Lipid Nanoparticle Compositions

Abstract

The present disclosure provides lipid nanoparticle (LNP) compositions of ionizable lipids, helper lipids, neutral lipids, and PEG lipids, which are useful for delivering biologically active agents, for example, for the preparation of engineered cells, to cells. The LNP compositions disclosed herein are useful in methods of gene editing, methods of delivering biologically active agents, and methods of modifying or cleaving DNA.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 176227 (filed April 17, 2021), U.S. Provisional Patent Application No. 63 / 254948 (filed October 12, 2021), U.S. Provisional Patent Application No. 63 / 274153 (filed November 1, 2021), and U.S. Provisional Patent Application No. 63 / 316568 (filed March 4, 2022), the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] Lipid nanoparticles formulated with ionizable lipids can function as cargo vehicles for delivering biologically active agents, particularly polynucleotides, such as RNA (including mRNA and guide RNA), into cells. LNP compositions containing ionizable lipids can facilitate the delivery of oligonucleotide agents across cell membranes and can also be used to introduce gene editing components and compositions into living cells. Biologically active agents that are particularly difficult to deliver to cells include proteins, nucleic acid-based agents, and their derivatives, particularly agents that contain relatively large oligonucleotides such as mRNA. Of particular interest are compositions for delivering promising gene editing technologies to cells, such as components of the CRISPR / Cas9 system, such as mRNAs encoding nucleases and associated guide RNAs (gRNAs).

[0003] There is a need for compositions for improving the delivery of nucleic acids (e.g., RNA) in vivo and in vitro. As an example, there is a need for compositions for delivering CRISPR / Cas components to eukaryotic cells (e.g., human cells). In particular, compositions for delivering mRNAs encoding CRISPR protein components and compositions for delivering CRISPR gRNAs are of particular interest. Also of particular interest are compositions that have properties useful for in vitro and in vivo delivery that can stabilize and deliver RNA components. Summary of the Invention

[0004] The present disclosure provides lipid compositions (e.g., lipid nanoparticle (LNP) compositions). Such lipid compositions may have advantageous properties for delivering biological agents, including, for example, nucleic acid cargoes (e.g., CRISPR / Cas gene editing components), to cells.

[0005] In some embodiments, the LNP composition comprises: a nucleic acid component, and The lipid component comprises: a) an ionizable lipid in an amount of about 25-45 mol % of the lipid component; b) neutral lipids in an amount of about 10-30 mol % of the lipid component; c) a helper lipid in an amount of about 25-65 mol % of the lipid component; and d) PEG lipid in an amount of about 1.5 to 3.5 mol% of the lipid component; The ionizable lipid is a compound of formula (I) [ka] (In the formula, X 1 is O, NH, or a direct bond, X 2 is C 2~3 is alkylene, R 3 is C 1~3 is alkyl, R2 is C 1~3 is alkyl, or R 2 is the nitrogen atom to which it is bonded and X 2 together with 2 to 3 carbon atoms of the formula (I) to form a 5- or 6-membered ring, or R 2 is R 3 and together with the nitrogen atom to which they are attached form a five-membered ring, Y 1 is C 6~10 is alkylene, Y 2 teeth [ka] is selected from R 4 is C 4~11 is alkyl, Z 1 is C 2~5 is alkylene, Z 2 teeth [ka] or absent, R 5 is C 6~8 Alkyl, C 5~10 Alkoxy (e.g., -OC 5~10 haloalkyl), -O(C 2~3 Alkyl)OC 6~10 Alkyl, -OC 6~10 Alkenyl (e.g., -OC 6~10 branched alkenyl), or -OC 6~10 is alkynyl, R 6 is C 6~8 Alkyl, C 5~10 Alkoxy (e.g., -OC 5~10 haloalkyl), -O(C 2~3 Alkyl)OC 6~10 Alkyl, -OC 6~10 Alkenyl (e.g., -OC 6~10 branched alkenyl), or -OC 6~10 is alkynyl, or R 5 is R 6 Together with the geminal C 6~8 Forming a six-membered acetal substituted with alkyl or a salt thereof.

[0006] In some embodiments, the LNP composition comprises: Biologically active agents, and The lipid component comprises: a) an ionizable lipid in an amount of about 25-45 mol % of the lipid component; b) neutral lipids in an amount of about 10-30 mol % of the lipid component; c) a helper lipid in an amount of about 25-65 mol % of the lipid component; and d) PEG lipid in an amount of about 1.5 to 3.5 mol% of the lipid component; The ionizable lipid is a compound of formula (I) [ka] (In the formula, X 1 is O, NH, or a direct bond, X 2 is C 2~3 is alkylene, R 3 is C 1~3 is alkyl, R 2 is C 1~3 is alkyl, or R 2 is the nitrogen atom to which it is bonded and X 2 together with 2 to 3 carbon atoms of the formula (I) to form a 5- or 6-membered ring, or R 2 is R 3 and together with the nitrogen atom to which they are attached form a five-membered ring, Y 1 is C 6~10 is alkylene, Y 2 teeth [ka] is selected from R 4 is C 4~11 is alkyl, Z 1 is C 2~5 is alkylene, Z 2 teeth [ka] or absent, R 5 is C 6~8 Alkyl or C 6~8 is an alkoxy; R 6 is C 6~8 Alkyl or C 6~8 (alkoxy) or a salt thereof.

[0007] In certain embodiments, the amount of ionizable lipid is about 29-44 mol% of the lipid component, the amount of neutral lipid is about 11-28 mol% of the lipid component, the amount of helper lipid is about 28-55 mol% of the lipid component, and the amount of PEG lipid is about 2.3-3.5 mol% of the lipid component.

[0008] In some embodiments, the amount of ionizable lipid is about 33 mol% of the lipid component, the amount of neutral lipid is about 15 mol% of the lipid component, the amount of helper lipid is about 49 mol% of the lipid component, and the amount of PEG lipid is about 3 mol% of the lipid component.

[0009] In certain embodiments, the ionizable lipid is [ka] or a salt thereof, wherein the neutral lipid is DSPC, the helper lipid is cholesterol, and the PEG lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

[0010] In certain embodiments, the LNPs have a Z-average diameter of less than about 145 nm, e.g., less than about 120 nm, less than about 115 nm, or less than about 100 nm. In certain embodiments, the LNPs have a number average diameter of more than about 50 nm, e.g., more than about 60 nm.

[0011] In certain embodiments, the LNPs have a polydispersity index of about 0.005 to about 0.75, for example, about 0.005 to about 0.1.

[0012] In some embodiments, the N / P ratio of the LNP composition is about 5 to about 7, preferably about 6.

[0013] In certain embodiments, the present disclosure relates to any LNP composition described herein, wherein the nucleic acid component is an RNA component.In some embodiments, the RNA component comprises an mRNA.In a preferred embodiment, the present disclosure relates to any LNP composition described herein, wherein the RNA component comprises an RNA-guided DNA binding agent, for example, a Cas nuclease mRNA, for example, a class 2 Cas nuclease mRNA, or a Cas9 nuclease mRNA.

[0014] In certain embodiments, the present disclosure relates to any LNP composition described herein, wherein the mRNA is modified mRNA.In some embodiments, the present disclosure relates to any LNP composition described herein, wherein the RNA component comprises gRNA nucleic acid.In certain embodiments, the present disclosure relates to any LNP composition described herein, wherein the gRNA nucleic acid is gRNA.

[0015] In certain preferred embodiments, the present disclosure relates to LNP compositions described herein, in which the RNA component comprises a class 2 Cas nuclease mRNA and a gRNA. In certain embodiments, the present disclosure relates to LNP compositions described herein, in which the gRNA nucleic acid is or encodes a dual guide RNA (dgRNA). In certain embodiments, the present disclosure relates to LNP compositions described herein, in which the gRNA nucleic acid is or encodes a single guide RNA (sgRNA).

[0016] In certain embodiments, the present disclosure relates to an LNP composition described herein comprising a guide RNA nucleic acid and a class 2 Cas nuclease mRNA, wherein the weight ratio of mRNA:guide RNA nucleic acid is about 2:1 to 1:4, preferably about 1:1.

[0017] In certain embodiments, the disclosure relates to LNP compositions described herein, wherein the gRNA is a modified gRNA, e.g., the modified gRNA includes a modification in one or more of the first five nucleotides at the 5' end, or the modified gRNA includes a modification in one or more of the last five nucleotides at the 3' end, or both.

[0018] In certain embodiments, the present disclosure relates to a method of delivering a biologically active agent to a cell, the method comprising contacting the cell with an LNP composition described herein.

[0019] In certain embodiments, the present disclosure relates to a method of cleaving DNA, comprising contacting a cell with an LNP composition described herein. In certain embodiments, the cleaving step comprises introducing a single-stranded DNA nick. In other embodiments, the cleaving step comprises introducing a double-stranded DNA break. In certain embodiments, the LNP composition comprises Class 2 Cas mRNA and a gRNA nucleic acid. In certain embodiments, the method further comprises introducing at least one template nucleic acid into the cell.

[0020] In certain embodiments, the disclosure relates to any of the methods of gene editing described herein, comprising administering the LNP composition to an animal (e.g., a human). In certain embodiments, the method comprises administering the LNP composition to a cell (e.g., a eukaryotic cell, particularly a human cell). In some embodiments, the cell is of a type useful for therapy (e.g., adoptive cell therapy (ACT)). Examples of ACT include autologous cell therapy and allogeneic cell therapy. In some embodiments, the cell is a stem cell, e.g., a hematopoietic stem cell, an induced pluripotent stem cell, or another multipotent or pluripotent cell. In some embodiments, the cell is a stem cell, e.g., a mesenchymal stem cell that can develop into bone, cartilage, muscle, or fat cells. In some embodiments, the stem cell comprises an ocular stem cell. In certain embodiments, the cells are selected from mesenchymal stem cells, hematopoietic stem cells (HSCs), mononuclear cells, endothelial progenitor cells (EPCs), neural stem cells (NSCs), limbic 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 transplantation.

[0021] In certain embodiments, the cell is a liver cell. In other embodiments, the cell is an immune cell, such as a leukocyte or lymphocyte, preferably a lymphocyte, more preferably a T cell, a B cell, or a NK cell, most preferably an activated T cell or a non-activated T cell.

[0022] In certain embodiments, the present disclosure relates to any of the methods of gene editing described herein, comprising administering an mRNA formulated in a first LNP composition and a second LNP composition comprising one or more of an mRNA, a gRNA, and a gRNA nucleic acid. In some embodiments, the first and second LNP compositions are administered simultaneously. In other embodiments, the first and second LNP compositions are administered sequentially. In certain embodiments, the mRNA and the gRNA nucleic acid are formulated in a single LNP composition. In some embodiments, the first LNP composition comprises a first gRNA, and the second LNP composition comprises a second gRNA, and the first and second gRNA comprise different guide sequences that are complementary to different targets.

[0023] In certain embodiments, the present disclosure relates to any method of gene editing described herein, in which a cell is contacted with an LNP composition in vitro. In certain embodiments, the present disclosure relates to any method of gene editing described herein, in which a cell is contacted with an LNP composition ex vivo. In certain embodiments, the present disclosure relates to any method of gene editing described herein, comprising contacting a tissue of an animal with an LNP.

[0024] In certain embodiments, the present disclosure relates to any of the methods of gene editing described herein, where the gene editing results in a gene knockout.

[0025] In some embodiments, the disclosure relates to any of the methods of gene editing described herein, where the gene editing results in a gene correction.

[0026] In certain embodiments, the disclosure relates to any of the methods of gene editing described herein, wherein the gene editing results in an insertion, hi some embodiments, the insertion is a gene insertion.

[0027] Provided herein are methods of genetically engineering T cells in vitro that overcome the hurdles of the preceding processes. In some embodiments, naive T cells are contacted in vitro with at least one lipid composition and genetically modified. In some embodiments, non-activated T cells are contacted in vitro with two or more lipid compositions and genetically modified. In some embodiments, activated T cells are contacted in vitro with two or more lipid compositions and genetically modified. In some embodiments, T cells are modified in a pre-activation step that includes contacting (non-activated) T cells with one or more lipid compositions, followed by activation of the T cells, followed by further modification of the T cells in a post-activation step that includes contacting the activated T cells with one or more lipid compositions. In some embodiments, non-activated T cells are contacted with 1, 2, or 3 lipid compositions. In some embodiments, activated T cells are contacted with 1-12 lipid compositions. In some embodiments, activated T cells are contacted with 1-8 lipid compositions, optionally 1-4 lipid compositions. In some embodiments, activated T cells are contacted with 1-6 lipid compositions. In some embodiments, the T cells are contacted with two lipid compositions. In some embodiments, the T cells are contacted with three lipid compositions. In some embodiments, the T cells are contacted with four lipid compositions. In some embodiments, the T cells are contacted with five lipid compositions. In some embodiments, the T cells are contacted with six lipid compositions. In some embodiments, the T cells are contacted with seven lipid compositions. In some embodiments, the T cells are contacted with eight lipid compositions. In some embodiments, the T cells are contacted with nine lipid compositions. In some embodiments, the T cells are contacted with ten lipid compositions. In some embodiments, the T cells are contacted with eleven lipid compositions. In some embodiments, the T cells are contacted with twelve lipid compositions.Sequential administration of such exemplary lipid compositions (optionally with further sequential or simultaneous administration in pre-activation and post-activation steps) takes advantage of the activation state of the T cells, providing unique advantages and healthier cells post-editing. In some embodiments, the engineered T cells have high editing efficiency at each target site, increased survival after editing, low toxicity against multiplicity of transfection, low translocations (e.g., no measurable inter-target translocations, no measurable target-to-target translocations), increased production of cytokines (e.g., IL-2, IFNγ, TNFα), continued proliferation upon repeated stimulation (e.g., upon repeated antigen stimulation), increased proliferation, and / or expression of phenotypic markers of memory cells (including, e.g., early stem cells). [Brief description of the drawings]

[0028] [Figure 1A] 1 is a graph showing the percentage of CD3− cells following delivery of Cas9 mRNA and sgRNA to activated CD3+ T cells using LNP compositions comprising Compound 6 with varying ratios of lipid components. [Figure 1B] 1 is a graph showing the percentage of CD3− cells following delivery of Cas9 mRNA and sgRNA to non-activated CD3+ T cells using LNP compositions comprising Compound 6 with varying ratios of lipid components. [Figure 2A] 1 is a graph showing the percentage of CD3− cells following delivery of Cas9 mRNA and sgRNA to activated CD3+ T cells using LNP compositions comprising Compound 6 with varying ratios of lipid components. [Figure 2B] 1 is a graph showing the percentage of CD3− cells following delivery of Cas9 mRNA and sgRNA to non-activated CD3+ T cells using LNP compositions comprising Compound 6 with varying ratios of lipid components. [Figure 3A]Graph showing the percentage of CD3− cells following delivery of Cas9 mRNA and sgRNA to activated CD3+ T cells using LNP compositions including Compound 6, Compound 8, and Compound 11 with the following nominal mol% ratios of lipid components: 35% ionizable lipid, 15% DSPC, 47.5% cholesterol, and 2.5% PEG-2k-DMG, and comparative LNP compositions including Compound 6, Compound 8, and Compound 11 with the following nominal mol% ratios of lipid components: 50% ionizable lipid, 10% DSPC, 38.5% cholesterol, and 1.5% PEG-2k-DMG. [Figure 3B] Graph showing the percentage of CD3− cells following delivery of Cas9 mRNA and sgRNA to non-activated CD3+ T cells using LNP compositions including Compound 6, Compound 8, and Compound 11 with the following nominal mol% ratios of lipid components: 35% ionizable lipid, 15% DSPC, 47.5% cholesterol, and 2.5% PEG-2k-DMG, and comparative LNP compositions including Compound 6, Compound 8, and Compound 11 with the following nominal mol% ratios of lipid components: 50% ionizable lipid, 10% DSPC, 38.5% cholesterol, and 1.5% PEG-2k-DMG. [Figure 4A] FIG. 13 shows the effect of various ratios of sgRNA:Cas9 mRNA on the percentage of CD3− cells following delivery of Cas9 mRNA and sgRNA to activated CD3+ T cells using an LNP composition comprising Compound 6 with the following nominal mol% ratios of lipid components: 35% ionizable lipid, 15% DSPC, 47.5% cholesterol, and 2.5% PEG-2k-DMG, and a comparative LNP composition with the following nominal mol% ratios of lipid components: 50% ionizable lipid, 10% DSPC, 38.5% cholesterol, and 1.5% PEG-2k-DMG. [Figure 4B]FIG. 13 shows the effect of various ratios of sgRNA:Cas9 mRNA on the percentage of CD3− cells after delivery of Cas9 mRNA and sgRNA to resting CD3+ T cells from two different donors using an LNP composition containing Compound 6 with a lipid component of the following nominal mol% ratios: 35% ionizable lipid, 15% DSPC, 47.5% cholesterol, and 2.5% PEG-2k-DMG, and a comparative LNP composition with a lipid component of the following nominal mol% ratios: 50% ionizable lipid, 10% DSPC, 38.5% cholesterol, and 1.5% PEG-2k-DMG. [Diagram 5] FIG. 13 is a graph showing the effect of various serum media conditions on GFP insertion efficiency in activated CD3+ T cells after delivery of Cas9 mRNA and sgRNA using an LNP composition comprising Compound 6 with lipid components in the following nominal mol% ratios: 35% ionizable lipid, 15% DSPC, 47.5% cholesterol, and 2.5% PEG-2k-DMG, and a comparative LNP composition with lipid components in the following nominal mol% ratios: 50% ionizable lipid, 10% DSPC, 38.5% cholesterol, and 1.5% PEG-2k-DMG. [Figure 6A] FIG. 13 shows the effect of various LNP composition concentrations on the percentage of CD3− cells following delivery of Cas9 mRNA and TRAC-targeting sgRNA to activated T cells using an LNP composition having a lipid component with the following nominal mol% ratios: 35% ionizable lipid, 15% DSPC, 47.5% cholesterol, and 2.5% PEG-2k-DMG, and a comparative LNP composition having a lipid component with the following nominal mol% ratios: 50% ionizable lipid, 10% DSPC, 38.5% cholesterol, and 1.5% PEG-2k-DMG. [Figure 6B]FIG. 13 shows the effect of various ratios of LNP composition concentrations on the percentage of HLA-DR-, HLA-DP-, and HLA-DQ- cells after delivery of Cas9 mRNA and CIITA-targeting sgRNA to activated T cells using an LNP composition having the following nominal mol% ratios of lipid components: 35% ionizable lipid, 15% DSPC, 47.5% cholesterol, and 2.5% PEG-2k-DMG, and a comparative LNP composition having the following nominal mol% ratios of lipid components: 50% ionizable lipid, 10% DSPC, 38.5% cholesterol, and 1.5% PEG-2k-DMG. [Figure 6C] FIG. 13 shows the effect of various LNP composition concentrations on the percentage of CD3− cells after delivery of Cas9 mRNA and TRBC-targeting sgRNA to activated T cells using an LNP composition having lipid components with the following nominal mol% ratios: 35% ionizable lipid, 15% DSPC, 47.5% cholesterol, and 2.5% PEG-2k-DMG, and a comparative LNP composition having lipid components with the following nominal mol% ratios: 50% ionizable lipid, 10% DSPC, 38.5% cholesterol, and 1.5% PEG-2k-DMG. [Figure 6D] FIG. 13 is a graph showing the effect of various LNP composition concentrations on the percentage of HLA-A- cells following delivery of Cas9 mRNA and HLA-A-targeting sgRNA to activated T cells using an LNP composition having a lipid component with the following nominal mol% ratios: 35% ionizable lipid, 15% DSPC, 47.5% cholesterol, and 2.5% PEG-2k-DMG, and a comparative LNP composition having a lipid component with the following nominal mol% ratios: 50% ionizable lipid, 10% DSPC, 38.5% cholesterol, and 1.5% PEG-2k-DMG. [Figure 7A]Graph showing the editing rates of HLA-A, CIITA, TCR and WT1 in CD8+ T cells after delivery of Cas9 mRNA and sgRNA targeting the CIITA, HLA-A, TRAC and TRBC loci by using LNP compositions at concentrations of 0.65ug / mL or 2.5ug / mL having lipid components with the following nominal mol% ratios: 35% ionizable lipid, 15% DSPC, 47.5% cholesterol and 2.5% PEG-2k-DMG, along with comparative LNP compositions and negative controls having lipid components with the following nominal mol% ratios: 50% ionizable lipid, 10% DSPC, 38.5% cholesterol and 1.5% PEG-2k-DMG. [Figure 7B] Graph showing the combined editing rates of HLA-A, CIITA, TCR and WT1 in CD8+ T cells after delivery of Cas9 mRNA and sgRNA targeting the CIITA, HLA-A, TRAC and TRBC loci by using LNP compositions at concentrations of 0.65ug / mL or 2.5ug / mL having lipid components with the following nominal mol% ratios: 35% ionizable lipid, 15% DSPC, 47.5% cholesterol and 2.5% PEG-2k-DMG, along with comparative LNP compositions and negative controls having lipid components with the following nominal mol% ratios: 50% ionizable lipid, 10% DSPC, 38.5% cholesterol and 1.5% PEG-2k-DMG. [Figure 8] FIG. 13 is a graph showing the effect of various LNP composition concentrations on percent editing following delivery of Cas9 mRNA and AAVS1-targeting sgRNA to NK cells using an LNP composition having a lipid component with the following nominal mol% ratios: 35% ionizable lipid, 15% DSPC, 47.5% cholesterol, and 2.5% PEG-2k-DMG, and a comparative LNP composition having a lipid component with the following nominal mol% ratios: 50% ionizable lipid, 10% DSPC, 38.5% cholesterol, and 1.5% PEG-2k-DMG. [Figure 9A]FIG. 13 is a graph showing the effect of various LNP composition concentrations on percent editing following delivery of Cas9 mRNA and AAVS1-targeting sgRNA to monocytes using an LNP composition having lipid components in the following nominal mol% ratios: 35% ionizable lipid, 15% DSPC, 47.5% cholesterol, and 2.5% PEG-2k-DMG, and a comparative LNP composition having lipid components in the following nominal mol% ratios: 50% ionizable lipid, 10% DSPC, 38.5% cholesterol, and 1.5% PEG-2k-DMG. [Figure 9B] FIG. 13 is a graph showing the effect of various LNP composition concentrations on percent editing following delivery of Cas9 mRNA and AAVS1-targeting sgRNA to macrophages using an LNP composition having lipid components in the following nominal mol% ratios: 35% ionizable lipid, 15% DSPC, 47.5% cholesterol, and 2.5% PEG-2k-DMG, and a comparative LNP composition having lipid components in the following nominal mol% ratios: 50% ionizable lipid, 10% DSPC, 38.5% cholesterol, and 1.5% PEG-2k-DMG. [Figure 10] FIG. 13 is a graph showing the effect of various LNP composition concentrations on percent editing following delivery of Cas9 mRNA and AAVS1-targeting sgRNA to B cells using an LNP composition having lipid components in the following nominal mol% ratios: 35% ionizable lipid, 15% DSPC, 47.5% cholesterol, and 2.5% PEG-2k-DMG, and a comparative LNP composition having lipid components in the following nominal mol% ratios: 50% ionizable lipid, 10% DSPC, 38.5% cholesterol, and 1.5% PEG-2k-DMG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present disclosure provides lipid compositions useful for delivering biologically active agents, including nucleic acids (e.g., CRISPR / Cas component RNA (mRNA and / or gRNA)) ("cargo") to cells, as well as methods of preparing and using such compositions. Such lipid compositions include ionizable lipids, neutral lipids, PEG lipids, and helper lipids. In some embodiments, the ionizable lipid is a compound of formula (I) or (II) as defined herein. In certain embodiments, the lipid composition can include a biologically active agent (e.g., an RNA component). In certain embodiments, the RNA component includes an mRNA. In some embodiments, the mRNA is an mRNA encoding a class 2 Cas nuclease. In certain embodiments, the RNA component includes a gRNA and, optionally, an mRNA encoding a class 2 Cas nuclease. In some embodiments, the lipid composition is a lipid nanoparticle (LNP) composition. "Lipid nanoparticle" or "LNP" refers, without any limitation, to a particle that includes multiple (i.e., two or more) lipid components physically bound to one another by intermolecular forces.

[0030] Methods of gene editing and methods of producing engineered cells using these lipid compositions are also provided. In some embodiments, the LNP compositions can be used to deliver biologically active agents to cells, tissues, or animals. In some embodiments, the cells are eukaryotic cells, particularly human cells. In some embodiments, the cells are hepatocytes. In some embodiments, the cells are of a type useful for therapy, e.g., adoptive cell therapy (ACT), e.g., autologous and allogeneic cell therapy. In some embodiments, the cells are stem cells, e.g., hematopoietic stem cells, induced pluripotent stem cells, or other multipotent or pluripotent cells. In some embodiments, the cells are stem cells, e.g., mesenchymal stem cells that can develop into bone, cartilage, muscle, or fat cells. In some embodiments, the stem cells include ocular stem cells. In certain embodiments, the cells are selected from mesenchymal stem cells, hematopoietic stem cells (HSCs), mononuclear cells, endothelial progenitor cells (EPCs), neural stem cells (NSCs), limbic 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 transplantation.

[0031] In some embodiments, the cell is an immune cell (e.g., a white blood cell or a lymphocyte). In a preferred embodiment, the immune cell is a lymphocyte. In certain embodiments, the lymphocyte is a T cell, a B cell, or a NK cell. In a preferred embodiment, the lymphocyte is a T cell. In certain embodiments, the lymphocyte is an activated T cell. In certain embodiments, the lymphocyte is a non-activated T cell.

[0032] In some embodiments, the LNP compositions and methods provided herein provide an editing efficiency of greater than about 80%, greater than about 90%, or greater than about 95%. In some embodiments, the LNP compositions and methods provide an editing efficiency of about 80-95%, about 90-95%, about 80-99%, about 90-99%, or about 95-99%.

[0033] Ionizable lipids The present disclosure provides ionizable lipids that can be used in LNP compositions.

[0034] In some embodiments, the ionizable lipid is a compound of formula (I) [ka] (In the formula, X 1 is O, NH, or a direct bond, X 2 is C 2~3 is alkylene, R 3 is C 1~3 is alkyl, R 2 is C 1~3 is alkyl, or R 2 is the nitrogen atom to which it is bonded and X 2 together with 2 to 3 carbon atoms of the formula (I) to form a 5- or 6-membered ring, or R 2 is R 3 and together with the nitrogen atom to which they are attached form a five-membered ring, Y 1 is C 6~10 is alkylene, Y 2 teeth [ka] is selected from R 4 is C 4~11 is alkyl, Z 1 is C 2~5 is alkylene, Z 2 teeth [ka] or absent, R 5 is C 6~8 Alkyl, C 5~10 Alkoxy (e.g., -OC5~10 haloalkyl), -O(C 2~3 Alkyl)OC 6~10 Alkyl, -OC 6~10 Alkenyl (e.g., -OC 6~10 branched alkenyl), or -OC 6~10 is alkynyl, R 6 is C 6~8 Alkyl, C 5~10 Alkoxy (e.g., -OC 5~10 haloalkyl), -O(C 2~3 Alkyl)OC 6~10 Alkyl, -OC 6~10 Alkenyl (e.g., -OC 6~10 branched alkenyl), or -OC 6~10 is alkynyl, or R 5 is R 6 Together with the geminal C 6~8 Forming a six-membered acetal substituted with alkyl or a salt thereof.

[0035] In some embodiments, the ionizable lipid is a compound having the structure of Formula I [ka] (In the formula, X 1 is O, NH, or a direct bond, X 2 is C 2~3 is alkylene, R 3 is C 1~3 is alkyl, R 2 is C 1~3 is alkyl, or R 2 is the nitrogen atom to which it is bonded and X 2 together with 2 to 3 carbon atoms of the formula (I) to form a 5- or 6-membered ring, or R 2 is R 3 and together with the nitrogen atom to which they are attached form a five-membered ring, Y 1 is C 6~10 is alkylene, Y 2 teeth [ka] is selected from R 4 is C 4~11 is alkyl, Z 1 is C 2~5 is alkylene, Z 2 teeth [ka] or absent, R 5 is C 6~8 Alkyl or C 6~8 is an alkoxy; R 6 is C 6~8 Alkyl or C 6~8 (alkoxy) or a salt thereof.

[0036] In some embodiments, the ionizable lipid is a compound of formula (II) [ka] (In the formula, X 1 is O, NH, or a direct bond, X 2 is C 2~3 is alkylene, Z 1 is C3 alkylene, and R 5 and R 6 are each C6 alkyl, or Z 1 is a direct bond, and R 5 and R 6 are each C8 alkoxy; R 8 teeth [ka] is) or a salt thereof.

[0037] In certain embodiments, X 1 is O; in other embodiments, X 1 is NH. In yet another embodiment, X 1 is a direct bond.

[0038] In certain embodiments, X 2 is C alkylene. In certain embodiments, X 2 is a C2 alkylene.

[0039] In certain embodiments, Z 1 is a direct bond, and R 5 and R 6 Each is C alkoxy. In another embodiment, Z 1 is C3 alkylene, and R 5 and R 6 Each is a C6 alkyl.

[0040] In certain embodiments, R 8 teeth [ka] In another embodiment, R 8 teeth [ka] It is.

[0041] In certain embodiments, the ionizable lipid is a salt.

[0042] Representative compounds of formula (I) include the following: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] or a salt thereof, such as a pharma- ceutically acceptable salt thereof. The compounds can be synthesized according to the methods described in WO2015 / 095340 (e.g., pages 84-86) and WO2020 / 219876 (e.g., pages 87-186), each of which is incorporated by reference in its entirety.

[0043] The compounds of formula (I) or (II) of the present disclosure can form salts depending on the pH of the medium in which they are present. For example, in a slightly acidic medium, the compounds of formula (I) or (II) can be protonated and have a positive charge. Conversely, in a slightly basic medium (e.g., blood, which has a pH of approximately 7.35), the compounds of formula (I) or (II) can be unprotonated and uncharged. In some embodiments, the compounds of formula (I) or (II) of the present disclosure can be protonated primarily at a pH of at least about 9. In some embodiments, the compounds of formula (I) or (II) of the present disclosure can be protonated primarily at a pH of at least about 10.

[0044] The pH at which a compound of formula (I) or (II) is primarily protonated is related to its inherent pKa. In some embodiments, the salt of a compound of formula (I) or (II) of the present disclosure has a pKa in the range of about 5.1 to about 8.0, more preferably about 5.5 to about 7.6. In some embodiments, the salt of a compound of formula (I) or (II) of the present disclosure has a pKa in the range of about 5.7 to about 8, about 5.7 to about 7.6, about 6 to about 8, about 6 to about 7.5, about 6 to about 7, about 6 to about 6.9, about 6 to about 6.5, about 6.1 to about 6.9, or about 6 to about 6.85. In some embodiments, the salt of a compound of formula (I) or (II) of the present disclosure has a pKa of about 6.0, about 6.1, about 6.1, about 6.2, about 6.3, about 6.4, about 6.6, about 6.7, about 6.8, or about 6.9. Alternatively, the salts of the compounds of formula (I) or (II) of the present disclosure have a pKa in the range of about 6 to about 8. The pKa of the salts of the compounds of formula (I) or (II) can be an important consideration when formulating LNPs. This is because LNPs formulated with certain lipids having a pKa in the range of about 5.5 to about 7.0 have been found to be effective for in vivo delivery of cargo, for example, to the liver. Furthermore, LNPs formulated with certain lipids having a pKa in the range of about 5.3 to about 6.4 have been found to be effective for in vivo delivery, for example, to tumors. See, for example, WO2014 / 136086. In some embodiments, the ionizable lipids are positively charged at acidic pH but neutral in blood.

[0045] Additional lipids Suitable "neutral lipids" for use in the lipid compositions of the present 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), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (P ... phosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, and combinations thereof. In certain embodiments, the neutral phospholipid may be selected from distearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidylethanolamine (DMPE), preferably distearoylphosphatidylcholine (DSPC).

[0046] "Helper lipids" include steroids, sterols, and alkylresorcinols. 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.

[0047] In some embodiments, the LNP composition comprises a polymeric lipid, such as a PEG lipid, which can affect the length of time that the nanoparticles can exist in vivo or ex vivo (e.g., in blood or medium). The PEG lipid can aid in the formulation process, for example, by reducing particle aggregation and controlling particle size. The PEG lipid used herein can modulate the pharmacokinetic properties of the LNP. Typically, the PEG lipid comprises a lipid portion and a polymer portion (PEG portion) based on PEG (sometimes referred to as poly(ethylene oxide)). Information regarding PEG lipids suitable for use in lipid compositions having compounds of formula (I) or (II) of the present disclosure, and the biochemistry of such lipids, can be found in Romberg et al., Pharmaceutical Research 25(1), 2008, pp. 55-71 and Hoekstra et al., Biochimica et Biophysica Acta 1660(2004) 41-52. Additional suitable PEG lipids are disclosed, for example, in WO2015 / 095340 (line 14, p. 31 to line 6, p. 37), WO2006 / 007712, and WO2011 / 076807 ("stealth lipids"), each of which is incorporated by reference in its entirety.

[0048] In some embodiments, the lipid moiety may be derived from a diacylglycerol or diacylglycamide, including those that include a dialkylglycerol or dialkylglycamide group having an alkyl chain length independently comprising from about C4 to about C40 saturated or unsaturated carbon atoms, where the chain may include one or more functional groups (e.g., amide or ester). In some embodiments, the alkyl chain length comprises from about C10 to C20. The dialkylglycerol or dialkylglycamide group may further comprise one or more substituted alkyl groups. The chain length may be symmetric or asymmetric.

[0049] Unless otherwise indicated, as used herein, the term "PEG" refers to any polyethylene glycol or other polyalkylene ether polymer, e.g., an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In certain embodiments, the PEG moiety is unsubstituted. Alternatively, the PEG moiety may be substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. For example, the PEG moiety may comprise a PEG copolymer, such as PEG-polyurethane or PEG-polypropylene (see, e.g., J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)), or alternatively, the PEG moiety may be a PEG homopolymer. In certain embodiments, the PEG moiety has a molecular weight of about 130 to about 50,000, e.g., about 150 to about 30,000, even about 150 to about 20,000. Similarly, the PEG moiety can have a molecular weight of about 150 to about 15,000, about 150 to about 10,000, about 150 to about 6,000, or even about 150 to about 5,000. In certain preferred embodiments, the PEG moiety has a molecular weight of about 150 to about 4,000, about 150 to about 3,000, about 300 to about 3,000, about 1,000 to about 3,000, or about 1,500 to about 2,500.

[0050] In certain preferred embodiments, the PEG moiety is "PEG-2K" (also referred to as "PEG-2000"), which has an average molecular weight of about 2,000 daltons. PEG-2K is represented herein by the following formula (III): [ka] where n is about 45, meaning that the number average degree of polymerization includes about 45 subunits. However, other PEG embodiments known in the art can be used, including, for example, embodiments with a number average degree of polymerization of about 23 subunits (n=23) and / or 68 subunits (n=68). In some embodiments, n can range from about 30 to about 60. In some embodiments, n can range from about 35 to about 55. In some embodiments, n can range from about 40 to about 50. In some embodiments, n can range from about 42 to about 48. In some embodiments, n can be 45. In some embodiments, R can be selected from H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R can be an unsubstituted alkyl, such as methyl.

[0051] In any of the embodiments described herein, the PEG lipid may be PEG-dilaurylglycerol, PEG-dimyristoylglycerol (PEG-DMG) (catalog number GM-020 (NOF, Tokyo, Japan)), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE) (catalog number DSPE-020CN (NOF, Tokyo, Japan)), PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, and PEG-distearoylglycamide, PEG-cholesterol (1-[8'-(cholest-5-ene-3[beta]-oxy)carboxamido-3'-,6'-dioxazone-1-yl)-1,1-dimethylethyl ether (DMG), PEG-dimyristoylglycerol (DMG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (DSPE), PEG-DSPE, ... [butanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMPE), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2k-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE) (catalog no. 880120C (Avanti Polar Lipids, Alabaster, Alabama, USA), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG2k-DSG; GS-020 (NOF Tokyo, Japan)), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA), and 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA). In certain such embodiments, the PEG lipid may be PEG2k-DMG. In some embodiments, the PEG lipid may be PEG2k-DSG. In other embodiments, the PEG lipid may be PEG2k-DSPE. In some embodiments, the PEG lipid may be PEG2k-DMA.In other embodiments, the PEG lipid may be PEG2k-C-DMA. In certain embodiments, the PEG lipid may be compound S027 disclosed in WO2016 / 010840 (paragraphs

[0240] to

[0244] ). In some embodiments, the PEG lipid may be PEG2k-DSA. In other embodiments, the PEG lipid may be PEG2k-C11. In some embodiments, the PEG lipid may be PEG2k-C14. In some embodiments, the PEG lipid may be PEG2k-C16. In some embodiments, the PEG lipid may be PEG2k-C18.

[0052] In a preferred embodiment, the PEG lipid comprises a glycerol group. In a preferred embodiment, the PEG lipid comprises a dimyristoyl glycerol (DMG) group. In a preferred embodiment, the PEG lipid comprises PEG-2k. In a preferred embodiment, the PEG lipid is PEG-DMG. In a preferred embodiment, the PEG lipid is PEG-2k-DMG. In a preferred embodiment, the PEG lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000. In a preferred embodiment, the PEG-2k-DMG is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

[0053] lipid composition Described herein is a lipid composition comprising at least one compound of formula (I) or (II), or a salt thereof (e.g., a pharma- ceutically acceptable salt thereof), at least one helper lipid, at least one neutral lipid, and at least one polymeric lipid. In some embodiments, the lipid composition comprises at least one compound of formula (I) or (II), or a salt thereof, at least one neutral lipid, at least one helper lipid, and at least one PEG lipid. In some embodiments, the neutral lipid is DSPC or DPME. In some embodiments, the helper lipid is cholesterol, 5-heptadecylresorcinol, or cholesterol hemisuccinate.

[0054] In a preferred embodiment, the ionizable lipid is [ka] In a preferred embodiment, the neutral lipid is DSPC. In a preferred embodiment, the helper lipid is cholesterol. In a preferred embodiment, the PEG lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000. In a particularly preferred embodiment, the ionizable lipid is [ka] where the neutral lipid is DSPC, the helper lipid is cholesterol, and the PEG lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

[0055] In some embodiments, the lipid composition further comprises one or more additional lipid components.

[0056] In some embodiments, the lipid composition is in the form of a liposome. In a preferred embodiment, the lipid composition is in the form of a lipid nanoparticle (LNP). In certain embodiments, the lipid composition is suitable for in vivo delivery. In certain embodiments, the lipid composition is suitable for delivery to an organ, such as the liver. In certain embodiments, the lipid composition is suitable for ex vivo delivery to a tissue. In certain embodiments, the lipid composition is suitable for in vitro delivery to a cell.

[0057] The lipid composition comprising the lipid of formula (I) or (II), or a pharma- ceutically acceptable salt thereof, can take various forms, including, but not limited to, particle-forming delivery agents, including microparticles, nanoparticles, and transfection agents, that are useful for delivering various molecules to cells. Certain compositions are effective for transfection or delivery of biologically active agents. A preferred biologically active agent is a nucleic acid, such as RNA. In further embodiments, the biologically active agent is selected from mRNA and gRNA. The gRNA can be a dgRNA or sgRNA. In certain embodiments, the cargo comprises an mRNA encoding an RNA-guided DNA-binding agent (e.g., a Cas nuclease, a class 2 Cas nuclease, or Cas9), a gRNA or a nucleic acid encoding a gRNA, or a combination of mRNA and gRNA.

[0058] Exemplary compounds of formula (I) or (II) for use in the lipid compositions described above are set forth in WO2020 / 219876 (incorporated by reference in its entirety). In certain embodiments, the compound of formula (I) is compound 1. In certain embodiments, the compound of formula (I) is compound 2. In certain embodiments, the compound of formula (I) is compound 3. In certain embodiments, the compound of formula (I) is compound 4. In certain embodiments, the compound of formula (I) is compound 5. In certain embodiments, the compound of formula (I) is compound 6. In certain embodiments, the compound of formula (I) is compound 7. In certain embodiments, the compound of formula (I) is compound 8. In certain embodiments, the compound of formula (I) is compound 9. In certain embodiments, the compound of formula (I) is compound 10. In certain embodiments, the compound of formula (I) is compound 11. In certain embodiments, the compound of formula (I) is compound 12. In certain embodiments, the compound of formula (I) is compound 13. In certain embodiments, the compound of formula (I) is compound 14. In certain embodiments, the compound of formula (I) is compound 15. In certain embodiments, the compound of formula (I) is compound 16. In certain embodiments, the compound of formula (I) is compound 17. In certain embodiments, the compound of formula (I) is compound 18. In certain embodiments, the compound of formula (I) is compound 19.

[0059] Generally, but not necessarily, the composition comprises one or more pharma- ceutically acceptable excipients. The term "excipient" includes any component other than the compound(s) of the present disclosure, other lipid component(s), and biologically active agent. The excipient can provide either functional properties (e.g., drug release rate control) and / or non-functional properties (e.g., processing aid or dilution) to the composition. The choice of excipient depends to a large extent 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.

[0060] Typically, parenteral formulations are aqueous or oily solutions or suspensions. If the formulation is aqueous, it will contain excipients such as sugars (including, but not limited to, glucose, mannitol, sorbitol, etc.), salts, carbohydrates, and buffers (preferably to provide a pH of 3 to 9), but in some applications it may be more conveniently formulated using sterile non-aqueous solutions or as a dry form for use with a suitable vehicle such as sterile pyrogen-free water (WFI).

[0061] LNP composition The lipid composition can be provided as an LNP composition, and the LNP compositions described herein can be provided as lipid compositions. The lipid nanoparticles can be, for example, microspheres (unilamellar and multilamellar vesicles, e.g., "liposomes": in some embodiments, substantially spherical, and in more particular embodiments, comprising a lamellar phase lipid bilayer that can include an aqueous core (e.g., containing a substantial portion of the RNA molecules), the dispersed phase in an emulsion, a micelle, or the internal phase in a suspension.

[0062] Described herein are LNP compositions comprising at least one compound of formula (I) or (II), or a salt thereof (e.g., a pharma- ceutically acceptable salt thereof), at least one helper lipid, at least one neutral lipid, and at least one polymeric lipid. In some embodiments, the LNP composition comprises at least one compound of formula (I) or (II), or a pharma- ceutically acceptable salt thereof, at least one neutral lipid, at least one helper lipid, and at least one PEG lipid. In some embodiments, the neutral lipid is DSPC or DPME. In some embodiments, the helper lipid is cholesterol, 5-heptadecylresorcinol, or cholesterol hemisuccinate.

[0063] In a preferred embodiment, the ionizable lipid is [ka] In a preferred embodiment, the neutral lipid is DSPC. In a preferred embodiment, the helper lipid is cholesterol. In a preferred embodiment, the PEG lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000. In a particularly preferred embodiment, the ionizable lipid is [ka] where the neutral lipid is DSPC, the helper lipid is cholesterol, and the PEG lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

[0064] The present disclosure provides lipid compositions described according to the respective molar ratios of the component lipids in the composition. All mol% figures are shown as a percentage of the lipid component of the lipid composition, more specifically the LNP composition. In some embodiments, the mol% of lipid relative to the lipid component is ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the designated, nominal, or measured mol% of lipid. In some embodiments, the mol% of lipid relative to the lipid component is ±4 mol%, ±3 mol%, ±2 mol%, ±1.5 mol%, ±1 mol%, ±0.5 mol%, ±0.25 mol%, or ±0.05 mol% of the designated, nominal, or measured mol% of lipid. In certain embodiments, the mol% of lipid varies by less than 15%, less than 10%, less than 5%, less than 1%, or less than 0.5% from the designated, nominal, or measured mol% of lipid. In some embodiments, the mol% numbers are based on nominal concentrations. As used herein, "nominal concentration" refers to a concentration based on the input amounts of materials combined to form the resulting composition. For example, if 100 mg of solute is added to 1 L of water, the nominal concentration is 100 mg / L. In some embodiments, the mol% numbers are based on measured concentrations, e.g., concentrations quantified by analytical methods. In some embodiments, the measured lipid concentrations of the lipid components can be quantified, for example, by performing chromatography (e.g., liquid chromatography) followed by a detection method (e.g., charged aerosol detection). In some embodiments, the measured lipid concentrations of the lipid components can be characterized by lipid analysis, AF4-MALS, NTA, and / or cryo-EM. All mol% numbers are presented as percentages of lipids of the lipid components.

[0065] The present disclosure provides LNP compositions described according to the respective molar ratios of lipids in the lipid component. In certain embodiments, the amount of ionizable lipid is about 25 mol% to about 45 mol%, the amount of neutral lipid is about 10 mol% to about 30 mol%, the amount of helper lipid is about 25 mol% to about 65 mol%, and the amount of PEG lipid is about 1.5 mol% to about 3.5 mol%. In certain embodiments, the amount of ionizable lipid is about 29-44 mol% of the lipid component, the amount of neutral lipid is about 11-28 mol% of the lipid component, the amount of helper lipid is about 28-55 mol% of the lipid component, and the amount of PEG lipid is about 2.3-3.5 mol% of the lipid component. In certain embodiments, the amount of ionizable lipids is about 29-38 mol% of the lipid component, the amount of neutral lipids is about 11-20 mol% of the lipid component, the amount of helper lipids is about 43-55 mol% of the lipid component, and the amount of PEG lipids is about 2.3-2.7 mol% of the lipid component. In certain embodiments, the amount of ionizable lipids is about 25-34 mol% of the lipid component, the amount of neutral lipids is about 10-20 mol% of the lipid component, the amount of helper lipids is about 45-65 mol% of the lipid component, and the amount of PEG lipids is about 2.5-3.5 mol% of the lipid component. In certain embodiments, the amount of ionizable lipids is about 30-43 mol% of the lipid component, the amount of neutral lipids is about 10-17 mol% of the lipid component, the amount of helper lipids is about 43.5-56 mol% of the lipid component, and the amount of PEG lipids is about 1.5-3 mol% of the lipid component. In certain embodiments, the amount of ionizable lipid is about 33 mol% of lipid component, the amount of neutral lipid is about 15 mol% of lipid component, the amount of helper lipid is about 49 mol% of lipid component, and the amount of PEG lipid is about 3 mol% of lipid component.In certain embodiments, the amount of ionizable lipid is about 32.9 mol% of lipid component, the amount of neutral lipid is about 15.2 mol% of lipid component, the amount of helper lipid is about 49.2 mol% of lipid component, and the amount of PEG lipid is about 2.7 mol% of lipid component.In certain embodiments, the amount of ionizable lipid is about 35 mol% of the lipid component, the amount of neutral lipid is about 15 mol% of the lipid component, the amount of helper lipid is about 47.5 mol% of the lipid component, and the amount of PEG lipid is about 2.5 mol% of the lipid component.

[0066] In certain embodiments, the amount of ionizable lipid is about 20-50 mol%, about 25-34 mol%, about 25-38 mol%, about 25-45 mol%, about 29-38 mol%, about 29-43 mol%, about 29-34 mol%, about 30-34 mol%, about 30-38 mol%, about 30-43 mol%, about 30-43 mol%, or about 33 mol%. In additional embodiments, the amount of ionizable lipid is 25-45 mol%, about 25-43 mol%, about 25-40 mol%, about 25-38 mol%, about 25-35 mol%, about 25-33 mol%, about 25-30 mol%, about 25-28 mol%, about 28-45 mol%, about 28-43 mol%, about 28-40 mol%, about 28-38 mol%, about 28-35 mol%, about 28-33 mol%, about 28-30 mol%, about 30-45 mol%, about 30-43 mol%, about 30-43 mol%, about 30-45 ... 0-40 mol%, about 30-38 mol%, about 30-35 mol%, about 30-33 mol%, about 32-45 mol%, about 32-43 mol%, about 32-40 mol%, about 32-38 mol%, about 32-35 mol%, about 35-45 mol%, about 35-43 mol%, about 35-40 mol%, about 35-38 mol%, about 37-45 mol%, about 37-43 mol%, about 37-40 mol%, about 40-45 mol%, about 40-43 mol%, or about 42-45 mol%. In some embodiments, the mol% of the ionizable lipid may be about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, or about 45 mol%. In some embodiments, the mol% of the ionizable lipid relative to the lipid component is ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the specified, nominal, or measured mol%. In some embodiments, the mol% of the ionizable lipid relative to the lipid component is ±4 mol%, ±3 mol%, ±2 mol%, ±1.5 mol%, ±1 mol%, ±0.5 mol%, or ±0.25 mol% of the specified, nominal, or measured mol%.In certain embodiments, the LNP lot-to-lot variation of ionizable lipid mol% is less than 15%, less than 10%, or less than 5%. In some embodiments, the mol% number is based on nominal concentration. In some embodiments, the mol% number is based on measured concentration.

[0067] In certain embodiments, the amount of neutral lipid is about 10-30 mol%, about 11-30 mol%, about 11-20 mol%, about 13-17 mol%, or about 15 mol%. In additional embodiments, the amount of neutral lipid is about 5-30 mol%, about 5-28 mol%, about 5-25 mol%, about 5-23 mol%, about 5-20 mol%, about 5-18 mol%, about 5-23 mol%, about 5-20 mol%, about 5-18 mol%, about 5-15 mol%, about 5-13 mol%, about 5-10 mol%, about 10-30 mol%, about 10-28 mol%, about 10-25 mol%. ol%, about 10-23 mol%, about 10-20 mol%, about 10-18 mol%, about 10-23 mol%, about 10-20 mol%, about 10-18 mol%, about 10-15 mol%, about 10 ~13mol%, about 12-30mol%, about 12-28mol%, about 12-25mol%, about 12-23mol%, about 12-20mol%, about 12-18mol%, about 12-23mol%, Approx. 12~20mol%, Approx. 12~18mol%, Approx. 12~15mol%, Approx. 15~30mol%, Approx. 15~28mol%, Approx. 15~25mol%, Approx. 15~23mol%, Approx. 15~20m ol%, about 15-18 mol%, about 15-23 mol%, about 15-20 mol%, about 15-18 mol%, about 17-30 mol%, about 17-28 mol%, about 17-25 mol%, about 17- 23 mol%, about 17-20 mol%, about 17-18 mol%, about 17-23 mol%, about 17-20 mol%, about 20-30 mol%, about 20-28 mol%, about 20-25 mol%, about 20-23 mol%, about 22-30 mol%, about 22-28 mol%, about 22-25 mol%, about 22-23 mol%, about 22-20 mol%, or about 22-18 mol%. In some embodiments, the mol% of the neutral lipids can be about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol%, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, or about 25 mol%.In some embodiments, the mol% of neutral lipids relative to the lipid component is ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the specified, nominal, or measured mol% of neutral lipids. In some embodiments, the mol% of neutral lipids relative to the lipid component is ±4 mol%, ±3 mol%, ±2 mol%, ±1.5 mol%, ±1 mol%, ±0.5 mol%, or ±0.25 mol% of the specified, nominal, or measured mol%. In certain embodiments, the lot-to-lot variation of the LNP is less than 15%, less than 10%, or less than 5%. In some embodiments, the mol% figures are based on nominal concentrations. In some embodiments, the mol% figures are based on measured concentrations.

[0068] In certain embodiments, the amount of helper lipid is about 35-50 mol%, about 35-65 mol%, about 35-55 mol%, about 38-50 mol%, about 38-55 mol%, about 38-65 mol%, about 40-50 mol%, about 40-65 mol%, about 43-65 mol%, about 43-55 mol%, or about 49 mol%. In additional embodiments, the amount of helper lipid is about 25-65 mol%, about 28-65 mol%, about 30-65 mol%, about 32-65 mol%, about 35-65 mol%, about 38-65 mol%, about 40-65 mol%, about 42-65 mol%, about 45-65 mol%, about 48-65 mol%, about 50-65 mol%, about 52-65 mol%, about 55-65 mol%, about 58-65 mol%, about 60-65 mol%, about 25-62 mol%, about 28-62 ... 0 to 62 mol%, approximately 32 to 62 mol%, approximately 35 to 62 mol%, approximately 38 to 62 mol%, approximately 40 to 62 mol%, approximately 42 to 62 mol%, approximately 45 to 62 mol%, approximately 48 to 62 mol%, approximately 50 to 62 mol%, approximately 52 to 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 40-6 0mol%, about 42-60mol%, about 45-60mol%, about 48-60mol%, about 50-60mol%, about 52-60mol%, about 55-60mol%, about 58-60mol%, about 25-58mol%, about 28-58mol%, about 30-58mol%, about 32-58mol%, about 35-58mol%, about 38-58mol%, about 40-58mol%, about 42-58mol%, about 45-58mol%, about 48-58mol%, about 50-58mol%, about 52-58mol% l%, 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- 55mol%, about 48-55mol%, about 50-55mol%, about 52-55mol%, about 25-53mol%, about 28-53mol%, about 30-53mol%, about 32-53mol%, about 35-53mol%, about 38-53mol%,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 ~50mol%, approximately 32~50mol%, approximately 35~50mol%, approximately 38~50mol%, approximately 40~50mol%, approximately 42~50mol%, approximately 45~50mol%, approximately 48~50m ol%, 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 ~45mol%, about 40~45mol%, about 42~45mol%, about 25~43mol%, about 28~43mol%, about 30~43mol%, about 32~43mol%, about 35~43mol%, about 38~43mol%, about 40~43mol%, about 25~40mol%, about 28~40mol%, about 30~40mol%, about 32~40mol%, about 35~40mol% , about 38-40 mol%, about 25-38 mol%, about 28-38 mol%, about 30-38 mol%, about 32-38 mol%, about 35-38 mol%, about 25-35 mol%, about 28-35 mol%, about 30-35 mol%, about 32-35 mol%, about 25-33 mol%, about 28-33 mol%, about 30-33 mol%, about 25-30 mol%, about 28-30 mol%, or about 25-28 mol%. In certain embodiments, the amount of helper lipid is adjusted based on the amount of ionizable lipid, neutral lipid, and / or PEG lipid to bring the lipid component to about 100 mol%. In some embodiments, the mol% of helper lipid relative to the lipid component is ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the specified, nominal, or measured mol% of helper lipid. In some embodiments, the mol% of helper lipid relative to the lipid component is ±4 mol%, ±3 mol%, ±2 mol%, ±1.5 mol%, ±1 mol%, ±0.5 mol%, or ±0.25 mol% of the specified, nominal, or measured mol%. In certain embodiments, the lot-to-lot variation of the LNP is less than 15%,In some embodiments, the mol% figures are based on nominal concentrations. In some embodiments, the mol% figures are based on measured concentrations.

[0069] In certain embodiments, the amount of 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 PEG lipid is 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 3.0-3.0 mol%, about 3.1-3.0 mol%, about 3.2-3.0 mol%, about 3.3-3.0 mol%, about 3.4-4.0 mol%, about 3.5-4.0 mol%, about 3.6-4.0 mol%, about 3.7-4.0 mol%, about 3.8-4.0 mol%, about 3.9-4.0 mol%, about 4.0 ... %, 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 3.5~4.0mol%, approximately 3.7~4.0mol%, 1.0~3.7mol%, approximately 1.2~3.7mol%, approximately 1.4~3.7mol%, approximately 1.5~3.7mol%, approximately 1.6~3.7mol%, approximately 1.7~3.7mol%, approximately 1.8~3 .7mol%, approximately 1.9~3.7mol%, approximately 2.0~3.7mol%, approximately 2.1~3.7mol%, approximately 2.2~3.7mol%, approximately 2.3~3.7mol%, approximately 2.4~3.7mol%, approximately 2.5~3.7mol%, approximately 2.6~3.7m ol%, about 2.7 to 3.7 mol%, about 2.8 to 3.7 mol%, about 2.9 to 3.7 mol%, about 3.0 to 3.7 mol%, about 3.1 to 3.7 mol%, about 3.2 to 3.7 mol%, about 3.3 to 3.7 mol%, about 3.4 to 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 2.7-3.5mol%, about 2.8-3.5mol%, about 2.9-3.5mol%, about 3.0-3.5mol%, about 3.1-3.5mol%, about 3.2-3.5mol%, about 3.3-3.5mol%, about 3.4-3.5mol%, 1.0-3.4mol%, about 1.2-3.4mol%, about 1.4-3.4mol%, about 1.5-3.4mol%, about 1.6-3.4mol%, about 1.7-3.4mol%, about 1.8-3.4mol%, about 1.9-3.4mol%, about 2.0-3.4mol%, about 2.1-3.4mol%, about 2.2-3.4mol%, about 2.3-3.4mol 1%, about 2.4-3.4mol%, about 2.5-3.4mol%, about 2.6-3.4mol%, about 2.7-3.4mol%, about 2.8-3.4mol%, about 2.9-3.4mol%, about 3.0-3.4mol%, about 3.1-3.4mol%, about 3.2-3.4mol%, about 3.3-3.4mol%, 1.0-3.3mol%, about 1.2-3.3mol%, about 1.4-3.3mol%, about 1.5-3.3mol%, about 1.6-3.3mol%, about 1.7-3.3mol%, about 1.8-3.3mol%, about 1.9-3.3mol%, about 2.0-3.3mol%, 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. 9~3.2mol%, about 2.0~3.2mol%, about 2.1~3.2mol%, about 2.2~3.2mol%, about 2.3~3.2mol%, about 2.4~3.2mol%, about 2.5~3.2mol%, about 2.6~3.2mol%, about 2.7~3.2mol%, about 2.8~3.2mol%, about 2.9~3.2mol%, about 3.0~3.2mol%, about 3.1~3.2mol%, 1.0~3.1mol%, about 1.2~3.1mol%, about 1.4~3.1mol%, about 1.5~3.1mol%, about 1.6~3.1mol%, about 1.7~3.1mol%, about 1.8~3.1mol%, about 1.9-3.1mol%, about 2.0-3.1mol%, about 2.1-3.1mol%, about 2.2-3.1mol%, about 2.3-3.1mol%, about 2.4-3.1mol%, about 2.5-3.1mol%, about 2.6-3.1mol%, about 2.7-3.1mol%, about 2.8~3.1mol%, approximately 2.9~3.1mol%, approximately 3.0~3.1mol%, 1.0~3.0mol%, approximately 1.2~3.0mol%, approximately 1.4~3.0mol%, approximately 1.5~3.0mol%, approximately 1.6~3.0mol%, approximately 1.7~3.0mol%, approximately 1.8~3.0mo l%, about 1.9-3.0 mol%, about 2.0-3.0 mol%, about 2.1-3.0 mol%, about 2.2-3.0 mol%, about 2.3-3.0 mol%, about 2.4-3.0 mol%, about 2.5-3.0 mol%, about 2.6-3.0 mol%, about 2.7-3.0 mol%, about 2.8 ~3.0mol%, approximately 2.9~3.0mol%, 1.0~2.9mol%, approximately 1.2~2.9mol%, approximately 1.4~2.9mol%, approximately 1.5~2.9mol%, approximately 1.6~2.9mol%, approximately 1.7~2.9mol%, approximately 1.8~2.9mol%, approximately 1.9~2.9mol%, 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. 2~2.8mol%, approx. 2.3~2.8mol%, approx. 2.4~2.8mol%, approx. 2.5~2.8mol%, approx. 2.6~2.8mol%, approx. 2.7~2.8mol%, 1.0~2.7mol%, approx. 1.2~2.7mol%, approx. 1.4~2.7mol%, approx. 1.5~2.7mol% , 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 2.5-2.7mol%, about 2.6-2.7mol%, 1.0-2.6mol%, about 1.2-2.6mol%, about 1.4-2.6mol%, about 1.5-2.6mol%, about 1.6-2.6mol%, about 1.7-2.6mol%, about 1.8-2.6mol%, about 1.9-2.6mol%, about 2.0-2.6mol%, about 2.1-2.6mol%, about 2.2-2.6mol%, about 2.3-2.6mol%, about 2.4-2.6mol%, about 2.5-2.6mol%, 1.0-2.5mol%, about 1.2-2.5mol%, about 1.4-2.5mol%, about 1.5-2.5mol 1%, about 1.6-2.5mol%, about 1.7-2.5mol%, about 1.8-2.5mol%, about 1.9-2.5mol%, about 2.0-2.5mol%, about 2.1-2.5mol%, about 2.2-2.5mol%, about 2.3-2.5mol%, about 2.4-2.5mol%, 1.0-2.4mol%, about 1.2-2.4mol%, about 1.4-2.4mol%, about 1.5-2.4mol%, about 1.6-2.4mol%, about 1.7-2.4mol%, about 1.8-2.4mol%, about 1.9-2.4mol%, about 2.0-2.4mol%, about 2.1-2.4mol%, 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. 8~2.2mol%, about 1.9~2.2mol%, about 2.0~2.2mol%, about 2.1~2.2mol%, about 2.2~2.2mol%, about 2.3~2.2mol%, about 2.4~2.2mol%, 1.0~2.1mol%, about 1.2~2.1mol%, about 1.4~2.1mol%, about 1.5~2.1mol%, about 1.6~2.1mol%, about 1.7~2.1mol%, about 1.8~2.1mol%, about 1.9~2.1mol%, about 2.0~2.1mol%, 1.0~2.0mol%, about 1.2~2.0mol%, about 1.4~2.0mol%, about 1.5~2.0 mol%, about 1.6-2.0 mol%, about 1.7-2.0 mol%, about 1.8-2.0 mol%, about 1.9-2.0 mol%, 1.0-1.9 mol%, about 1.2-1.9 mol%, about 1.4-1.9 mol%, about 1.5-1.9 mol%, about 1.6-1.9 mol%, about 1.7-1.9 mol%, about 1.8-1.9 mol%, 1.0-1.8 mol%, about 1.2-1.8 mol%, about 1.4-1.8 mol%, about 1.5-1.8 mol%, about 1.6-1.8 mol%, about 1.7-1.8 mol%, 1.0-1.7 mol%, about 1.2-1. 7 mol%, about 1.4-1.7 mol%, about 1.5-1.7 mol%, about 1.6-1.7 mol%, 1.0-1.6 mol%, about 1.2-1.6 mol%, about 1.4-1.6 mol%, about 1.5-1.6 mol%, 1.0-1.5 mol%, about 1.2-1.5 mol%, about 1.4-1.5 mol%, about 1.5-1.5 mol%, about 1.6-1.5 mol%, about 1.7-1.5 mol%, about 1.7-1.7 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%. In some embodiments, the PEG lipid mol% can be about 1.5 mol%, about 1.6 mol%, about 1.7 mol%, about 1.8 mol%, about 1.9 mol%, about 2.0 mol%, about 2.1 mol%, about 2.2 mol%, about 2.3 mol%, about 2.4 mol%, about 2.5 mol%, about 2.6 mol%, about 2.7 mol%, about 2.8 mol%, about 2.9 mol%, about 3.0 mol%, about 3.1 mol%, about 3.2 mol%, about 3.3 mol%, about 3.4 mol%, or about 3.5 mol%. In some embodiments, the PEG lipid mol% relative to the lipid component will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the specified, nominal, or measured PEG lipid mol%. In some embodiments, the mol% of PEG lipid relative to the lipid component is within ±4 mol%, ±3 mol%, ±2 mol%, ±1.5 mol%, ±1 mol%, ±0.5 mol%, or ±0.5 mol% of the specified, nominal, or measured mol%.25 mol%. In certain embodiments, the lot-to-lot variation of the LNP is less than 15%, less than 10%, or less than 5%. In some embodiments, the mol% number is based on a nominal concentration. In some embodiments, the mol% number is based on an actual measured concentration.

[0070] In certain embodiments, the amount of helper lipid is about 30-40 mol%, the amount of PEG lipid is about 2.5-3.5 mol%, or the amount of helper lipid is about 33-37 mol%, the amount of PEG lipid is about 2.9-3.3 mol%, or the amount of helper lipid is about 35 mol%, the amount of PEG lipid is about 3 mol%. In certain embodiments, the amount of helper lipid is about 37-47 mol%, the amount of PEG lipid is about 2.0-3.0 mol%, or the amount of helper lipid is about 40-45 mol%, the amount of PEG lipid is about 2.2-2.8 mol%, or the amount of helper lipid is about 42 mol%, the amount of PEG lipid is about 2.2-2.8 mol%. In certain embodiments, the amount of helper lipid is about 47-57 mol% and the amount of PEG lipid is about 2.0-3.0 mol%, or the amount of helper lipid is about 50-55 mol% and the amount of PEG lipid is about 2.3-2.7 mol%, or the amount of helper lipid is about 52 mol% and the amount of PEG lipid is about 2.3-2.7 mol%.

[0071] In certain embodiments, the lipid composition (e.g., LNP composition) comprises a lipid component and a nucleic acid component (also referred to as an aqueous component) (e.g., an RNA component), and the molar ratio of the compound of formula (I) or (II):nucleic acid can be measured. The present disclosure also provides lipid compositions having a defined molar ratio between the positively charged amine group (N) of the pharma- ceutically acceptable salt of the compound of formula (I) or (II) and the negatively charged phosphate group (P) of the nucleic acid to be encapsulated. This can be mathematically represented by the formula N / P. In some embodiments, the lipid composition (e.g., LNP composition) can comprise a lipid component comprising a compound of formula (I) or (II) or a pharma- ceutically acceptable salt thereof, with an N / P ratio of about 3-10, and a nucleic acid component. In some embodiments, the LNP composition can comprise a lipid component comprising a compound of formula (I) or (II) or a pharma- ceutically acceptable salt thereof, with an N / P ratio of about 3-10, and an RNA component. For example, the N / P ratio can be about 4 to 7, about 5 to 7, or about 6 to 7. In some embodiments, the N / P ratio can be about 6, such as 6±1, or 6±0.5. In some embodiments, the N / P ratio can be about 7, such as 7±1, or 7±0.5.

[0072] In some embodiments, the aqueous component comprises a biologically active agent. In some embodiments, the aqueous component comprises a polypeptide, optionally in combination with a nucleic acid. In some embodiments, the aqueous component comprises a nucleic acid (e.g., RNA). In some embodiments, the aqueous component is a nucleic acid component. In some embodiments, the nucleic acid component comprises DNA, which may be referred to as a DNA component. In some embodiments, the nucleic acid component comprises RNA. In some embodiments, the aqueous component (e.g., RNA component) can comprise an mRNA (e.g., an mRNA encoding an RNA-guided DNA binder). In some embodiments, the RNA-guided DNA binder is a Cas nuclease. In certain embodiments, the aqueous component can comprise an mRNA encoding a Cas nuclease (e.g., Cas9). In certain embodiments, the biologically active agent is a Cas nuclease mRNA. In certain embodiments, the biologically active agent is a class 2 Cas nuclease mRNA. In certain embodiments, the biologically active agent is a Cas9 nuclease mRNA. In certain embodiments, the aqueous component can comprise a modified RNA. In some embodiments, the aqueous component can comprise a guide RNA nucleic acid. In certain embodiments, the aqueous component can comprise a gRNA. In certain embodiments, the aqueous component can comprise a dgRNA. In certain embodiments, the aqueous component can comprise a modified gRNA. In some compositions that include an mRNA encoding an RNA-guided DNA-binding agent, the composition further comprises a gRNA nucleic acid (e.g., a gRNA). In some embodiments, the aqueous component comprises an RNA-guided DNA-binding agent and a gRNA. In some embodiments, the aqueous component comprises a Cas nuclease mRNA and a gRNA. In some embodiments, the aqueous component comprises a class 2 Cas nuclease mRNA and a gRNA.

[0073] In certain embodiments, the lipid composition (e.g., LNP composition) can include an mRNA encoding a Cas nuclease (e.g., a class 2 Cas nuclease), a compound of formula (I) or (II) or a pharma- ceutically acceptable salt thereof, a helper lipid, and optionally a neutral lipid and a PEG lipid. In certain compositions comprising an mRNA encoding a Cas nuclease (e.g., a class 2 Cas nuclease), the helper lipid is cholesterol. In other compositions comprising an mRNA encoding a Cas nuclease (e.g., a class 2 Cas nuclease), the neutral lipid is DSPC. In additional embodiments comprising an mRNA encoding a Cas nuclease (e.g., a class 2 Cas nuclease, e.g., Cas9), the PEG lipid is PEG2k-DMG. In certain compositions comprising an mRNA encoding a Cas nuclease (e.g., a class 2 Cas nuclease) and a compound of formula (I) or (II) or a pharma- ceutically acceptable salt thereof. In certain compositions, the composition further comprises a gRNA (e.g., a dgRNA or an sgRNA).

[0074] In some embodiments, the lipid composition (e.g., LNP composition) can include a gRNA. In certain embodiments, the composition can include a compound of formula (I) or (II) or a pharma- ceutically acceptable salt thereof, a gRNA, a helper lipid, and optionally a neutral lipid and a PEG lipid. In certain LNP compositions that include a gRNA, the helper lipid is cholesterol. In some compositions that include a gRNA, the neutral lipid is DSPC. In additional embodiments that include a gRNA, the PEG lipid is PEG2k-DMG. In certain compositions, the gRNA is selected from a dgRNA and an sgRNA.

[0075] In certain embodiments, the lipid composition (e.g., LNP composition) comprises an RNA-guided DNA-binding agent and an mRNA encoding a gRNA (which may be an sgRNA) in the aqueous component, and comprises a compound of formula (I) or (II) in the lipid component. For example, the LNP composition can comprise a compound of formula (I) or (II) or a pharma- ceutically acceptable salt thereof, an mRNA encoding a Cas nuclease, a gRNA, a helper lipid, a neutral lipid, and a PEG lipid. In certain compositions comprising an mRNA and a gRNA encoding a Cas nuclease, the helper lipid is cholesterol. In some compositions comprising an mRNA and a gRNA encoding a Cas nuclease, the neutral lipid is DSPC. In additional embodiments comprising an mRNA and a gRNA encoding a Cas nuclease, the PEG lipid is PEG2k-DMG.

[0076] In certain embodiments, the lipid composition (e.g., LNP composition) comprises an RNA-lead DNA binder (e.g., class 2 Cas mRNA and at least one gRNA). In some embodiments, the gRNA is an sgRNA. In some embodiments, the RNA-lead DNA binder is a Cas9 mRNA. In certain embodiments, the LNP composition comprises a ratio of gRNA:RNA-lead DNA binder mRNA (e.g., class 2 Cas nuclease mRNA) of about 1:1 to about 1:2. In some embodiments, the weight ratio is about 25:1 to about 1:25, about 10:1 to about 1:10, about 8:1 to about 1:8, about 4:1 to about 1:4, about 2:1 to about 1:2, about 2:1 to about 1:4, or about 1:1 to about 1:2.

[0077] The lipid compositions (e.g., LNP compositions) disclosed herein can be used in the methods disclosed herein to deliver CRISPR / Cas9 components and insert template nucleic acids (e.g., DNA templates). The template nucleic acid can be delivered separately from the lipid composition comprising a compound of formula (I) or (II) or a pharma- ceutically acceptable salt thereof. In some embodiments, the template nucleic acid can be single-stranded or double-stranded, depending on the desired repair mechanism. The template can have regions of homology with the target DNA, for example, within the target DNA sequence and / or in sequences adjacent to the target DNA.

[0078] In some embodiments, the LNP composition is formed by mixing an aqueous RNA solution with an organic solvent-based lipid solution. Suitable solutions or solvents include or can include water, PBS, Tris buffer, NaCl, citrate buffer, acetate buffer, ethanol, chloroform, diethyl ether, cyclohexane, tetrahydrofuran, methanol, isopropanol. For example, the organic solvent can be 100% ethanol. A pharma- ceutically acceptable buffer can be used, such as a buffer for in vivo administration of the LNP composition. In certain embodiments, a buffer is used to maintain the pH of the LNP-containing composition at pH 6.5 or higher. In certain embodiments, a buffer is used to maintain the pH of the LNP-containing composition at pH 7.0 or higher. In certain embodiments, the composition has a pH in the range of about 7.2 to about 7.7. In additional embodiments, the composition has a pH in the range of about 7.3 to about 7.7 or 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, or 7.7. The pH of the composition can be measured using 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 can include up to 10% cryoprotectant (e.g., sucrose). In certain embodiments, the composition can include Tris-saline sucrose (TSS). In certain embodiments, the LNP composition can include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% cryoprotectant. In certain embodiments, the LNP composition can include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% sucrose. In some embodiments, the LNP composition can include a buffer. In some embodiments, the buffer can include phosphate buffer (PBS), Tris buffer, citrate buffer, and mixtures thereof. In certain exemplary embodiments, the buffer includes NaCl.In certain embodiments, the buffer lacks NaCl. Exemplary amounts of NaCl can range from about 20 mM to about 45 mM. Exemplary amounts of NaCl can range from about 40 mM to about 50 mM. In some embodiments, the amount of NaCl is about 45 mM. In some embodiments, the buffer is a Tris buffer. Exemplary amounts of Tris can range from about 20 mM to about 60 mM. Exemplary amounts of Tris can 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 composition comprise 5% sucrose and 45 mM NaCl in a Tris buffer. In other exemplary embodiments, the composition comprises sucrose in an amount of about 5% w / v, about 45 mM NaCl, and about 50 mM Tris at pH 7.5. The amounts of salt, buffer, and cryoprotectant can be varied to maintain the osmolality of the overall composition. For example, the final osmolality can be maintained below 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.

[0079] In some embodiments, microfluidic mixing, T-mixing, or cross-mixing of aqueous RNA and lipid solutions in organic solvents is used. In certain embodiments, the flow rate, junction size, junction geometry, junction shape, tubing diameter, solutions, and / or RNA and lipid concentrations can be varied. The LNP or LNP composition can be concentrated or purified, for example, via dialysis, centrifugal filters, tangential flow filtration, or chromatography. The LNP composition can be stored, for example, as a suspension, emulsion, or lyophilized powder. In some embodiments, the LNP composition is stored at 2-8°C, and in certain embodiments, the LNP composition is stored at room temperature. In additional embodiments, the LNP composition is stored frozen, for example, at -20°C or -80°C. In other embodiments, the LNP composition is stored at a temperature ranging from about 0°C to about -80°C. The frozen LNP composition can be thawed, for example, on ice, at room temperature, or at 25°C, prior to use.

[0080] Preferred lipid compositions (e.g., LNP compositions) are biodegradable, for example, in that they do not accumulate to cytotoxic levels in vivo at therapeutically effective doses.In some embodiments, the compositions do not induce a natural immune response that leads to substantial adverse effects at therapeutic dose levels.In some embodiments, the compositions provided herein do not induce toxicity at therapeutic dose levels.

[0081] In some embodiments, the concentration of LNP in the LNP composition is about 1-10 μg / mL, about 2-10 μg / mL, about 2.5-10 μg / mL, about 1-5 μg / mL, about 2-5 μg / mL, about 2.5-5 μg / mL, about 0.04 μg / mL, about 0.08 μg / mL, about 0.16 μg / mL, about 0.25 μg / mL, about 0.63 μg / mL, about 1.25 μg / mL, about 2.5 μg / mL, or about 5 μg / mL.

[0082] In some embodiments, dynamic light scattering ("DLS") can be used to characterize the polydispersity index (PDI) and size of the LNPs of the present disclosure. DLS measures the scattering of light resulting from subjecting a sample to a light source. The PDI determined from a DLS measurement corresponds to the distribution of particle sizes (around the average particle size) in a population, with a completely homogenous population having a PDI of zero.

[0083] In some embodiments, the LNPs disclosed herein have a PDI of about 0.005 to about 0.75. In some embodiments, the LNPs disclosed herein have a PDI of about 0.005 to about 0.1. In some embodiments, the LNPs disclosed herein have a PDI of 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 LNPs have a PDI of about 0.01 to about 0.5. In some embodiments, the LNPs have a PDI of about zero to about 0.4. In some embodiments, the LNPs have a PDI of about zero to about 0.35. In some embodiments, the LNPs have a PDI that can range from about zero to about 0.3. In some embodiments, the LNPs have a PDI that can range from about zero to about 0.25. In some embodiments, the LNPs PDI can range from about zero to about 0.2. In some embodiments, the LNPs have a PDI of about zero to about 0.05. In some embodiments, the LNPs have a PDI of about zero to about 0.01, hi some embodiments, the LNPs have a PDI of 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.

[0084] LNP size can be measured by various analytical methods known in the art. In some embodiments, LNP size can be measured using asymmetric flow field fractionation-multi-angle light scattering (AF4-MALS). In certain embodiments, LNP size can be measured by separating particles in a composition by hydrodynamic radius and then measuring the molecular weight, hydrodynamic radius, and root-mean-square radius of the separated particles. In some embodiments, LNP size and particle concentration can be measured by nanoparticle tracking analysis (NTA, Malvern Nanosight). In certain embodiments, LNP samples are appropriately diluted and injected onto a microscope slide. A camera records the scattered light as the particles are slowly injected through the field of view. After a video is captured, nanoparticle tracking analysis processes the video by tracking pixels and calculating the diffusion coefficient. This diffusion coefficient can be converted to the hydrodynamic radius of the particle. Such methods can also count the number of individual particles to obtain particle concentration. In some embodiments, size, morphology, and structural characteristics of LNPs can be quantified by cryo-electron microscopy ("cryo-EM").

[0085] 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 about 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 LNPs have a size of about 60 to about 100 nm. In some embodiments, the LNPs have a size of about 75 to about 150 nm. In some embodiments, the LNPs have a size of about 75 to about 120 nm. In some embodiments, the LNPs have a size of about 75 to about 100 nm. In some embodiments, the LNPs have a size of about 50 to about 145 nm, about 50 to about 120 nm, about 50 to about 120 nm, about 50 to about 115 nm, about 50 to about 100 nm, about 60 to about 145 nm, about 60 to about 120 nm, about 60 to about 115 nm, or about 60 to about 100 nm. In some embodiments, the LNPs have a size of less than about 145 nm, less than about 120 nm, less than about 115 nm, less than about 100 nm, or less than about 80 nm. In some embodiments, the LNPs have a size of greater than about 50 nm or greater than about 60 nm. In some embodiments, the particle size is the Z-average particle size. In some embodiments, the particle size is the number average particle size. In some embodiments, the particle size is the size of an individual LNP. Unless otherwise indicated, all sizes referred to herein are the average size (diameter) of fully formed nanoparticles as measured by dynamic light scattering on a Malvern Zetasizer or Wyatt NanoStar. Dilute the nanoparticle sample in phosphate-buffered saline (PBS) to achieve a count rate of approximately 200-400 kcps.

[0086] In some embodiments, the LNP compositions are formed with an average encapsulation efficiency ranging from about 50% to about 100%. In some embodiments, the LNP compositions are formed with an average encapsulation efficiency ranging from about 50% to about 95%. In some embodiments, the LNP compositions are formed with an average encapsulation efficiency ranging from about 70% to about 90%. In some embodiments, the LNP compositions are formed with an average encapsulation efficiency ranging from about 90% to about 100%. In some embodiments, the LNP compositions are formed with an average encapsulation efficiency ranging from about 75% to about 95%. In some embodiments, the LNP compositions are formed with an average encapsulation efficiency ranging from about 90% to about 100%. In some embodiments, the LNP compositions are formed with an average encapsulation efficiency ranging from about 95% to about 100%. In some embodiments, the LNP compositions are formed with an average encapsulation efficiency ranging from about 98% to about 100%. In some embodiments, the LNP compositions are formed with an average encapsulation efficiency ranging from about 99% to about 100%.

[0087] cargo Cargo delivered via the LNP compositions described herein includes biologically active agents, which can be nucleic acids (e.g., mRNA and gRNA). In certain embodiments, the cargo may be one or more biologically active agents, such as mRNA, gRNA, expression vectors, RNA-guided DNA binding agents, antibodies (e.g., monoclonal, chimeric, humanized, nanobodies, and fragments thereof), cholesterol, hormones, peptides, proteins, chemotherapeutic agents and other types of anti-neoplastic agents, small molecule drugs, vitamins, cofactors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, antisense DNA or RNA compositions, chimeric DNA:RNA compositions, allozymes, aptamers, ribozymes, decoys and analogs thereof, plasmids and other types of vectors, as well as small nucleic acid molecules, RNAi agents, short interfering nucleic acids (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and "self-replicating RNA" (encoding replicase enzyme activity and in The biologically active agents may be or include: molecules capable of directing their own replication or amplification in vivo, peptide nucleic acids (PNAs), locked nucleic acid ribonucleotides (LNAs), morpholino nucleotides, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), sisiRNAs (small internal segmented interfering RNAs), and iRNAs (asymmetric interfering RNAs). The above list of biologically active agents is illustrative only and is not intended to be limiting. Such compounds may be purified or partially purified, naturally occurring or synthetic, and may be chemically modified.

[0088] The cargo delivered via the LNP composition can be RNA (e.g., an mRNA molecule encoding a protein of interest). Examples include mRNA for expressing a protein such as green fluorescent protein (GFP), an RNA-guided DNA binder, or a Cas nuclease. LNP compositions are provided that include a Cas nuclease mRNA, e.g., a class 2 Cas nuclease mRNA that allows expression of a class 2 Cas nuclease (e.g., Cas9 or Cpf1 (also called Cas12a) protein) in a cell. Additionally, the cargo can include one or more gRNAs or a nucleic acid encoding a gRNA. A template nucleic acid (e.g., for repair or recombination) can be included in the composition or used in the methods described herein. In a subembodiment, the cargo includes an mRNA encoding Streptococcus pyogenes Cas9 and, optionally, an S. pyogenes gRNA. In a further subembodiment, the cargo includes an mRNA encoding Neisseria meningitidis Cas9 and, optionally, an Nme (Neisseria meningitidis) gRNA.

[0089] "mRNA" refers to a polynucleotide and includes an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by ribosomes and aminoacylated tRNAs). An mRNA can include a phosphate-sugar backbone that includes ribose residues or analogs thereof (e.g., 2'-methoxyribose residues). In some embodiments, the sugars of the mRNA phosphate-sugar backbone consist essentially of ribose residues, 2'-methoxyribose residues, or combinations thereof. Generally, an mRNA does not include a substantial amount of thymidine residues (e.g., 0 residues, or 30, 20, 10, 5, 4, 3, or 2 thymidine residues, or a thymidine content of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%). An mRNA can include modified uridines at some or all of the uridine positions.

[0090] Genome editing tools In some embodiments, the LNP composition is a lipid-nucleic acid assembly, also referred to as a lipid-nucleic acid composition. In some embodiments, the lipid-nucleic acid composition or LNP composition comprises a genome editing tool or a nucleic acid encoding the same. As used herein, the term "genome editing tool" (or "gene editing tool") is any component of a "genome editing system" (or "gene editing system") that is necessary or useful for generating edits in a cell's genome. In some embodiments, the present disclosure provides a method for delivering a genome editing tool of a genome editing system (e.g., a zinc finger nuclease system, a TALEN system, a meganuclease system, or a CRISPR / Cas system) to a cell (or a population of cells). Genome editing tools include, for example, nucleases that can make single-stranded or double-stranded breaks in the DNA or RNA of a cell, e.g., the genome of a cell. Genome editing tools, e.g., nucleases or nickases, can optionally modify the genome of a cell without cleaving the nucleic acid. Genome editing nucleases or nickases can be encoded by mRNA. Such nucleases include, for example, RNA-guided DNA binders and CRISPR / Cas components. Genome editing tools include fusion proteins, including, for example, nickases fused with effector domains (e.g., editor domains). Genome editing tools include any item (e.g., guide RNA, sgRNA, dgRNA, donor nucleic acid, etc.) necessary or useful to achieve genome editing goals.

[0091] A variety of suitable gene editing systems, including genome editing tools for delivery with lipid nucleic acid assembly compositions, are described herein, including, but not limited to, CRISPR / Cas systems, zinc finger nuclease (ZFN) systems, and transcription activator-like effector nuclease (TALEN) systems. Generally, gene editing systems involve the use of engineered cleavage systems to induce double-strand breaks (DSBs) or nicks (e.g., single-strand breaks, or SSBs) within target DNA sequences. Cleavage or nicking can occur by using specific nucleases (e.g., engineered ZFNs, TALENs), or by using CRISPR / Cas systems with engineered guide RNAs to guide specific cleavage or nicking of target DNA sequences. Additionally, targeted nucleases based on the Argonaute system have been developed (e.g., from T. thermophilus known as "TtAgo"; see Swarts et al (2014) Nature 507(7491):258-261), which may also have the potential to be used in genome editing and gene therapy.

[0092] In certain embodiments, the compositions of the present disclosure include one or more DNA modifying agents (e.g., DNA cleavage agents). A variety of DNA modifying agents can be included in the LNP compositions described herein. For example, DNA modifying agents include nucleases (both sequence specific and non-specific), topoisomerases, methylases, acetylases, chemicals, pharmaceuticals, and other agents. In some embodiments, proteins that bind to a given DNA sequence or set of sequences can be used to induce DNA modifications, such as strand cleavage. Proteins can be used to induce DNA modifications, such as strand cleavage, for example, when radioactive decay causes strand cleavage. 125Proteins can be modified by a number of means, such as the incorporation of I or modification with cross-linking reagents such as 4-azidophenacyl bromide, which forms cross-links with DNA upon exposure to UV light. Such protein-DNA cross-links can be subsequently converted to double-stranded DNA breaks by treatment with piperidine. Another approach to DNA modification involves the production of antibodies against specific proteins that are bound to one or more DNA sites, such as transcription factors or architectural chromatin proteins, which are used to isolate DNA from nucleoprotein complexes.

[0093] In certain embodiments, the composition of the present disclosure comprises one or more DNA cleavage agents. DNA cleavage agents include technologies such as zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), mito-TALEN, and meganuclease system. TALEN and ZFN technologies use a strategy of tethering the catalytic domain of an endonuclease to a modular DNA binding protein to induce double-strand breaks (DSBs) in target DNA at specific genomic loci. Additional DNA cleavage agents include small interfering RNA, microRNA, anti-microRNA, antagonists, small hairpin RNA, and aptamers (RNA, DNA, or peptide-based (including affimers)).

[0094] In some embodiments, the gene editing system is a TALEN system. Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences of DNA. They are created by fusing a TAL effector DNA binding domain to a DNA cleavage domain (a nuclease that cleaves DNA strands). Transcription activator-like effectors (TALEs) can be engineered to bind to desired DNA sequences and promote DNA cleavage at specific locations (see, for example, Boch, 2011, Nature Biotech). Restriction enzymes can be introduced into cells for use in gene editing or for in situ genome editing, a technique known as genome editing with engineered nucleases. Such methods and compositions for use therein are known in the art. See, for example, WO2019147805, WO2014040370, WO2018073393, the contents of which are incorporated herein in their entirety.

[0095] In some embodiments, the gene editing system is a zinc finger system. Zinc finger nuclease (ZFN) is an artificial restriction enzyme that is generated by fusing a zinc finger DNA binding domain with a DNA cleavage domain. The zinc finger domain can be engineered to target specific desired DNA sequences, enabling the zinc finger nuclease to target unique sequences in complex genomes. The non-specific cleavage domain of the type IIs restriction endonuclease FokI is typically used as the cleavage domain in ZFN. The cleavage is repaired by endogenous DNA repair machinery, allowing ZFN to precisely modify the genome of higher organisms. Such methods and compositions for use therein are known in the art. See, for example, WO2011091324, the contents of which are incorporated herein in their entirety.

[0096] In preferred embodiments, the compositions of the disclosure comprise an mRNA encoding an RNA-guided DNA-binding agent (e.g., a Cas nuclease). In certain embodiments, the compositions of the disclosure comprise an mRNA encoding a class 2 Cas nuclease, such as S. pyogenes Cas9.

[0097] As used herein, "RNA-guided DNA binder" refers to a polypeptide or complex of polypeptides having RNA and DNA binding activity, or a DNA-binding subunit of such a complex, where the DNA-binding activity is sequence-specific and dependent on the sequence of the RNA. Exemplary RNA-guided DNA binders include Cas cleavage / nickases and their inactivated forms ("dCas DNA binders"). As used herein, "Cas nucleases" encompass Cas cleavage, Cas nickase, and dCas DNA binders. Cas cleavage / nickases and dCas DNA binders include the Csm or Cmr complex of type III CRISPR systems, Cas10, its Csm1 or Cmr2 subunits, the Cascade complex of type I CRISPR systems, its Cas3 subunit, and class 2 Cas nucleases. As used herein, "class 2 Cas nucleases" are single-chain polypeptides having RNA-guided DNA binding activity. Class 2 Cas nucleases include Class 2 Cas cleavases / nickases that additionally have RNA-guided DNA cleavase or nickase activity (e.g., H840A, D10A, or N863A variants), and Class 2 dCas DNA binders in which the cleavase / nickase activity has been inactivated. Class 2 Cas nucleases that can be used with the LNP compositions described herein include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof. The Cpf1 protein (Zetsche et al., Cell, 163:1-13 (2015)) is homologous to Cas9 and contains a RuvC-like nuclease domain. The Cpf1 sequence of Zetsche is incorporated by reference in its entirety.See, e.g., Tables 2 and 4 of Zetsche. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11):722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).

[0098] Non-limiting examples of species from which Cas nucleases may be derived include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus 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, Methanohlobium 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、、、Acaryochloris marina is included。.

[0099] In some embodiments, the Cas nuclease is a Cas9 nuclease from Streptococcus pyogenes. In other embodiments, the Cas nuclease is a Cas9 nuclease from Streptococcus thermophilus. In still other embodiments, the Cas nuclease is a Cas9 nuclease from Neisseria meningitidis. In some embodiments, the Cas nuclease is a Cas9 nuclease from Staphylococcus aureus. In some embodiments, the Cas nuclease is a Cpf1 nuclease from Francisella novicida. In other embodiments, the Cas nuclease is a Cpf1 nuclease from Acidaminococcus sp. In still other embodiments, the Cas nuclease is a Cpf1 nuclease from Lachnospiraceae bacterium ND2006. In further embodiments, the Cas nuclease is a Cpf1 nuclease from Francisella tularensis, Lachnospiraceae bacterium, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium, Parcubacteria bacterium, Smithella, Acidaminococcus, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi, Leptospira inadai, Porphyromonas crevioricanis, Prevotella disiens, or Porphyromonas macacae. In some embodiments, the Cas nuclease is a Cpf1 nuclease from Acidaminococcus or Lachnospiraceae.

[0100] 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 two or more RuvC domains and / or two or more HNH domains. In some embodiments, the Cas9 nuclease is wild-type Cas9. In some embodiments, the Cas9 is capable of inducing a double-stranded break in the target DNA. In other embodiments, the Cas nuclease may cleave dsDNA, may cleave a single strand of dsDNA, or may not have DNA cleavage or nickase activity.

[0101] In some embodiments, chimeric Cas nucleases are used in which one domain or region is replaced with a portion of a different protein. In some embodiments, the Cas nuclease domain may be replaced with a domain from a different nuclease, such as Fok1. In some embodiments, the Cas nuclease may be a modified nuclease.

[0102] 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 a 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 protein may be from a type III CRISPR / Cas system. In some embodiments, the Cas nuclease may have RNA cleavage activity.

[0103] In some embodiments, the RNA-guided DNA-binding agent has single-stranded nickase activity, i.e., it can cleave one DNA strand, resulting in a single-stranded 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., it cleaves one strand of the DNA double helix, but not the other. In some embodiments, the Cas nickase is a version of a Cas nuclease (e.g., a Cas nuclease described above) in which the endonuclease active site has been inactivated, for example, by one or more modifications (e.g., point mutations) in the catalytic domain. See, e.g., U.S. Pat. No. 8,889,356 for a discussion of Cas nickases and exemplary catalytic domain modifications. In some embodiments, the Cas nickase (e.g., Cas9 nickase) has an inactivated RuvC or HNH domain. In some embodiments, the RNA-guided DNA-binding agent is modified to include only one functional nuclease domain. For example, the drug protein can be modified such that one of the nuclease domains is mutated or completely or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, a nickase with a RuvC domain having reduced activity is used. In some embodiments, a nickase with an inactive RuvC domain is used. In some embodiments, a nickase with an HNH domain having reduced activity is used. In some embodiments, a nickase with an inactive HNH domain is used.

[0104] In some embodiments, conserved amino acids in the Cas protein nuclease domain are substituted to reduce or modify nuclease activity. In some embodiments, the Cas nuclease can include 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 can include an amino acid substitution in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015). Further exemplary amino acid substitutions include D917A, E1006A, and D1255A (based on the Francisella novicida U112 Cpf1 (FnCpf1) sequence (UniProtKB: A0Q7Q2(CPF1_FRATN))).

[0105] In some embodiments, the mRNA encoding the nickase is provided in combination with a pair of guide RNAs that are complementary to the sense and antisense strands of the target sequence, respectively. In this embodiment, the guide RNA guides the nickase to the target sequence and introduces a DSB by generating a nick on the opposite strand of the target sequence (i.e., double nicking). In some embodiments, the use of double nicking can improve specificity and reduce off-target effects. In some embodiments, the nickase is used with two separate guide RNAs that target opposite strands of DNA to generate a double nick in the target DNA. In some embodiments, the nickase is used with two separate guide RNAs that are selected to be in close proximity to generate a double nick in the target DNA.

[0106] In some embodiments, the RNA-guided DNA-binding agent lacks cleavase and nickase activity. In some embodiments, the RNA-guided DNA-binding agent comprises a dCas DNA-binding polypeptide. While the dCas polypeptide has DNA-binding activity, it essentially lacks catalytic activity (cleavase / nickase activity). In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, the RNA-guided DNA-binding agent lacking cleavase and nickase activity, or the dCas DNA-binding polypeptide, is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) whose endonuclease active site has been inactivated, for example, by one or more modifications (e.g., point mutations) in its catalytic domain. See, e.g., US2014 / 0186958A1 and US2015 / 0166980A1.

[0107] In some embodiments, the RNA-guided DNA binding agent comprises an APOBEC3 deaminase. In some embodiments, the APOBEC3 deaminase is APOBEC3A (A3A). In some embodiments, the A3A is human A3A. In some embodiments, the A3A is wild-type A3A.

[0108] In some embodiments, the RNA-guided DNA binding agent comprises an editor. An exemplary editor is BC22n, which comprises H. sapiens APOBEC3A fused to S. pyogenes-D10ACas9 nickase by an XTEN linker. In some embodiments, the editor is provided with a uracil glycosylase inhibitor ("UGI"). In some embodiments, the editor is fused to a UGI. In some embodiments, the mRNA encoding the editor and the mRNA encoding the UGI are formulated together in a LNP. In other embodiments, the editor and the UGI are provided in separate LNPs.

[0109] In some embodiments, the RNA-guided DNA binding agent comprises (eg, is or comprises a fusion polypeptide) one or more heterologous functional domains.

[0110] In some embodiments, the heterologous functional domain can facilitate the transport of the RNA-guided DNA binding agent into the cell nucleus. For example, the heterologous functional domain can be a nuclear localization signal (NLS).

[0111] In some embodiments, the heterologous functional domain may be capable of modifying the intracellular half-life of the RNA-guided DNA binder. In some embodiments, the half-life of the RNA-guided DNA binder may be increased. In some embodiments, the half-life of the RNA-guided DNA binder may be decreased. In some embodiments, the heterologous functional domain may be capable of increasing the stability of the RNA-guided DNA binder. In some embodiments, the heterologous functional domain may be capable of decreasing the stability of the RNA-guided DNA binder. In some embodiments, the heterologous functional domain may act as a signal peptide for protein degradation. In some embodiments, the protein degradation may be mediated by a proteolytic enzyme (e.g., a proteasome, a lysosomal protease, or a calpain protease). In some embodiments, the heterologous functional domain may comprise a PEST sequence. In some embodiments, the RNA-guided DNA binder may be modified by the addition of ubiquitin or polyubiquitin chains. In some embodiments, the ubiquitin may be a ubiquitin-like protein (UBL). Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon stimulated gene-15 (ISG15)), ubiquitin-related modifier-1 (URM1), neuronal-precursor-cell expressed developmentally downregulated protein-8 (NEDD8; also called Rub1 in S. cerevisiae), human leukocyte antigen F-related (FAT10), autophagy-8 (ATG8) and -12 (ATG12), Fau ubiquitin-like protein (FUB1), membrane-anchored UBL (MUB), ubiquitin fold modifier-1 (UFM1), and ubiquitin-like protein-5 (UBL5).

[0112] In some embodiments, the heterologous functional domain may be a marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, epitope tags, and reporter gene sequences. In some embodiments, the marker domain may be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (e.g., mKa te, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent protein (mOrange, mKO, Kusabira-Orange, monomeric Kusabira-Orange, mTangerine, tdTomato), or any other suitable fluorescent protein. In other embodiments, the marker domain may be a purification tag and / or an epitope tag.Non-limiting exemplary tags include glutathione-S-transferase (GST), chitin-binding protein (CBP), maltose-binding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis, 8xHis, biotin carboxyl carrier protein (BCCP), poly-His, and calmodulin. Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent proteins.

[0113] In additional embodiments, the heterologous functional domain can target the RNA-guided DNA binding agent to a particular organelle, cell type, tissue, or organ. In some embodiments, the heterologous functional domain can target the RNA-guided DNA binding agent to mitochondria.

[0114] In further embodiments, the heterologous functional domain may be an effector domain (e.g., an editor domain). When an RNA-guided DNA binding agent is guided to a target sequence, for example when a Cas nuclease is guided to a target sequence by a gRNA, the effector domain (e.g., an editor domain) can modify or affect the target sequence. In some embodiments, the effector domain (e.g., an editor domain) can be selected from a nucleic acid binding domain, a nuclease domain (e.g., a non-Cas nuclease domain), an epigenetic modification domain, a transcription activation domain, or a transcription repression domain. In some embodiments, the heterologous functional domain is a nuclease (e.g., a FokI nuclease). See, e.g., U.S. Pat. No. 9,023,649. In some embodiments, the heterologous functional domain is a transcription activator or a transcription repressor. For example, Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression,” Cell 152:1173-83 (2013); Perez-Pinera et al., “RNA-guided gene activation by CRISPR-Cas9- based transcription factors,” Nat.Methods 10:973-6 (2013); Mali et al., “CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering,” Nat.Biotechnol.31:833-8 (2013); Gilbert et al., “CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes,” Cell 154:442-51 (2013).Therefore, the RNA-guided DNA binding agent essentially becomes a transcription factor that can be guided to bind to a desired target sequence using a guide RNA. In some embodiments, the DNA modification domain is a methylation domain (e.g., a demethylation or methyltransferase domain). In some embodiments, the effector domain is a DNA modification domain (e.g., a base editing domain). In certain embodiments, the DNA modification domain is a nucleic acid editing domain (e.g., a deaminase domain) that introduces specific modifications to DNA. See, for example, WO2015 / 089406 and US2016 / 0304846. The nucleic acid editing domain, deaminase domain, and Cas9 variants described in WO2015 / 089406 and US2016 / 0304846 are each incorporated herein by reference in their entirety.

[0115] The nuclease may include at least one domain that interacts with a guide RNA ("gRNA"). Additionally, the nuclease may be guided to a target sequence by the gRNA. In a Class 2 Cas nuclease system, the gRNA interacts with the nuclease and the target sequence to guide binding to the target sequence. In some embodiments, the gRNA provides the specificity of target cleavage, and the nuclease is versatile and can pair with different gRNAs to cleave different target sequences. Class 2 Cas nucleases can pair with gRNA scaffold structures of the types, orthologs, and exemplary species listed above.

[0116] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a gRNA together with an RNA-guided DNA-binding agent (e.g., a Cas nuclease, e.g., a Cas cleavase, a Cas nickase, or a dCas DNA-binding agent (e.g., Cas9)). In some embodiments, the gRNA guides the RNA-guided DNA-binding agent (e.g., Cas9) to a target sequence, the gRNA hybridizes to the target sequence, and the binding agent binds to the target sequence. If the binding agent is a cleavase or nickase, binding may occur after cleavage or nicking.

[0117] In some embodiments of the present disclosure, the cargo in the LNP composition comprises at least one gRNA comprising a guide sequence that guides an RNA-guided DNA binding agent, which may be a nuclease (e.g., a Cas nuclease, such as Cas9), to a target DNA. The gRNA can guide a Cas nuclease or a class 2 Cas nuclease to a target sequence on a target nucleic acid molecule. In some embodiments, the gRNA binds to a class 2 Cas nuclease and provides specificity for cleavage by the nuclease. In some embodiments, the gRNA and the Cas nuclease can form a ribonucleoprotein (RNP), e.g., a CRISPR / Cas complex (e.g., a CRISPR / Cas9 complex). In some embodiments, the CRISPR / Cas complex can be a type II CRISPR / Cas9 complex. In some embodiments, the CRISPR / Cas complex can be a type V CRISPR / Cas complex (e.g., a Cpf1 / gRNA complex). The Cas nuclease and the cognate gRNA can pair. The gRNA scaffold structure that pairs with each Class 2 Cas nuclease varies depending on the specific CRISPR / Cas system.

[0118] "Guide RNA", "gRNA" and simply "guide" are used interchangeably herein to refer to the cognate guide nucleic acid of an RNA-guided DNA-binding agent. Guide RNA can include modified RNA as described herein. gRNA can be, for example, either a single guide RNA or a combination of crRNA and trRNA (aka tracrRNA). crRNA and trRNA can be associated as a single RNA molecule (as single guide RNA, sgRNA) or, for example, in two separate RNA strands (dual guide RNA, dgRNA). In some systems, gRNA can be a crRNA (aka CRISPR RNA). "Guide RNA" or "gRNA" refer to each type. trRNA can be a naturally occurring sequence or a trRNA sequence that has modifications or variations compared to the naturally occurring sequence.

[0119] In some embodiments, the mRNA encoding the RNA-guided DNA-binding agent is formulated in a first LNP composition and the gRNA nucleic acid is formulated in a second LNP composition. In some embodiments, the first and second LNP compositions are administered simultaneously. In other embodiments, the first and second LNP compositions are administered sequentially. In some embodiments, the first and second LNP compositions are combined prior to the preincubation step. In other embodiments, the first and second LNP compositions are preincubated separately.

[0120] In some embodiments, the cargo can comprise a DNA molecule. In some embodiments, the nucleic acid can comprise a nucleotide sequence encoding a crRNA. In some embodiments, the nucleotide sequence encoding the crRNA comprises a targeting sequence flanked by all or a portion of a repeat sequence from a naturally occurring CRISPR / Cas system. In some embodiments, the nucleic acid can comprise a nucleotide sequence encoding a tracr RNA. In certain embodiments, the crRNA and the tracr RNA can be encoded by two separate nucleic acids. In other embodiments, the crRNA and the tracr RNA can be encoded by a single nucleic acid. In some embodiments, the crRNA and the tracr RNA can be encoded by opposing strands of a single nucleic acid. In other embodiments, the crRNA and the tracr RNA can be encoded by the same strand of a single nucleic acid. In some embodiments, the gRNA nucleic acid encodes a sgRNA. In some embodiments, the gRNA nucleic acid encodes a Cas9 nuclease sgRNA. In some embodiments, the gRNA nucleic acid encodes a Cpf1 nuclease sgRNA.

[0121] The nucleotide sequence encoding the guide RNA can be operably linked to at least one transcriptional or regulatory control sequence (e.g., promoter, 3'UTR, or 5'UTR). In one example, the promoter can be a tRNA promoter (e.g., tRNALys3 or tRNA chimera). See Mefferd et al., RNA. 2015 21:1683-9; Scherer et al., Nucleic Acids Res. 2007 35:2620-2628. In some embodiments, the promoter can be recognized by RNA polymerase III (Pol III). Non-limiting examples of Pol III promoters also include the U6 promoter and the H1 promoter. In some embodiments, the nucleotide sequence encoding the guide RNA can be operably linked to a mouse or human U6 promoter. In some embodiments, the gRNA nucleic acid is a modified nucleic acid. In some embodiments, the gRNA nucleic acid comprises modified nucleosides or nucleotides. In some embodiments, the gRNA nucleic acid comprises a 5'-end modification (e.g., a modified nucleoside or nucleotide) to stabilize and prevent nucleic acid integration. In other embodiments, the gRNA nucleic acid comprises double-stranded DNA with a 5'-end modification on each strand. In some embodiments, the gRNA nucleic acid comprises an inverted dideoxy-T or an inverted abasic nucleoside or nucleotide as the 5'-end modification. In some embodiments, the gRNA nucleic acid comprises a label, such as biotin, desthiobiotin-TEG, digoxigenin, and a fluorescent marker (including, for example, FAM, ROX, TAMRA, and AlexaFluor).

[0122] As used herein, "guide sequence" refers to a sequence in a gRNA that is complementary to a target sequence and directs the gRNA to the target sequence for binding and / 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 may be, for example, 20 base pairs in length for Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs / orthologues. Shorter or longer sequences (e.g., 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides in length) can also be used as a guide. In some embodiments, the target sequence is, for example, within a gene or on a chromosome, and is complementary to the guide sequence. In some embodiments, the complementarity or identity between a guide sequence and a corresponding target sequence can be about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the guide sequence and the target region can be 100% complementary or identical over a region of at least 15, 16, 17, 18, 19, or 20 consecutive nucleotides. In other embodiments, the guide sequence and the target region can include at least one mismatch. For example, the guide sequence and the target sequence can include one, two, three, or four mismatches, where the total length of the target sequence is at least 17, 18, 19, 20, or more base pairs. In some embodiments, the guide sequence and the target region can include one to four mismatches, where the guide sequence includes at least 17, 18, 19, 20, or more nucleotides. In some embodiments, the guide sequence and the target region can contain 1, 2, 3, or 4 mismatches, where the guide sequence comprises 20 nucleotides.

[0123] In certain embodiments, multiple LNP compositions can be used cooperatively and / or for separate purposes. In some embodiments, cells can be contacted with a first and a second LNP composition described herein. In some embodiments, the first and second LNP compositions each independently comprise one or more of an mRNA, a gRNA, and a guide RNA nucleic acid. In some embodiments, the first and second LNP compositions are administered simultaneously. In some embodiments, the first and second LNP compositions are administered sequentially.

[0124] In some embodiments, a method for generating multiple genome edits in a cell (sometimes referred to herein as "multiplexing" or "multiple gene editing" or "multiple genome editing") is provided. The ability to engineer multiple attributes into a single cell depends on the ability to efficiently perform multiple target gene edits (including knockouts and locus insertions) while retaining viability and the desired cellular phenotype. In some embodiments, the method includes culturing the cell in vitro, contacting the cell with two or more lipid nucleic acid assembly compositions, each lipid nucleic acid assembly composition comprising a nucleic acid genome editing tool capable of editing a target site, and propagating the cell in vitro. This method results in cells with multiple and distinct genome edits. In certain embodiments, the first LNP composition comprises a first gRNA and the second LNP composition comprises a second gRNA, and the first and second gRNAs comprise different guide sequences that are complementary to different targets. In such embodiments, the LNP composition may enable multiple gene editing. In some embodiments, the cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 lipid nucleic acid ensemble compositions. In some embodiments, the cells are contacted with at least 6 lipid nucleic acid ensemble compositions.

[0125] Since the nucleic acid substrate of the Cas protein is a double-stranded nucleic acid, the target sequence of the RNA-guided DNA-binding protein (e.g., Cas protein) includes both the plus and minus strands of genomic DNA (i.e., a given sequence and its reverse complement). Thus, when a guide sequence is described as being "complementary to a target sequence," it is understood that the guide sequence can guide the gRNA to bind to the reverse complement of the target sequence. Thus, in some embodiments, when the guide sequence binds to the reverse complement of the target sequence, the guide sequence is identical to a certain nucleotide in the target sequence (e.g., the target sequence without the PAM), except that T is replaced by U in the guide sequence.

[0126] In some embodiments, the gRNA described herein targets a gene that reduces or eliminates surface expression of T cell receptor, MHC class I, or MHC class II. In certain embodiments, the gRNA described herein targets TRAC. In some embodiments, the gRNA described herein targets TRBC. In further embodiments, the gRNA described herein targets CIITA. In other embodiments, the gRNA described herein targets HLA-A. In some embodiments, the gRNA described herein targets HLA-B. In other embodiments, the gRNA described herein targets HLA-C. In further embodiments, the gRNA described herein targets B2M. In some embodiments, a method for generating multiple genome edits in an in vitro cultured cell is provided, the method comprising: a) contacting the cell in vitro with at least a first lipid composition comprising a first nucleic acid to generate a contacted cell; b) contacting the cell in vitro with at least a second lipid composition comprising a second nucleic acid, the second nucleic acid being different from the first nucleic acid; and c) growing the cell in vitro. In certain embodiments, a method is provided for generating multiple genome edits in an in vitro cultured cell, the method comprising: a) contacting the cell in vitro with at least a first lipid composition comprising a first nucleic acid to generate a contacted cell, b) culturing the contacted cell in vitro to purify the cultured contacted cell, c) contacting the cultured contacted cell in vitro with at least a second lipid composition comprising a second nucleic acid, where the second nucleic acid is different from the first nucleic acid, and d) growing the cell in vitro. In a further embodiment, the method further comprises contacting the cell in vitro with at least a third lipid composition comprising a third nucleic acid, where the third nucleic acid is different from the first and second nucleic acids.In a further embodiment, the method further comprises contacting the cell in vitro with at least a fourth lipid composition comprising a fourth nucleic acid, wherein the fourth nucleic acid is different from the first, second, and third nucleic acid. In yet a further embodiment, the method further comprises contacting the cell in vitro with at least a fifth lipid composition comprising a fifth nucleic acid, wherein the fifth nucleic acid is different from the first, second, third, and fourth nucleic acid. In an additional embodiment, the method further comprises contacting the cell in vitro with at least a sixth lipid composition comprising a sixth nucleic acid, wherein the sixth nucleic acid is different from the first, second, third, fourth, and fifth nucleic acid. In certain embodiments, at least two lipid compositions are administered sequentially. In some embodiments, at least two lipid compositions are administered simultaneously. In some embodiments, the proliferation of the cells indicates an increase in viability.

[0127] In some embodiments, the nucleic acid of any of the aforementioned methods for generating multiple genome edits in in vitro cultured cells is RNA (e.g., gRNA). In certain embodiments, at least one of the lipid compositions comprises a gRNA targeting TRAC. In some embodiments, at least one of the lipid compositions comprises a gRNA targeting TRBC. In further embodiments, at least one of the lipid compositions comprises a gRNA targeting a gene that reduces or eliminates surface expression of MHC class I. In yet further embodiments, at least one of the lipid compositions comprises a gRNA targeting a gene that reduces or eliminates surface expression of MHC class II. In certain embodiments, at least one of the lipid compositions comprises a gRNA targeting TRAC, and at least one of the lipid compositions comprises a gRNA targeting TRBC. In some embodiments, at least one of the lipid compositions comprises a gRNA targeting HLA-A, and optionally, the cells are homozygous for HLA-B and homozygous for HLA-C. In some embodiments, at least one of the lipid compositions comprises a gRNA targeting CIITA. In certain embodiments, at least one of the lipid compositions comprises a gRNA targeting TRAC, at least one of the lipid compositions comprises a gRNA targeting TRBC, at least one of the lipid compositions comprises a gRNA targeting HLA-A, and at least one of the lipid compositions comprises a gRNA targeting CIITA. In certain embodiments, at least one of the lipid compositions comprises a gRNA targeting TRAC, at least one of the lipid compositions comprises a gRNA targeting TRBC, a gRNA targeting HLA-A, and at least one of the lipid compositions comprises a gRNA that reduces or eliminates surface expression of MHC class II.In certain embodiments, at least one of the lipid compositions comprises a gRNA targeting TRAC, at least one of the lipid compositions comprises a gRNA targeting TRBC, at least one of the lipid compositions comprises a gRNA targeting a gene that reduces or eliminates surface expression of MHC class I, and at least one of the lipid compositions comprises a gRNA targeting CIITA. In certain embodiments, at least one of the lipid compositions comprises a gRNA targeting a gene that reduces or eliminates surface expression of T cell receptor, at least one of the lipid compositions comprises a gRNA targeting HLA-A, and at least one of the lipid compositions comprises a gRNA targeting CIITA. In certain embodiments, at least one of the lipid compositions comprises a gRNA targeting TRAC, at least one of the lipid compositions comprises a gRNA targeting HLA-A, and at least one of the lipid compositions comprises a gRNA targeting CIITA. In certain embodiments, at least one of the lipid compositions comprises a gRNA targeting a gene that reduces or eliminates surface expression of MHC class I, and at least one of the lipid compositions comprises a gRNA targeting CIITA.

[0128] In certain embodiments, at least one of the above lipid compositions comprises the nucleic acid genome editing tool described herein. In some embodiments, the further lipid composition comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9.

[0129] In some embodiments, the method of the disclosure further comprises contacting the cell with a donor nucleic acid. In some embodiments, the additional lipid composition comprises a donor nucleic acid. The donor nucleic acid can be inserted into a target sequence. In some embodiments, the donor nucleic acid sequence is provided as a vector. In some embodiments, the donor nucleic acid encodes a targeting receptor. In certain embodiments, the donor nucleic acid comprises a region that has homology with a corresponding region of a T cell receptor sequence. A "targeting receptor" is a polypeptide that is present on the surface of a cell (e.g., a T cell) and allows the cell to bind to a target site (e.g., a specific cell or tissue within an organism). In some embodiments, the targeting receptor is a CAR. In some embodiments, the targeting receptor is a universal CAR (UniCAR). In some embodiments, the targeting receptor is a TCR. In some embodiments, the targeting receptor is a T cell receptor fusion construct (TRuC). In some embodiments, the targeting receptor is a B cell receptor (BCR) (e.g., one expressed on a B cell). In some embodiments, the targeting receptor is a chemokine receptor. In some embodiments, the targeting receptor is a cytokine receptor.

[0130] In some embodiments, the in vitro genome editing method provides high editing efficiency at multiple target sites in T cells. In some embodiments, engineered T cells are generated in which an endogenous TCR is knocked out. In some embodiments, engineered T cells are generated in which expression of an endogenous TCR is knocked out. In some embodiments, engineered T cells are generated in which expression of two genes is reduced and / or knocked out. In some embodiments, engineered T cells are generated in which three genes are knocked down and / or knocked out. In some embodiments, engineered T cells are generated in which four genes are knocked down and / or knocked out. In some embodiments, engineered T cells are generated in which five genes are knocked down and / or knocked out. In some embodiments, engineered T cells are generated in which six genes are knocked down and / or knocked out. In some embodiments, engineered T cells are generated in which seven genes are knocked down and / or knocked out. In some embodiments, engineered T cells are generated in which eight genes are knocked down and / or knocked out. In some embodiments, engineered T cells are generated in which nine genes are knocked down and / or knocked out. In some embodiments, engineered T cells are generated in which 10 genes are knocked down and / or knocked out. In some embodiments, engineered T cells are generated in which 11 genes are knocked down and / or knocked out.

[0131] In some embodiments, engineered T cells are generated in which the endogenous TCR is knocked out and a transgenic TCR is inserted and expressed. In some embodiments, the engineered T cells are primary human T cells. In some embodiments, the tgTCR targets Wilms Tumor 1 (WT1). In some embodiments, the WT1 tgTCR is inserted into a high percentage (e.g., greater than 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) of T cells using the lipid compositions of the present disclosure.

[0132] β2M or B2M are used interchangeably herein in reference to the nucleic acid sequence or protein sequence of β-2 microglobulin. The human gene has the accession number NC_000015 (range 44711492..44718877), reference GRCh38.p13. B2M protein is associated with MHC class I molecules as a heterodimer on the surface of nucleated cells and is required for the expression of MHC class I proteins.

[0133] CIITA or CIITA or C2TA are used interchangeably herein and refer to the nucleic acid or protein sequence of class II major histocompatibility complex transactivator, human gene accession number NC_000016.10 (range 10866208..10941562), reference GRCh38.p13, incorporated herein by reference. The CIITA protein in the nucleus functions as a positive regulator of MHC class II gene transcription and is required for the expression of MHC class II proteins.

[0134] MHC or MHC molecule(s) or MHC protein or MHC complex(es) refers to major histocompatibility complex molecule(s), including, for example, MHC class I and MHC class II molecules. In humans, MHC molecules are referred to as human leukocyte antigen complexes or HLA molecules or HLA proteins. The use of the terms MHC and HLA is not intended to be limiting, and as used herein, the term MHC can be used to refer to human MHC molecules, i.e., HLA molecules. Thus, the terms MHC and HLA are used interchangeably herein.

[0135] As used herein in the context of HLA-A protein, HLA-A refers to an MHC class I protein molecule that is a heterodimer containing a heavy chain (encoded by the HLA-A gene) and a light chain (i.e., beta 2 microglobulin). As used herein in the context of nucleic acids, the term HLA-A or HLA-A gene refers to a gene that encodes the heavy chain of the HLA-A protein molecule. The HLA-A gene is also referred to as HLA class I histocompatibility A alpha chain, and the human gene has accession number NC_000006.12 (29942532..29945870), which is incorporated herein by reference. The HLA-A gene is known to have hundreds of different versions (also referred to as alleles) in the population (and one individual may receive two different alleles of the HLA-A gene). All alleles of HLA-A are encompassed by the terms HLA-A and HLA-A gene.

[0136] As used herein in the context of nucleic acids, HLA-B refers to the gene encoding the heavy chain of the HLA-B protein molecule. HLA-B is also referred to as HLA class I histocompatibility B alpha chain, and the human gene accession number is NC_000006.12 (31353875..31357179), which is incorporated herein by reference.

[0137] As used herein in the context of nucleic acids, HLA-C refers to the gene encoding the heavy chain of the HLA-C protein molecule. HLA-C is also referred to as HLA class I histocompatibility C alpha chain, and the human gene accession number is NC_000006.12 (31268749..31272092), which is incorporated herein by reference.

[0138] The length of the targeting sequence may depend on the CRISPR / Cas system and components used. For example, different class 2 Cas nucleases from different bacterial species have different optimal target sequence lengths. Thus, the targeting sequence may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length. In some embodiments, the length of the targeting sequence is 0, 1, 2, 3, 4, or 5 nucleotides longer or shorter than the guide sequence of a naturally occurring CRISPR / Cas system. In certain embodiments, the Cas nuclease and the gRNA scaffold are derived from the same CRISPR / Cas system. In some embodiments, the targeting sequence may comprise or consist of 18-24 nucleotides. In some embodiments, the targeting sequence can comprise or consist of 19-21 nucleotides, hi some embodiments, the targeting sequence can comprise or consist of 20 nucleotides.

[0139] In some embodiments, the sgRNA is a "Cas9 sgRNA" capable of mediating RNA-guided DNA cleavage by a Cas9 protein. In some embodiments, the sgRNA is a "Cpf1 sgRNA" capable of mediating RNA-guided DNA cleavage by a Cpf1 protein. In certain embodiments, the gRNA comprises sufficient crRNA and tracr RNA to form an active complex with a Cas9 protein and mediate RNA-guided DNA cleavage. In certain embodiments, the gRNA comprises sufficient crRNA to form an active complex with a Cpf1 protein and mediate RNA-guided DNA cleavage. See Zetsche 2015.

[0140] Certain embodiments also provide nucleic acids (e.g., expression cassettes) encoding the gRNAs described herein. "Guide RNA nucleic acid" is used herein to refer to gRNAs (e.g., sgRNA or dgRNA) and gRNA expression cassettes (nucleic acids encoding one or more gRNAs).

[0141] modified RNA In certain embodiments, the lipid composition (eg, the LNP composition) comprises a modified nucleic acid (including a modified RNA).

[0142] Modified nucleosides or nucleotides may be present in an RNA (e.g., gRNA or mRNA). A gRNA or mRNA that includes one or more modified nucleosides or nucleotides is referred to as, for example, a "modified" RNA to account for the presence of one or more non-natural and / or naturally occurring components or structures that are used in place of, or in addition to, the canonical A, G, C, and U residues. In some embodiments, a modified RNA is synthesized with non-canonical nucleosides or nucleotides, and is referred to herein as "modified."

[0143] Modified nucleosides and nucleotides can include one or more of: (i) alteration (e.g., substitution) of one or both of the non-linked phosphate oxygens in the phosphodiester backbone linkages and / or one or both of the linked phosphate oxygens (exemplary backbone modifications); (ii) alteration (e.g., substitution) of a component of the ribose sugar, for example, of the 2' hydroxyl on the ribose sugar (exemplary sugar modifications); (iii) wholesale replacement of a phosphate moiety with a "dephosphorylated" linker (exemplary backbone modifications); (iv) modification or substitution of a naturally occurring nucleobase (including non-canonical nucleobases) (exemplary base modifications); (v) substitution or modification of the ribose-phosphate backbone (exemplary backbone modifications); (vi) modification of the 3' or 5' end of a polynucleotide, for example, removal, modification, or substitution of a terminal phosphate group, or conjugation of a moiety, cap, or linker (such 3' or 5' cap modifications can include sugar and / or backbone modifications); and (vii) modification or replacement of the sugar (exemplary sugar modifications). Certain embodiments include a 5'-end modification to the mRNA, gRNA, or nucleic acid. Certain embodiments include a modification to the mRNA, gRNA, or nucleic acid. Certain embodiments include a 3'-end modification to the mRNA, gRNA, or nucleic acid. The modified RNA can include modifications at the 5'-end and the 3'-end. The modified RNA can include one or more modified residues at a non-terminal position. In certain embodiments, the gRNA includes at least one modified residue. In certain embodiments, the mRNA includes at least one modified residue. In certain embodiments, the modified gRNA includes a modification in one or more of the first five nucleotides at the 5'-end. In certain embodiments, the modified gRNA includes a modification in one or more of the first five nucleotides at the 5'-end. The LNP composition of claim 52 or 53, wherein the modified gRNA includes a modification in one or more of the last five nucleotides at the 3'-end.

[0144] Unmodified nucleic acids may be prone to degradation, for example, by intracellular nucleases or nucleases found in serum. For example, nucleases may hydrolyze phosphodiester bonds of nucleic acids. Thus, in one aspect, the RNAs (e.g., mRNA, gRNA) described herein may include one or more modified nucleosides or nucleotides, for example, to introduce stability against intracellular nucleases or serum-based nucleases. In some embodiments, the modified RNA molecules described herein can exhibit reduced innate immune responses when introduced into a cell population, both in vivo and ex vivo. The term "innate immune response" includes cellular responses to exogenous nucleic acids, including single-stranded nucleic acids, which involve the expression and release of cytokines, particularly interferons, and the induction of cell death.

[0145] Thus, in some embodiments, the RNA or nucleic acid comprises at least one modification that confers increased or enhanced stability to the nucleic acid, including, for example, improved resistance to nuclease digestion in vivo. As used herein, the terms "modification" and "modified", when such terms relate to the nucleic acids provided herein, preferably include at least one modification that enhances stability and makes the RNA or nucleic acid more stable (e.g., more resistant to nuclease digestion) than the wild-type or naturally occurring version of the RNA or nucleic acid. As used herein, terms such as "stable" and "stability", when such terms relate to the nucleic acids described herein, particularly in RNA, refer to increased or enhanced resistance to degradation by, for example, nucleases (i.e., endonucleases or exonucleases) that can normally degrade such RNA. Increased stability can include, for example, increased or enhanced residence of such RNA or nucleic acid in a target cell, tissue, subject, and / or cytoplasm by being less susceptible to hydrolysis or other destruction by endogenous enzymes (e.g., endonucleases or exonucleases) or conditions within the target cell or tissue. The stabilized RNA or nucleic acid molecules provided herein exhibit a longer half-life than their naturally occurring unmodified counterparts (e.g., wild-type versions of the molecules). The terms "modification" and "modified" as used in relation to the mRNA of the LNP compositions disclosed herein also contemplate alterations that improve or enhance translation of the mRNA nucleic acid, including, for example, the inclusion of sequences that function in the initiation of protein translation (e.g., Kozak consensus sequences). (Kozak, M., Nucleic Acids Res 15(20):8125-48(1987)).

[0146] In some embodiments, RNA or nucleic acid undergoes chemical or biological modification to make it more stable.Exemplary modifications to RNA or nucleic acid include base depletion (e.g., by deletion or by substitution of one nucleotide with another nucleotide) or base modification (e.g., chemical modification of base).As used herein, the term "chemical modification" includes modification that introduces a chemical property different from that found in naturally occurring RNA or nucleic acid, such as covalent modification (e.g., inclusion of modified nucleotide (e.g., nucleotide analog, or pendant group (e.g., deoxynucleoside) that is not naturally found in such RNA or nucleic acid molecule).

[0147] In some embodiments of backbone modification, the phosphate group of the modified residue can be modified by replacing one or more of the oxygens with different substituents.Furthermore, the modified residue, e.g., the modified residue present in the modified nucleic acid, can include wholesale replacement of the unmodified phosphate moiety with a modified phosphate group as described herein.In some embodiments, the backbone modification of the phosphate backbone can include modifications that result in either an uncharged linker or a charged linker with an asymmetric charge distribution.

[0148] Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. The phosphorus atom of an unmodified phosphate group is achiral. However, the phosphorus atom can be made chiral by replacing one of the non-bridging oxygens with one of the atoms or groups of atoms listed above. The stereochemical phosphorus atom can have either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). The backbone can also be modified by replacing the bridging oxygens (i.e., the oxygens that connect the phosphate to the nucleoside) with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates). This replacement can occur at one or both of the bond oxygens. The phosphate group can be replaced by a non-phosphorus-containing connector in certain backbone modifications. In some embodiments, the charged phosphate group can be replaced by a neutral moiety. Examples of moieties that can replace the phosphate group include, but are not limited to, methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formatel, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, and methyleneoxymethylimino.

[0149] mRNA In some embodiments, a composition or formulation disclosed herein comprises an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA-binding agent (e.g., a Cas nuclease or a class 2 Cas nuclease described herein). In some embodiments, an mRNA comprising an ORF encoding an RNA-guided DNA-binding agent (e.g., a Cas nuclease or a class 2 Cas nuclease) is provided, used, or administered. The mRNA can comprise one or more of a 5' cap, a 5' untranslated region (UTR), a 3' UTR, and a polyadenine tail. The mRNA can comprise a modified open reading frame, for example, to encode a nuclear localization sequence or to encode a protein using alternative codons.

[0150] The mRNA in the LNP compositions of the present disclosure can encode a cell surface or intracellular polypeptide. The mRNA in the LNP compositions of the present disclosure can encode, for example, a secreted hormone, an enzyme, a receptor, a polypeptide, a peptide, or other protein of interest that is normally secreted. In some embodiments, the mRNA can optionally have chemical or biological modifications that, for example, improve the stability and / or half-life of such mRNA or improve or otherwise facilitate protein production.

[0151] In addition, suitable modifications include altering one or more nucleotides of a codon so that the codon codes for the same amino acid, but is more stable than the codon found in the wild-type version of the mRNA. For example, an inverse relationship between RNA stability and a higher number of cytidine (C) and / or uridine (U) residues has been demonstrated, and RNA lacking C and U residues has been found to be stable to most RNases (Heidenreich, et al. J Biol Chem 269, 2131-8 (1994)). In some embodiments, the number of C and / or U residues in the mRNA sequence is reduced. In another embodiment, the number of C and / or U residues is reduced by replacing one codon that codes for a particular amino acid with another codon that codes for the same or related amino acid. Contemplated modifications to mRNA nucleic acid also include the incorporation of pseudouridine. Incorporation of pseudouridine into mRNA nucleic acid can enhance stability and translational capacity, as well as reduce immunogenicity in vivo. See, e.g., Kariko, K., et al., Molecular Therapy 16(11):1833-1840 (2008). mRNA substitutions and modifications can be carried out by methods readily known to those of skill in the art.

[0152] The constraints of reducing the number of C and U residues in a sequence may be greater in the coding regions of an mRNA compared to the untranslated regions (i.e., it is probably not possible to remove all C and U residues present in the message while still retaining the capacity of the message to code for the desired amino acid sequence). However, the degeneracy of the genetic code provides the opportunity to allow a reduction in the number of C and / or U residues present in a sequence while maintaining the same coding capacity (i.e., depending on which amino acids are coded for by the codons, several different possibilities for modification of an RNA sequence may be possible).

[0153] The term modification also includes, for example, the incorporation of non-nucleotide linkages or modified nucleotides into the mRNA sequence (e.g., modifications to either or both of the 3' and 5' ends of an mRNA molecule encoding a functional secreted protein or enzyme). Such modifications include the addition of bases to the mRNA sequence (e.g., inclusion of a polyA tail or a longer polyA tail), alteration of the 3' or 5' UTR, complexing the mRNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and the inclusion of elements that alter the structure of the mRNA molecule (e.g., those that form secondary structures).

[0154] PolyA tails are believed to stabilize natural messengers. Therefore, long polyA tails can be added to mRNA molecules to make them more stable. PolyA tails can be added using a variety of techniques recognized in the art. For example, polyA polymerase can be used to add long polyA tails to synthetic or in vitro transcribed mRNAs (Yokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256). Transcription vectors can also encode long polyA tails. Additionally, polyA tails can be added by direct transcription from PCR products. In some embodiments, the length of the polyA tail is at least about 90, 200, 300, 400, at least 500 nucleotides. In certain embodiments, the length of the polyA tail is adjusted to control the stability of the modified mRNA molecule and thus the transcription of the protein. For example, the length of the polyA tail can affect the half-life of an mRNA molecule, and thus the length of the polyA tail can be adjusted to modify the level of resistance of the mRNA to nucleases, thereby controlling the time course of protein expression in a cell. In some embodiments, the stabilized mRNA molecule is sufficiently resistant to in vivo degradation (e.g., by nucleases) that it can be delivered to a target cell without a transfer vehicle.

[0155] In certain embodiments, mRNA can be modified by incorporating 3' and / or 5' untranslated (UTR) sequences not naturally found in wild-type mRNA. In some embodiments, 3' and / or 5' flanking sequences naturally adjacent to the mRNA and encoding a second, unrelated protein can be incorporated into the nucleotide sequence of the mRNA molecule encoding a therapeutic or functional protein to modify it. For example, 3' or 5' sequences from a stable mRNA molecule (e.g., globin, actin, GAPDH, tubulin, histones, or citric acid cycle enzymes) can be incorporated into the 3' and / or 5' regions of the sense mRNA nucleic acid molecule to increase the stability of the sense mRNA molecule. See, e.g., US2003 / 0083272.

[0156] A more detailed description of mRNA modifications can be found on pages 57-68 of US2017 / 0210698A1, the contents of which are incorporated herein.

[0157] Template nucleic acid The methods disclosed herein can include using a template nucleic acid. The template can be used to modify or insert a nucleic acid sequence at or near a target site of an RNA-guided DNA-binding protein (e.g., a Cas nuclease, such as a class 2 Cas nuclease). In some embodiments, the method includes introducing a template into a cell. In some embodiments, a single template can be provided. In other embodiments, two or more templates can be provided so that editing can occur at two or more target sites. For example, different templates can be provided to edit a single gene in a cell, or to edit two different genes in a cell.

[0158] In some embodiments, the template can be used in homologous recombination. In some embodiments, the template sequence or a part of the template sequence can be integrated into the target nucleic acid molecule as a result of homologous recombination. In other embodiments, the template can be used for homologous recombination repair, which involves DNA strand invasion at the break site in the nucleic acid. In some embodiments, the template sequence can be included in the edited target nucleic acid molecule as a result of homologous recombination repair. In yet other embodiments, the template can be used for gene editing mediated by non-homologous end joining. In some embodiments, the template sequence does not have similarity to the nucleic acid sequence near the break site. In some embodiments, the template or a part of the template sequence is integrated. In some embodiments, the template includes flanking inverted terminal repeat (ITR) sequences.

[0159] In some embodiments, the template sequence can correspond to, comprise, or consist of an endogenous sequence of the target cell. It can also correspond to, comprise, or consist of an exogenous sequence of the 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 the cell or that is at a different location than its native location in the genome of the cell. In some embodiments, the endogenous sequence can be a genomic sequence of the cell. In some embodiments, the endogenous sequence can be a chromosomal sequence or an extrachromosomal sequence. In some embodiments, the endogenous sequence can be a plasmid sequence of the cell.

[0160] In some embodiments, the template comprises ssDNA or dsDNA that includes flanking inverted terminal repeat (ITR) sequences. In some embodiments, the template is provided as a vector, a plasmid, a minicircle, a nanocircle, or a PCR product.

[0161] In some embodiments, the nucleic acid is purified. In some embodiments, the nucleic acid is purified using a precipitation method (e.g., LiCl precipitation, alcohol precipitation, or equivalent method, e.g., methods described herein). In some embodiments, the nucleic acid is purified using a chromatography-based method (e.g., an HPLC-based method or equivalent method, e.g., methods described herein). In some embodiments, the nucleic acid is purified using both a precipitation method (e.g., LiCl precipitation) and an HPLC-based method. In some embodiments, the nucleic acid is purified by tangential flow filtration (TFF).

[0162] Cell type In some embodiments, the cell is an immune cell. As used herein, "immune cell" refers to a cell of the immune system, including, for example, lymphocytes (e.g., T cells, B cells, natural killer cells ("NK cells" and NKT cells, or iNKT cells), monocytes, macrophages, mast cells, dendritic cells, or granulocytes (e.g., neutrophils, eosinophils, and basophils). In some embodiments, the cell is a primary immune cell. In some embodiments, the immune system cell can be selected from CD3+, CD4+, and CD8+ T cells, regulatory T cells (Tregs), B cells, NK cells, and dendritic cells (DCs). 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.

[0163] As used herein, a T cell may be defined as a cell that expresses a T cell receptor ("TCR" or "αβ TCR" or "γδ TCR"), although in some embodiments, the TCR of a T cell may be genetically modified to reduce its expression (e.g., by genetic modification to the TRAC or TRBC gene), such that expression of the protein CD3 can be used as a marker to identify T cells by standard flow cytometry methods. CD3 is a multi-subunit signaling complex that associates with the TCR. Thus, T cells may be referred to as CD3+. In some embodiments, a T cell is a cell that expresses the CD3+ marker and either the CD4+ marker or the CD8+ marker.

[0164] In some embodiments, the T cells express the glycoprotein CD8 and are therefore CD8+ by standard flow cytometry and may be referred to as "cytotoxic" T cells. In some embodiments, the T cells express the glycoprotein CD4 and are therefore CD4+ by standard flow cytometry and may be referred to as "helper" T cells. CD4+ T cells can differentiate into subsets and may be referred to as Th1 cells, Th2 cells, Th9 cells, Th17 cells, Th22 cells, T regulatory ("Treg") cells, or T follicular helper cells ("Tfh"). Each CD4+ subset releases specific cytokines that may have either inflammatory or anti-inflammatory, survival or protective functions. T cells can be isolated from a subject by CD4+ or CD8+ selection methods.

[0165] In some embodiments, the T cells are memory T cells. In the body, memory T cells have encountered antigen. Memory T cells may be located in secondary lymphoid organs (central memory T cells) or in recently infected tissues (effector memory T cells). Memory T cells may be CD8+ T cells. Memory T cells may be CD4+ T cells. As used herein, "central memory T cells" may be defined as antigen-experienced T cells, and may express, for example, CD62L and CD45RO. Central memory T cells may be detected as CD62L+ and CD45RO+. Central memory T cells also express CCR7, and therefore may be detected as CCR7+ by standard flow cytometry methods.

[0166] As used herein, "early stem cell memory T cells" (or "Tscm") can be defined as T cells that express CD27 and CD45RA and are therefore CD27+ and CD45RA by standard flow cytometry methods. Tscm do not express the CD45 isoform CD45RO, and therefore when stained for this isoform by standard flow cytometry methods, Tscm are also CD45RO-. Thus, CD45RO-CD27+ cells are also early stem cell memory T cells. Tscm cells further express CD62L and CCR7 and therefore can be detected as CD62L+ and CCR7+ by standard flow cytometry methods. Early stem cell memory T cells have been shown to correlate with increased persistence and therapeutic efficacy of cell therapy products.

[0167] In some embodiments, the cell is a B cell. As used herein, a "B cell" can be defined as a cell that expresses CD19 and / or CD20, and / or B cell maturation antigen ("BCMA"), and thus the B cell is CD19+, and / or CD20+, and / or BCMA+ by standard flow cytometry. The B cell is further CD3 and CD56 negative by standard flow cytometry. The B cell can be a plasma cell. The B cell can be a memory B cell. The B cell can be a naive B cell. The B cell can be IgM+ or have a class switched B cell receptor (e.g., IgG+, IgA+).

[0168] Cells used in ACT therapy include, for example, mesenchymal stem cells (e.g., isolated from bone marrow (BM), peripheral blood (PB), placenta, umbilical cord (UC), or fat), hematopoietic stem cells (HSC; isolated, for example, from BM), mononuclear cells (e.g., isolated from BM or PB), endothelial progenitor cells (EPC; isolated from BM, PB, and UC), neural stem cells (NSC), limbal stem cells (LSC), or tissue-specific primary or derived cells (TSC). Cells used in ACT therapy further include induced pluripotent stem cells (iPSC), ocular stem cells, pluripotent stem cells (PSC), embryonic stem cells (ESC), cells for organ or tissue transplantation (e.g., islet cells, cardiomyocytes, thyroid cells, thymus cells, nerve cells, skin cells, retinal cells, chondrocytes, muscle cells, and keratinocytes) that can be induced to differentiate into other cell types (including, for example, islet cells, neural cells, and blood cells).

[0169] In some embodiments, the cells are human cells (e.g., cells from a subject). In some embodiments, the cells are isolated from a human subject (e.g., a human donor). In some embodiments, the cells are isolated from human donor PBMCs or leukopacks. In some embodiments, the cells are from a subject having a condition, disorder, or disease. In some embodiments, the cells are from a human donor having Epstein-Barr Virus ("EBV").

[0170] In some embodiments, the cells are mononuclear cells (e.g., from bone marrow or peripheral blood). In some embodiments, the cells are peripheral blood mononuclear cells ("PBMCs"). In some embodiments, the cells are PBMCs (e.g., lymphocytes or monocytes). In some embodiments, the cells are peripheral blood lymphocytes ("PBLs").

[0171] In some embodiments, the method is carried out ex vivo. As used herein, "ex vivo" refers to an in vitro method that allows cells to be transferred to a subject, for example, as an ACT therapy. In some embodiments, the ex vivo method is an in vitro method that involves an ACT therapy cell or cell population.

[0172] In some embodiments, the cells are maintained in culture. In some embodiments, the cells are transplanted into a patient. In some embodiments, the cells are removed from a subject, genetically modified ex vivo, and then administered back to the same patient. In some embodiments, the cells are removed from a subject, genetically modified ex vivo, and then administered to a subject other than the one from which they were removed.

[0173] In some embodiments, the cells are from a cell line. In some embodiments, the cell line is from a human subject. In some embodiments, the cell line is a lymphoblastoid cell line ("LCL"). The cells can be cryopreserved and thawed. The cells do not have to have been previously cryopreserved.

[0174] In some embodiments, the cells are from a cell bank. In some embodiments, the cells are genetically modified and then transferred to a cell bank. In some embodiments, the cells are removed from a subject, genetically modified ex vivo, and transferred to a cell bank. In some embodiments, the genetically modified cell population is transferred to a cell bank. In some embodiments, the genetically modified immune cell population is transferred to a cell bank. In some embodiments, a genetically modified immune cell population comprising a first and a second subpopulation, the first and second subpopulations having at least one common genetic modification and at least one different genetic modification, is transferred to a cell bank.

[0175] In some embodiments, T cells are activated by polyclonal activation (or "polyclonal stimulation") (not antigen-specific stimulation). In some embodiments, T cells are activated by CD3 stimulation (e.g., provision of an anti-CD3 antibody). In some embodiments, T cells are activated by CD3 and CD28 stimulation (e.g., provision of an anti-CD3 antibody and an anti-CD28 antibody). In some embodiments, T cells are activated (e.g., via CD3 / CD28 stimulation) using ready-to-use reagents for activating T cells. In some embodiments, T cells are activated by CD3 / CD28 stimulation provided by beads. In some embodiments, T cells are activated via CD3 / CD28 stimulation, one or more components of which are soluble and / or one or more components of which are bound to a solid surface (e.g., a plate or beads). In some embodiments, T cells are activated by antigen-independent mitogens (e.g., including lectins (e.g., concanavalin A ("ConA") or PHA)).

[0176] In some embodiments, one or more cytokines are used for T cell activation. IL-2 is provided for T cell activation and / or to promote T cell survival. In some embodiments, the cytokine(s) for T cell activation are cytokines that bind to the common gamma chain (γc) receptor. In some embodiments, IL-2 is provided for T cell activation. In some embodiments, IL-7 is provided for T cell activation. In some embodiments, IL-15 is provided for T cell activation. In some embodiments, IL-21 is provided for T cell activation. In some embodiments, a combination of cytokines (e.g., including IL-2, IL-7, IL-15, and / or IL-21) is provided for T cell activation.

[0177] In some embodiments, T cells are activated by exposing the cells to an antigen (antigen stimulation). T cells are activated by an antigen when the antigen is presented as a peptide within a major histocompatibility complex (MHC) molecule (peptide-MHC complex). Cognate antigens can be presented to the T cells by co-culturing the T cells with antigen-presenting cells (feeder cells) and the antigen. In some embodiments, T cells are activated by co-culture with antigen-pulsed antigen-presenting cells. In some embodiments, the antigen-presenting cells are pulsed with a peptide of the antigen.

[0178] In some embodiments, the T cells can be activated for 12-72 hours. In some embodiments, the T cells can be activated for 12-48 hours. In some embodiments, the T cells can be activated for 12-24 hours. In some embodiments, the T cells can be activated for 24-48 hours. In some embodiments, the T cells can be activated for 24-72 hours. In some embodiments, the T cells can be activated for 12 hours. In some embodiments, the T cells can be activated for 48 hours. In some embodiments, the T cells can be activated for 72 hours.

[0179] While the invention will be described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. Instead, the present disclosure is intended to cover all alternatives, modifications, and equivalents (including equivalents of specific features) that may be included within the invention as defined by the appended claims.

[0180] The general description and detailed description above, as well as the following examples, are merely illustrative and explanatory, and are not intended to limit the teachings. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter in any manner. In the event that a document incorporated by reference conflicts with a term defined herein, the present specification shall control. All ranges provided in this application include both end values ​​unless otherwise stated.

[0181] definition It should be noted that as used in this application, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes a plurality of compositions, reference to "a cell" includes a plurality of cells, etc. The use of "or" is inclusive and means "and / or" unless specifically stated otherwise.

[0182] Unless otherwise noted herein above, embodiments described herein as "comprising" various components are also contemplated as "consisting" or "consisting essentially" of the described components, embodiments described herein as "consisting" of various components are also contemplated as "comprising" or "consisting essentially" of the described components, embodiments described herein as "about" various components are also contemplated as "in" the described components, and embodiments described herein as "consisting essentially" of various components are also contemplated as "consisting" of or "including" the described components (this interchangeability does not apply to the use of these terms in the claims).

[0183] Numerical ranges are inclusive of the numbers defining the range. Measurements and measurables are understood to be approximations taking into account significant figures and errors associated with measurement. The terms "about" and "approximately" as used in this application have their meaning as understood in the art. Use of one and the other does not necessarily imply a different range. Unless otherwise indicated, numerical values ​​used in this application, with or without modifiers such as "about" or "approximately", should be understood to encompass normal deviations and / or variations that would be understood by one of ordinary skill in the relevant art. In certain embodiments, the term "approximately" or "about" can refer to a range of values ​​that falls in either direction (greater or smaller) of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or less of a stated reference value, unless otherwise specified or clear from the context (except where such number would exceed 100% of possible values).

[0184] As used herein, the term "contact" refers to establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a nanoparticle composition means that the mammalian cell and the nanoparticle share a physical connection. Methods for contacting a cell with an external entity, both in vivo and ex vivo, are well known in the biological arts. For example, contacting a mammalian cell with a nanoparticle composition disposed within a mammal can be performed by various routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and can involve various amounts of the nanoparticle composition. Additionally, multiple mammalian cells can be contacted with the nanoparticle composition.

[0185] As used herein, the term "deliver" means to provide an entity to a destination. For example, delivering a therapeutic and / or prophylactic agent to a subject can involve administering to the subject (e.g., via intravenous, intramuscular, intradermal, or subcutaneous routes) a nanoparticle composition comprising the therapeutic and / or prophylactic agent. Administering a nanoparticle composition to a mammal or mammalian cells can include contacting one or more cells with the nanoparticle composition.

[0186] As used herein, "encapsulation efficiency" refers to the amount of therapeutic and / or prophylactic agent that becomes part of the nanoparticle composition relative to the total initial amount of therapeutic and / or prophylactic agent used to prepare the nanoparticle composition. For example, if 97 mg of therapeutic and / or prophylactic agent is encapsulated within the nanoparticle composition out of a total of 100 mg of therapeutic and / or prophylactic agent initially provided in the composition, then the encapsulation efficiency can be taken as 97%. As used herein, "encapsulation" can refer to complete, substantial, or partial entrapment, restraint, surrounding, or encasement.

[0187] As used herein, the terms "editing efficiency", "editing percentage", "indel efficiency" and "percent indel" 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 position in a genome can 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 the percentage of cells (e.g., CD3) that no longer contain a gene after treatment relative to the number of cells (e.g., CD3+ cells) that originally contained the gene.

[0188] As used herein, "knockdown" refers to a reduction in the expression of a particular gene product (e.g., protein, mRNA, or both). Protein knockdown can be measured by detecting the total intracellular amount of the protein from a sample, such as a tissue, fluid, or cell population of interest. It can also be measured by measuring a surrogate, marker, or activity of the protein. Methods for measuring mRNA knockdown are known and include sequencing of mRNA isolated from a sample of interest. In some embodiments, "knockdown" can refer to any loss of expression of a particular gene product, such as a reduction in the amount of mRNA transcribed, or a reduction in the amount of protein expressed by a cell population, including an in vivo population such as found in a tissue.

[0189] As used herein, "knockout" refers to the loss of expression from a particular gene or of a particular protein in a cell. Knockout can be measured, for example, by detecting the total cellular amount of the protein in a cell, tissue, or cell population. Knockout can also be detected, for example, at the genomic or mRNA level.

[0190] As used herein, the term "biodegradable" is used to refer to a material that, when introduced into a cell, degrades by cellular mechanisms (e.g., enzymatic degradation) or hydrolysis to components that the cell can reuse or discard without significant toxic effect(s) to the cell. In certain embodiments, the components produced by degradation of the biodegradable material do not induce inflammation and / or other adverse effects in vivo. In some embodiments, the biodegradable material is enzymatically degraded. Alternatively or additionally, in some embodiments, the biodegradable material is hydrolytically degraded.

[0191] As used herein, "N / P ratio" refers to the molar ratio of ionizable nitrogen atom-containing lipid (e.g., a compound of formula I) to phosphate groups in RNA, for example, in a nanoparticle composition comprising a lipid component and RNA.

[0192] The composition may also include one or more salts of the compounds. The salt may be a pharma- ceutically acceptable salt. As used herein, "pharma-ceutically acceptable salt" refers to a derivative of the disclosed compound in which the parent compound is modified by converting an existing acid or base moiety into its salt form (e.g., by reacting a free base group with a suitable organic acid). Examples of pharma-ceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic acid salts of acidic residues such as carboxylic acids, and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, and the like. Representative salts include, for example, salts of the phenylalanine, ...The pharma- ceutically acceptable salts of the present disclosure can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods.In general, such salts can be prepared by reacting the free acid or base forms of these compounds with stoichiometric amounts of appropriate bases or acids in water or organic solvents, or in a mixture of the two, and generally non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17. th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.

[0193] As used herein, "polydispersity index" refers to a ratio that represents the uniformity of the particle size distribution of a system. A small value, for example less than 0.3, indicates a narrow particle size distribution. In some embodiments, the polydispersity index may be less than 0.1.

[0194] As used herein, "transfection" refers to the introduction of a species (e.g., RNA) into a cell. Transfection can occur, for example, in vitro, ex vivo, or in vivo.

[0195] As used herein, the term "alkyl" refers to a branched or unbranched saturated hydrocarbon group having 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, and tetracosyl. The alkyl group may be cyclic or noncyclic. The alkyl group may be branched or unbranched (i.e., linear). Additionally, the alkyl group may be substituted or unsubstituted. For example, the alkyl group may be substituted with one or more groups, including but not limited to alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfoxo, sulfonate, carboxylate, or thiol, as described herein. A "lower alkyl" group is an alkyl group containing 1 to 6 (eg, 1 to 4) carbon atoms.

[0196] As used herein, the term "alkenyl" refers to an aliphatic group containing at least one carbon-carbon double bond, and is intended to include both "unsubstituted alkenyl" and "substituted alkenyl," the latter of which refers to an alkenyl moiety having a substituent replacing a hydrogen on one or more carbons of the alkenyl group. Such substituents may be present on one or more carbons that are included or not included in one or more double bonds. Furthermore, such substituents include all of those contemplated below for alkyl groups, except where stability would be inhibited. For example, it is contemplated that an alkenyl group may be substituted with one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups. Exemplary alkenyl groups include, but are not limited to, vinyl (-CH=CH2), allyl (-CH2CH=CH2), cyclopentenyl (-CH7), and 5-hexenyl (-CH2CH2CH2CH2CH=CH2).

[0197] An "alkylene" group refers to a divalent alkyl radical that may be branched or unbranched (i.e., linear). Any of the monovalent alkyl groups described above can be converted to an alkylene by removing a second hydrogen atom from the alkyl. Exemplary alkylenes include, but are not limited to, C 2~4 Alkylene and C 2~3 Examples of alkylene groups include, but are not limited to, -CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH2CH(CH3)-, -CH2C(CH3)2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and the like. Additionally, alkylene groups can be substituted or unsubstituted. For example, alkylene groups can be substituted with one or more groups, including but not limited to, alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfoxo, sulfonate, carboxylate, or thiol, as described herein.

[0198] The term "alkenylene" includes divalent linear or branched unsaturated acyclic hydrocarbyl groups having at least one carbon-carbon double bond, and in one embodiment no carbon-carbon triple bonds. Any of the monovalent alkenyl groups described above can be converted to an alkenylene by removing a second hydrogen atom from the alkenyl. Representative alkenylenes include C 2~6 Alkenylene is exemplified.

[0199] "C x~y The term "alkyl" when used with a chemical moiety such as alkyl or alkylene is meant to include groups that contain x to y carbons in the chain. For example, "C x~y The term "alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain and branched alkyl and alkylene groups, containing x to y carbons in the chain.

[0200] The term "alkoxy" refers to an alkyl group, preferably a lower alkyl group, having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like.

[0201] Incorporation by Reference The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein are incorporated herein by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application all material and information contained in such articles, patents, patent applications, or other physical and electronic documents. EXAMPLES

[0202] Example 1: Materials and Methods Example 1.1 Lipid Nanoparticle ("LNP") Formulations LNP components were dissolved in 100% ethanol at various molar ratios. RNA cargo (e.g., Cas9 mRNA and sgRNA combination) was dissolved in 25 mM citric acid, 100 mM NaCl, pH 5.0, resulting in an RNA cargo concentration of approximately 0.45 mg / mL. LNPs were formulated with a lipid amine:RNA phosphate (N:P) molar ratio of approximately 6 and a sgRNA:Cas9 mRNA weight ratio of 1:2, unless otherwise specified.

[0203] LNPs were prepared using various amine lipids in a four-component lipid system. Unless otherwise specified, the LNPs included an ionizable lipid, compound 6 ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (also known as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate), DSPC, cholesterol, and PEG2k-DMG.

[0204] LNPs were prepared using a cross-flow technique that utilizes impingement jet mixing of lipids in ethanol with two volumes of RNA solution and one volume of water. Lipids in ethanol were mixed with two volumes of RNA solution through a mixing cross. A fourth water stream was mixed with the outlet stream of the cross through an in-line tee (see FIG. 2 of WO2016010840). LNPs were held at room temperature for 1 hour and further diluted with water (approximately 1:1 v / v). LNPs were concentrated using tangential flow filtration, for example on a flat sheet cartridge (Sartorius, 100 kD MWCO), and optionally buffer exchanged into 50 mM Tris, 45 mM NaCl, 5% (w / v) sucrose, pH 7.5 (TSS) using a PD-10 desalting column (GE). Alternatively, LNPs were optionally concentrated using a 100 kDa Amicon spin filter and buffer exchanged into TSS using a PD-10 desalting column (GE). The resulting mixture was then filtered using a 0.2 μM sterile filter, and the final LNPs were stored at 4° C. or −80° C. until further use.

[0205] Example 1.2 In Vitro Transcription ("IVT") of Nuclease mRNA Capped and polyadenylated mRNA containing N1-methylpseudo-U was generated by in vitro transcription using linearized plasmid DNA template and T7 RNA polymerase. Plasmid DNA containing the T7 promoter, sequence for transcription, and polyadenylation region was linearized by incubation with XbaI for 2 hours at 37°C using the following conditions: 200 ng / μL plasmid, 2 U / μL XbaI (NEB), and 1× reaction buffer. XbaI was inactivated by heating the reaction at 65°C for 20 minutes. Linearized plasmid was purified from enzymes and buffer salts. IVT reactions to generate modified mRNA were performed in the following conditions: 50 ng / μL linearized plasmid; 2–5 mM each of GTP, ATP, CTP, and N1-methylpseudo-UTP (Trilink); 10–25 mM ARCA (Trilink); 5 U / μL T7 RNA polymerase (NEB); 1 U / μL mouse RNase inhibitor (NEB); 0.004 U / μL inorganic E. coli pyrophosphatase (NEB); and 1× reaction buffer by incubating at 37°C for 1.5–4 h. TURBO DNase (ThermoFisher) was added to a final concentration of 0.01 U / μL, and the reaction was incubated for an additional 30 min to remove the DNA template. mRNA was purified using the MegaClear Transcription Clean-up kit (ThermoFisher) or RNeasy Maxi kit (Qiagen) according to the manufacturer's protocol.

[0206] Alternatively, mRNA was purified by a precipitation protocol, optionally followed by HPLC-based purification. Briefly, after DNase digestion, mRNA was purified using LiCl precipitation, ammonium acetate precipitation, and sodium acetate precipitation. For HPLC-purified mRNA, after LiCl precipitation and reconstitution, mRNA was purified by RP-IP HPLC (see, e.g., Kariko, et al. Nucleic Acids Research, 2011, Vol. 39, No. 21 e142). Fractions selected for pooling were combined and desalted by sodium acetate / ethanol precipitation as described above. In a further alternative method, mRNA was purified by LiCl precipitation method followed by further purification by tangential flow filtration. RNA concentration was quantified by measuring light absorption at 260 nm (Nanodrop) and transcripts were analyzed by capillary electrophoresis with a Bioanlayzer (Agilent).

[0207] Streptococcus pyogenes ("Spy") Cas9 mRNA was generated from plasmid DNA encoding an open reading frame according to SEQ ID NOs: 1-3 (see sequences in Table 35). It is understood that when the sequences cited in this paragraph are referred to below in relation to RNA, Ts should be replaced with Us (which may be modified nucleosides as described above). The messenger RNA used in the examples includes a 5' cap and a 3' polyadenylation sequence (e.g., up to 100 nt) and is set forth in Table 35.

[0208] Guide RNAs are chemically synthesized by methods known in the art.

[0209] Example 1.4 Formulation Analysis Dynamic light scattering ("DLS") was used to characterize the polydispersity index ("pdi") and size of the LNPs of the present disclosure. DLS measures the scattering of light resulting from subjecting a sample to a light source. The PDI determined from DLS measurements corresponds to the distribution of particle sizes (around the average particle size) in a population, with a completely homogenous population having a PDI of zero.

[0210] Asymmetric flow field fractionation-multi-angle light scattering (AF4-MALS) is used to separate particles in a composition by hydrodynamic radius, and then to measure the molecular weight, hydrodynamic radius, and root-mean-square radius of the separated particles. This allows for the assessment of secondary properties such as Burchard-Stockmeyer plots (the ratio of root-mean-square ("rms") radius:hydrodynamic radius over time, which indicates the inner core density of the particle) and rms conformation plots (logarithm of rms radius vs. logarithm of molecular weight, where the slope of the resulting linear fit gives the degree of compression vs. elongation).

[0211] Cryo-electron microscopy ("cryo-EM") can be used to quantitate the particle size, morphology, and structural characteristics of LNPs.

[0212] The lipid composition analysis of the LNPs was determined using liquid chromatography and charged aerosol detection (LC-CAD), which compared the measured and nominal lipid contents.

[0213] The LNP compositions were analyzed for mean particle size, polydispersity index (pdi), total RNA content, RNA encapsulation efficiency, and zeta potential. LNP compositions can be further characterized by lipid analysis, AF4-MALS, NTA, and / or cryo-EM. Mean particle size and polydispersity were measured by dynamic light scattering (DLS) using a Malvern Zetasizer DLS instrument. LNP samples were diluted with PBS buffer and then measured by DLS. Z-average diameter was reported along with number-average diameter and pdi. Z-average refers to the intensity-weighted average hydrodynamic size of a population of particles, measured by dynamic light scattering. Number-average refers to the particle number-weighted average hydrodynamic size of a population of particles measured by dynamic light scattering. The Malvern Zetasizer instrument was also used to measure the zeta potential of the LNPs. Samples were diluted 1:17 (50 μL in 800 μL) with 0.1× PBS, pH 7.4 prior to measurement.

[0214] Encapsulation efficiency was calculated as (total RNA-free RNA) / total RNA. Total RNA concentration and free RNA were quantified using a fluorescence-based assay (Ribogreen®; ThermoFisher Scientific). LNP samples were appropriately diluted with 1×TE buffer containing 0.2% Triton-X100 to quantify total RNA or 1×TE buffer to quantify free RNA. Standard curves were prepared by utilizing the starting RNA solution used to make the compositions and diluted with 1×TE buffer + / - 0.2% Triton-X100. Diluted RiboGreen® dye (following manufacturer's instructions) was then added to each of the standards and samples and incubated for approximately 10 minutes at room temperature in the absence of light. Samples were read using a SpectraMax M5 microplate reader (Molecular Devices) with excitation, autocutoff, and emission wavelengths set at 488 nm, 515 nm, and 525 nm, respectively. Total RNA and free RNA were quantified from the appropriate standard curves.

[0215] AF4-MALS is used to investigate molecular weight and size distribution, as well as second order statistics from these calculations. LNPs are appropriately diluted and injected into the AF4 separation channel using an HPLC autosampler, concentrated, and then eluted with an exponential gradient of cross-flow across the channel. All fluids are driven by HPLC pumps and a Wyatt Eclipse instrument. Particles eluted from the AF4 channel flow through a UV detector, a multi-angle light scattering detector, a quasi-elastic light scattering detector, and a differential refractive index detector. Raw data is processed by using the Debeye model to quantify molecular weight and rms radius from the detector signals.

[0216] CAD is a destructive mass-based detector that detects all non-volatile compounds, and the signal is consistent regardless of analyte structure.

[0217] The lipid components in the LNPs were quantitatively analyzed by HPLC coupled with a charged aerosol detector (CAD). Chromatographic separation of the four lipid components was achieved by reversed-phase HPLC. The HPLC lipid analysis provided the actual mole percent (mol-%) lipid levels of each component of the LNP compositions described in the following examples, as shown in Table 1. [Table 2-1] [Table 2-2]

[0218] Example 1.5 Preparation of T Cells Leukapheresis of healthy human donors was obtained commercially (Hemacare). T cells were isolated by negative selection using the EasySep Human T Cell Isolation Kit (Stem Cell Technology; Cat. No. 17951) or by CD4 / CD8 positive selection using StraightFrom® Leukopak® CD4 / CD8 microbeads (Milteny; Cat. No. 130-122-352) on a MultiMACS™ Cell24 Separator Plus instrument according to the manufacturer's instructions. T cells were cryopreserved in Cryostor CS10 freezing medium (Cat. No. 07930) for future use.

[0219] Once thawed, T cells were cultured in complete T cell growth medium consisting of CTS OpTmizer basal medium (Gibco; A3705001) supplemented with 1x GlutaMAX, 10 mM HEPES buffer (10 mM), and 1% pen-strep (Gibco; 15140-122), and further supplemented with 200 IU / mL IL2 (Peprotech; 200-02), 5 ng / mL IL7 (Peprotech; 200-07), 5 ng / mL IL15 (Peprotech; 200-15), and 2.5% human serum (Gemini; 100-512). After overnight resting, T cells at a density of 10^6 cells / mL were activated with T cell TransAct reagent (1:100 dilution, Miltenyi) and incubated at 37°C for 24 or 48 hours. After incubation, cells at a density of 0.5×10^6 / mL were used for editing purposes.

[0220] Unless otherwise indicated, the same process was used for non-activated T cells with the following exceptions: Once thawed, non-activated T cells were cultured in CTS complete growth medium consisting of CTS OpTmizer basal medium (Thermofisher; A10485-01), 1% pen-strep (Corning; 30-002-CI), 1× GlutaMAX (Thermofisher; 35050061), 10 mM HEPES (Thermofisher; 15630080), and further supplemented with 200 U / mL IL2 (Peprotech; 200-02), 5 ng / mL IL7 (Peprotech; 200-07), 5 ng / mL IL15 (Peprotech; 200-15), and 5% human AB serum (Gemini; 100-512) and incubated for 24 hours without activation. T cells were plated at a cell density of 10^6 / mL in 100uL of the above-mentioned CTS OpTmizer basal medium containing 2.5% human serum and cytokines for editing applications.

[0221] Example 1.6 LNP Transfection of T Cells T cells were transfected with LNPs prepared as described above in Example 1. Materials used for LNP transfection are described in Example 1. LNP dose response curve (DRC) transfection was performed on a Hamilton Microlab STAR liquid handling system. The liquid handler was provided with: (a) 4x the highest desired dose of LNPs in the top row of deep wells of a 96 deep well plate, (b) ApoE3 diluted in culture medium at 20μg / mL, (c) complete T cell growth medium composed of CTS OpTmizer basal medium as described above in Example 1, and (d) T cells plated at a density of 10^6 / ml in 100uL in a 96 well flat bottom tissue culture plate. The liquid handler first performed an 8-point 2-fold serial dilution of LNPs from the 4x dose of LNPs in the deep well plate. An equal volume of ApoE3 medium was then added to each well. 100μL of the LNP-ApoE mixture was then added to each T cell plate. The final concentration of LNPs at the highest dose was 5μg / mL. The final concentration of ApoE3 was 5μg / mL, and T cells were at a final density of 0.5×10^6 cells / mL. Plates were incubated at 37℃, 5% CO2 for 24 or 48 hours for activated and on-activated T cells, respectively. After incubation, LNP-treated T cells were harvested and analyzed for on-target editing or Cas9 protein expression detection. The remaining cells were cultured for 7–10 days after LNP treatment, and protein surface expression was assessed by flow cytometry.

[0222] Example 1.8 Flow cytometry analysis The T cell receptor alpha chain, encoded by TRAC, is required for the assembly and translocation of the T cell receptor / CD3 complex to the cell surface. Editing was assayed by the increase in the percentage of CD3-negative cells after editing. To assay cell surface proteins by flow cytometry, T cells were suspended in 100 μL of antibody cocktail (1:100 PE-anti-human CD3 [Biolegend; Cat. No. 300441], 1:200 FITC anti-human CD4 [Biolegend; Cat. No. 300538], 1:200 APC anti-human CD8a [Biolegend; Cat. No. 301049], FACS buffer [PBS + 2% FBS + 2 mM EDTA]) and incubated for 30 min at 4°C. T cells were washed and then resuspended in FACS buffer (PBS + 2% FBS + 2 mM EDTA). T cells were then processed with a Cytoflex instrument (Beckman Coulter). Data analysis was performed using the FlowJo software package (v.10.6.1 or v.10.7.1). Briefly, T cells were gated on lymphocytes and then on single cells. These single cells were gated on CD4+ / CD8+ status from which CD8+ / CD3- cells were selected. The percentage of CD8+ / CD3- cells was quantified to determine the percentage of the cell population in which the edited target locus resulted in TCR knockout. Using a linear regression model, dose-response curves for TCR KO were generated using Prism GraphPad (v.9.0). The half-effective concentration (EC 50 ) and maximum percentage of CD3- were calculated for each LNP.

[0223] Example 1.9. Next generation sequencing (NGS) and analysis of editing efficiency To quantitatively determine the editing efficiency at the target position in the genome, sequencing was used to identify the presence of insertions and deletions introduced by gene editing. PCR primers were designed based on the target site in the gene of interest (e.g., TRAC) to amplify the genomic segment of interest. Primer sequence design was performed as standard in the field.

[0224] An additional PCR was performed according to the manufacturer's protocol (Illumina) to add sequencing chemistry. Amplicons were sequenced on an Illumina MiSeq instrument. After removing reads with low quality scores, reads were aligned to a reference genome (e.g., hg38). Result files containing reads were mapped to the reference genome (BAM files), reads overlapping the target region of interest were selected, and the number of reads containing insertions or deletions ("indels") relative to the number of wild-type reads was calculated.

[0225] Example 2: Screening of Compound 6 Compositions in T Cells 2.1 Characterization of ionizable lipids of LNPs in CD3+ T cells To assess the efficiency of editing, T cells were treated with LNP compositions containing various mole percentages of lipid components encapsulating Cas9 mRNA and sgRNA targeting the TRAC gene, and loss of T cell receptor surface protein was assessed by flow cytometry.

[0226] Generally, LNPs were prepared as in Example 1, using the lipid compositions shown in Table 1 (expressed as molar ratios of ionizable compound 6 / DSPC / cholesterol / PEG, respectively). The LNPs delivered mRNA encoding Cas9 (SEQ ID NO:4) and sgRNA targeting human TRAC (SEQ ID NO:10). The cargo weight ratio of sgRNA:Cas9 mRNA was 1:2.

[0227] The LNP compositions were analyzed for Z-average particle size, number average particle size, polydispersity (pdi), total RNA content, and RNA encapsulation efficiency as described in Example 1. The results are shown in Table 2. Lipid analysis showed the observed mol-% lipid levels of each component as shown in Table 1. [Table 3]

[0228] The LNPs in Table 2 were evaluated to quantify the effect of LNP composition ratio on the editing efficiency of CD3 positive (CD3+) T cells. T cells from two donors (lot numbers W0106 and W0186) were prepared and transfected as described in Example 1 for activated and non-activated T cells, respectively. Seven days after transfection, edited T cells were harvested and phenotyped by flow cytometry as described in Example 1.

[0229] The percentage of CD3 negative (CD3-) T cells was measured after treatment with LNP concentrations of 0.04 μg / mL, 0.08 μg / mL, 0.16 μg / mL, 0.25 μg / mL, 0.63 μg / mL, 1.25 μg / mL, 2.5 μg / mL, and 5 μg / mL. The mean percent of CD3 negative T cells, maximum percent of CD3 negative, and EC50 for each LNP dose are shown in Table 3 and Figure 1A for activated T cells and in Table 4 and Figure 1B for non-activated T cells. Approximate values ​​are marked with a "tilde" and values ​​that could not be quantified by the analysis software are marked with "ND". [Table 4-1] [Table 4-2] [Table 4-3] [Table 5-1] [Table 5-2] [Table 5-3]

[0230] Example 3: Screening of Selected Compound 6 LNP Compositions in T Cells 3.1 Evaluation of selected LNP compositions in edited CD3+ T cells To assess the efficacy of LNP editing, T cells were treated with LNP compositions containing compound 6 and varying mole percentages of lipid components encapsulating Cas9 mRNA and an sgRNA targeting the TRAC gene, and loss of T cell receptor surface proteins was assessed by flow cytometry.

[0231] Generally, LNPs were prepared as described in Example 1 with the lipid compositions shown in Table 5 (expressed as molar ratios of ionizable compound 6 / DSPC / cholesterol / PEG, respectively). The LNPs delivered mRNA encoding Cas9 mRNA (SEQ ID NO:4) and sgRNA targeting human TRAC (SEQ ID NO:10). The cargo weight ratio of sgRNA:Cas9 mRNA was 1:2.

[0232] The mean particle size, polydispersity (pdi), total RNA content, and RNA encapsulation efficiency of the LNP compositions were analyzed as described in Example 1. The results are shown in Table 5. Lipid analysis showed the observed mol-% lipid levels as shown in Table 1. [Table 6]

[0233] T cells from two donors (lot numbers W0106 and W790) were prepared and transfected as described in Example 1. Six days after transfection, edited activated and non-activated T cells were harvested and phenotyped by flow cytometry as described in Example 1. The percentage of CD3 negative T cells was measured for T cells treated with LNP concentrations of 0.04 μg, 0.08 μg, 0.16 μg, 0.31 μg, 0.63 μg, 1.25 μg, 2.5 μg, and 5 μg. The mean percent of CD3 negative T cells, calculated CD3 maximum percent, and EC50 for each LNP dose are shown in Table 6 and Figure 2A for activated T cells and Table 7 and Figure 2B for non-activated T cells. [Table 7-1] [Table 7-2] [Table 7-3] [Table 8-1] [Table 8-2] [Table 8-3]

[0234] Example 4. Screening of Compound 6 Compositions in T Cells 4.1 Characterization of ionizable lipids of LNPs in CD3+ T cells To assess the efficacy of LNP editing, T cells were treated with LNP compositions containing alternative molar ratios of lipid components encapsulating Cas9 mRNA and sgRNA targeting the TRAC gene, and loss of T cell receptor surface proteins was assessed by flow cytometry.

[0235] Generally, LNPs were prepared as in Example 1, using the lipid compositions shown in Table 8 (expressed as molar ratios of ionizable compound 6 / DSPC / cholesterol / PEG, respectively). The LNPs delivered mRNA encoding Cas9 (SEQ ID NO:4) and sgRNA targeting human TRAC (SEQ ID NO:10). The cargo weight ratio of sgRNA:Cas9 mRNA was 1:2 or 1:1. Comparative composition 21 had a cargo ratio of 1:2, and the remaining LNPs had a cargo ratio of 1:1.

[0236] The mean particle size, polydispersity (pdi), total RNA content, and RNA encapsulation efficiency of the LNP compositions were analyzed as described in Example 1. The results are shown in Table 8. Lipid analysis showed the observed mol-% lipid levels as shown in Table 1. [Table 9]

[0237] T cells from one donor (W0106) were prepared, activated, and transfected as described in Example 1. Seven days after transfection, edited activated and non-activated T cells were harvested and phenotyped by flow cytometry as described in Example 1.

[0238] The percentage of CD3 negative T cells was measured after treatment with LNP concentrations of 0.04 μg, 0.08 μg, 0.16 μg, 0.25 μg, 0.63 μg, 1.25 μg, 2.5 μg, and 5 μg. The mean percent of CD3 negative T cells, maximum percentage of CD3, and EC50 for each LNP dose are shown in Tables 9 and 10. [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6] [Table 10-7] [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4] [Table 11-5] [Table 11-6]

[0239] As can be seen in Tables 9 and 10, LNPs with lower amounts of ionizable lipids and higher amounts of DSPC, cholesterol, and PEG lipids had surprisingly favorable EC50 values ​​compared to the comparative compositions.

[0240] Example 5: Screening for ionizable lipids in T cells 5.1 Characterization of LNP compositions containing various ionizable lipids To evaluate the effect of other ionizable lipids in LNPs on editing, T cells were treated with LNP compositions in which one of three ionizable lipids was formulated in two different component ratios. Generally, LNPs were prepared as described in Example 1 with the lipid compositions shown in Table 11 (each expressed as the molar ratio of ionizable lipid compound / DSPC / cholesterol / PEG). Compound 6, compound 8, and compound 11 were each formulated in LNPs with the following nominal mol% ratios of lipid components: 35% ionizable lipid, 15% DSPC, 47.5% cholesterol, and 2.5% PEG-2k-DMG, and in comparative LNPs with the following nominal mol% ratios of lipid components: 50% ionizable lipid, 10% DSPC, 38.5% cholesterol, and 1.5% PEG-2k-DMG. The LNPs encapsulated Cas9 mRNA and sgRNA targeting the TRAC gene. Editing was assessed for loss of T-cell receptor surface proteins by flow cytometry.

[0241] The LNPs delivered mRNA encoding Cas9 (SEQ ID NO:5) and an sgRNA targeting human TRAC (SEQ ID NO:10). The cargo weight ratio of sgRNA:Cas9 mRNA in the tested LNPs was 1:1.

[0242] The mean particle size, polydispersity (pdi), total RNA content, and RNA encapsulation efficiency of the LNP compositions were analyzed as described in Example 1. The results are shown in Table 11. Lipid analysis showed the observed mol-% lipid levels as shown in Table 1. [Table 12]

[0243] T cells from a single donor (W0106) were prepared, activated, and transfected generally as described in Example 1, except that the non-activated T cells were rested for 48 hours prior to transfection. Seven days after transfection, edited activated T cells were harvested and phenotyped by flow cytometry as described in Example 1.

[0244] The percentage of CD3 negative T cells was measured after treatment with LNP concentrations of 0.04 μg, 0.08 μg, 0.16 μg, 0.25 μg, 0.63 μg, 1.25 μg, 2.5 μg, and 5 μg. The mean percent of CD3 negative T cells, maximum percent of CD3, and EC50 for each LNP dose are shown in Table 12 and Figure 3A for activated T cells and in Table 13 and Figure 3B for non-activated T cells. [Table 13-1] [Table 13-2] [Table 14-1] [Table 14-2]

[0245] Example 6: Cargo Ratio Assessment of Selected LNP Compositions To evaluate the effect of cargo ratio on LNP editing efficacy, CD3+ T cells were treated with LNP compositions containing various ratios of Cas9 mRNA and sgRNA targeting the TRAC gene. Editing was evaluated for loss of T cell receptor surface protein by flow cytometry.

[0246] 6.1 Cargo ratio assessment in activated T cells The effect of changing cargo ratios on the editing efficiency of selected LNPs listed in Table 14 was tested in activated CD3-positive T cells. Generally, LNPs were prepared as described in Example 1 with the lipid compositions shown in Table 14 (respectively expressed as molar ratios of ionizable compound 6 / DSPC / cholesterol / PEG). LNPs delivered mRNA encoding Cas9 mRNA (SEQ ID NO:5) and sgRNA targeting TRAC (SEQ ID NO:10). Editing was assessed for loss of T cell receptor surface protein by flow cytometry. The cargo weight ratio of sgRNA:Cas9 mRNA was 1:1, 1:2, 2:1, or 4:1.

[0247] The mean particle size, polydispersity (pdi), total RNA content, and RNA encapsulation efficiency of the LNP compositions were analyzed as described in Example 1. The results are shown in Table 14. Lipid analysis showed the observed mol-% lipid levels as shown in Table 1. [Table 15]

[0248] T cells were harvested from a single donor (Lot No. W0106), prepared, and transfected as described in Example 1. Seven days after transfection, T cells were phenotyped by flow cytometry analysis as described in Example 1. The percentage of CD3 negative T cells was measured after treatment with LNP concentrations of 0.04 μg, 0.08 μg, 0.16 μg, 0.25 μg, 0.63 μg, 1.25 μg, 2.5 μg, and 5 μg. The mean percent of CD3 negative T cells, maximum percentage of CD3, and EC50 at each LNP dose are shown in Table 15 and Figure 4A. [Table 16-1] [Table 16-2]

[0249] 6.2 Charge ratio evaluation in non-activated T cells The effect of varying cargo ratios on LNP editing efficiency of selected LNPs described in Example 6.1 was tested in non-activated CD3 positive T cells. Selected LNP compositions described in Example 6.1 were used in this study.

[0250] T cells from two donors (lot numbers W106, W790) were prepared and transfected as described in Example 1. Seven days after transfection, edited, resting T cells were phenotyped by flow cytometry analysis as described in Example 1.

[0251] The percentage of CD3 negative T cells was measured at LNP concentrations of 0.04 μg, 0.08 μg, 0.16 μg, 0.25 μg, 0.63 μg, 1.25 μg, 2.5 μg, and 5 μg. The mean percent of CD3 negative T cells, maximum percent of CD3, and EC50 for each LNP dose are shown in Tables 16 and 17 and in FIG. 4B. [Table 17-1] [Table 17-2] [Table 18-1] [Table 18-2]

[0252] Example 7: LNP composition activity assessed under serum media conditions To evaluate the editing efficacy of LNPs, LNP compositions were tested in vitro to evaluate the effect of alternative media conditions on insertion efficiency in CD3 positive T cells. T cells were treated with LNP compositions containing various molar ratios of lipid components encapsulating Cas9 mRNA and sgRNA targeting the TRAC gene. AAV6 viral constructs delivered a homology-directed repair template (HDRT) encoding a GFP reporter flanked by homology arms for site-specific integration into the TRAC locus. TRAC gene disruption was evaluated for loss of T cell receptor surface protein by flow cytometry. Insertion was evaluated for GFP emission by flow cytometry.

[0253] Generally, LNPs were prepared as in Example 1, using the lipid compositions shown in Table 18 (expressed as molar ratios of ionizable compound 6 / DSPC / cholesterol / PEG, respectively). The LNPs delivered mRNA encoding Cas9 (SEQ ID NO:4) and an sgRNA targeting human TRAC (SEQ ID NO:10). The cargo weight ratio of sgRNA:Cas9 mRNA was 1:2.

[0254] The mean particle size, polydispersity (pdi), total RNA content, and RNA encapsulation efficiency of the LNP compositions were analyzed as described in Example 1. The results are shown in Table 18. Lipid analysis showed the observed mol-% lipid levels as shown in Table 1. [Table 19]

[0255] Single donor derived T cells (Lot No. W0106) were prepared as described in Example 1 with the following media modifications: T cells were plated in media supplemented with either 2.5% human AB serum (HABS), 2.5% CTS immune cell SR (Gibco; Catalog No. A25961-01) serum replacement (SR), 5% serum replacement (SR), or a combination of 2.5% human AB serum and 2.5% serum replacement. T cells were activated 24 hours after thawing as described in Example 1.5. Two days after activation, T cells were transfected with LNP as described in Example 1.6 at LNP concentrations of 0.31 μg / ml, 0.63 μg / ml, 1.25 μg / ml, and 2.5 μg / ml. AAV6 encoding a homology-directed repair template (HDRT) encoding a GFP reporter (Vigene; SEQ ID NO: 7) flanked by homology arms for site-specific integration into the TRAC locus was added to each well at a multiplicity of infection (MOI) of 3 x 10e5 viral particles / cell. A small molecule inhibitor of DNA-dependent protein kinase was added at 0.25 uM. The inhibitor, hereafter referred to as "DNAPKI compound 4", is 9-(4,4-difluorocyclohexyl)-7-methyl-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-7,9-dihydro-8H-purin-8-one, and is shown below. [ka]

[0256] DNAPKI compound 4 was prepared as follows.

[0257] General information All reagents and solvents were purchased from commercial vendors and used as received or synthesized according to the cited procedures. All intermediates and final compounds were purified using flash column chromatography on silica gel. NMR spectra were recorded on Bruker or Varian 400 MHz spectrometers, and NMR data were collected at ambient temperature in CDCl3. Chemical shifts are reported in ppm relative to CDCl3 (7.26). 1H NMR data are reported as follows: chemical shift, multiplicity (br = broad, s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, dt = doublet of triplets, m = multiplet), coupling constants, and integrals. MS data were recorded on a Waters SQD2 mass spectrometer equipped with an electrospray ionization (ESI) source. The purity of the final compounds was quantified by UPLC-MS-ELS using a Waters Acquity H-Class liquid chromatography system equipped with a SQD2 mass spectrometer equipped with a photodiode array (PDA) detector and an evaporative light scattering (ELS) detector.

[0258] Intermediate 1a: (E)-N,N-dimethyl-N'-(4-methyl-5-nitropyridin-2-yl)formimidamide [ka] To a solution of 4-methyl-5-nitro-pyridin-2-amine (5 g, 1.0 equiv.) in toluene (0.3 M) was added DMF-DMA (3.0 equiv.). The mixture was stirred at 110° C. for 2 h. The reaction mixture was concentrated under reduced pressure to give a residue that was purified by column chromatography to give the product as a yellow solid (59%). 1 H NMR (400 MHz, (CD3)2SO) δ 8.82 (s, 1H), 8.63 (s, 1H), 6.74 (s, 1H), 3.21 (m, 6H).

[0259] Intermediate 1b: (E)-N-hydroxy-N'-(4-methyl-5-nitropyridin-2-yl)formimidamide [ka] To a solution of intermediate 1a (4 g, 1.0 equiv.) in MeOH (0.2 M) was added NH2OH-HCl (2.0 equiv.). The reaction mixture was stirred at 80° C. for 1 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was partitioned between H2O and EtOAc, followed by extraction twice with EtOAc. The organic phase was concentrated under reduced pressure to give a residue that was purified by column chromatography to give the product as a white solid (66%). 1H NMR (400 MHz, (CD3)2SO) δ 10.52 (d, J = 3.8 Hz, 1H), 10.08 (dd, J = 9.9, 3.7 Hz, 1H), 8.84 (d, J = 3.8 Hz, 1H), 7.85 (dd, J = 9.7, 3.8 Hz, 1H), 7.01 (d, J = 3.9 Hz, 1H), 3.36 (s, 3H).

[0260] Intermediate 1c: 7-Methyl-6-nitro-[1,2,4]triazolo[1,5-a]pyridine [ka] To a solution of intermediate 1b (2.5 g, 1.0 equiv.) in THF (0.4 M) was added trifluoroacetic anhydride (1.0 equiv.) at 0° C. The mixture was stirred at 25° C. for 18 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give the product as a white solid (44%). 1 H NMR (400 MHz, CDCl3) δ 9.53 (s, 1H), 8.49 (s, 1H), 7.69 (s, 1H), 2.78 (d, J = 1.0 Hz, 3H).

[0261] Intermediate 1d: 7-Methyl-[1,2,4]triazolo[1,5-a]pyridin-6-amine [ka] To a mixture of Pd / C (10% w / w, 0.2 equiv.) in EtOH (0.1 M) was added intermediate 1c (1.0 equiv.) and ammonium formate (5.0 equiv.). The mixture was heated at 105° C. for 2 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give the product as a light brown solid. 1 H NMR (400 MHz, (CD3)2SO) δ 8.41 (s, 2H), 8.07 (d, J = 9.0 Hz, 2H), 7.43 (s, 1H), 2.22 (s, 3H).

[0262] Intermediate 1e: 8-methylene-1,4-dioxaspiro[4.5]decane [ka] To a solution of methyl(triphenyl)phosphonium bromide (1.15 equiv.) in THF (0.6 M) was added n-BuLi (1.1 equiv.) dropwise at -78°C, and the mixture was stirred at 0°C for 1 h. Then 1,4-dioxaspiro[4.5]decan-8-one (50 g, 1.0 equiv.) was added to the reaction mixture. The mixture was stirred at 25°C for 12 h. The reaction mixture was poured into aqueous NH4Cl at 0°C, diluted with H2O, and extracted three times with EtOAc. The combined organic layers were concentrated under reduced pressure to give a residue that was purified by column chromatography to give the product as a colorless oil (51%). 1 H NMR (400 MHz, CDCl3) δ 4.67 (s, 1H), 3.96 (s, 4 H), 2.82 (t, J = 6.4 Hz, 4 H), 1.70 (t, J = 6.4 Hz, 4 H).

[0263] Intermediate 1f: 7,10-dioxadispiro[2.2.4 6 .2 3 ]Dodecane [ka] To a solution of intermediate 4a (5 g, 1.0 equiv.) in toluene (3M) was added ZnEt2 (2.57 equiv.) dropwise at -40°C, and the mixture was stirred at -40°C for 1 h. Diiodomethane (6.0 equiv.) was then added dropwise to the mixture under N2 at -40°C. The mixture was then stirred under N2 atmosphere at 20°C for 17 h. The reaction mixture was poured into aqueous NH4Cl at 0°C and extracted twice with EtOAc. The combined organic phase was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated in vacuo. The residue was purified by column chromatography to give the product as a pale yellow oil (73%).

[0264] Intermediate 1g: spiro[2.5]octan-6-one [ka] To a solution of intermediate 4b (4 g, 1.0 equiv.) in 1:1 THF / H2O (1.0 M) was added TFA (3.0 equiv.). The mixture was stirred under N2 atmosphere at 20° C. for 2 h. The reaction mixture was concentrated under reduced pressure to remove THF and the pH of the residue was adjusted to 7 with 2 M NaOH (aq.). The mixture was poured into water and extracted three times with EtOAc. The combined organic phase was washed with brine, dried over anhydrous Na2SO4, filtered and the filtrate was concentrated in vacuo. The residue was purified by column chromatography to give the product as a pale yellow oil (68%). 1 H NMR (400 MHz, CDCl3) δ 2.35 (t, J = 6.6 Hz, 4H), 1.62 (t, J = 6.6 Hz, 4H), 0.42 (s, 4H).

[0265] Intermediate 1h: N-(4-methoxybenzyl)spiro[2.5]octan-6-amine [ka] To a mixture of intermediate 4c (2 g, 1.0 equiv.) and (4-methoxyphenyl)methanamine (1.1 equiv.) in DCM (0.3 M) was added AcOH (1.3 equiv.). The mixture was stirred at 20° C. for 1 h under N2 atmosphere. NaBH(OAc)3 (3.3 equiv.) was then added to the mixture at 0° C., and the mixture was stirred at 20° C. for 17 h under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to remove DCM, and the resulting residue was diluted with H2O and extracted three times with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give the product as a grey solid (51%). 1 H NMR (400 MHz, (CD3)2SO) δ 7.15 - 7.07 (m, 2H), 6.77 - 6.68 (m, 2H), 3.58 (s, 3H), 3.54 (s, 2H), 2.30 (ddt, J = 10.1, 7.3, 3.7 Hz, 1H), 1.69 - 1.62 (m, 2H), 1.37 (td, J = 12.6, 3.5 Hz, 2H), 1.12 - 1.02 (m, 2H), 0.87 - 0.78 (m, 2H), 0.13 - 0.04 (m, 2H).

[0266] Intermediate 1i: spiro[2.5]octan-6-amine [ka] To a suspension of Pd / C (10% w / w, 1.0 equiv) in MeOH (0.25 M) was added intermediate 4d (2 g, 1.0 equiv) and the mixture was stirred under H2 atmosphere at 80° C. and 50 Psi for 24 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue which was purified by column chromatography to give the product as a white solid. 1H NMR (400 MHz, (CD3)2SO) δ 2.61 (tt, J = 10.8, 3.9 Hz, 1H), 1.63 (ddd, J = 9.6, 5.1, 2.2 Hz, 2H), 1.47 (td, J = 12.8, 3.5 Hz, 2H), 1.21 - 1.06 (m, 2H), 0.82 - 0.72 (m, 2H), 0.14 - 0.05 (m, 2H).

[0267] Intermediate 1j: Ethyl 2-chloro-4-(spiro[2.5]octan-6-ylamino)pyrimidine-5-carboxylate [ka] To a mixture of ethyl 2,4-dichloropyrimidine-5-carboxylate (2.7 g, 1.0 equiv.) and Intermediate 1i (1.0 equiv.) in ACN (0.5-0.6 M) was added K2CO3 (2.5 equiv.) in one portion under N2. The mixture was stirred at 20 °C for 12 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give the product as a white solid (54%). 1 H NMR (400 MHz, (CD3)2SO) δ 8.64 (s, 1H), 8.41 (d, J = 7.9 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H), 4.08 (d, J = 9.8 Hz, 1H), 1.90 (dd, J = 12.7, 4.8 Hz, 2H), 1.64 (t, J = 12.3 Hz, 2H), 1.52 (q, J = 10.7, 9.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H), 1.12 (d, J = 13.0 Hz, 2H), 0.40 - 0.21 (m, 4H).

[0268] Intermediate 1k: 2-chloro-4-(spiro[2.5]octan-6-ylamino)pyrimidine-5-carboxylic acid [ka] To a solution of intermediate 1j (2 g, 1.0 equiv.) in 1:1 THF / H2O (0.3 M) was added LiOH (2.0 equiv.). The mixture was stirred at 20° C. for 12 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was adjusted to pH 2 with 2 M hydrochloric acid and the precipitate was collected by filtration, washed with water and run under vacuum. The product was used directly in the next step without further purification (82%). 1 H NMR (400 MHz, (CD3)2SO) δ 13.54 (s, 1H), 8.38 (d, J = 8.0 Hz, 1H), 8.35 (s, 1H), 3.82 (qt, J = 8.2, 3.7 Hz, 1H), 1.66 (dq, J = 12.8, 4.1 Hz, 2H), 1.47 - 1.34 (m, 2H), 1.33 - 1.20 (m, 2H), 0.86 (dt, J = 13.6, 4.2 Hz, 2H), 0.08 (dd, J = 8.3, 4.8 Hz, 4H).

[0269] Intermediate 1l: 2-chloro-9-(spiro[2.5]octan-6-yl)-7,9-dihydro-8H-purin-8-one [ka] To a mixture of intermediate 1k (1.5 g, 1.0 equiv.) and Et3N (1.0 equiv.) in DMF (0.3 M) was added DPPA (1.0 equiv.). The mixture was stirred at 120° C. for 8 h under N2 atmosphere. The reaction mixture was poured into water. The precipitate was collected by filtration, washed with water, and dried under vacuum to give a residue that was used directly in the next step without further purification (67%). 1H NMR (400 MHz, (CD3)2SO) δ 11.68 (s, 1H), 8.18 (s, 1H), 4.26 (ddt, J = 12.3, 7.5, 3.7 Hz, 1H), 2.42 (qd, J = 12.6, 3.7 Hz, 2H), 1.95 (td, J = 13.3, 3.5 Hz, 2H), 1.82 - 1.69 (m, 2H), 1.08 - 0.95 (m, 2H), 0.39 (tdq, J = 11.6, 8.7, 4.2, 3.5 Hz, 4H).

[0270] Intermediate 1m: 2-chloro-7-methyl-9-(spiro[2.5]octan-6-yl)-7,9-dihydro-8H-purin-8-one [ka] To a mixture of intermediate 1l (1.0 g, 1.0 equiv.) and NaOH (5.0 equiv.) in 1:1 THF / H2O (0.3-0.5 M) was added MeI (2.0 equiv.). The mixture was stirred at 20 °C under N2 atmosphere for 12 h. The reaction mixture was concentrated under reduced pressure to give a residue which was purified by column chromatography to give the product as a pale yellow solid (67%). 1 H NMR (400 MHz, CDCl3) δ 7.57 (s, 1H), 4.03 (tt, J = 12.5, 3.9 Hz, 1H), 3.03 (s, 3H), 2.17 (qd, J = 12.6, 3.8 Hz, 2H), 1.60 (td, J = 13.4, 3.6 Hz, 2H), 1.47 - 1.34 (m, 2H), 1.07 (s, 1H), 0.63 (dp, J = 14.0, 2.5 Hz, 2H), -0.05 (s, 4H).

[0271] DNAPKI compound 4: 7-methyl-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-9-(spiro[2.5]octan-6-yl)-7,9-dihydro-8H-purin-8-one [ka] A mixture of intermediate 1m (1.0 equiv.) and intermediate 1d (1.0 equiv.) was added with Pd(dppf)Cl2 (0.2 equiv.), XantPhos (0.4 equiv.), and Cs2CO3 (2.0 equiv.) in DMF (0.2-0.3 M) degassed and purged with N2 three times, and the mixture was stirred at 130 °C under N2 atmosphere for 12 h. The mixture was then poured into water and extracted three times with DCM. The combined organic phase was washed with brine, dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo. The residue was purified by column chromatography to give the product as an off-white solid. 1 H NMR (400 MHz, (CD3)2SO) δ 9.09 (s, 1H), 8.73 (s, 1H), 8.44 (s, 1H), 8.16 (s, 1H), 7.78 (s, 1H), 4.21 (t, J = 12.5 Hz, 1H), 3.36 (s, 3H), 2.43 (s, 3H), 2.34 (dt, J = 13.0, 6.5 Hz, 2H), 1.93 - 1.77 (m, 2H), 1.77 - 1.62 (m, 2H), 0.91 (d, J = 13.2 Hz, 2H), 0.31 (t, J = 7.1 Hz, 2H). MS: 405.5 m / z [M+H].

[0272] Five days after transfection, T cells were phenotyped by flow cytometry analysis as described in Example 1 to evaluate the insertion efficiency of the LNP composition. Table 19 shows the percentage of CD3 negative cells. T cell receptor alpha chain encoded by TRAC is required for the assembly and translocation of T cell receptor / CD3 complex to the cell surface. Therefore, disruption of the TRAC gene by genome editing leads to the loss of CD3 protein on the cell surface of T cells. The average percentage of GFP positive T cells in each medium condition is shown in Table 20 and Figure 5. Cells expressing GFP protein indicate successful insertion into the genome. [Table 20] [Table 21]

[0273] Example 8: Evaluation of LNP compositions with various sgRNAs To evaluate the editing efficacy in multiple targets, T cells were treated with LNP compositions containing various molar ratios of lipid components encapsulating Cas9 mRNA and sgRNA targeting TRAC (SEQ ID NO: 10), TRBC (SEQ ID NO: 11), HLA-A (SEQ ID NO: 13), or CIITA (SEQ ID NO: 12) genes. Editing of TRAC or TRBC was assayed by the increase in the percentage of CD3-negative cells after editing. The T cell receptor alpha chain and the T cell receptor beta chain, encoded by TRAC or TRBC, respectively, are required for the assembly and translocation of the T cell receptor / CD3 complex to the cell surface. Thus, disruption of either the TRAC or TRBC gene by genome editing leads to the loss of CD3 protein on the cell surface of T cells. Disruption of CIITA leads to the loss of HLA-DR DP DQ alleles on the cell surface, since CIITA is a transcription factor directly involved in the expression of HLA-class II genes.

[0274] Healthy human donor leukocytes from three donors (W0662, 110046967, 110044591) were obtained commercially (STEMCELL Technologies). T cells were isolated by negative selection using the EasySep Human T Cell Isolation Kit (Stem Cell Technology; Catalog No. 17951) or by CD4 / CD8 positive selection using StraightFrom® Leukopak® CD4 / CD8 microbeads (Milteny; Catalog No. 130-122-352) on a MultiMACS™ Cell24 Separator Plus instrument according to the manufacturer's instructions. T cells were cryopreserved in Cryostor CS10 freezing medium (Cat. No. 07930) for future use.

[0275] Once thawed, T cells were cultured in complete T cell growth medium consisting of CTS OpTmizer basal medium (Gibco; A3705001) supplemented with 1x GlutaMAX, 10 mM HEPES buffer (10 mM), and 1% pen-strep (Gibco; 15140-122), and further supplemented with 200 IU / mL IL2 (Peprotech; 200-02), 5 ng / mL IL7 (Peprotech; 200-07), 5 ng / mL IL15 (Peprotech; 200-15), and 2.5% human serum (Gemini; 100-512). After overnight resting, T cells at a density of 10^6 / mL were activated with T cell TransAct reagent (1:100 dilution, Miltenyi) and treated with LNPs using the following method:

[0276] T cells were transfected with LNPs prepared as described in Example 1, with lipid compositions expressed as the molar ratio of ionizable Compound 6 / DSPC / Cholesterol / PEG shown in Table 21. The LNPs delivered mRNA encoding Cas9 (SEQ ID NO:4) and sgRNA as indicated. The cargo weight ratio of sgRNA:Cas9 mRNA was 1:2 in the 50 / 10 / 35.5 / 1.5 (Compound 6 / DSPC / Cholesterol / PEG) LNP composition and 1:1 in the 35 / 15 / 47.5 / 2.5 (Compound 6 / DSPC / Cholesterol / PEG) composition. The N:P ratio was approximately 6 unless otherwise indicated.

[0277] Activated T cells were transfected with LNPs formulated as described above in Example 1. LNP transfections were performed in the order and timing after activation as described for each composition in Table 26. T cells containing sgRNA targeting CIITA (SEQ ID NO: 12) were treated with LNPs immediately after activation, T cells containing sgRNA targeting HLA-A (SEQ ID NO: 13) were treated 24 hours after activation, T cells containing sgRNA targeting TRAC (SEQ ID NO: 10) were treated 48 hours after activation, and T cells containing sgRNA targeting TRBC (SEQ ID NO: 11) were treated 72 hours after activation. LNP-treated cells were incubated at 37°C, 5% CO2 until use.

[0278] For each LNP treatment, T cells were plated in a 96-well plate in a volume of 100 μL. An 8-point 2-fold serial dilution of the LNPs shown in Table 21 was performed using ApoE complete T cell growth medium composed of CTS OpTmizer basal medium, as previously described in Example 1, starting with 5 μg / mL as the highest LNP final dose. 100 μL of the LNP-ApoE mixture was then added to the cells plated in the well, resulting in a final density of 0.5×10^6 / mL and a final ApoE concentration of 5 μg / mL in a final volume of 200 μL. The cells were cultured at 37° C., 5% CO2 for 24 hours, then split 1:4 onto fresh medium.

[0279] After incubation, LNP-treated T cells were harvested and analyzed for on-target editing. Plates were incubated at 37°C, 5% CO2 until day 11 and then harvested for flow cytometry analysis.

[0280] Flow cytometry analysis To assay cell surface proteins by flow cytometry, T cells were incubated with antibodies targeting CD4 (Biolegend; Cat. No. 300538) and CD8a (Biolegend; Cat. No. 301049) mixed with antibodies targeting CD3 (Biolegend; Cat. No. 300441) or HLA-DR, DP, DQ (Biolegend; Cat. No. 361712) or HLA-A*02 (Biolegend; Cat. No. 343320). T cells were then processed on a Cytoflex instrument (Beckman Coulter). Data analysis was performed using the FlowJo software package (v.10.6.1 or v.10.7.1). Briefly, T cells were gated on lymphocytes and then on single cells. These single cells were gated on CD4+ / CD8+ status, from which CD8+ / CD3-, CD8+ / HLA-DR, DP, DQ-, or CD8+ / HLA-A*02- cells were quantified.

[0281] The percentage of CD8+ / CD3-, CD8+ / HLA-DR, DP, DQ-, and CD8+ / HLA-A2- cells was quantified to determine the percentage of cell populations in which the edited target locus resulted in TCR knockout. A linear regression model was used to generate dose-response curves for TCR KO using Prism GraphPad (v.9.0). The half-effective concentration (EC 50 ) and maximum percentage of CD3- were calculated for each LNP.

[0282] The Z-average and number-average particle size, polydispersity (pdi), total RNA content, and RNA encapsulation efficiency of the LNP compositions were analyzed as described in Example 1. The results are shown in Table 21. [Table 22]

[0283] The percentage of CD3 negative T cells was measured after treatment with LNP concentrations of 0.04 μg / mL, 0.08 μg / mL, 0.16 μg / mL, 0.31 μg / mL, 0.63 μg / mL, 1.25 μg / mL, 2.5 μg / mL, and 5 μg / mL. Assays were performed in triplicate. The mean percent of CD3 negative T cells, standard deviation of CD3 values, and EC50 for each LNP dose are shown in Tables 22-25 and Figures 6A-6D. [Table 23] [Table 24] [Table 25] [Table 26]

[0284] Example 9: Sequential delivery of multiple LNP compositions for multiple gene disruptions and insertions T cells were engineered with a series of gene disruptions and insertions. Healthy donor cells were treated sequentially with four LNP compositions, each co-formulated with mRNA encoding Cas9 (SEQ ID NO: 4) and sgRNA targeting either TRAC (SEQ ID NO: 10), TRBC (SEQ ID NO: 11), CIITA (SEQ ID NO: 12), or HLA-A (SEQ ID NO: 13). LNPs were formulated according to the groups shown in Table 26 with Compound 6, DSPC, cholesterol, and PEG2k-DMG in a molar ratio of 35:15:47.5:2.5 (Groups 1 and 2), or Compound 6, DSPC, cholesterol, and PEG2k-DMG in a molar ratio of 50:10:38.5:1.5 (Group 3), at the indicated doses, respectively.

[0285] Group 1 and Group 2 have different LNP concentrations. The lipid nucleic acid assembly was formulated with a lipid amine:RNA phosphate (N:P) molar ratio of about 6 and a gRNA:mRNA weight ratio of 1:2. AAV was used to deliver a homologous recombination repair template to site-specifically integrate transgenic WT1-targeting TCR (SEQ ID NO: 14) into the TRAC cleavage site. Groups of LNP were prepared daily and delivered sequentially to T cells as described in Table 26.

[0286] 9.1 Preparation of T cells T cells from three HLA-A*02:01+ serotypes were isolated from leukapheresis products (STEMCELL Technologies) from two healthy donors. T cells were isolated using the EasySep Human T Cell Isolation Kit (STEMCELL Technologies; Catalog No. 17951) according to the manufacturer's protocol and cryopreserved using a Cryostor CS10 (STEMCELL Technologies; Catalog No. 07930). The day before initiating T cell editing, cells were thawed and placed overnight in T cell activation medium (TCAM:CTS OpTmizer [Thermofisher; Catalog No. A3705001] supplemented with 2.5% human AB serum [Gemini; Catalog No. 100-512], 1X GlutaMAX [Thermofisher; Catalog No. 35050061], 10 mM HEPES [Thermofisher; Catalog No. 15630080], 200 U / mL IL-2 [Peprotech; Catalog No. 200-02], 5 ng / mL IL-7 [Peprotech; Catalog No. 200-07], and 5 ng / mL IL-15 [Peprotech; Catalog No. 200-15]).

[0287] 9.2 LNP Treatment and T Cell Expansion LNPs were thawed each day, diluted into ApoE-containing medium, and delivered to T cells as follows. [Table 27]

[0288] On day 1, the LNPs shown in Table 26 were incubated in TCAM containing 5 μg / mL rhApoE3 (Peprotech; 350-02). Meanwhile, T cells were harvested, washed, and resuspended in TCAM with T cell TransAct human reagent (Miltenyi; 130-111-160) diluted 1:50 at a density of 2x10^6 cells / mL. T cells and LNP-ApoE medium were mixed in a 1:1 ratio, and T cells were plated in culture flasks overnight.

[0289] On day 2, the LNPs shown in Table 26 were incubated at a concentration of 25ug / mL in TCAM containing 20ug / mL rhApoE3 (Peprotech; 350-02). The LNP-ApoE solution was then added to the appropriate culture medium at a 10:1 ratio.

[0290] On the third day, the TRAC-LNPs shown in Table 26 were incubated in TCAM containing 5 μg / mL rhApoE3 (Peprotech; 350-02). Meanwhile, T cells were harvested, washed, and resuspended in TCAM at a density of 1×10^6 cells / mL. T cells and LNP-ApoE medium were mixed in a 1:1 ratio, and T cells were plated in a culture flask. WT1 (SEQ ID NO: 14) AAV was then added to each group at an MOI of 3×10^5 GC / cell. DNAPKI compound 4 was added to each group at a concentration of 0.25 μM.

[0291] On day 4, the LNPs shown in Table 26 were incubated in TCAM containing 5ug / mL rhApoE3 (Peprotech; 350-02). Meanwhile, T cells were harvested, washed, and resuspended in TCAM at a density of 1x10^6 cells / mL. T cells and LNP-ApoE medium were mixed in a 1:1 ratio, and T cells were plated in a culture flask.

[0292] On days 5–13, T cells were transferred to 24-well GREX plates (Wilson Wolf; 80192) in T cell expansion medium (TCEM:CTS OpTmizer [Thermofisher; catalog no. A3705001] supplemented with 5% human AB serum [Gemini; catalog no. 100-512], 1X GlutaMAX [Thermofisher; catalog no. 35050061], 10 mM HEPES [Thermofisher; catalog no. 15630080], 200 U / mL IL-2 [Peprotech; catalog no. 200-02], 5 ng / mL IL-7 [Peprotech; catalog no. 200-07], and 5 ng / mL IL-15 [Peprotech; catalog no. 200-15]) and expanded according to the manufacturer's protocol. Briefly, T cells were expanded for 8 days with medium changes every 2–3 days.

[0293] 9.3 Quantification of T cell editing by flow cytometry and NGS After expansion, edited T cells were assayed by flow cytometry to determine the percentage of cells expressing HLA-A*02:01 knockout, HLA-DR-DP-DQ knockdown with CIITA knockout, WT1-TCR insertion (CD3+Vb8+), and residual endogenous (CD3+Vb8-). T cells were incubated with an antibody cocktail targeting the following molecules: Vb8 (Biolegend; Catalog No. 348104), HLA-A2 (Biolegend; Catalog No. 343320), HLA-DRDPDQ (Biolegend; Catalog No. 361712), CD4 (Biolegend; Catalog No. 300538), CD8 (Biolegend; Catalog No. 301046), CD3 (Biolegend; Catalog No. 317336), CCR7 (Biolegend; Catalog No. 353214), CD62L (Biolegend; Catalog No. 304820), CD45RA (Biolegend; Catalog No. 304134), CD45RO (Biolegend; Catalog No. 304230), CD56 (Biolegend; Catalog No. 318328), and Viakrome (Beckman). Cells were then washed, processed on a CytoFlex LX instrument (Beckman Coulter; catalog number C36628), and analyzed using the FlowJo software package. T cells were gated on size and CD4 / CD8 status before editing and insertion rates were determined.

[0294] LNP compositions used for delivery of Cas9 mRNA and sgRNA targeting the CIITA, HLA-A, TRAC, and TRBC loci were analyzed as described in Examples 1 and 8.

[0295] The percentage of cells expressing the relevant cell surface proteins after sequential T cell manipulation is shown in Table 27 and Figure 7A for CD8+ T cells. The percentage of T cells with all intended edits (i.e., WT1-TCR insertion combined with HLA-A and CIITA knockout, or CD3+Vb8+HLA-A-HLA-DRDPDQ-%) is shown for each group in Figure 7B. High levels of HLA-A and CIITA knockout and WT1-TCR insertion were observed in edited samples from all groups, resulting in more than 75% fully edited CD8+ T cells. The low dose used in Group 1 (0.65 μg / mL) showed similar efficacy in editing T cells across all targets, as did the higher LNP doses (2.5 μg / mL) of Group 2 and Group 3 formulations. [Table 28]

[0296] Example 10. Editing in NK cells 10.1.LNP composition LNPs were formulated as described in Example 1. The LNPs were formulated with a lipid amine:RNA phosphate (N:P) molar ratio of about 6, a sgRNA:Cas9 mRNA weight ratio (cargo ratio) of 1:2 for composition 64, or a cargo weight ratio of 1:1 for composition 65, and compound 6. The LNPs delivered mRNA encoding Cas9 (SEQ ID NO:4) and an sgRNA (SEQ ID NO:15) targeting the human AAVS1 gene.

[0297] The lipid components in LNP were quantitatively analyzed by HPLC coupled with a charged aerosol detector (CAD). Chromatographic separation of the four lipid components was achieved by reversed-phase HPLC. The HPLC lipid analysis provided the actual mole percent (mol-%) lipid levels of each component of the LNP compositions described in the following examples, as shown in Table 28. [Table 29]

[0298] The LNP compositions were analyzed for Z-average and number-average particle size, polydispersity (PDI), total RNA content, and RNA encapsulation efficiency according to the methods described in Example 1. The results are shown in Table 29. [Table 30]

[0299] NK cells were isolated from commercially available leukopacks from healthy donors using the EasySep Human NK Cell Isolation Kit (STEMCELL; Cat. No. 17955) according to the manufacturer's protocol. After isolation, NK cells were cryopreserved until required. After cell thawing, NK cells were placed overnight in CTS™ OpTmizer™ T cell growth medium (Gibco; Catalog No. A10221-01) containing 5% human AB serum (GemCell; Catalog No. 100-512), 500 U / mL IL-2 (Peprotech; Catalog No. 200-02), 5 ng / ml IL-15 (Peprotech; Catalog No. 200-15), 10 ml Glutamax (Gibco; Catalog No. 35050-61), 10 ml HEPES (Gibco; Catalog No. 15630-080), and 1% penicillin-streptomycin (ThermoFisher; Catalog No. 15140-122). The rested NK cells were then cultured at a 1:1 ratio with irradiated K562 cells expressing 41BBL (SEQ ID NO: 16) and membrane-bound IL21 (SEQ ID NO: 17) as feeder cells for NK activation for 3 days in CTS™ OpTmizer™ T Cell Expansion Medium as described above.

[0300] Three days after activation, NK cells were treated with LNPs delivering mRNA encoding Cas9 (SEQ ID NO: 4) and sgRNA (SEQ ID NO: 15) targeting the AAVS1 locus. A 12-point dose-response curve was generated by performing a two-fold serial dilution series starting with 10 μg / mL LNPs mixed with 2.5 μg / ml ApoE3 (Peprotech; 350-02) in the above CTS™ OpTmizer™ T cell growth medium containing 2.5% human AB serum and 0.25 μM DNAPKI compound 4. As shown in Table 30, the final concentrations of total RNA cargo in the LNPs were 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08, 0.04, 0.02, 0.01, 0.005, and 0 μg / ml (untreated control). The mixed LNPs were added to NK cells at 1x10^6 cells / ml in a 1:1 ratio in triplicate.

[0301] Seven days after LNP treatment, genomic DNA was isolated from the cells and NGS analysis was performed as described in Example 1.

[0302] The mean percent editing, standard deviation, and EC50 of the LNP compositions at the indicated concentrations are shown in Table 30, and the dose-response curves are shown in FIG. [Table 31]

[0303] Example 11. Editing of monocytes and macrophages CD14+ cells were isolated from commercially available leukopacks (Hemacare) using the StraightFrom® Leukopak® CD14 Bead Kit for Human (Miltenyi Biotec; Catalog 130-117-020) on a MultiMACS™ Cell24 Separator Plus instrument (Miltenyi Biotec) according to the manufacturer's protocol. CD14+ cells were thawed and cultured in triplicate at 50,000 cells / well in OpTmizer basal medium as described in Example 1 with 10 ng / mL GM-CSF (Stemcell; 78140.1) at a cell density of 5×10^5 / mL in 96-well non-tissue culture plates (Falcon; 351172). Every 2–3 days, replace 50% of the OpTmizer medium per well with 20 ng / mL fresh cytokine medium (GM-CSF (Stemcell; 78140.1), 2.5% human serum (HS) OpTmizer (Gibco; A3705001)).

[0304] Cells were treated with LNPs prepared as described in Example 10. LNPs were preincubated with 10 μg / ml ApoE3 (Peprotech 350-02) for 15 min at 37° C. Preincubated LNPs were added to cells at a 1:1 v / v ratio to give final total RNA cargo amounts ranging from 0 to 1.25 μg / mL.

[0305] After incubation, 50 μL of LNP concentration was added to monocytes on the same day that CD14+ cells were plated onto non-tissue culture plates (Falcon; 351172) and to macrophages after 5 days of incubation on non-tissue culture plates (Falcon; 351172). Monocyte and macrophage plates were incubated at 37° C. until use.

[0306] Six days after LNP treatment, genomic DNA was isolated from monocytes as described in Example 1, and macrophage engineered cells were harvested for NGS as described in Example 1.

[0307] The mean percent editing, standard deviation, and EC50 for each LNP composition at the indicated concentrations are shown for monocytes in Table 31 and for macrophages in Table 32. Dose-response curves in monocytes and macrophages are shown in Figures 9A and 9B, respectively. [Table 32] [Table 33]

[0308] Example 12. Editing B cells 12.1. Isolation and culture of B cells and medium preparation B cells (Hemacare) were cultured in Stemspan SFEM medium (StemCell Technologies; Catalog No. 09650) supplemented with 1% penicillin-streptomycin (ThermoFisher; Catalog No. 15140122), 1 μg / ml CpG ODN 2006 (Invivogen; Catalog No. tlrl-2006-1), 50 ng / ml IL-2 (Peprotech; Catalog No. 200-02), 50 ng / ml IL-10 (Peprotech; Catalog No. 200-10), and 10 ng / ml IL-15 (Peprotech; Catalog No. 200-15). Two media components at various concentrations were also used to supplement the medium: 1. Human serum AB (Gemini Bioproducts; Catalog No. 100-512, Lot No. H94X00K, 2.5% and 5%), and 2. MEGACD40L (Enzo Life Sciences; Catalog No. ALX-522-110-0000, 1 ng / ml and 100 ng / ml). The B cell culture medium composition used for the preparation of B cells is shown in Table 33. [Table 34]

[0309] B cells were isolated by CD19 positive selection from leukopacks (Hemacare) from healthy human donors using the StraightFrom Leukopack CD19 MicroBead Kit (Miltenyi, 130-117-021) on a MultiMACS Cell24 Separator Plus instrument according to the manufacturer's instructions. Isolated CD19+ B cells were frozen and stored in liquid nitrogen until required.

[0310] When ready for use, B cells were thawed and activated the same day with B cell medium 1.

[0311] Two days after thawing and activation of B cells, B cells were cultured in medium 2 and treated with LNPs delivering Cas9 mRNA (SEQ ID NO: 4) and gRNA targeting AAVS1 (SEQ ID NO: 15). Several concentrations of each LNP were tested to generate an 8-point dose-response curve by setting up a 1:2 serial dilution starting at 20 μg / ml total RNA cargo (4x the final dose). 4 μg / ml ApoE3 (4x the final dose) was then added in B cell medium 2 before adding B cells to the LNP-APOE3 mixture in a 1:1 ratio v / v, resulting in final doses of 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078 μg / ml total RNA cargo, as shown in Table 34. Cells were treated with LNPs prepared and analyzed as described in Example 10, or no LNPs as a control.

[0312] Three days after LNP treatment, cells were washed and resuspended in B cell medium 3. Seven days after LNP treatment, genomic DNA was isolated from cells and NGS analysis was performed as described in Example 1.

[0313] The mean percent edited and standard deviations for the LNP compositions at the indicated concentrations are shown in Table 34, and the dose-response curves are shown in Figure 10. "Naive B cells" were not treated with the LNP compositions. [Table 35]

[0314] In the tables below and throughout, the terms "mA," "mC," "mU," or "mG" are used to denote 2'-O-Me modified nucleotides.

[0315] In the table below, "*" is used to indicate a PS modification. In this application, the terms A*, C*, U*, or G* may be used to indicate a nucleotide that is linked to the next (e.g., 3') nucleotide with a PS linkage.

[0316] It should be understood that when a DNA sequence (containing T) is referred to in relation to RNA, T should be replaced with U (which may or may not be modified depending on the context), and vice versa.

[0317] In the table below, the peptide sequences are shown using the single letter amino acid code. [Table 36-1] [Table 36-2] [Table 36-3] [Table 36-4] [Table 36-5] [Table 36-6] [Table 36-7] [Table 36-8] [Table 36-9]

Table 36-10

Table 36-11

Table 36-12

Table 36-13

Table 36-14

Table 36-15

Table 36-16

Table 36-17

Table 36-18

Table 36-19

Claims

1. 1. A lipid nanoparticle (LNP) composition comprising: Biologically active agents, and The lipid component comprises: a) an ionizable lipid in an amount of about 25-45 mol % of said lipid component; b) neutral lipids in an amount of about 10-30 mol % of said lipid component; c) a helper lipid in an amount of about 25-65 mol % of the lipid component; and d) PEG lipids in an amount of about 1.5-3.5 mol % of the lipid component; The ionizable lipid is a compound of formula (I) 【Chemistry 1】 (In the formula, X １ is O, NH, or a direct bond; X ２ is C ２～３ is alkylene, R ３ is C １～３ is alkyl, R ２ is C １～３ is alkyl, or R ２ is the nitrogen atom to which it is attached and X ２ together with 2 to 3 carbon atoms of the formula (I) to form a 5- or 6-membered ring; or R ２ is R ３ and together with the nitrogen atom to which they are attached form a five-membered ring, Y １ is C ６～１０ is alkylene, Y ２ teeth 【Chemistry 2】 is selected from R ４ is C ４～１１ is alkyl, Z １ is C ２～５ is alkylene, Z ２ teeth 【Chemistry 3】 or absent, R ５ is C ６～８ Alkyl, C ５～１０ Alkoxy (e.g., —OC ５～１０ haloalkyl), -O(C ２～３ Alkyl)OC ６～１０ Alkyl, -OC ６～１０ Alkenyl (e.g., —OC ６～１０ branched alkenyl), or -OC ６～１０ alkynyl, R ６ is C ６～８ Alkyl, C ５～１０ Alkoxy (e.g., —OC ５～１０ haloalkyl), -O(C ２～３ Alkyl)OC ６～１０ Alkyl, -OC ６～１０ Alkenyl (e.g., —OC ６～１０ branched alkenyl), or -OC ６～１０ is alkynyl, or R ５ is R ６ Together with Germinal C ６～８ forming a six-membered acetal ring substituted with alkyl or a salt thereof.

2. The ionizable lipid is a compound of formula (II) 【Chemistry 4】 (In the formula, X １ is O, NH, or a direct bond; X ２ is C ２～３ is alkylene, Z １ is C ３ alkylene, R ５ and R ６ are C ６ alkyl, or Z １ is a direct bond, R ５ and R ６ are C ８ is an alkoxy; R ８ teeth 【Chemistry 5】 (It is) or a salt thereof.

3. The ionizable lipid is 【Chemistry 6-1】 【Chemistry 6-2】 【Chemistry 6-3】 【Chemistry 6-4】 or a salt thereof.

4. The ionizable lipid is 【Chemistry 7】 or a salt thereof.

5. The ionizable lipid is 【Chemistry 8】 or a salt thereof.

6. The LNP composition of claim 1 , wherein the neutral lipid is DSPC or DPME.

7. The LNP composition of claim 1 , wherein the neutral lipid is DSPC.

8. 2. The LNP composition of claim 1, wherein the helper lipid is selected from cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate.

9. The LNP composition of claim 1 , wherein the helper lipid is cholesterol.

10. 2. The LNP composition of claim 1, wherein the PEG lipid comprises dimyristoyl glycerol (DMG).

11. The LNP composition of claim 1, wherein the PEG lipid comprises PEG-2k.

12. The LNP composition of claim 1 , wherein the PEG lipid is PEG-DMG.

13. The LNP composition of claim 1, wherein the PEG lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

14. The ionizable lipid is 【Chemistry 9】 2. The LNP composition of claim 1, wherein the neutral lipid is DSPC, the helper lipid is cholesterol, and the PEG lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

15. 2. The LNP composition of claim 1, wherein the amount of said ionizable lipid is about 29-38 mol% of said lipid component.

16. 2. The LNP composition of claim 1, wherein the amount of said ionizable lipid is about 30-43 mol% of said lipid component.

17. 2. The LNP composition of claim 1, wherein the amount of the ionizable lipid is about 35 mol% of the lipid component.

18. 2. The LNP composition of claim 1, wherein the amount of neutral lipid is about 11-20 mol% of the lipid component.

19. 2. The LNP composition of claim 1, wherein the amount of neutral lipid is about 15 mol% of the lipid component.

20. The LNP composition of claim 1, wherein the amount of the helper lipid is about 43-65 mol% of the lipid component.

21. The LNP composition of claim 1, wherein the amount of the helper lipid is about 43-55 mol% of the lipid component.

22. 2. The LNP composition of claim 1, wherein the amount of helper lipid is about 47.5 mol% of the lipid component.

23. The LNP composition of claim 1, wherein the amount of the PEG lipid is about 2.0-3.5 mol% of the lipid component.

24. 2. The LNP composition of claim 1, wherein the amount of PEG lipid is about 2.3-2.7 mol% of the lipid component.

25. 2. The LNP composition of claim 1, wherein the amount of PEG lipid is about 2.5 mol% of the lipid component.

26. The LNP composition of claim 1, wherein the amount of the ionizable lipid is about 29-44 mol% of the lipid component, the amount of the neutral lipid is about 11-28 mol% of the lipid component, the amount of the helper lipid is about 28-55 mol% of the lipid component, and the amount of the PEG lipid is about 2.3-3.5 mol% of the lipid component.

27. The LNP composition of claim 1, wherein the amount of the ionizable lipid is about 29-38 mol% of the lipid component, the amount of the neutral lipid is about 11-20 mol% of the lipid component, the amount of the helper lipid is about 43-55 mol% of the lipid component, and the amount of the PEG lipid is about 2.3-2.7 mol% of the lipid component.

28. The LNP composition of claim 1, wherein the amount of the ionizable lipid is about 30-43 mol% of the lipid component, the amount of the neutral lipid is about 10-17 mol% of the lipid component, the amount of the helper lipid is about 43.5-56 mol% of the lipid component, and the amount of the PEG lipid is about 1.5-3 mol% of the lipid component.

29. 2. The LNP composition of claim 1, wherein the amount of the ionizable lipid is about 35 mol% of the lipid component, the amount of the neutral lipid is about 15 mol% of the lipid component, the amount of the helper lipid is about 47.5 mol% of the lipid component, and the amount of the PEG lipid is about 2.5 mol% of the lipid component.

30. The LNP composition of claim 1, wherein the LNP composition is in the form of lipid nanoparticles (LNPs), and the LNPs have a polydispersity index of about 0.005 to about 0.

75.

31. The LNP composition of claim 1, wherein the biologically active agent comprises a nucleic acid component, the nucleic acid component comprising an mRNA encoding an RNA-guided DNA-binding agent.

32. The LNP composition of claim 31 , wherein the mRNA comprises a class 2 Cas nuclease mRNA.

33. The LNP composition of claim 31 , wherein the mRNA comprises a Cas9 nuclease mRNA.

34. 32. The LNP composition of claim 31 , wherein the nucleic acid component comprises a guide RNA nucleic acid.

35. 35. The LNP composition of claim 34, wherein the guide RNA nucleic acid is a dual guide RNA (dgRNA) or a single guide (sgRNA).

36. The LNP composition of claim 34, wherein the guide RNA nucleic acid is a modified gRNA comprising a modification in one or more of the first five nucleotides at the 5' end and / or the 3' end.

37. The LNP composition of claim 34, wherein the nucleic acid component comprises a guide RNA nucleic acid, the mRNA comprises a class 2 Cas nuclease mRNA, and the weight ratio of the mRNA:the guide RNA nucleic acid is about 2:1 to 1:

4.

38. 38. The LNP composition of claim 37, wherein the weight ratio of said guide RNA nucleic acid: said Class 2 Cas nuclease mRNA is about 1:

1.

39. The LNP composition of claim 31, wherein the N / P ratio of the LNP composition is about 5 to about 7.

40. An ex vivo method of gene editing, cleaving DNA, or delivering a biologically active agent to a cell, comprising contacting the cell with the LNP composition of any one of claims 1 to 39.

41. 41. The method of claim 40, further comprising introducing at least one template nucleic acid into the cell.

42. 41. The method of claim 40, wherein the LNP composition is a first LNP composition, and the method further comprises contacting the cell with a second LNP composition comprising one or more of an mRNA and a gRNA nucleic acid.

43. 41. The method of claim 40, wherein the cell is a stem cell.

44. 44. The method of claim 43, wherein the stem cells are hematopoietic stem cells (HSCs) or induced pluripotent stem cells (iPSCs).

45. 41. The method of claim 40, wherein the cell is an immune cell.

46. 46. ​​The method of claim 45, wherein the immune cell is a leukocyte or lymphocyte.

47. 1. An ex vivo method for generating multiple genome edits in a cell or population of cells, comprising: contacting the cell or population of cells in vitro with at least a first LNP composition of any one of claims 1-39 and a second LNP composition of any one of claims 1-39, thereby generating a plurality of genome edits in the cell or population of cells; the biologically active agent of the first LNP composition comprises a first guide RNA (gRNA) directed to a first target sequence, and optionally a nucleic acid genome editing tool; The method, wherein the biologically active agent of the second LNP composition comprises a second gRNA directed to a second target sequence, and optionally a nucleic acid genome editing tool.

48. 48. The method of claim 47, wherein the cells are stem cells or the population of cells is a population of stem cells.

49. 48. The method of claim 47, wherein the cell is an immune cell or the population of cells is a population of immune cells.

50. A method according to any one of claims 1 to 39 for the manufacture of a medicament for gene editing in a subject. Use of the LNP composition described in.

51. Use of an LNP composition described in any one of claims 1 to 39 in the manufacture of a pharmaceutical for cleaving DNA in a subject.

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