The LNP of claim 73, wherein the LNP has a 20-fold to 100-fold higher gene editing potency than the control composition.

75. The LNP of any one of claims 42-74, wherein the targeting ligand targets a liver cell.

76. The LNP of any one of claims 42-74, wherein the targeting ligand targets non-liver cell, e.g., a bone marrow cell or a bone marrow derived cell.NTLA-0118USP01-RNP-LNP RFEM: 5640-10677. The LNP of claim 76, wherein the bone marrow cell or bone marrow derived cell is selected from a Hematopoietic Stem Cell (HSC), Multipotent Progenitor Cell (MPP), Common Myeloid Progenitor (CMP), Common Lymphoid Progenitor (CLP), Granulocyte-Macrophage Progenitor (GMP), Megakaryocyte-Erythroid Progenitor (MEP), Erythroid Precursor Cells (Erythroblasts), Meg akary oblast, Granulocyte Progenitors (Myeloblasts), Monoblast, Promyelocyte, Myelocyte, Metamyelocyte, Band Cells, Mature Granulocytes, Mature Monocytes, Erythrocytes, Mature Megakaryocytes, Plasma Cells, B Lymphocytes, or T Lymphocytes.

78. The LNP of claim 76 or claim 77, wherein the bone marrow cell or bone marrow derived cell marker is CD34, CD38, CD45, CD117, CD123, or CD135.

79. The LNP of any preceding claim, wherein the cargo further comprises a nucleic acid of interest.

80. The LNP of claim 79, wherein the nucleic acid of interest comprises a template nucleic acid.

81. The LNP of claim 80, wherein the template nucleic acid comprises an endogenous sequence of a cell or the template nucleic acid comprises an exogenous sequence.

82. The LNP of claim 81, wherein the exogenous sequence comprises a protein or RNA coding sequence operably linked to an endogenous or exogenous promoter sequence, an exon sequence, an intron sequence, a regulatory sequence, a transcriptional control sequence, a translational control sequence, a splicing site, or a non-coding sequence.

83. The LNP of claim 80, wherein the template nucleic acid comprises a nucleic acid sequence that is complementary to at least a portion of a target nucleic acid.

84. The LNP of any one of claims 80-83, wherein the template nucleic acid comprises a ssDNA or dsDNA containing flanking invert-terminal repeat (ITR) sequences, a plasmid, minicircle, nanocircle, or a PCR product.

85. The LNP of any preceding claim, wherein the gRNA is a modified gRNA.NTLA-0118USP01-RNP-LNP RFEM: 5640-10686. The LNP of any preceding claim, wherein the gRNA is a single-guide RNA (sgRNA) or a dual-guide RNA (dgRNA).

87. The LNP of any preceding claim, wherein the gRNA is 60 to 200 nucleotides in length or 60 to 150 nucleotides in length.

88. The LNP of any preceding claim, wherein the gRNA comprises a 3’ end modification, a modification in the hairpin region, and a 5’ end modification.

89. The LNP of claim 88, 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.

90. The LNP of any one of claims 88 or 89, wherein the 3’ and / or 5’ end modification comprises a protective end modification, such as a modified nucleotide selected from 2’- O-methyl (2'-0Me) modified nucleotide, 2’-O-(2-methoxyethyl) (2’- O-MOE) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, an inverted abasic modified nucleotide, or combinations thereof.

91. The LNP of claim 90, wherein the modification in the hairpin region comprises a modified nucleotide selected from 2’-O-methyl (2’-0Me) modified nucleotide, a 2’- fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, or combinations thereof.

92. A composition comprising the LNP of any preceding claim, wherein the composition comprises a carrier.

93. The composition of claim 92, wherein the composition comprises a buffer having a pH of about 6.5 to about 8.0 at 25 °C.

94. The composition of claim 93, wherein the composition comprises a buffer having a pH of about 7.0 to about 7.8, or a pH of about 7.4 at 25 °C.

95. The composition of any one of claims 92-94, wherein the composition further comprises a cryoprotectant, a tonicity modifying agent, or a combination thereof.NTLA-0118USP01-RNP-LNP RFEM: 5640-10696. The composition of claim 95, wherein the composition comprises a cryoprotectant.

97. The composition of claim 96, wherein the cryoprotectant comprises sucrose or trehalose.

98. The composition of any one of claims 95-97, wherein the cryoprotectant is from 5-12% (v / v) of the composition.

99. The composition of any one of claims 92-98, wherein the encapsulation efficiency is about 80% to about 100%, about 85% to 100%, about 90% to 100%, about 95% to about 100%, about 96%, about 96%, about 98%, or about 99%.

100. The composition of claim 92-99, wherein the encapsulation efficiency is about 80% to about 99%.

101. The composition of any one of claims 92-100, wherein the LNP has a particle hydrodynamic size Z-average of 60-70 nm, 71-80 nm, 81-90, nm, 91-100 nm, or 101-120 nm.

102. The composition of claim 101, wherein the LNP has a particle hydrodynamic size Z-average of 81-90 nm, 91-100 nm, or 101-120 nm.

103. The composition of any one of claims 92-102, wherein the composition has a nitrogen to phosphorus ratio (N: P) ratio of about 3:1 to about 9:1, about 4:1 to about 7:1, or about 6: 1 to about 8:1.

104. The composition of any one of claims 92-103, wherein the composition is a pharmaceutical composition for delivery to a patient in need thereof.

105. A method for genetically engineering a cell, comprising:a) contacting the cell with the LNP of any one of claims 1-91 or the composition of any one of claims 92-104, andb) introducing a single stranded DNA nick or introducing a double-stranded DNA break in the genome of the cell.NTLA-0118USP01-RNP-LNP RFEM: 5640-106106. The method of claim 105, wherein the genetic engineering comprises forming indels in the genome of the cell.

107. The method of any one of claims 105-106, wherein the genetic engineering inserts a nucleotide in the genomic sequence.

108. The method of any one of claims 106-107, wherein the genetic engineering deletes a nucleotide in the genomic sequence.

109. The method of any one of claims 106-107, wherein the genetic engineering introduces a change in an amino acid sequence encoded by a locus in which the genomic sequence is present.

110. The method of any one of claims 106-107, wherein the genetic engineering does not result in a change in an amino acid sequence encoded by a locus in which the genomic sequence is present.

111. The method of any one of claims 105-110, wherein the contacting is in vitro.

112. The method of any one of claims 105-110, wherein the contacting is in vivo.

113. The method of any one of claims 105-110, wherein the contacting is ex vivo.

114. The method of any one of claims 105-110, wherein the cell is a eukaryotic cell.

115. The method of any one of claims 105-110, wherein the cell is a plant cell.

116. The method of any one of claims 105-110, wherein the cell is an animal cell.

117. The method of any one of claims 105-110 and 116, wherein the cell is a mammalian cell.

118. A method for genetically engineering a cell in a subject comprising administering a therapeutically effective amount of a composition comprising the LNP of any one of claims 1-91 or the composition of any one of claims 92-104 to the subject to the subject.NTLA-0118USP01-RNP-LNP RFEM: 5640-106119. The method of claim 118, wherein the cell is a liver cell, a bone cell, a bone maiTow cell or a bone marrow derived cell, a cancerous cell, a cell associated with a disease or disorder, or any combination thereof.

120. The method of claim 119, wherein the cell is a liver cell.

121. The method of claim 119, wherein the cell is a bone marrow cell or a bone maiTow derived cell.

122. The method of claim 121, wherein the bone marrow cell or bone marrow derived cell is selected from a Hematopoietic Stem Cell (HSC), Multipotent Progenitor Cell (MPP), Common Myeloid Progenitor (CMP), Common Lymphoid Progenitor (CLP), Granulocyte-Macrophage Progenitor (GMP), Megakaryocyte-Erythroid Progenitor (MEP), Erythroid Precursor Cells (Erythroblasts), Meg akary oblast, Granulocyte Progenitors (Myeloblasts), Monoblast, Promyelocyte, Myelocyte, Metamyelocyte, Band Cells, Mature Granulocytes, Mature Monocytes, Erythrocytes, Mature Megakaryocytes, Plasma Cells, B Lymphocytes, or T Lymphocytes.

123. The method of claim 121 or claim 122, wherein the bone marrow cell or bone marrow derived cell is selected from a CD34+ cell, CD38+ cell, CD45+ cell, GDI 17+ cell, CD123+ cell, or a CD135+ cell.

124. The method of claim 121, wherein the cell is an immune cell, a T cell, a resident T cell, a B cell, a natural killer (NK) cell.

125. The method of any one of claims 118-124, wherein the method comprises administering the composition at a LNP concentration of about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 5 mg / kg, or about 0.3 mg / kg to about 3 mg / kg to a subject.

126. The method of any one of claims 118-125, comprising administering the composition systemically, parenterally, or intratumorally to a subject.

127. The method of any one of claims 118-126, comprising administering the composition via injection or infusion to a subject.NTLA-0118USP01-RNP-LNP RFEM: 5640-106128. The method of any one of claims 118-127, wherein the subject has a genetic disease or disorder.

129. The method of any one of claims 118-128, comprising administering the composition in an amount sufficient to obtain at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% gene editing.

130. The method of any one of claims 118-129, further comprising introducing at least one template nucleic acid into the cell.

131. The method of any one of claims 118-130, wherein the genetic engineering comprises introducing a gene knockout.

132. The method of any one of claims 118-131, wherein the genetic engineering comprises introducing a gene modification.

133. The method of claim 118-130, wherein the gene modification cuts, edits, blocks, marks or labels the gene.

134. The method of claim 118-130, wherein the gene modification inserts, deletes, or substitutes a base in the gene.

135. The method of claim 118-130, wherein the gene modification comprises an insertion or deletion of more than one base in the gene.

136. The method of any one of claims 118-130, wherein the genetic engineering targets a transthyretin (TTR) gene.

137. The method of claim 136, wherein serum TTR is reduced by 20 to 100-fold, 25 to 75-fold, or 30 to 50-fold.

138. The method of any one of claims 118-137, wherein the method comprises administering a first lipid nanoparticle (LNP) composition and a second LNPcomposition.NTLA-0118USP01-RNP-LNP RFEM: 5640-106139. The method of claim 138, wherein the first LNP composition and the second LNP composition comprise different cargo.

140. The method of claim 138 or claim 139, wherein the first and second LNP compositions are administered simultaneously.

141. The method of claim 138 or claim 139, wherein the first and second LNP compositions are administered sequentially.

142. A container, vial, syringe, injector pen, or kit comprising at least one dose of the LNP of any one of claims 1-91 or the composition of any one of claims 92-104.

143. A method of preparing a lipid nanoparticle (LNP) encapsulating a cargo comprising a ribonucleoprotein (RNP), wherein the method comprises:(i) mixing a first solution comprising a RNP in a preformulation buffer, and a second solution comprising a lipid component dissolved in ethanol, to prepare an RNP-LNP solution;(ii) mixing the RNP-LNP solution downstream with a third solution; and (iii) diluting the solution in (ii) with a fourth solution to prepare a diluted RNP- LNP solution;wherein the preformulation buffer has a pH about 5.5 to about 7.0 when measured at 25 °C and wherein the lipid component comprises an ionizable lipid having a pKa of about 6.0 to about 7.5.

144. A method of preparing a lipid nanoparticle (LNP) encapsulating a cargo comprising a ribonucleoprotein (RNP), wherein the method comprises:(i) mixing a first solution comprising a RNP in a preformulation buffer, and a second solution comprising a lipid component dissolved in ethanol, to prepare an RNP-LNP solution;(ii) mixing the RNP-LNP solution downstream with a third solution; and (iii) diluting the solution in (ii) with a fourth solution to prepare a diluted RNP- LNP solution;wherein the preformulation buffer has a pKa of about 5.5 to about 7.0.NTLA-0118USP01-RNP-LNP RFEM: 5640-106145. The method of any one of claims 143-144, wherein the LNP has a particle hydrodynamic size Z-average of 120 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, or about 60 nm.

146. The method of any one of claims 143-145, wherein the LNP has a particle hydrodynamic size Z-average of 60-80 nm, 80-100 nm, or 100-120 nm.

147. The method of any one of claims 143-146, wherein the encapsulation efficiency is about 80% to about 100%, about 85% to 100%, about 90% to 100%, about 95% to about 100%, about 96%, about 96%, about 98%, or about 99%.

148. The method of any one of claims 143-147, wherein the encapsulation efficiency is about 90% to about 100% or about 95% to 100%.

149. The method of any one of claims 143-148, wherein the first solution and second solution are mixed perpendicularly in a mixing cross.

150. The method of any one of claims 143-149, wherein the lipid component comprises an ionizable lipid, a neutral lipid, a helper lipid, and a structural PEG-lipid.

151. The method of any one of claims 143-150, wherein the method further comprises incubating the RNP-LNP solution before step (iii) at room temperature for about one hour.

152. The method of any one of claims 143-151, wherein the method further comprises step (iv) buffer exchanging the diluted RNP-LNP solution with a formulation buffer.

153. The method of any one of claims 143-152, wherein the preformulation buffer has a pH of about 6.0 to about 7.0, about 6.0 to about 6.5, or about 6.5 to about 7.0 when measured at 25 °C.

154. The method of any one of claims 143-152, wherein the preformulation buffer has a pH of about 5.5 to about 6.5 when measured at 25 °C.NTLA-0118USP01-RNP-LNP RFEM: 5640-106155. The method of any one of claims 143-152, wherein the preformulation buffer has a pH of about 6.0 when measured at 25 °C.

156. The method of any one of claims 143-155, wherein the first solution comprising the RNP is mixed with the second solution comprising the lipid component at a ratio of about 4:1 to about 1:1, about 3:1 to about 1:1, or about 2:1 (v / v).

157. The method of any one of claims 143-156, wherein the ionizable lipid has a pKa of about 6.0 to about 6.5, about 6.5 to about 7.0, about 7.0 to about 7.5.

158. The method of any one of claims 143-157, wherein the ionizable lipid has a pKa of about 0.5 units above the preformulation buffer pH.

159. The method of any one of claims 143-158, wherein the preformulation buffer has a pKa of about 6.0 to about 7.0, about 6.0 to about 6.5, or about 6.5 to about 7.0.

160. The method of any one of claims 143-159, wherein the preformulation buffer has a pKa of about 6.0 to about 6.5.

161. The method of any one of claims 143-160, wherein the preformulation buffer comprises a buffer agent at a concentration of 25 to 100 mM, 30 to 75 mM, 40 mM to 60 mM, or about 50 mM.

162. The method of any one of claims 143-161, wherein the preformulation buffer comprises KC1 or NaCl.

163. The method of any one of claims 143-162, wherein the preformulation buffer comprises KC1.

164. The method of any one of claims 143-163, wherein the preformulation buffer comprises KC1 or NaCl at a concentration of about 10 mM to about 100 mM.NTLA-0118USP01-RNP-LNP RFEM: 5640-106165. The method of claim 164, wherein the preformulation buffer comprises KC1 or NaCl at a concentration of about 18 mM to about 65 mM.

166. The method of claim 165, the preformulation buffer comprises KC1 or NaCl at a concentration of about 20 mM to about 55 mM.

167. The method of claim 166, wherein the preformulation buffer comprises KC1 or NaCl at a concentration of about 21 mM.

168. The method of claim 167, wherein the preformulation buffer comprises KC1 or NaCl at a concentration of about 50 mM,169. The method of any one of claims 161-168, wherein the buffer agent comprises histidine, BES, MOPS, succinate, MES, Bis-Tris, ADA, ACES, PIPES, citrate acetate, sodium citrate, phosphate buffer, or a combination thereof.

170. The method of any one of claims 143-169, wherein the third solution comprises an MES buffer, a BIS-Tris buffer, or a combination thereof.

171. The method of any one of claims 143-170, wherein the fourth solution comprises water.

172. The method of any one of claims 143-171, wherein the fourth solution comprises KC1 at a concentration of about 20 mM to about 30 mM KC1, or about 25 mM KC1.

173. The method of claim 172, wherein the fourth solution comprises an MES buffer, a BIS-Tris buffer, or a combination thereof.

174. The method of claim 173, wherein the fourth solution comprises the BIS-Tris buffer at a concentration of about 30 mM to about 40 mM, or about 33-38 or about 35.6 mM.

175. The method of claim 173, wherein the fourth solution comprises the MES buffer at a concentration of about 30 mM to about 40 mM, or about 33 to about 38 or about 35.6 mM.NTLA-0118USP01-RNP-LNP RFEM: 5640-106176. The method of any one of claims 143-175, wherein the solution in (ii) is mixed with the fourth solution at a ratio of about 4: 1 to about 1:1, about 3: 1 to about 1:1, about 2:1 to about 1:1, or about 1:1 (v / v).

177. The method of claim 152, wherein the formulation buffer comprises a buffer concentration of about 20 mM to about 100 mM, about 25 mM to about 75 mM, about 30 mM to 70 mM, about 35 mM to 65 mM, about 40 mM to 60 mM, about 43 mM to 57 mM, about 45 mM to 55 mM, about 48 mM to 52 mM or about 50 mM.

178. The method of any one of claims 152 or 177, wherein the formulation buffer has a pH of about 5 to about 7, about 5.5 to about 6.5, or about 6.0.

179. The method of any one of claims 152 or 177-178, wherein the formulation buffer comprises one or more excipients.

180. The method of claim 179, wherein the one or more excipients are selected from a salt, a sugar, or a combination thereof.

181. The method of claim 180, wherein the salt is sodium chloride.

182. The method of claim 181, wherein the sodium chloride is at a concentration of about 10 mM to about 150 mM, about 15 mM to about 100 mM, about 20 mM to about 75 mM, about 30 mM to about 50 mM, or about 45 mM.

183. The method of claim 180, wherein the sugar is at a concentration of about 1% to about 20%, about 2% to about 15%, about 3% to about 10%, or about 5% (w / v).

184. The method of claim 183, wherein the sugar is sucrose.

185. The method of any one of claims 143-184, wherein the RNP comprises a Type II CRISPR-Cas nuclease polypeptide (Cas nuclease) and a guide RNA (gRNA).

186. The method of any one of claims 185, wherein a ratio of gRNA: Cas nuclease is in a range of about 1:1 to about 16:1, about 1.1:1 to about 12:1, about 1.2:1 to about 10:1,NTLA-0118USP01-RNP-LNP RFEM: 5640-106about 1.4:1 to about 8:1, about 1.6:1 to about 6:1, about 1.8:1 to about 5:1, about 2:1 to 4:1, or about 2:1.

187. The method of any one of claims 143-186, wherein the LNP has a molar ratio of ionizable lipid to Type II Cas nuclease polypeptide of 4,000:1 to 2,400:1, or about 3,800:1, or 3,792:1.

188. The method of claim 187, wherein the LNP has a molar ratio of ionizable lipid to Type II Cas nuclease polypeptide of about 3,800:1, or 3,792:1.

189. The method of claim 188, wherein the LNP has a molar ratio of ionizable lipid to Type II Cas nuclease polypeptide of 3,792:1.

190. The method of any one of claims 143-189, wherein the LNP has a particle hydrodynamic size Z-average of 60-80 nm, 80-100 nm, or 100-120 nm.

191. The method of claim 185, wherein the Type II Cas nuclease polypeptide is a CRISPR-Cas9 polypeptide.

192. The method of any one of claims 143-191, wherein the lipid component comprises:a) the helper lipid in an amount from about 30 to about 50 mol % of the lipid component;b) the neutral lipid in an amount from about 0 to about 25 mol % of the lipid component;c) the structural PEG-lipid in an amount from about 0.5 to about 5 mol % of the lipid component; andd) the ionizable lipid in an amount of from about 30 to about 60 mol % of the lipid component.

193. The method of claim 186, wherein the ionizable lipid is present in an amount from about 32 to about 40 mol % of the lipid component.

194. The method of claim 186, wherein the neutral lipid is present in an amount from about 5 to about 20 mol % of the lipid component.NTLA-0118USP01-RNP-LNP RFEM: 5640-106195. The method of claim 186, wherein the ionizable lipid, the neutral lipid, the helper lipid, and the structural PEG-lipid are in a ratio of 32 to 40: 5 to 20: 40 to 50: 1.5 to 3.5.

196. The method of claim 186, wherein the ionizable lipid, the neutral lipid, the helper lipid, and the structural PEG-lipid are in a ratio of 35: 15:47.5:2.5.

197. The method of claim 192, wherein the ionizable lipid, the neutral lipid, the helper lipid, and the structural PEG-lipid arc in a ratio of 50:9:38:3, a ratio of 45:7:45:3, a ratio of 50:10:38.5:1.5, or a ratio of 50:10:37.8:2.2.

198. The method of any one of claims 143-191, wherein the lipid component comprises:a) the helper lipid in an amount from about 25 to about 65 mol % of the lipid component;b) the neutral lipid in an amount from about 0 to about 25 mol % of the lipid component;c) the structural PEG-lipid in an amount from about 0.5 to about 5 mol % of the lipid component; andd) the ionizable lipid in an amount of from about 40 to about 60 mol % of the lipid component.

199. The method of any one of claims 143-191 and 198, wherein the ionizable lipid, the neutral lipid, the helper lipid, and the structural PEG-lipid are in a ratio of 40 to 50: 20 to 25: 25 to 35: 1.5 to 3.5.

200. The method of any one of claims 143-191 and 198-199, wherein the ionizable lipid, the neutral lipid, the helper lipid, and the structural PEG-lipid are in a ratio of 47:22.5:28:2.5.

201. The method of any one of claims 143-191, wherein lipid component comprises:a) the helper lipid in an amount from about 27 to about 62 mol % or about 30 to about 60 mol% of the lipid component;NTLA-0118USP01-RNP-LNP RFEM: 5640-106b) the neutral lipid in an amount from about 2 to about 23 mol % or about 4 to about 20 mol% of the lipid component;c) the structural PEG-lipid in an amount from about 0.7 to about 5 mol % or about 0.8 to about 5 mol% of the lipid component; andd) the ionizable lipid in an amount of from about 42 to about 58 mol % or about 44 to about 56 mol% of the lipid component.

202. The method of any one of claims 143-201, wherein the ionizable lipid is a compound or a salt thereof represented by a structure selected from the group consisting of:O, O'-(2-((((2-(diethylamino)ethyl)carbamoyl)oxy)methyl)propane-l,3-diyl) di(heptadecan-9-yl) diglutarate,oO, O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-l,3-diyl) di(heptadecan-9-yl) diglutarate,NTLA-0118USP01-RNP-LNP RFEM: 5640-1063-(((2-(azepan-1-yl)ethyl)carbamoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propyl heptadecan-9-yl glutarate, and(9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate.

203. The method of any one of claims 143-201, 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, l-[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), l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N- [methoxy (polyethylene glycol)-2000] (PEG2K-DMPE), 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (PEG2K-DMG), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2K-DSPE), l,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-l,2-tetradecyoxypropylamine (C14 Ether), methoxy-NTLA-0118USP01-RNP-LNP RFEM: 5640-106PEG2000-carbamoyl-l,2-tridecyoxypropylamine (Cl 3 Ether), methoxy-PEG2000- carbamoyl-l,2-didecyoxypropylamine (C12 Ether), or a combination thereof.

204. The method of claim 202, wherein the structural PEG- lipid is 1,2-dimyristoyl-rac- glycero-3-methoxypolyethylene glycol 2000 (PEG2K-DMG), 1,2-distearoyl-sn-glycero- 3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2K-DSPE), methoxy-PEG2000-carbamoyl-l,2-didecyoxypropylamine (Cl 2 Ether), methoxy - PEG2000-carbamoyl-l,2-tetradecyoxypropylamine (C14 Ether), methoxy-PEG2000- carbamoyl-l,2-tridecyoxypropylamine (Cl 3 Ether), or a combination thereof.

205. The method of any one of claims 143-203, wherein the neutral lipid is selected from dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), 1 -palmitoyl- 2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC), l,2-diarachidoyl-sn-glycero-3- phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1 -myristoyl-2-palmitoyl phosphatidylcholine (MPPC), l-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), lysophosphatidylethanolamine, or a combination thereof.

206. The method of claim 204, wherein the neutral lipid is DSPC or DMPE.

207. The method of claim 205, wherein the neutral lipid is DSPC.

208. The method of any one of claims 143-206, wherein the LNP does not include 1,2- dioleoyl- 3 -trimethylammonium-propane (DOTAP).NTLA-0118USP01-RNP-LNP RFEM: 5640-106209. The method of any one of claims 143-207, wherein the helper lipid is cholesterol, 5 -heptadecylresorcinol, or cholesterol hemisuccinate.

210. The method of claim 208, wherein the helper lipid is cholesterol.

211. The method of any one of claims 143-210, wherein the lipid component comprises:(i) 3-(((2-(azepan- 1 -yl)ethyl)carbamoyl)oxy)-2-((((9Z, 12Z)-octadeca-9, 12- dicnoyl)oxy)mcthyl)propyl hcptadccan-9-yl glutarate; DSPC; cholesterol; and Cl 3 Ether structural PEG-lipid;(ii) O, O'-(2-((((2-(diethylamino)ethyl)carbamoyl)oxy)methyl)propane-l,3-diyl) di(heptadecan-9-yl) diglutarate; DSPC; cholesterol; and C13 Ether structural PEG-lipid;(iii)2-((4-(((2- (ethyl(methyl)amino)ethyl)carbamoyl)oxy)dodecanoyl)oxy)propane-l,3-diyl (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate); DSPC; cholesterol; and C13 Ether structural PEG-lipid; or(iv)O, O’-(2-((((3-(dicthylamino)propoxy)carbonyl)oxy)mcthyl)propanc-l,3-diyl) di(heptadecan-9-yl) diglutarate; DSPC; cholesterol; and Cl 3 Ether structural PEG-lipid.

212. The method of claim 211, wherein the ionizable lipid, the neutral lipid, the helper lipid, and the structural PEG-lipid are in a ratio of about 50:9:38:3.

213. The method of claim 210 or claim 211, wherein the gRNA: Cas nuclease ratio is about 2:1.

214. The method of any one of claims 204-206, wherein the LNP has a molar ratio of ionizable lipid to Type II Cas nuclease polypeptide of about 3,800:1 or about 3792:1.

215. The method of any one of claims 143-214, wherein the lipid component further comprises an anchor PEG-lipid comprising a first coupling moiety.NTLA-0118USP01-RNP-LNP RFEM: 5640-106216. The method of claim 215, wherein the anchor PEG-lipid is selected from DSPE- PEG(IOOO), DSPE-PEG(2000), DSPE-PEG(3400), DSPE-PEG(5000), or a combination thereof.

217. The method of claim 216, wherein the anchor PEG-lipid and the 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(IOOO) 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.

218. The method of any one of claims 215-217, wherein the lipid component comprises:a) the helper lipid in an amount from about 25 to about 65 mol % of the lipid component;b) the neutral lipid in an amount from about 0 to about 25 mol % of the lipid component;c) the structural PEG-lipid in an amount from about 0.5 to about 5 mol % of the lipid component;d) the ionizable lipid in an amount of from about 40 to about 60 mol % of the lipid component; ande) the anchor PEG-lipid in an amount from about 0.001 to about 1.5 mol % of the lipid component.

219. The method of any one of claims 215-218, wherein a targeting ligand is attached to the first coupling moiety via a second coupling moiety.

220. The method of any one of claims 215-219, wherein the amount of the anchor PEG-lipid is the same as an anchor PEG-lipid density.

221. The method of any one of claims 215-220, wherein the targeting ligand has a density that is the same or less than the anchor PEG-lipid density.NTLA-0118USP01-RNP-LNP RFEM: 5640-106222. The method of any one of claims 215-221, wherein the targeting ligand is selected from an antibody, an antibody fragment, a small molecule, or a peptide.

223. The method of claim 222, wherein the targeting ligand is an antibody.

224. The method of claim 223, wherein the anchor PEG-lipid is in an amount from about 0.005 to about 0.045 mol% of the lipid component.

225. The method of claim 224, wherein the targeting ligand is an antibody fragment.

226. The method of claim 225, 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, a disulfide-linked Fv (sdFv), a Fd fragment consisting of VH and CHI 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.

227. The method of claim 225 or claim 226, wherein the anchor PEG-lipid is in an amount from about 0.005 to about 0.075 mol% of the lipid component.

228. The method of claim 222, wherein the targeting ligand is a peptide.

229. The method of claim 228, wherein the anchor PEG-lipid is in an amount from about 0.1 to about 0.9 mol% of the lipid component.

230. The method of any one of claims 215-217, wherein lipid component comprises:a) the helper lipid in an amount from about 27 to about 62 mol % or about 30 to about 60 mol% of the lipid component;b) the neutral lipid in an amount from about 2 to about 23 mol % or about 4 to about 20 mol% of the lipid component;c) the structural PEG-lipid in an amount from about 0.7 to about 5 mol % or about 0.8 to about 5 mol% of the lipid component;d) the ionizable lipid in an amount of from about 42 to about 58 mol % or about 44 to about 56 mol% of the lipid component; andNTLA-0118USP01-RNP-LNP RFEM: 5640-106e) the anchor PEG-lipid in an amount from about 0.015 to about 1 mol% or about 0.02 to about 0.9 mol % of the lipid component.

231. The method of claim 230, wherein the anchor PEG-lipid comprises about 0.05 to about 0.2 mol % of the lipid component.

232. The method of claim 231, wherein the anchor PEG-lipid comprises about 0.03 to about 0.1 mol % of the lipid component.

233. The method of claim 232, wherein the anchor PEG-lipid comprises about 0.25 to about 0.7 mol % of the lipid component.

234. The method of claim 233, wherein the anchor PEG-lipid comprises about 0.042 to 0.48 mol% or about 0.045 mol%.

235. The method of any one of claims 215-234, wherein the lipid component comprises: 3-(((2-(azepan-l-yl)ethyl)carbamoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12- dienoyl)oxy)methyl)propyl heptadecan-9-yl glutarate; DSPC; cholesterol; and C13 Ether structural PEG-lipid and DSPE-PEG-SpyTag anchor PEG-lipid.

236. The method of claim 235, wherein the ionizable lipid, the neutral lipid, the helper lipid, the structural PEG-lipid, and the anchor PEG-lipid are in a ratio of 50:9:38:2.955:0.045.

237. The method of any one of claims 215-236, wherein the gRNA: Cas nuclease ratio is about 2:1.

238. The method of any one of claims 215-237, wherein the LNP has a molar ratio of ionizable lipid to Type II Cas nuclease polypeptide of 4,000:1 to 2,400:1, or about 3,800:1.

239. The method of any one of claims 215-238, 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.NTLA-0118USP01-RNP-LNP RFEM: 5640-106240. The method of any one of claims 215-239, wherein first coupling moiety and the second coupling moiety form a covalent bond.

241. The method of any one of claims 215-240, wherein first coupling moiety and the second coupling moiety form a non-covalent bond.

242. The method of any one of claims 215-235 and 236-241, wherein the first coupling moiety comprises dibenzocyclooctyne (DBCO), cysteine, a bioconjugation protein, streptavidin, protein G, protein G-dcrivcd 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.

243. The method of any one of claims 215-235 and 236-241, 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.

244. The method of any one of claims 215-243, wherein a molar ratio of the structural PEG-lipid to the anchor PEG-lipid is between about 2:1 to about 300:1.

245. The method of claim 244, wherein a molar ratio of the structural PEG-lipid to the anchor PEG-lipid is between about 2:1 to about 300:1, about 3:1 to about 100:1, or about 5:1 to about 50:1.

246. The method of any one of claims 143-245, wherein the LNP has a gene editing potency of 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100- fold higher than a control composition’s gene editing potency, wherein the control composition comprises (i) a mixture of the Cas9 / gRNA RNP that is not encapsulated in an LNP and (ii) a corresponding LNP that lacks the cargo encapsulated therein.

247. The method of claim 246, wherein the LNP has a 20-fold to 100-fold higher gene editing potency than the control composition.NTLA-0118USP01-RNP-LNP RFEM: 5640-106248. The method of any one of claims 243-247, wherein the cargo further comprises a nucleic acid of interest.

249. The method of claim 248, wherein the nucleic acid of interest comprises a template nucleic acid.

250. The method of claim 249, wherein the template nucleic acid comprises an endogenous sequence of a cell or the template nucleic acid comprises an exogenous sequence.

251. The method of claim 250, wherein the exogenous sequence comprises a protein or RNA coding sequence operably linked to an endogenous or exogenous promoter sequence, an exon sequence, an intron sequence, a regulatory sequence, a transcriptional control sequence, a translational control sequence, a splicing site, or a non-coding sequence.

252. The method of claim 251, wherein the template nucleic acid comprises a nucleic acid sequence that is complementary to at least a portion of a target nucleic acid.

253. The method of claim 252, wherein the template nucleic acid comprises a ssDNA or dsDNA containing flanking invert-terminal repeat (ITR) sequences, a plasmid, minicircle, nanocircle, or a PCR product.

254. The method of any one of claims 143-253, wherein the gRNA is a modified gRNA.

255. The method of claim 254, wherein the gRNA is a single-guide RNA (sgRNA) or a dual-guide RNA (dgRNA).

256. The method of any one of claims 143-255, wherein the gRNA is 60 to 200 nucleotides in length or 60 to 150 nucleotides in length.

257. The method of any one of claims 143-256, wherein the gRNA comprises a 3’ end modification, a modification in the hairpin region, and a 5’ end modification.NTLA-0118USP01-RNP-LNP RFEM: 5640-106258. The method of claim 257, 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.

259. The method of claim 258, wherein the 3’ and / or 5’ end modification comprises a protective end modification, such as a modified nucleotide selected from 2'-O-methyl (2’- OMe) modified nucleotide, 2’-O-(2-methoxyethyl) (2’- O-MOE) modified nucleotide, a 2’ -fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, an inverted abasic modified nucleotide, or combinations thereof.

260. The method of claim 259, wherein the modification in the hairpin region comprises a modified nucleotide selected from 2’-O-methyl (2’-OMe) modified nucleotide, a 2’ -fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, or combinations thereof.

261. The method of any one of claims 143-253, wherein the LNP has a polydispersity index (PDI) in a range of 0.001-0.2, 0.001-0.15, 0.001-0.1, 0.03-0.7, 0.07-0.10, 0.11-0.15, 0.15-0.2, 0.003-0.009, 0.01-0.02, or 0.001-0.05.

262. A lipid nanoparticle (LNP) or composition comprising LNPs prepared by the method of any one of claims 143-261.

263. The method of any one of claims 143-262, wherein the cell has about 30% to about 90%, about 35% to about 85% indels, about 40% to about 80%, about 45% to about 75% indels, about 50% to about 70%, about 55% to about 65% indels, about 60% to about 63%, or about 61% to about 62% indels after the contacting step.

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