In vitro cell delivery methods

Abstract

Compositions and methods are provided for multiplex delivery and gene editing in vitro.

Description

[Background technology]

[0001] The ability to introduce multiple gene edits into cells in vitro is of interest for gene editing and clinical therapeutic applications. For example, adoptive cell therapy approaches using genetically modified immune cells have become an attractive modality for treating various conditions and diseases, including cancer, to reconstitute cell lineages and immune system defenses. However, the clinical application of cell product therapy has been difficult, in part, due to the complex genetic engineering requirements. The ability to engineer multiple attributes into a single cell depends on the ability to efficiently perform edits in multiple target genes, including knockouts and gene loci insertions, while maintaining viability and the desired cell phenotype.

[0002] While CRISPR / Cas9 genome editing has been demonstrated to be highly efficient, simultaneous editing at different loci has been reported to result in poorer cell survival, increased translocations that may compromise the quality and safety of the cellular product, and decreased gene editing efficiency with increasing number of edits. Existing cell manipulation techniques, including electroporation, present limitations in providing the necessary cell quality and yield using sequential editing processes due to cumulative toxicity to cells. Furthermore, certain cell types, including T cells, have proven particularly challenging for persistent multiple editing in vitro.

[0003] Therefore, there is a need for safer processes for delivering multiple genome editing tools to cells and for performing gene editing.

[0004] Methods provided herein involve the use of lipid nucleic acid assembly compositions (e.g., lipid nanoparticles ("LNPs")) for safer delivery of genome editing tools and for multiplex genome editing applications that offer substantial advantages over conventional methods.

[0005] In some embodiments, the methods produce cells with a lower toxicity profile, fewer translocations, and higher survival and expansion, thereby shortening the time required for manufacturing and increasing yield. In some embodiments, the methods provide highly efficient multiplex editing of T cells in vitro to replace endogenous T cell receptors (TCRs) with therapeutic TCRs, resulting in engineered T cells with increased cytokine production, a favorable early stem cell memory phenotype, and continued proliferation upon antigen-specific stimulation. [Brief explanation of the drawings]

[0006] [Figure 1] Shown is the fold expansion of T cells treated with electroporation (EP) or lipid nanoparticles (LNP), with or without AAV, after 10 days of culture after editing. [Figure 2] Shown are the percentages of CD3+Vb8+TCR T cells (gated on CD8+ and CD4+) treated with electroporation (EP) or lipid nanoparticles (LNP) with or without AAV at day 7 post-editing. [Figure 3] Shown are the percentages of residual endogenous TCR-expressing (CD3+Vb8-) T cells (gated on CD8+ and CD4+) treated with electroporation (EP) or lipid nanoparticles (LNP) with or without AAV at day 7 post-editing. [Figure 4] Staining of early stem cell memory phenotype CD8+ T cells by flow cytometry (CD27+, CD45RA+) in EP-treated and LNP-treated T cells is shown. [Figure 5] IL-2 secretion of WT1 TCR-engineered T cells (EP-treated vs. LNP-treated) in co-culture with OCI-AML2 cells pulsed with VLD peptide is shown. [Figure 6] IFNγ secretion of WT1 TCR-engineered T cells (EP-treated vs. LNP-treated) in co-culture with K562 HLA-A*02:01-positive cells is shown. [Figure 7]Specific lysis of K562 HLA-A*02:01 positive cells by WT1 TCR engineered T cells (EP-treated vs. LNP-treated) is shown. [Figure 8] Proliferation (as cumulative fold change) after repeated stimulation of EP-treated versus LNP-treated WT1 TCR-engineered T cells when co-cultured with OCI-AML3 target cells pulsed with VLD peptide is shown. [Figure 9] Shown is the expansion of T cells after editing by electroporation ("EP"), simultaneous LNP ("SIM"), sequential process 1 (2.5 μg / ml LNP) ("BF2.5"; TRBC target, then TRAC target), sequential process 2 (5 μg / ml LNP) ("BF5"; TRBC target, then TRAC target), and sequential process 3 (2.5 μg / ml LNP) ("AF"; TRAC target, then TRBC target). [Figure 10] The insertion rate (%Vb8+, CD3+) of transgenic TCR (tgTCR) after editing by electroporation ("EP"), simultaneous LNP ("SIM"), sequential process 1 (2.5 μg / ml LNP) ("BF2.5"; TRBC target, then TRAC target), sequential process 2 (5 μg / ml LNP) ("BF5"; TRBC target, then TRAC target), and sequential process 3 (2.5 μg / ml LNP) ("AF"; TRAC target, then TRBC target) is shown. [Figure 11] Shown is the percentage of CD8+ T cells retaining endogenous TCR after editing by electroporation ("EP"), simultaneous LNP ("SIM"), sequential process 1 (2.5 μg / ml LNP) ("BF2.5"; TRBC target, then TRAC target), sequential process 2 (5 μg / ml LNP) ("BF5"; TRBC target, then TRAC target), and sequential process 3 (2.5 μg / ml LNP) ("AF"; TRAC target, then TRBC target). [Figure 12]Shown is the percentage of engineered T cells associated with a memory phenotype (CD27+) after editing by electroporation ("EP"), simultaneous LNP ("SIM"), sequential process 1 (2.5 μg / ml LNP) ("BF2.5"; TRBC target, then TRAC target), sequential process 2 (5 μg / ml LNP) ("BF5"; TRBC target, then TRAC target), and sequential process 3 (2.5 μg / ml LNP) ("AF"; TRAC target, then TRBC target). [Figure 13A] The percentages of TRAC-TRBC translocation cells and cells with TCR insertion into the TRBC locus are shown in engineered T cells after editing by electroporation ("EP"), simultaneous LNP ("SIM"), sequential process 1 (2.5 μg / ml LNP) ("BF2.5"; TRBC target, then TRAC target), sequential process 2 (5 μg / mL LNP) ("BF5"; TRBC target, then TRAC target), and sequential process 3 (2.5 μg / mL LNP) ("AF"; TRAC target, then TRBC target). The translocation detected by the TRAC probe is shown in Figure 13A, and the TRBC probe is shown in Figure 13B. [Figure 13B] (As mentioned above.) [Figure 14A] The percentages of TRBC-TRAC translocation cells and cells with TCR insertion into the TRBC locus are shown in engineered T cells after editing by electroporation ("EP"), simultaneous LNP ("SIM"), sequential process 1 (2.5 μg / ml LNP) ("BF2.5"; TRBC target, then TRAC target), sequential process 2 (5 μg / mL LNP) ("BF5"; TRBC target, then TRAC target), and sequential process 3 (2.5 μg / mL LNP) ("AF"; TRAC target, then TRBC target). The translocation detected by the TRAC probe is shown in Figure 14A, and the TRBC probe is shown in Figure 14B. [Figure 14B] (As mentioned above.) [Figure 14C]The percentage of TRAC-TRBC translocated cells in engineered T cells after editing by electroporation ("EP"), simultaneous LNP ("SIM"), sequential process 1 (2.5 μg / ml LNP) ("BF2.5"; TRBC target, then TRAC target), sequential process 2 (5 μg / mL LNP) ("BF5"; TRBC target, then TRAC target), and sequential process 3 (2.5 μg / mL LNP) ("AF"; TRAC target, then TRBC target) is shown. Translocations detected by the TRAC probe are shown in Figure 14C, and the TRBC probe is shown in Figure 14D. [Figure 14D] (As mentioned above.) [Figure 14E] The percentage of TRBC-TRAC translocated cells in engineered T cells after editing by electroporation ("EP"), simultaneous LNP ("SIM"), sequential process 1 (2.5 μg / ml LNP) ("BF2.5"; TRBC target, then TRAC target), sequential process 2 (5 μg / mL LNP) ("BF5"; TRBC target, then TRAC target), and sequential process 3 (2.5 μg / mL LNP) ("AF"; TRAC target, then TRBC target) is shown. Translocations detected by the TRAC probe are shown in Figure 14E, and the TRBC probe is shown in Figure 14F. [Figure 14F] (As mentioned above.) [Figure 15A-C] Figure 15 shows T cell-mediated cytotoxicity of WT1 TCR-engineered T cells as assessed by luciferase-based target cell killing assay. Engineered T cells were cocultured with K562 cells (Figures 15A and 15D), K562-A2.1 cells (Figures 15B and 15E), and 697-luc cells (Figures 15C and 15F). [Figure 15D-F] (As mentioned above.) [Figure 16] Shown is the rate of tgTCR insertion (Vb8+, CD3+) of engineered T cells as assessed by flow cytometry (EP-treated vs. LNP-treated). [Figure 17]The percentage of CD8+ T cells with inserted GFP (CD3-, GFP+) or retention of endogenous TCR (CD3+) after editing as assessed by flow cytometry is shown (EP-treated vs. LNP-treated). [Figure 18] The percentage of engineered T cells associated with a memory phenotype (CD27+, CD45RO-) after editing is shown (EP-treated vs. LNP-treated). [Figure 19] Liquid tumor burden in NOG-hIL-2 mice after treatment with engineered T cells is shown, and bioluminescence was used as a measure of leukemic tumor burden. [Figure 20] 1 shows the percent survival of NOG-hIL-2 mice after treatment with engineered T cells. [Figure 21A] The percentage of beta-2 microglobulin (B2M) negative cells by flow cytometry (FIG. 21A) and percent B2M editing by NGS (FIG. 21B) are shown in response to LNP dose. [Figure 21B] (As mentioned above.) [Figure 22A] The percentage of TRAC-negative cells by flow cytometry (FIG. 22A) and percent of TRAC indels by NGS (FIG. 22B) are shown in response to LNP dose. [Figure 22B] (As mentioned above.) [Figure 23A] The percentage of editing by NGS before (Figure 23A) and after (Figure 23B) MACS processing is shown. [Figure 23B] (As mentioned above.) [Figure 24A] Protein expression of engineered T cells by flow cytometry before (FIG. 24A) and after (FIG. 24B) MACS treatment is shown. [Figure 24B] (As mentioned above.) [Figure 25] Figure 1 shows the perturbation of chromosome structure in engineered cells by KromaTiD dGH assay. [Figure 26]Shown are the mean editing percentages (expressed as % indels) of T cells edited using mRNA and gRNA delivery with different ionizable lipid formulations. [Figure 27] Figure 1 shows the time to reach an editing plateau in T cells edited using mRNA and gRNA delivery with different ionizable lipid formulations. [Figure 28] The percentage of CD3 − cells by flow cytometry in T cells treated with LNP and different serum factors is shown. [Figure 29] The frequency of B2M-negative T cells (treated with lipoplexes) by flow cytometry is shown. [Figure 30] The editing frequency (indels) of lipoplex-treated T cells is shown. [Figure 31] 1 shows the effect of media composition on percent editing in activated T cells, demonstrating delivery of Cas9 mRNA and gRNA by LNPs. [Figure 32] 1 shows the effect of media composition on percent editing in resting T cells, demonstrating delivery of Cas9 mRNA and gRNA by LNPs. [Figure 33] 1 shows the editing frequency in lymphoblastoid cells treated with LNPs delivering mRNA and gRNA of RNA-guided DNA binders. [Figure 34] Shown is the percentage of B2M-negative lymphoblastoid cells treated with LNPs delivering mRNA and gRNA of RNA-guided DNA binders. [Figure 35] Shown is the percentage of engineered T cells with multiple insertions (TCR insertion and GFP insertion) by flow cytometry after co-delivery with LNPs. [Figure 36] Shown is the percentage of engineered T cells with residual TCR or residual HLA-ABC expression by flow cytometry after co-delivery with LNPs. [Figure 37] A heatmap of transcript levels in engineered T cells is shown. [Figure 38A]Figure 38A shows an experimental schematic and leukemic blast levels in mice treated with engineered WT1 T cells and controls. Figure 38A shows a timeline and schematic of the in vivo experiment. Figure 38B shows the proliferation of AML leukemic blasts upon treatment of mice with engineered WT1 T cells generated by electroporation or LNP processes, compared with T cells transduced with an irrelevant MART1-TCR or another control (leukemic blasts only) without any treatment. Leukemia incidence was measured over time as cells per microliter of blood. Figure 38C shows the percentage of AML cells per total viable cells in the bone marrow of treated groups of mice. Figure 38D shows the percentage of AML cells per total viable cells in the spleen of treated groups of mice. [Figure 38B] (As mentioned above.) [Figure 38C] (As mentioned above.) [Figure 38D] (As mentioned above.) [Figure 39A] Figure 39 shows the editing profile of T cells when treated with various levels of BC22n (as used herein, "BC22n" refers to BC22 without UGI) mRNA and Cas9 mRNA. Cells were edited with individual guide RNAs G015995 (Figure 39A), G016017 (Figure 39B), G016206 (Figure 39C), and G018117 (Figure 39D). [Figure 39B] (As mentioned above.) [Figure 39C] (As mentioned above.) [Figure 39D] (As mentioned above.) [Figure 40A] Figure 40 shows the editing profiles of T cells simultaneously edited with four guides using varying levels of BC22n mRNA or Cas9 mRNA. The editing profile at each edited locus is represented separately: G015995 (Figure 40A), G016017 (Figure 40B), G016206 (Figure 40C), and G018117 (Figure 40D). [Figure 40B] (As mentioned above.) [Figure 40C] (As mentioned above.) [Figure 40D] (As mentioned above.) [Figure 41A] Figure 41A shows the phenotypic analysis results as a percentage of cells negative for antibody binding, accompanied by an increase in total RNA for both BC22 and Cas9 samples. Figure 41A shows the percentage of B2M-negative cells when B2M guide G015995 was used for editing. Figure 41B shows the percentage of B2M-negative cells when multiple guides were used for editing. Figure 41C shows the percentage of CD3-negative cells when TRAC guide G016017 was used for editing. Figure 41D shows the percentage of CD3-negative cells when TRBC guide G016206 was used for editing. Figure 41E shows the percentage of CD3-negative cells when multiple guides were used for editing. Figure 41F shows the percentage of MHCII-negative cells when CIITA guide G018117 was used for editing. Figure 41G shows the percentage of MHCII-negative cells when multiple guides were used for editing. Figure 41H shows the percentage of triple (B2M, CD3, MHCII) negative cells when multiple guides were used for editing. [Figure 41B] (As mentioned above.) [Figure 41C] (As mentioned above.) [Figure 41D] (As mentioned above.) [Figure 41E] (As mentioned above.) [Figure 41F] (As mentioned above.) [Figure 41G] (As mentioned above.) [Figure 41H] (As mentioned above.) [Figure 42] Figure 1 shows cell viability relative to untreated cells after electroporation or LNP delivery of BC22n or Cas9 editor and single or multiple guides. [Figure 43] Total γH2AX spot intensity per nucleus after electroporation or LNP delivery of BC22n or Cas9 editor and single or multiple guides is shown. [Figure 44]1 shows the percentage of editing at the locus of interest after LNP delivery of the BC22n or Cas9 editor and single or multiple guides. [Figure 45] Shown are the percentages of cells negative for the specified surface proteins after LNP delivery of BC22n or Cas9 editor and single or multiple guides. [Figure 46] Shown is the percentage of interchromosomal translocations among total unique molecules after LNP delivery of BC22n or Cas9 editor and multiple guides. [Figure 47A] Figure 47 shows the results of sequential editing in CD8+ T cells. Figure 47A shows the percentage of HLA-A positive cells. Figure 47B shows the percentage of MHC class II positive cells. Figure 47C shows the percentage of WT1 TCR positive CD3+, Vb8+ cells. Figure 47D shows the percentage of CD3+, Vb8 low cells, which exhibit mismatched TCRs. Figure 47E shows the percentage of CD3+, vb8- cells, which exhibit only endogenous TCRs. Figure 47F shows the percentage of CD3+, Vb8+ cells that are positive for WT1 TCR and negative for HLA-A and MHC class II. [Figure 47B] (As mentioned above.) [Figure 47C] (As mentioned above.) [Figure 47D] (As mentioned above.) [Figure 47E] (As mentioned above.) [Figure 47F] (As mentioned above.) [Figure 48A] Figure 48 shows the results of sequential editing in CD4+ T cells. Figure 48A shows the percentage of HLA-A positive cells. Figure 48B shows the percentage of MHC class II positive cells. Figure 48C shows the percentage of WT1 TCR positive CD3+, Vb8+ cells. Figure 48D shows the percentage of CD3+, Vb8 low cells, which exhibit mismatched TCRs. Figure 48E shows the percentage of CD3+, vb8- cells, which exhibit only endogenous TCRs. Figure 48F shows the percentage of CD3+, Vb8+ cells that are positive for WT1 TCR and negative for HLA-A and MHC class II. [Figure 48B] (As mentioned above.) [Figure 48C] (As mentioned above.) [Figure 48D] (As mentioned above.) [Figure 48E] (As mentioned above.) [Figure 48F] (As mentioned above.) [Figure 49A] Shown are the indel percentages after sequential editing of T cells for CIITA (Figure 49A), HLA-A (Figure 49B), TRBC1 (Figure 49C), and TRBC2 (Figure 49D) in T cells. [Figure 49B] (As mentioned above.) [Figure 49C] (As mentioned above.) [Figure 49D] (As mentioned above.) [Figure 50A] The percentage of CD3eta+, Vb8- cells, representing the population of T cells without gene disruption at the TRAC or TRBC1 / 2 loci, is shown. [Figure 50B] The percentage of CD3eta+, Vb8+ cells representing the population of T cells with WT1 TCR insertion in TRAC is shown. [Figure 50C] The percentage of HLA-A2- cells, representing the population of T cells with effective gene disruption at the HLA locus, is shown. [Figure 50D] The percentage of HLA-DRDPDQ- cells, representing the population of T cells with an effective gene disruption at the CIITA locus, is shown. [Figure 50E] The percentage of GFP+ cells is shown, representing the population of T cells with GFP insertion at the AAVS1 locus. [Figure 50F] The percentage of Vb8+GFP+HLA-A-HLA-DRDPDQ- cells, representing the population of T cells carrying the five genome edits, is shown. [Figure 51A] Shown is the percentage of CD3-negative cells, representing the population of T cells with effective gene disruption at the TRBC1 / 2 locus, after treating activated T cells with LNPs pre-incubated with different levels of Apo protein. [Figure 51B] Shown is the percentage of CD3-negative cells, representing the population of T cells with effective gene disruption at the TRBC1 / 2 locus, after treatment of rested T cells with LNPs pre-incubated with different levels of Apo protein. [Figure 52A] Shown is the percentage of CD3 negative cells, representing the population of T cells with effective gene disruption at the TRAC locus, following treatment of resting T cells at 0 hours with co-formulated or mRNA-only first LNPs formulated with PEG-2kDMG, and treatment with gRNA-only second LNPs at 0 hours or 72 hours. [Figure 52B] Shown is the percentage of CD3 negative cells, representing the population of T cells with effective gene disruption at the TRAC locus, following treatment of resting T cells at 0 hours with co-formulated or mRNA-only first LNPs formulated with PEG-Lipid H, and treatment with gRNA-only second LNPs at 0 hours or 72 hours. [Figure 53A] 1 shows the percentage of CD3 − cells, representing the population of T cells with effective gene disruption at the TRAC locus, following treatment of activated T cells with LNPs formulated with various lipid molar ratios. [Figure 53B] 1 shows the percentage of CD3 − cells, representing the population of T cells with effective gene disruption at the TRAC locus, following treatment of resting T cells with LNPs formulated with various lipid molar ratios. [Figure 54] Shown is the percentage of CD3− cells, representing the population of T cells with effective gene disruption at the TRAC locus, following treatment of activated T cells with LNPs formulated with various w / w ratios of mRNA and sgRNA. [Figure 55A] Figure 55A shows the percentage of CD3- cells, representing the population of T cells with effective gene disruption at the TRAC locus, after treatment of resting T cells with LNPs formulated with various w / w ratios of mRNA and sgRNA. Figure 55A shows donor 1. Figure 55B shows donor 2. [Figure 55B] (As mentioned above.) [Figure 56A] Figure 56 shows the percentage of CD86+ cells among CD20+ cells, representing the activated B cell population, after culture under various media conditions. Figure 56A shows cells cultured in IMDM-based media. Figure 56B shows cells cultured in StemSpan-based media. [Figure 56B] (As mentioned above.) [Figure 56C] Figure 56 shows the percentage of LDLR+ cells among CD20+ B cells after culture under various media conditions. Figure 56C shows cells cultured in IMDM-based media. Figure 56D shows cells cultured in StemSpan-based media. [Figure 56D] (As mentioned above.) [Figure 57A] Figure 57 shows the fold expansion of B cells cultured in medium containing 1, 10, or 100 ng / ml CD40L on day 14. Figure 57A shows cells stimulated for primary activation only. Figure 57B shows cells stimulated for secondary activation (plasmablast differentiation). [Figure 57B] (As mentioned above.) [Figure 58A] Figure 58A shows the average percent editing determined by NGS in B cells after editing with LNPs formulated with the specified lipids. Figure 58B shows B cells cultured in IMDM. Figure 58C shows B cells cultured in StemSpan. [Figure 58B] (As mentioned above.) [Figure 59] Shown is the percentage of B2M-negative cells, representing the population of B cells with effective gene disruption, after treatment with LNP formulated with lipid A or lipid D and pre-incubated with ApoE3 or ApoE4. [Figure 60A] Figure 60 shows the percent B2M negative cells, representing the population of B cells with effective gene disruption, after treatment with LNP formulated with lipid A or lipid D. Figure 60A shows LNP treatment from 1 day before activation to 5 days after activation. Figure 60B shows treatment with LNP formulated with lipid A from 6 to 10 days after activation. [Figure 60B] (As mentioned above.) [Figure 61] 1 shows the percentage of B2M-negative cells, representing the population of B cells with effective gene disruption, after editing with DNAPK inhibitors Compound 1 or Compound 4. [Figure 62] Percent editing assessed by NGS in NK cells treated with LNPs formulated with the indicated lipids is shown. [Figure 63] Percent editing assessed by NGS in NK cells treated with various doses of LNPs is shown 14 days after LNP treatment. [Figure 64] Shown is the percentage of NK cells with high GFP expression (GFP++) after editing to insert GFP into the AAVS1 locus. [Figure 65A] 1 shows the mean percent editing in AAVS1 assessed by NGS after treatment with LNP and various doses of the DNAPK inhibitors Compound 1 or Compound 4. [Figure 65B] Shown is the percentage of NK cells with high GFP expression (GFP++) after editing to insert GFP into the AAVS1 locus with DNAPK inhibitors Compound 1 or Compound 4. [Figure 66] 1 shows the relative Cas9 protein expression in macrophage cells after editing with various lipid compositions relative to lipid A. [Figure 67] Shown is the percentage of B2M-negative cells, representing the population of cells with effective gene disruption, after editing in macrophage or monocyte cells. [Figure 68] Percent editing assessed by NGS in macrophage cells after treatment with LNPs 0–8 days after thawing is shown. [Figure 69A] The mean percentage of negative cells after sequential LNP treatments is shown. Figure 69A shows the percentage of HLA-DR, DP, DQ negative cells, representing effective disruption of the CIITA locus. Figure 69B shows the percentage of B2M negative cells. [Figure 69B] (As mentioned above.) [Figure 70]CD68+, CD11b+, HLA-ABC- cell percentages after editing with LNP formulated with lipid A or lipid D are shown. DETAILED DESCRIPTION OF THE INVENTION

[0007] The present disclosure provides platform methods using lipid-nucleic acid assembly compositions for delivering nucleic acids, such as genome editing tools, to cells and for in vitro multiplexed genome editing. The methods provide, for example, the ability to deliver multiple genome editing tools to cells without significant cellular side effects. The methods also provide, for example, multiple in vitro genome editing within a single cell without significant loss of cell viability, whereas previous methods, for example, using electroporation, were hampered by their toxicity to cells. In some embodiments, the platform relates to manufacturing methods for preparing cells in vitro for subsequent therapeutic administration to a subject. In some embodiments, the platform relates to multiplexed genome editing via simultaneous or sequential administration of lipid-nucleic acid assembly compositions containing genome editing tools. The platform is relevant to any cell type, but is particularly advantageous for preparing cells that require multiple genome edits for full therapeutic applicability, for example, in primary immune cells. The present methods may exhibit improved properties compared to previous delivery techniques, for example, the present methods provide efficient delivery of nucleic acids, such as genome editing tools, while reducing loss of cell viability and / or cell death caused by the transfection process itself, e.g., due to high levels of DNA damage, including translocations, caused by previous transfection methods. As provided herein, the platform methods are applied to "cells" in vitro or to "cell populations" (or "populations of cells") in vitro. When reference is made herein to delivery or gene editing methods for "cells," it is understood that these methods can be used for delivery to or gene editing of "cell populations."

[0008] In some embodiments, provided herein are methods for delivering two or more lipid nucleic acid assembly compositions comprising nucleic acids, e.g., genome editing tools, to cells in vitro. In some embodiments, the methods comprise administering multiple nucleic acid assembly compositions sequentially and / or simultaneously. In some embodiments, the methods comprise pre-incubating a serum factor with the lipid nucleic acid assembly composition. In some embodiments, the lipid nucleic acid assembly composition comprises a nucleic acid, an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid. In some embodiments, the methods further comprise contacting cells in vitro with the pre-incubated lipid nucleic acid assembly composition. In some embodiments, the methods further comprise culturing the cells in vitro. In some embodiments, the methods result in delivery of genome editing tools to cells without significant loss of cell viability.

[0009] In some embodiments, provided herein are methods for producing genetically engineered primary immune cells, e.g., T cells or B cells, in vitro. In some embodiments, the primary immune cells are cultured in vitro and a lipid-nucleic acid assembly composition comprising a nucleic acid genome editing tool is provided. In some embodiments, more than one such composition is provided for the primary immune cells. In some embodiments, the method results in the production of genetically engineered primary immune cells. In some embodiments, the method results in the production of genetically engineered primary immune cells with more than one genetic modification.

[0010] In some embodiments, provided herein are methods utilizing lipid nucleic acid assemblies, e.g., lipid nanoparticle (LNP)-based compositions, with useful properties, particularly for the delivery of CRISPR-Cas gene editing components. Lipid nucleic acid assembly compositions facilitate the delivery of nucleic acids across cell membranes, and in certain embodiments, they introduce gene editing components and compositions into living cells. In some embodiments, the methods provide for the delivery of guide RNA, e.g., via an LNP composition, along with an RNA-guided DNA-binding agent, such as a CRISPR-Cas system, to substantially reduce or knock out the expression of a specific gene. In some embodiments, the methods provide for the delivery of guide RNA, along with an RNA-guided DNA-binding agent, such as a CRISPR-Cas system, via a lipid nucleic acid assembly, e.g., an LNP composition, and a donor nucleic acid (also referred to herein as a "template nucleic acid" or "exogenous nucleic acid"), e.g., DNA encoding a desired protein that can be inserted into a target sequence. Some embodiments do both.

[0011] Of particular interest are methods for delivering components of the CRISPR / Cas gene editing system to immune cells, such as mononuclear cells, including lymphocytes, particularly T cells, in culture. Provided herein is a method for delivering RNA containing components of the CRISPR / Cas system to immune cells, such as mononuclear cells, including lymphocytes, particularly T cells. The method comprises delivering nucleic acids to cells, including lymphocytes, cultured in vitro, particularly T cells, and contacting the cells with a lipid nanoparticle (LNP) composition that provides mRNA encoding a protein. Additionally, methods for in vitro gene editing in immune cells, such as lymphocytes and particularly T cells, and methods for producing engineered cells are provided.

[0012] In some embodiments, provided herein are compositions of cell populations comprising edited cells. In some embodiments, such cell populations comprise edited cells comprising multiple genome edits per cell. The present disclosure provides cell populations comprising edited cells, wherein the population of cells comprises edited cells comprising a single genome edit. In some embodiments, the present disclosure provides cell populations comprising edited cells comprising at least two genome edits. In some embodiments, cell populations comprising edited cells have, for example, low levels of translocations and can be expanded after initiation of editing, making them suitable as cell therapy products.

[0013] In some embodiments, described herein are compositions and methods for adoptive cell transfer (ACT) therapy, such as immuno-oncology, including cells modified with one or more specific target sequences of interest in their genomes, including those modified by the introduction of a CRISPR system comprising a gRNA molecule that targets the target sequence, and methods for making and using them. For example, the disclosure provides methods for genome editing of immune cells, e.g., T cells engineered to lack endogenous TCR expression, e.g., T cells suitable for further engineering to insert a nucleic acid of interest, e.g., T cells further engineered to express a TCR, such as a transgenic TCR (tgTCR), and useful for ACT therapy; and genome editing of B cells, e.g., B cells engineered to lack endogenous B cell receptor (BCR) expression, e.g., B cells suitable for further engineering to insert a nucleic acid of interest, e.g., B cells further engineered to express a BCR, such as a transgenic BCR (tgBCR), or for the expression of an antibody; e.g., adapted for ACT therapy. The present invention relates to and provides gRNA molecules, CRISPR systems, cells, and methods useful for genome editing of NK cells, or monocytes, or macrophages, or iPSCs, or primary cells, or progenitor cells described herein that have been engineered to lack endogenous molecules to improve activity, e.g., NK cells, or monocytes, or macrophages, or iPSCs, or primary cells, or progenitor cells described herein that are suitable for manipulation to insert a nucleic acid of interest, e.g., NK cells, or monocytes, or macrophages, or iPSCs, or primary cells, or progenitor cells described herein that have been further engineered to express a heterologous protein sequence and are useful for ACT therapy.

[0014] In some embodiments, the present methods provide a novel process for genetically engineering T cells useful as adoptive cell therapy. For example, in some embodiments, T cells are genetically modified in vitro to reduce expression of multiple target genes, including, for example, endogenous T cell receptor genes, and further modified to insert a transgenic TCR in the form of donor nucleic acid. In some embodiments, T cells particularly desirable for use as adoptive cell therapy require multiple gene edits. The ability to genetically engineer T cells in vitro with the numerous modifications to their genomes disclosed herein has previously proven technically challenging. In addition to the obstacles associated with multiple gene editing discussed above, T cells are particularly difficult to genetically engineer in culture, for example, and can become exhausted.

[0015] Provided herein are methods for genetically engineering T cells in vitro that overcome the obstacles of previous processes. In some embodiments, naive T cells are contacted in vitro with at least one lipid-nucleic acid assembly composition and genetically engineered. In some embodiments, non-activated T cells are contacted in vitro with two or more lipid-nucleic acid assembly compositions and genetically engineered. In some embodiments, activated T cells are contacted in vitro with two or more lipid-nucleic acid assembly compositions and genetically engineered. In some embodiments, T cells are modified in a pre-activation step involving contacting (non-activated) T cells with one or more lipid-nucleic acid assembly compositions, followed by activation of the T cells, followed by further modification of the T cells in a post-activation step involving contacting the activated T cells with one or more lipid-nucleic acid assembly compositions. In some embodiments, non-activated T cells are contacted with one, two, or three lipid-nucleic acid assembly compositions. In some embodiments, activated T cells are contacted with one to twelve lipid-nucleic acid assembly compositions. In some embodiments, activated T cells are contacted with 1 to 8 lipid nucleic acid assembly compositions, optionally 1 to 4 lipid nucleic acid assembly compositions. In some embodiments, activated T cells are contacted with 1 to 6 lipid nucleic acid assembly compositions. In some embodiments, T cells are contacted with 2 lipid nucleic acid assembly compositions. In some embodiments, T cells are contacted with 3 lipid nucleic acid assembly compositions. In some embodiments, T cells are contacted with 4 lipid nucleic acid assembly compositions. In some embodiments, T cells are contacted with 5 lipid nucleic acid assembly compositions. In some embodiments, T cells are contacted with 6 lipid nucleic acid assembly compositions. In some embodiments, T cells are contacted with 7 lipid nucleic acid assembly compositions. In some embodiments, T cells are contacted with 8 lipid nucleic acid assembly compositions. In some embodiments, T cells are contacted with 9 lipid nucleic acid assembly compositions. In some embodiments, T cells are contacted with 10 lipid nucleic acid assembly compositions.In some embodiments, T cells are contacted with 11 lipid-nucleic acid assembly compositions. In some embodiments, T cells are contacted with 12 lipid-nucleic acid assembly compositions. Such exemplary sequential administration of lipid-nucleic acid assembly compositions (optionally with additional sequential or simultaneous administration in pre-activation and post-activation steps) takes advantage of the activation state of T cells, providing unique advantages and healthier cells after editing. In some embodiments, the engineered T cells have advantageous properties, such as high editing efficiency at each target site, increased survival after editing, low toxicity despite transfection variability, low translocation (e.g., no measurable target-to-target translocation), increased production of cytokines (e.g., IL-2, IFNγ, TNFα), continued proliferation with repeated stimulation (e.g., with repeated antigen stimulation), increased expansion, and expression of memory cell phenotypic markers, including early stem cells.

[0016] I. Definition Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings:

[0017] "Polynucleotide" and "nucleic acid" are used herein to refer to polymeric compounds comprising nucleosides or nucleoside analogs (including traditional RNA, DNA, mixed RNA-DNA, and polymers of their analogs) having nitrogenous heterocyclic bases or base analogs linked together along the backbone. The nucleic acid "backbone" can be composed of various linkages, including one or more of sugar phosphodiester linkages, peptide-nucleic acid linkages ("peptide nucleic acid" or PNA, PCT No. 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar moiety of the nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions, e.g., 2' methoxy or 2' halide substitutions. The nitrogenous bases can be the traditional bases (A, G, C, T, U), their analogs (e.g., modified uridines such as 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine), derivatives of inosine, purine, or pyrimidine (e.g., N 4-methyldeoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with a substituent at the 5- or 6-position (e.g., 5-methylcytosine), purine bases with a substituent at the 2-, 6-, or 8-position, 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and O 4 5,378,825 and PCT Publication No. 93 / 13121). For a general discussion, see The Biochemistry of the Nucleic Acids, vol. 5-36, edited by Adams et al., 11 th ed., 1992). Nucleic acids may contain one or more "abasic" residues, where the backbone does not contain a nitrogenous base at any position in the polymer (U.S. Pat. No. 5,585,481). Nucleic acids may contain only conventional RNA or DNA sugars, bases, and linkages, or may contain both conventional components and substitutions (e.g., polymers containing conventional bases with 2' methoxy linkages, or both conventional bases and one or more base analogs). Nucleic acids include "locked nucleic acids" (LNAs), which are analogs containing one or more LNA nucleotide monomers that have a bicyclic furanose unit locked to an RNA-mimetic sugar structure, enhancing hybridization affinity for complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). RNA and DNA have different sugar moieties and may differ by the presence of uracil or its analogs in RNA and thymine or its analogs in DNA.

[0018] "Guide RNA," "gRNA," and simply "guide" are used interchangeably herein to refer to a guide that directs an RNA-guided DNA-binding agent to a target DNA, and can be either a crRNA (also known as CRISPR RNA) or a combination of crRNA and trRNA (also known as tracrRNA). The crRNA and trRNA can associate as a single-stranded RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). "Guide RNA" or "gRNA" refers to each type. The trRNA can be a naturally occurring sequence or a trRNA sequence that has modifications or variations compared to the naturally occurring sequence.

[0019] As used herein, a "guide sequence" refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct the guide RNA to the target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA-binding agent. A "guide sequence" may also be referred to as a "targeting sequence" or a "spacer sequence." A guide sequence may be 20 base pairs in length, for example, in the case of Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs / orthologs. 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 degree of complementarity or identity between a guide sequence and its corresponding target sequence may be about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the guide sequence and target sequence may be 100% complementary or identical. In other embodiments, the guide sequence and target sequence may contain at least one mismatch. For example, the guide sequence and target sequence may contain 1, 2, 3, or 4 mismatches, and 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 target sequence may contain 1 to 4 mismatches, and the guide sequence comprises at least 17, 18, 19, 20, or more nucleotides. In some embodiments, the guide sequence and target sequence may contain 1, 2, 3, or 4 mismatches, and the guide sequence comprises 20 nucleotides.

[0020] Because the nucleic acid substrate of an RNA-guided DNA binder is a double-stranded nucleic acid, the target sequence of the RNA-guided DNA binder includes both the plus and minus strands of genomic DNA (i.e., the given sequence and the reverse complement of the sequence). Thus, when a guide sequence is said to be "complementary to a target sequence," it should be understood that the guide RNA can be directed 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 certain nucleotides of the target sequence (e.g., the target sequence without the PAM) except for the substitution of U for T in the guide sequence.

[0021] As used herein, "RNA-guided DNA binding agent" refers to a polypeptide or polypeptide complex 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 binding agents include Cas cleavase / nickases and their inactivated forms (dCas DNA binders). As used herein, "Cas nucleases," also referred to as "Cas proteins," encompass Cas cleavases, Cas nickases, and dCas DNA binders. Cas cleavases / nickases and dCas DNA binders include the Csm or Cmr complexes of type III CRISPR systems, Cas10, Csm1, or their Cmr2 subunits, the Cascade complex of type I CRISPR systems, its Cas3 subunit, and class 2 Cas nucleases. As used herein, a "class 2 Cas nuclease" is a single-chain polypeptide with RNA-guided DNA-binding activity. Class 2 Cas nucleases include Class 2 Cas cleavases / nickases that also have RNA-guided DNA cleavage or nickase activity (e.g., H840A, D10A, or N863A variants), as well as Class 2 dCas DNA binders with inactivated cleavage / nickase activity. Class 2 Cas nucleases 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 sequences of Zetsche are incorporated by reference in their entirety. See, e.g., Tables S1 and S3 of Zetsche.See, for example, Makarova et al., Nat Rev Microbiol, 13(11):722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).

[0022] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a guide RNA comprising 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 guide RNA guides the RNA-guided DNA-binding agent, such as Cas9, to a target sequence, the guide RNA hybridizes to the target sequence, and an agent binds to the target sequence, where the agent is a cleavase or nickase and can perform cleavage or nicking after binding.

[0023] As used herein, the term "editor" refers to an agent comprising a polypeptide capable of making modifications to bases (e.g., A, T, C, G, or U) in a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, the editor is capable of deaminating a base in a nucleic acid. In some embodiments, the editor is capable of deaminating a base in a DNA molecule. In some embodiments, the editor is capable of deaminating a cytosine (C) in DNA. In some embodiments, the editor is a fusion protein comprising an RNA-guided nickase fused to a cytidine deaminase domain. In some embodiments, the editor is a fusion protein comprising an RNA-guided nickase fused to an APOBEC3A deaminase (A3A). In some embodiments, the editor comprises a Cas9 nickase fused to an APOBEC3A deaminase (A3A).

[0024] As used herein, a first sequence is considered to "contain a sequence having at least X% identity" with a second sequence if alignment of the first sequence to the second sequence shows that X% or more of the positions of the second sequence are matched overall by the first sequence. For example, the sequence AAGA contains a sequence having 100% identity with the sequence AAG, because the alignment gives 100% identity in that there is a match at all three positions in the second sequence. Differences between RNA and DNA (generally, the replacement of thymidine with uridine, or vice versa), and the presence of nucleoside analogs such as modified uridines, do not contribute to differences in identity or complementarity between polynucleotides, as long as the related nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all thymidine, uridine, or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as their complement). Thus, for example, the sequence 5'-AXG, where X is any modified uridine, such as pseudouridine, N1-methylpseudouridine, or 5-methoxyuridine, is considered 100% identical to AUG, in that both are perfectly complementary to the same sequence (5'-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well known in the art. Those skilled in the art will understand what algorithm and parameter setting is appropriate for a given pair of sequences to be aligned. Generally, for sequences of similar length and predicted identity greater than 50% for amino acids or 75% for nucleotides, the Needleman-Wunsch algorithm, using the default settings of the Needleman-Wunsch algorithm interface provided by EBI at the www.ebi.ac.uk web server, is generally appropriate.

[0025] "mRNA" is used herein to refer to a polynucleotide that contains 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 a combination thereof.

[0026] As used herein, "indel" refers to an insertion / deletion mutation consisting of a number of nucleotides that are either inserted or deleted, for example, at the site of a double-strand break (DSB) in a target nucleic acid.

[0027] 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 cellular amount of 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 some loss of expression of a particular gene product, e.g., a reduction in the amount of mRNA transcribed, or a reduction in the amount of protein expressed by a cell population (including in vivo populations such as those found in tissues).

[0028] As used herein, "knockout" refers to the loss of expression from a particular gene in a cell or the loss of a particular protein in a cell. Knockout can be measured either by detecting the total cellular amount of the protein in a cell, tissue, or cell population.

[0029] As used herein, "target sequence" refers to a nucleic acid sequence within a target gene that has complementarity to the guide sequence of a gRNA. The interaction between the target sequence and the guide sequence directs the RNA-guided DNA-binding agent to bind and potentially (depending on the activity of the agent) nick or cleave within the target sequence.

[0030] As used herein, "treatment" refers to any administration or application of therapy for a disease or disorder in a subject, including inhibiting the disease, halting its progression, alleviating one or more symptoms of the disease, curing the disease, or preventing one or more symptoms of the disease, including the recurrence of symptoms.

[0031] As used herein, a "cell population comprising edited cells," or a "population of cells comprising edited cells," or the like, refers to a cell population that includes edited cells, although not all cells in the population must be edited. A cell population that includes edited cells may also include unedited cells. The percentage of edited cells in a cell population that includes edited cells can be determined by counting the number of cells in the population that are edited, as determined by standard cell counting methods. For example, in some embodiments, a cell population that includes edited cells that include a single genome edit has at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells in the population that have a single edit. In some embodiments, a cell population that includes edited cells that include at least two genome edits has at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the cells in the population that have at least two genome edits.

[0032] The term "about" or "approximately" refers to an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, or on variations that do not materially affect the properties of the described subject matter, or on the tolerances accepted in the art, e.g., within 10%, 5%, 2%, or 1%. Accordingly, unless otherwise indicated, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of at least the number of reported significant digits and by applying ordinary rounding techniques.

[0033] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings. 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. On the contrary, the invention is defined by the appended claims and is intended to cover all alternatives, modifications, and equivalents that may be included within the invention by the included embodiments.

[0034] Before describing the present teachings in detail, it should be understood that the present disclosure is not limited to particular compositions or process steps, as such may vary. It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "a conjugate" includes plural conjugates, a reference to "a cell" includes plural cells, and so forth.

[0035] Numerical ranges are inclusive of the numbers defining the range. Measured and measurable values ​​are understood to be approximate, taking into account significant orders of magnitude and the error associated with the measurements. Additionally, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be limiting. It should be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the teachings herein.

[0036] Unless otherwise stated herein, embodiments herein that recite various components as "comprising" are also assumed to "consist of" or "consist essentially of" the recited components. Embodiments herein that recite various components as "consisting of" are also assumed to "comprising" or "consisting essentially of" the recited components. Embodiments herein that recite various components as "consisting essentially of" are also assumed to "consist of" or "comprising" the recited components (this interchangeability does not apply to the use of these terms in the claims). The term "or" is used in its inclusive sense, i.e., equivalent to "and / or," unless the context clearly dictates otherwise.

[0037] The section headings used herein are for organizational purposes only and should not be construed as limiting the desired subject matter in any way. In the event that any material incorporated by reference conflicts with any term defined herein or any other explicit content of this specification, the present specification shall control. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. To the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

[0038] II. Multiplex Delivery and Genome Editing A. Multiplex delivery In some embodiments, methods are provided for delivering multiple lipid-nucleic acid assembly compositions to cells in vitro. In some embodiments, the multiplex delivery method results in cells that can be expanded into a cell population. In some embodiments, expansion of cells into a cell population is a marker of successful multiplex delivery. Similarly, methods are provided for delivering multiple lipid-nucleic acid assembly compositions to cells in vitro to produce an expanded cell population with increased survival. Such methods are useful, for example, in the production / manufacturing of cells for use in cell therapy, where cell therapy, as used herein, refers to the transfer of live, intact cells into a subject to treat a disease or disorder. Cell therapy approaches include therapeutic cell transplantation, including ACT therapy. Cell therapy includes autologous (cells derived from the subject) and allogeneic (cells derived from a donor) cell therapy.

[0039] In some embodiments, the multiplex delivery method involves delivering at least two lipid-nucleic acid assembly compositions to in vitro cultured cells. In some embodiments, the cells are contacted in vitro with at least a first lipid-nucleic acid assembly composition comprising a first nucleic acid, thereby producing contacted cells, and the contacted cells are cultured, thereby producing cultured contacted cells. The cultured contacted cells are contacted with at least a second lipid-nucleic acid assembly composition comprising a second nucleic acid, where the second nucleic acid is different from the first nucleic acid. The resulting cells are then expanded in vitro. In some embodiments, the delivery method results in an expanded cell population, e.g., a cell population with increased survival. In some embodiments, the expanded cells have a viability of at least 70%. The "first" and "second" nucleic acids may comprise guide RNAs (gRNAs).

[0040] In some embodiments, a method for delivering a lipid-nucleic acid assembly composition to in vitro cultured cells is provided, comprising: a) contacting the cells in vitro with at least a first lipid-nucleic acid assembly composition comprising a first nucleic acid, thereby producing contacted cells; b) culturing the contacted cells in vitro, thereby producing cultured contacted cells; c) contacting the cultured contacted cells in vitro with at least a second lipid-nucleic acid assembly composition comprising a second nucleic acid, wherein the second nucleic acid is different from the first nucleic acid; and d) expanding the cells in vitro, wherein the expanded cells exhibit increased viability. In some embodiments, the expanded cells have a viability of at least 70%. In some embodiments, the cells are contacted with 2 to 12 lipid-nucleic acid assembly compositions. In some embodiments, the cells are contacted with 2 to 8 lipid-nucleic acid assembly compositions. In some embodiments, the cells are contacted with 2 to 6 lipid-nucleic acid assembly compositions. In some embodiments, the cells are contacted with 3 to 8 lipid-nucleic acid assembly compositions. In some embodiments, cells are contacted with 3 to 6 lipid nucleic acid assembly compositions. In some embodiments, cells are contacted with 4 to 6 lipid nucleic acid assembly compositions. In some embodiments, cells are contacted with 4 to 12 lipid nucleic acid assembly compositions. In some embodiments, cells are contacted with 4 to 8 lipid nucleic acid assembly compositions. In some embodiments, cells are contacted with 6 to 12 lipid nucleic acid assembly compositions. In some embodiments, cells are contacted with 3, 4, 5, or 6 lipid nucleic acid assembly compositions. In some embodiments, cells are contacted with 6 lipid nucleic acid assembly compositions. In some embodiments, cells are contacted with 8 or fewer lipid nucleic acid assembly compositions simultaneously. In some embodiments, cells are contacted with 6 or fewer lipid nucleic acid assembly compositions simultaneously. In some embodiments, cells are T cells. In some embodiments, cells are non-activated cells. In some embodiments, cells are activated cells.In some embodiments, the cells of (a) are activated after contact with at least one lipid nucleic acid assembly composition.

[0041] In some embodiments, "increased survival" is demonstrated by at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% cell viability after transfection or expanded cells (referring to the viability of the population of cells resulting from the expanded cells, including edited cells). In some embodiments, lipid-nucleic acid assembly methods may reduce cell death compared to known techniques, such as electroporation. In some embodiments, lipid-nucleic acid assembly methods may cause less than 5%, less than 10%, less than 20%, less than 30%, or less than 40% cell death. In some embodiments, lipid-nucleic acid assembly methods deliver nucleic acids, such as RNA, without significant loss of cell viability, whereas previous methods, for example, using electroporation, were hampered by their toxicity to cells. In some embodiments, lipid-nucleic acid assembly methods result in engineered T cell populations with favorable initial stem cell memory phenotypes, improved cell expansion and / or cell phenotypes, such as cytokine production, proliferation profiles after repeated antigen stimulation, and / or chromosomal translocation rates.

[0042] In some embodiments, cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 lipid-nucleic acid assembly compositions. In some embodiments, cells are contacted with at least 6 lipid-nucleic acid assembly compositions. In some embodiments, cells are contacted with 12 or fewer lipid-nucleic acid assembly compositions. In some embodiments, cells are contacted with 2-12 lipid-nucleic acid assembly compositions. In some embodiments, cells are contacted with 2-8 lipid-nucleic acid assembly compositions. In some embodiments, cells are contacted with 2-6 lipid-nucleic acid assembly compositions. In some embodiments, cells are contacted with 3-8 lipid-nucleic acid assembly compositions. In some embodiments, cells are contacted with 3-6 lipid-nucleic acid assembly compositions. In some embodiments, cells are contacted with 4-6 lipid-nucleic acid assembly compositions. In some embodiments, cells are contacted with 4-12 lipid-nucleic acid assembly compositions. In some embodiments, cells are contacted with 4-8 lipid-nucleic acid assembly compositions. In some embodiments, cells are contacted with 6-12 lipid-nucleic acid assembly compositions. In some embodiments, cells are contacted with 3, 4, 5, or 6 lipid nucleic acid assembly compositions. In some embodiments, cells are contacted with 8 or fewer lipid nucleic acid assembly compositions simultaneously. In some embodiments, cells are contacted with 6 or fewer lipid nucleic acid assembly compositions simultaneously.

[0043] In some embodiments, the cells are contacted with two lipid nucleic acid assembly compositions. In some embodiments, the cells are contacted with three lipid nucleic acid assembly compositions. In some embodiments, the cells are contacted with four lipid nucleic acid assembly compositions. In some embodiments, the cells are contacted with five lipid nucleic acid assembly compositions. In some embodiments, the cells are contacted with six lipid nucleic acid assembly compositions.

[0044] In some embodiments, contact between the cells and the lipid nucleic acid assembly compositions is sequential (one after the other). In some embodiments, contact between the cells and the lipid nucleic acid assembly compositions is simultaneous (contact is simultaneous or near simultaneous). In some embodiments, multiple lipid nucleic acid assembly compositions are administered sequentially. In some embodiments, the lipid nucleic acid assembly compositions are administered simultaneously. In some embodiments, the lipid nucleic acid assembly compositions are administered sequentially and simultaneously. For example, in some embodiments, three lipid nucleic acid compositions are provided, two lipid nucleic acid compositions are administered first simultaneously, the cells are cultured for a period of time, and then the third lipid nucleic acid composition is administered (i.e., sequentially after the administration of the first two compositions). In another embodiment, three lipid nucleic acid compositions are provided, one lipid nucleic acid composition is administered first, the cells are cultured for a period of time, and then two lipid nucleic acid compositions are administered simultaneously (and sequentially after the administration of the first composition). Thus, simultaneous and sequential administration of lipid nucleic acid assembly compositions can overlap in certain embodiments.

[0045] In some embodiments, a method for delivering lipid nucleic acid assembly compositions to in vitro cultured cells is provided, comprising: a) contacting the cells in vitro with at least a first lipid nucleic acid assembly composition comprising a first nucleic acid, thereby producing contacted cells; b) culturing the contacted cells in vitro, thereby producing cultured contacted cells; c) contacting the cultured contacted cells in vitro with at least a second lipid nucleic acid assembly composition comprising a second nucleic acid, wherein the second nucleic acid is different from the first nucleic acid; and d) expanding the cells in vitro, wherein the expanded cells exhibit increased survival, wherein the cells are contacted with at least six lipid nucleic acid assembly compositions. In some embodiments, the expanded cells have a viability of at least 70%. In some embodiments, at least four lipid nucleic acid assembly compositions comprise a guide RNA and at least one lipid nucleic acid assembly composition comprises a first genome editing tool, thereby producing multiple genome edits in the cells. In some embodiments, at least six lipid nucleic acid assembly compositions are administered simultaneously. In some embodiments, the first genome editing tool is an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the RNA-guided DNA binding agent comprises APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, the method comprises contacting a cell with a lipid nucleic acid composition comprising a second genome editing tool. In some embodiments, the second genome editing tool is UGI. In some embodiments, the second genome editing tool is a donor nucleic acid. In some embodiments, the method comprises contacting a cell with a lipid nucleic acid composition comprising a third genome editing tool. In some embodiments, the third genome editing tool is an RNA-guided DNA binding agent. In some embodiments, the third genome editing tool is UGI. In some embodiments, the third genome editing tool is a donor nucleic acid.In some embodiments, the genome editing tool (e.g., the first genome editing tool, the second genome editing tool, the third genome editing tool) is mRNA. In some embodiments, the cell is a T cell. In some embodiments, the cell is a non-activated cell. In some embodiments, the cell is an activated cell. In some embodiments, the cell of (a) is activated after contact with at least one lipid-nucleic acid assembly composition.

[0046] In some embodiments, a method for delivering a lipid nanoparticle (LNP) composition to a population of in vitro cultured cells is provided, comprising: a) contacting the population of cells in vitro with at least a first LNP composition comprising a first nucleic acid, thereby producing a population of contacted cells; b) culturing the contacted population of cells in vitro, thereby producing a population of cultured contacted cells; c) contacting the population of cells or the cultured population of contacted cells in vitro with at least a second LNP composition comprising a second nucleic acid, wherein the second nucleic acid is different from the first nucleic acid; and d) expanding the population of cells in vitro, wherein the expanded population of cells exhibits at least 70% viability. In some embodiments, the expanded population of cells has at least 70% viability after 24 hours of expansion. In some embodiments, the expanded population of cells has at least 80% viability after 24 hours of expansion. In some embodiments, the expanded population of cells has at least 90% viability after 24 hours of expansion. In some embodiments, the expanded population of cells has a viability of at least 95% at 24 hours of expansion. In some embodiments, the population of cells and the cultured contacted population of cells are contacted with a total of 2 to 12 LNP compositions. In some embodiments, the population of cells and the cultured contacted population of cells are contacted with a total of 2 to 8 LNP compositions. In some embodiments, the population of cells and the cultured contacted population of cells are contacted with a total of 2 to 6 LNP compositions. In some embodiments, the population of cells and the cultured contacted population of cells are contacted with a total of 3 to 8 LNP compositions. In some embodiments, the population of cells and the cultured contacted population of cells are contacted with a total of 3 to 6 LNP compositions. In some embodiments, the population of cells and the cultured contacted population of cells are contacted with a total of 4 to 6 LNP compositions. In some embodiments, the population of cells and the cultured contacted population of cells are contacted with a total of 6 to 12 LNP compositions.In some embodiments, the population of cells and the cultured contacted population of cells are contacted with a total of three LNP compositions. In some embodiments, the population of cells and the cultured contacted population of cells are contacted with a total of four LNP compositions. In some embodiments, the population of cells and the cultured contacted population of cells are contacted with a total of six LNP compositions. In some embodiments, the population of cells and the cultured contacted population of cells are contacted with a total of three LNP compositions. In some embodiments, the population of cells are contacted with the LNP compositions simultaneously. In some embodiments, the population of cells are contacted with six or fewer LNP compositions simultaneously. In some embodiments, the population of cells are contacted with two or fewer LNP compositions simultaneously.

[0047] In some embodiments, a method for delivering a lipid nanoparticle (LNP) composition to a population of in vitro cultured cells is provided, comprising: a) contacting the population of cells in vitro with at least a first LNP composition comprising a first nucleic acid, thereby producing a population of contacted cells; b) culturing the contacted population of cells in vitro, thereby producing a population of cultured contacted cells; c) contacting the population of cells or the cultured population of contacted cells in vitro with at least a second LNP composition comprising a second nucleic acid, wherein the second nucleic acid is different from the first nucleic acid; and d) expanding the population of cells in vitro, wherein at least 70%, 80%, 90%, or 95% of the cells in the population of cells are viable 24 hours after final contact with the LNP composition. In some embodiments, at least 70% of the cells in the population of cells are viable 24 hours after final contact with the LNP composition. In some embodiments, at least 80% of the cells in the population of cells are viable 24 hours after last contact with an LNP composition. In some embodiments, at least 90% of the cells in the population of cells are viable 24 hours after last contact with an LNP composition. In some embodiments, at least 95% of the cells in the population of cells are viable 24 hours after last contact with an LNP composition. In some embodiments, the population of cells and the cultured contacted cell population are contacted with a total of 2 to 12 LNP compositions. In some embodiments, the population of cells and the cultured contacted cell population are contacted with a total of 2 to 8 LNP compositions. In some embodiments, the population of cells and the cultured contacted cell population are contacted with a total of 2 to 6 LNP compositions. In some embodiments, the population of cells and the cultured contacted cell population are contacted with a total of 3 to 8 LNP compositions. In some embodiments, the population of cells and the cultured contacted cell population are contacted with a total of 3 to 6 LNP compositions. In some embodiments, the population of cells and the cultured contacted cell population are contacted with a total of 4 to 6 LNP compositions.In some embodiments, the population of cells and the cultured contacted cell population are contacted with a total of 6 to 12 LNP compositions. In some embodiments, the population of cells and the cultured contacted cell population are contacted with a total of 3 LNP compositions. In some embodiments, the population of cells and the cultured contacted cell population are contacted with a total of 4 LNP compositions. In some embodiments, the population of cells and the cultured contacted cell population are contacted with a total of 6 LNP compositions. In some embodiments, the population of cells and the cultured contacted cell population are contacted with a total of 3 LNP compositions. In some embodiments, the population of cells are contacted with the LNP compositions simultaneously. In some embodiments, the population of cells are contacted with 6 or fewer LNP compositions simultaneously. In some embodiments, the population of cells are contacted with 2 or fewer LNP compositions simultaneously.

[0048] In some embodiments, a method for delivering lipid-nucleic acid assembly compositions to in vitro cultured cells is provided, comprising: a) contacting the cells in vitro with at least a first lipid-nucleic acid assembly composition comprising a first nucleic acid, thereby producing contacted cells; b) culturing the contacted cells in vitro, thereby producing cultured contacted cells; c) contacting the cultured contacted cells in vitro with at least a second lipid-nucleic acid assembly composition comprising a second nucleic acid, wherein the second nucleic acid is different from the first nucleic acid; and d) expanding the cells in vitro, wherein the expanded cells exhibit increased survival, and one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRAC. In some embodiments, one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRBC. In some embodiments, one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting a gene that reduces or eliminates surface expression of MHC class I. In some embodiments, one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting B2M. In some embodiments, one of the lipid nucleic acid assembly compositions comprises a gRNA targeting HLA-A, and optionally the cells are homozygous for HLA-B and homozygous for HLA-C. In some embodiments, one of the lipid nucleic acid assembly compositions comprises a gRNA targeting a gene that reduces or eliminates surface expression of MHC class II. In some embodiments, one of the lipid nucleic acid assembly compositions comprises a gRNA targeting CIITA.

[0049] In some embodiments, a method for delivering a lipid-nucleic acid assembly composition to in vitro cultured cells is provided, comprising: a) contacting the cells in vitro with at least a first lipid-nucleic acid assembly composition comprising a first nucleic acid, thereby producing contacted cells; b) culturing the contacted cells in vitro, thereby producing cultured contacted cells; c) contacting the cultured contacted cells in vitro with at least a second lipid-nucleic acid assembly composition comprising a second nucleic acid, wherein the second nucleic acid is different from the first nucleic acid; and d) expanding the cells in vitro, wherein the expanded cells exhibit increased survival, and wherein the first and second lipid-nucleic acid compositions each comprise a gRNA selected from: a) a gRNA targeting TRAC, b) a gRNA targeting TRBC, a gRNA targeting B2M, or a gRNA targeting HLA-A, and d) a gRNA targeting CIITA. In some embodiments, the additional lipid-nucleic acid assembly composition comprises an RNA-guided DNA-binding agent. In some embodiments, the RNA-guided DNA-binding agent is Cas9. In some embodiments, the additional lipid-nucleic acid assembly composition comprises a donor nucleic acid.

[0050] In some embodiments, a method for delivering lipid-nucleic acid assembly compositions to in vitro cultured cells is provided, comprising: a) contacting the cells in vitro with at least a first lipid-nucleic acid assembly composition comprising a first nucleic acid, thereby producing contacted cells; b) culturing the contacted cells in vitro, thereby producing cultured contacted cells; c) contacting the cultured contacted cells in vitro with at least a second lipid-nucleic acid assembly composition comprising a second nucleic acid, wherein the second nucleic acid is different from the first nucleic acid; and d) expanding the cells in vitro, wherein the expanded cells exhibit increased survival, and wherein one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRAC and one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRBC. In some embodiments, the additional lipid-nucleic acid assembly composition comprises an RNA-guided DNA-binding agent. In some embodiments, the RNA-guided DNA-binding agent is Cas9. In some embodiments, the additional lipid-nucleic acid assembly composition comprises a donor nucleic acid.

[0051] In some embodiments, a method for delivering a lipid-nucleic acid assembly composition to in vitro cultured cells is provided, comprising: a) contacting the cells in vitro with at least a first lipid-nucleic acid assembly composition comprising a first nucleic acid, thereby producing contacted cells; b) culturing the contacted cells in vitro, thereby producing cultured contacted cells; c) contacting the cultured contacted cells in vitro with at least a second lipid-nucleic acid assembly composition comprising a second nucleic acid, wherein the second nucleic acid is different from the first nucleic acid; and d) expanding the cells in vitro, wherein the expanded cells exhibit increased survival, and wherein one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRAC, one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRBC, and a further lipid-nucleic acid assembly composition comprises a gRNA targeting B2M. In some embodiments, the further lipid-nucleic acid assembly composition comprises an RNA-guided DNA-binding agent. In some embodiments, the RNA-guided DNA-binding agent is Cas9. In some embodiments, the further lipid-nucleic acid assembly composition comprises a donor nucleic acid.

[0052] In some embodiments, a method for delivering a lipid-nucleic acid assembly composition to in vitro cultured cells is provided, comprising: a) contacting the cells in vitro with at least a first lipid-nucleic acid assembly composition comprising a first nucleic acid, thereby producing contacted cells; b) culturing the contacted cells in vitro, thereby producing cultured contacted cells; c) contacting the cultured contacted cells in vitro with at least a second lipid-nucleic acid assembly composition comprising a second nucleic acid, wherein the second nucleic acid is different from the first nucleic acid; and d) expanding the cells in vitro, wherein the expanded cells exhibit increased survival, and wherein one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRAC, one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRBC, and a further lipid-nucleic acid assembly composition comprises a gRNA targeting HLA-A, and optionally the cells are homozygous for HLA-B and homozygous for HLA-C. In some embodiments, the further lipid-nucleic acid assembly composition comprises an RNA-guided DNA-binding agent. In some embodiments, the RNA-guided DNA-binding agent is Cas9. In some embodiments, the additional lipid-nucleic acid assembly composition comprises a donor nucleic acid.

[0053] In some embodiments, a method for delivering lipid-nucleic acid assembly compositions to in vitro cultured cells is provided, comprising: a) contacting the cells in vitro with at least a first lipid-nucleic acid assembly composition comprising a first nucleic acid, thereby producing contacted cells; b) culturing the contacted cells in vitro, thereby producing cultured contacted cells; c) contacting the cultured contacted cells in vitro with at least a second lipid-nucleic acid assembly composition comprising a second nucleic acid, wherein the second nucleic acid is different from the first nucleic acid; and d) expanding the cells in vitro, wherein the expanded cells exhibit increased survival, and wherein one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRAC, one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRBC, a further lipid-nucleic acid assembly composition comprises a gRNA targeting B2M, and a further lipid-nucleic acid assembly composition comprises a gRNA targeting CIITA. In some embodiments, the further lipid-nucleic acid assembly composition comprises an RNA-guided DNA-binding agent. In some embodiments, the RNA-guided DNA-binding agent is Cas9. In some embodiments, the additional lipid-nucleic acid assembly composition comprises a donor nucleic acid.

[0054] In some embodiments, a method is provided for delivering lipid nucleic acid assembly compositions to in vitro cultured cells, the method comprising: a) contacting the cells in vitro with at least a first lipid nucleic acid assembly composition comprising a first nucleic acid, thereby producing contacted cells; b) culturing the contacted cells in vitro, thereby producing cultured contacted cells; c) contacting the cultured contacted cells in vitro with at least a second lipid nucleic acid assembly composition comprising a second nucleic acid, wherein the second nucleic acid is different from the first nucleic acid; and d) expanding the cells in vitro, wherein the expanded cells exhibit increased survival, and wherein one of the lipid nucleic acid assembly compositions comprises a gRNA targeting TRAC, one of the lipid nucleic acid assembly compositions comprises a gRNA targeting TRBC, and a further lipid nucleic acid assembly composition comprises a gRNA targeting HLA-A, and optionally the cells are homozygous for HLA-B and homozygous for HLA-C, and the further lipid nucleic acid assembly composition comprises a gRNA targeting CIITA. In some embodiments, the additional lipid nucleic acid assembly composition comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the additional lipid nucleic acid assembly composition comprises a donor nucleic acid.

[0055] In some embodiments, the donor nucleic acid encodes a targeting receptor. A "targeting receptor" is a polypeptide present on the surface of a cell, e.g., a T cell, that enables the cell to bind to a target site, e.g., a specific cell or tissue in 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., expressed on B cells). In some embodiments, the targeting receptor is a chemokine receptor. In some embodiments, the targeting receptor is a cytokine receptor.

[0056] β2M or B2M are used interchangeably herein to refer to the nucleic acid or protein sequence of β-2 microglobulin, the human gene having accession number NC_000015 (range 44711492..44718877), reference GRCh38.p13. The B2M protein associates 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.

[0057] CIITA or CIITA or C2TA are used interchangeably herein to refer to the nucleic acid or protein sequence of the class II major histocompatibility complex transactivator, the human gene having accession number NC_000016.10 (range 10866208..10941562), reference GRCh38.p13. The nuclear CIITA protein functions as a positive regulator of MHC class II gene transcription and is required for MHC class II protein expression.

[0058] MHC, or MHC molecule, or MHC protein, or MHC complex refers to a 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 meant 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.

[0059] As used herein in the context of HLA-A protein, HLA-A refers to an MHC class I protein molecule that is a heterodimer consisting of 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 the gene encoding the heavy chain of the HLA-A protein molecule. The HLA-A gene is also referred to as the HLA class I histocompatibility, A alpha chain, and the human gene has the accession number NC_000006.12 (29942532..29945870). The HLA-A gene is known to have hundreds of different versions (also referred to as alleles) throughout the population (individuals 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.

[0060] 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 has the accession number NC_000006.12 (31353875..31357179).

[0061] 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 has the accession number NC_000006.12 (31268749..31272092).

[0062] The term homozygous refers to having two identical alleles of a particular gene.

[0063] Any cell type described herein can be used in the delivery methods, including cells useful in ACT therapy, such as stem cells, progenitor cells, and primary cells.

[0064] In some embodiments, the lipid nucleic acid assembly composition is pretreated with serum factors before contacting the cells. In some embodiments, the lipid nucleic acid assembly composition is pretreated with human serum before contacting the cells. In some embodiments, the lipid nucleic acid assembly composition is pretreated with ApoE before contacting the cells. In some embodiments, the lipid nucleic acid assembly composition is pretreated with recombinant ApoE3 or ApoE4 before contacting the cells. In some embodiments, the cells are serum starved before contacting the lipid nucleic acid assembly composition.

[0065] In some embodiments, the multiplex method involves pre-incubating the serum factors and lipid-nucleic acid assembly composition for about 30 seconds to overnight. In some embodiments, the pre-incubation step involves pre-incubating the serum factors and lipid-nucleic acid assembly composition for about 1 minute to 1 hour. In some embodiments, it involves pre-incubating for about 1 to 30 minutes. In other embodiments, it involves pre-incubating for about 1 to 10 minutes. Yet further embodiments involve pre-incubating for about 5 minutes.

[0066] In some embodiments, the pre-incubation step occurs at about 4° C. In some embodiments, the pre-incubation step occurs at about 25° C. In certain embodiments, the pre-incubation step occurs at about 37° C. The pre-incubation step may include a buffer such as sodium bicarbonate or HEPES.

[0067] B. Multiplex Genome Editing In some embodiments, methods are provided for producing multiple genome edits in cells in vitro (sometimes referred to herein and elsewhere as "multiplexing" or "multiple gene editing" or "multiple genome editing"). In some embodiments, the methods include culturing cells in vitro, contacting the cells 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 expanding the cells in vitro. The methods result in cells with more than one genome edit, where the genome edits are different. In some embodiments, the methods result in cells with a single genome edit.

[0068] The terms "genome editing" and "gene editing" are used interchangeably herein. The terms "genome editing tool" and "gene editing tool" are also used interchangeably herein. The terms "nucleic acid genome editing tool" and "genome editing tool" may also be used interchangeably herein.

[0069] In some embodiments, a method for producing multiple genome edits in in vitro cultured cells is provided, comprising: a) contacting the cells in vitro with at least a first lipid nanoparticle (LNP) composition and a second LNP composition, wherein the first LNP composition comprises a first guide RNA (gRNA) directed to a first target sequence and optionally a nucleic acid genome editing tool, and the second LNP composition comprises a second gRNA directed to a second target sequence different from the first target sequence and optionally a nucleic acid genome editing tool; and b) expanding the cells in vitro, thereby producing multiple genome edits in the cells. In some embodiments, the cells are contacted with at least one LNP composition comprising a genome editing tool. In some embodiments, the genome editing tool comprises a nucleic acid encoding an RNA-guided DNA binder. In some embodiments, the cells are further contacted with a donor nucleic acid for insertion at the target sequence. In some embodiments, the LNP compositions are administered sequentially. In some embodiments, the LNP compositions are administered simultaneously. In some embodiments, the population of cells is contacted with 2 to 12 LNP compositions. In some embodiments, the population of cells is contacted with 2 to 8 LNP compositions. In some embodiments, the population of cells is contacted with 2 to 6 LNP compositions. In some embodiments, the population of cells is contacted with 3 to 8 LNP compositions. In some embodiments, the population of cells is contacted with 3 to 6 LNP compositions. In some embodiments, the population of cells is contacted with 4 to 6 LNP compositions. In some embodiments, the population of cells is contacted with 6 to 12 LNP compositions. In some embodiments, the population of cells is contacted with 3 LNP compositions. In some embodiments, the population of cells is contacted with 4 LNP compositions. In some embodiments, the population of cells is contacted with 6 LNP compositions. In some embodiments, the population of cells is contacted with 3 LNP compositions. In some embodiments, the population of cells is contacted with 6 LNP compositions. In some embodiments, the population of cells is contacted with 3 LNP compositions simultaneously.In some embodiments, the population of cells is contacted with no more than six LNP compositions simultaneously. In some embodiments, the population of cells is contacted with no more than two LNP compositions simultaneously.

[0070] In some embodiments, a method for producing multiple genome edits in in vitro cultured cells is provided, comprising: a) contacting the cells in vitro with at least a first lipid nanoparticle (LNP) composition and a second LNP composition, wherein the first lipid LNP composition comprises a first guide RNA (gRNA) directed to a first target sequence and optionally a nucleic acid genome editing tool, and the second LNP composition comprises a second gRNA directed to a second target sequence different from the first target sequence and optionally a nucleic acid genome editing tool; and b) culturing the cells ex vivo, thereby producing multiple genome edits in the cells. In some embodiments, the cells are contacted with at least one LNP composition comprising a genome editing tool. In some embodiments, the genome editing tool comprises a nucleic acid encoding an RNA-guided DNA binder. In some embodiments, the cells are further contacted with a donor nucleic acid for insertion at the target sequence. In some embodiments, the LNP compositions are administered sequentially. In some embodiments, the LNP compositions are administered simultaneously. In some embodiments, the population of cells is contacted with 2 to 12 LNP compositions. In some embodiments, the population of cells is contacted with 2 to 8 LNP compositions. In some embodiments, the population of cells is contacted with 2 to 6 LNP compositions. In some embodiments, the population of cells is contacted with 3 to 8 LNP compositions. In some embodiments, the population of cells is contacted with 3 to 6 LNP compositions. In some embodiments, the population of cells is contacted with 4 to 6 LNP compositions. In some embodiments, the population of cells is contacted with 6 to 12 LNP compositions. In some embodiments, the population of cells is contacted with 3 LNP compositions. In some embodiments, the population of cells is contacted with 4 LNP compositions. In some embodiments, the population of cells is contacted with 6 LNP compositions. In some embodiments, the population of cells is contacted with 3 LNP compositions. In some embodiments, the population of cells is contacted with 6 LNP compositions. In some embodiments, the population of cells is contacted with 3 LNP compositions simultaneously.In some embodiments, the population of cells is contacted with no more than six LNP compositions simultaneously. In some embodiments, the population of cells is contacted with no more than two LNP compositions simultaneously.

[0071] In some embodiments, a method for gene editing in a population of cells is provided, comprising: a) contacting the population of cells in vitro with a first lipid nanoparticle (LNP) composition comprising a first genome editing tool and a second LNP composition comprising a second genome editing tool; and b) culturing the population of cells in vitro, wherein at least 70%, 80%, 90%, or 95% of the cells in the population of cells are viable 24 hours after last contact with the LNP composition, thereby editing the population of cells. In some embodiments, at least 70% of the cells in the population of cells are viable 24 hours after last contact with the LNP composition. In some embodiments, at least 80% of the cells in the population of cells are viable 24 hours after last contact with the LNP composition. In some embodiments, at least 90% of the cells in the population of cells are viable 24 hours after last contact with the LNP composition. In some embodiments, at least 95% of the cells in the population of cells are viable 24 hours after last contact with the LNP composition. In some embodiments, the first genome editing tool comprises a guide RNA. In some embodiments, the method further comprises contacting the cell in vitro with a third LNP composition comprising a genome editing tool, wherein at least two LNP compositions comprise a gRNA. In some embodiments, at least one LNP composition comprises an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is Cas9. In some embodiments, the method further comprises contacting the cell with a donor nucleic acid for insertion at the target sequence. In some embodiments, the second genome editing tool is an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is S. pyogenes Cas9.

[0072] In some embodiments, a method for gene editing in a cell is provided, comprising: a) contacting the cell in vitro with at least six lipid-nucleic acid assembly compositions, wherein at least two to four of the lipid-nucleic acid assembly compositions each comprise a guide RNA (gRNA), and at least one lipid-nucleic acid assembly composition comprises a first genome editing tool; and b) expanding the cell in vitro, thereby editing the cell. In some embodiments, the first genome editing tool comprises a guide RNA. In some embodiments, the method further comprises contacting the cell in vitro with a third lipid-nucleic acid assembly composition comprising a genome editing tool, wherein at least two lipid-nucleic acid assembly compositions comprise a gRNA. In some embodiments, at least one lipid-nucleic acid assembly composition comprises an RNA-guided DNA-binding agent. In some embodiments, the RNA-guided DNA-binding agent is Cas9. In some embodiments, the method further comprises contacting the cell with a donor nucleic acid. In some embodiments, the second genome editing tool is Cas9. In some embodiments, the cell is a T cell. In some embodiments, the cell is a non-activated cell. In some embodiments, the cells are activated cells. In some embodiments, the cells of (a) are activated after contact with at least one lipid-nucleic acid assembly composition.

[0073] In some embodiments, cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 lipid-nucleic acid assembly compositions. In some embodiments, this results in cells with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more genome edits, e.g., based on different gRNAs.

[0074] In some embodiments, cells are contacted with one or more lipid-nucleic acid assembly compositions comprising one or more genome editing tools in a single lipid-nucleic acid assembly composition. In some embodiments, the single lipid-nucleic acid assembly composition comprises multiple guide RNAs. In some embodiments, the single lipid-nucleic acid assembly composition comprises 2-8, 2-6, 2-5, 2-4, 3-5, or 3-6 guide RNAs. In some embodiments, the single lipid-nucleic acid assembly composition comprises 3-5 or 3-6 guide RNAs. In certain embodiments, lipid-nucleic acid assembly compositions comprising more than one guide RNA further comprise an RNA-guided DNA binding agent. In certain embodiments, lipid-nucleic acid assembly compositions comprising more than one guide RNA do not comprise an RNA-guided DNA binding agent.

[0075] In some embodiments, contact between the cells and the lipid nucleic acid assembly compositions is sequential (one after the other). In some embodiments, contact between the cells and the lipid nucleic acid assembly compositions is simultaneous (contact is simultaneous or near simultaneous). In some embodiments, multiple lipid nucleic acid assembly compositions are administered sequentially. In some embodiments, the lipid nucleic acid assembly compositions are administered simultaneously. In some embodiments, the lipid nucleic acid assembly compositions are administered sequentially and simultaneously. In some embodiments, three lipid nucleic acid compositions are provided, two lipid nucleic acid compositions are administered first simultaneously, the cells are cultured for a period of time, and then the third lipid nucleic acid composition is administered (i.e., sequentially after the administration of the first two compositions). In another embodiment, three lipid nucleic acid compositions are provided, one lipid nucleic acid composition is administered first, the cells are cultured for a period of time, and then two lipid nucleic acid compositions are administered simultaneously (and sequentially after the administration of the first composition). Thus, simultaneous and sequential administration of lipid nucleic acid assembly compositions can overlap in certain embodiments. In some embodiments, the first and second lipid nucleic acid assembly compositions each comprise a gRNA directed to a target sequence, and optionally each also comprise an RNA-guided DNA binding agent. In some embodiments, the first and second lipid nucleic acid assembly compositions each comprise a gRNA directed to a target sequence and may further comprise an RNA-guided DNA binding agent. In other words, the RNA-guided DNA binding agent may, in some embodiments, be provided to a cell by means other than a gRNA-containing lipid nucleic acid assembly composition. In some embodiments, the gRNA and the RNA-guided DNA binding agent may be co-encapsulated in a lipid nucleic acid assembly composition. In some embodiments, the gRNA and the RNA-guided DNA binding agent may be provided to a cell in separate lipid nucleic acid assembly compositions. In some embodiments, a lipid nucleic acid assembly comprising an RNA-guided DNA binding agent is first administered simultaneously with the guide RNA, either in the same lipid nucleic acid assembly or in a different lipid nucleic acid assembly, followed by sequential administration of the guide RNA without further administration of the RNA-guided DNA binding agent.In some embodiments, a lipid nucleic acid assembly comprising an RNA-guided DNA binding agent is first administered simultaneously with the guide RNA, either in the same lipid nucleic acid assembly or in a different lipid nucleic acid assembly, followed by sequential administration of the guide RNA with an additional RNA-guided DNA binding agent, optionally where the second RNA-guided DNA binding agent is different from the first RNA-guided DNA binding agent.

[0076] In some embodiments, the cells are frozen between sequential contacting or editing steps.

[0077] In some embodiments, the lipid nucleic acid assembly composition is pretreated with serum factors before contacting the cells. In some embodiments, the lipid nucleic acid assembly composition is pretreated with human serum before contacting the cells. In some embodiments, the lipid nucleic acid assembly composition is pretreated with a serum replacement, for example, a commercially available serum replacement, preferably a serum replacement suitable for ex vivo use. In some embodiments, the lipid nucleic acid assembly composition is pretreated with ApoE before contacting the cells. In some embodiments, the lipid nucleic acid assembly composition is pretreated with recombinant ApoE3 or ApoE4 before contacting the cells. In some embodiments, the cells are serum starved before contacting the lipid nucleic acid assembly composition.

[0078] In some embodiments, the multiplex method involves pre-incubating the serum factors and lipid-nucleic acid assembly composition for about 30 seconds to overnight. In some embodiments, the pre-incubation step involves pre-incubating the serum factors and lipid-nucleic acid assembly composition for about 1 minute to 1 hour. In some embodiments, it involves pre-incubating for about 1 to 30 minutes. In other embodiments, it involves pre-incubating for about 1 to 10 minutes. Yet further embodiments involve pre-incubating for about 5 minutes.

[0079] In some embodiments, the pre-incubation step occurs at about 4° C. In some embodiments, the pre-incubation step occurs at about 25° C. In some embodiments, the pre-incubation step occurs at about 37° C. The pre-incubation step may include a buffer such as sodium bicarbonate or HEPES.

[0080] In some embodiments, lipid-nucleic acid assembly compositions are provided to "non-activated" cells. "Non-activated" cells refer to cells that have not been stimulated in vitro. In some embodiments, "non-activated" T cells may have been stimulated in vivo (e.g., by an antigen) while in the body, but if the cells have not been stimulated in vitro in culture, the cells may be referred to herein as non-activated. "Activated" cells are also useful in the methods disclosed herein and can refer to cells that have been stimulated in vitro. Agents for activating cells in vitro are provided herein and are known in the art, particularly for activating T cells or B cells.

[0081] In some embodiments, T cells are cultured in culture medium prior to contact with the lipid nucleic acid assembly composition. In some embodiments, T cells are cultured with one or more proliferative cytokines, e.g., one or more or all of IL-2, IL-15, and IL-21, and / or one or more agents that provide activation via CD3 and / or CD28.

[0082] In some embodiments, the T cells are activated prior to contact with the lipid nucleic acid assembly composition, activated during contact with the lipid nucleic acid assembly composition, and / or activated after contact with the lipid nucleic acid assembly composition.

[0083] In some embodiments, the cells are T cells, and the method further comprises an activation step between the first and second contacting steps. In some embodiments, non-activated T cells are contacted with 1, 2, or 3 nucleic acid assembly compositions. In some embodiments, activated T cells are contacted with 1 to 8 lipid-nucleic acid assembly compositions, optionally 1 to 4 lipid-nucleic acid assembly compositions. In some embodiments, T cells are contacted with at least 6 lipid-nucleic acid assembly compositions. In some embodiments, T cells are contacted with 12 or fewer lipid-nucleic acid assembly compositions. In some embodiments, T cells are contacted with 2 to 12 lipid-nucleic acid assembly compositions. In some embodiments, T cells are contacted with 2 to 8 lipid-nucleic acid assembly compositions. In some embodiments, T cells are contacted with 2 to 6 lipid-nucleic acid assembly compositions. In some embodiments, T cells are contacted with 3 to 8 lipid-nucleic acid assembly compositions. In some embodiments, T cells are contacted with 3 to 6 lipid-nucleic acid assembly compositions. In some embodiments, T cells are contacted with 4 to 6 lipid-nucleic acid assembly compositions. In some embodiments, T cells are contacted with 4 to 12 lipid nucleic acid assembly compositions. In some embodiments, T cells are contacted with 4 to 8 lipid nucleic acid assembly compositions. In some embodiments, T cells are contacted with 6 to 12 lipid nucleic acid assembly compositions. In some embodiments, T cells are contacted with 3, 4, 5, or 6 lipid nucleic acid assembly compositions. In some embodiments, T cells are contacted simultaneously with 8 or fewer lipid nucleic acid assembly compositions. In some embodiments, T cells are contacted simultaneously with 6 or fewer lipid nucleic acid assembly compositions. In some embodiments, activated T cells are contacted with at least 6 lipid nucleic acid assembly compositions. In some embodiments, activated T cells are contacted with 12 or fewer lipid nucleic acid assembly compositions. In some embodiments, activated T cells are contacted with 2 to 12 lipid nucleic acid assembly compositions.In some embodiments, activated T cells are contacted with 4 to 12 lipid nucleic acid assembly compositions. In some embodiments, activated T cells are contacted with 4 to 8 lipid nucleic acid assembly compositions. In some embodiments, activated T cells are contacted with 8 or fewer lipid nucleic acid assembly compositions simultaneously. In some embodiments, activated T cells are contacted with 6 or fewer lipid nucleic acid assembly compositions simultaneously.

[0084] In some embodiments, T cells are contacted with at least a first lipid-nucleic acid assembly composition comprising a first nucleic acid genome editing tool targeting a first target sequence, activated, and the activated T cells are contacted with at least a second lipid-nucleic acid assembly composition comprising a second nucleic acid genome editing tool targeting a second target sequence. The activated T cells can be further contacted with additional lipid-nucleic acid assembly compositions. In some embodiments, T cells are contacted with two lipid-nucleic acid assembly compositions, activated, and the activated cells are contacted with a third lipid-nucleic acid assembly composition, and optionally the activated cells are contacted with an additional lipid-nucleic acid assembly composition. In some embodiments, T cells are contacted with three lipid-nucleic acid assembly compositions, activated, and the activated cells are contacted with a third lipid-nucleic acid assembly composition, and optionally the activated cells are contacted with an additional lipid-nucleic acid assembly composition. The activation step can improve the results of multiple genome editing compared to the same method without the activation step.

[0085] In some embodiments, a method for producing multiple genome edits in in vitro cultured T cells is provided, comprising: a) contacting the T cells in vitro with (i) a first lipid-nucleic acid assembly composition comprising a guide RNA (gRNA) directed to a first target sequence, and optionally (ii) one or two additional lipid-nucleic acid assembly compositions, wherein each additional lipid-nucleic acid assembly composition comprises a gRNA and / or genome editing tool directed to a target sequence that is different from the first target sequence; b) activating the T cells in vitro; c) contacting the activated T cells in vitro with (i) an additional nucleic acid assembly composition comprising an additional guide RNA directed to a target sequence that is different from the target sequence of (a), and optionally (ii) one or more lipid-nucleic acid assembly compositions, wherein each lipid-nucleic acid assembly composition comprises a guide RNA and / or genome editing tool directed to a target sequence that is different from the target sequence of (a) and that is different from each other; and d) expanding the cells in vitro, thereby producing multiple genome edits in the T cells. In some embodiments, the methods comprise contacting T cells with 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 lipid-nucleic acid assembly compositions, optionally 4-12 or 4-8 lipid-nucleic acid assembly compositions. In some embodiments, the methods comprise contacting cells or T cells with 4-12 or 4-8 lipid-nucleic acid assembly compositions. In some embodiments, the T cells of step (a) are contacted with two lipid-nucleic acid assembly compositions, and the lipid-nucleic acid assembly compositions are administered sequentially or simultaneously. In some embodiments, the T cells of step (a) are contacted with three lipid-nucleic acid assembly compositions, and the lipid-nucleic acid assembly compositions are administered (i) sequentially, (ii) simultaneously, or (iii) simultaneously (two compositions) and sequentially (one composition administered before or after).In some embodiments, the T cells of step (c) are contacted with between 1 and 8 lipid nucleic acid assembly compositions, optionally between 1 and 4 lipid nucleic acid assembly compositions, and the lipid nucleic acid assembly compositions are administered (i) sequentially, (ii) simultaneously, or (iii) simultaneously (at least two compositions) and sequentially (administered before or after at least one composition).

[0086] In some embodiments, a method for gene editing in a cell is provided, comprising: a) contacting the cell in vitro with a first lipid-nucleic acid assembly composition comprising a first genome editing tool and a second lipid-nucleic acid assembly composition comprising a second genome editing tool; and b) expanding the cell in vitro, thereby editing the cell, wherein one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRAC. In some embodiments, one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRBC. In some embodiments, one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting a gene that reduces or eliminates surface expression of MHC class I. In some embodiments, one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting B2M. In some embodiments, one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting HLA-A, and optionally, the cell is homozygous for HLA-B and homozygous for HLA-C. In some embodiments, one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting a gene that reduces or eliminates surface expression of MHC class II. In some embodiments, one of the lipid-nucleic acid assembly compositions comprises a gRNA that targets CIITA.

[0087] In some embodiments, a method for gene editing in a cell is provided, comprising: a) contacting the cell in vitro with a first lipid-nucleic acid assembly composition comprising a first genome editing tool and a second lipid-nucleic acid assembly composition comprising a second genome editing tool; and b) expanding the cell in vitro, thereby editing the cell, wherein the first and second lipid-nucleic acid compositions each comprise a gRNA selected from a) a gRNA targeting TRAC, b) a gRNA targeting TRBC, c) a gRNA targeting B2M or a gRNA targeting HLA-A, and d) a gRNA targeting CIITA. In some embodiments, the additional lipid-nucleic acid assembly composition comprises an RNA-guided DNA-binding agent. In some embodiments, the RNA-guided DNA-binding agent is Cas9. In some embodiments, the additional lipid-nucleic acid assembly composition comprises a donor nucleic acid.

[0088] In some embodiments, a method for gene editing in a cell is provided, comprising: a) contacting the cell in vitro with a first lipid-nucleic acid assembly composition comprising a first genome editing tool and a second lipid-nucleic acid assembly composition comprising a second genome editing tool; and b) expanding the cell in vitro, thereby editing the cell, wherein one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRAC and one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRBC. In some embodiments, the additional lipid-nucleic acid assembly composition comprises an RNA-guided DNA-binding agent. In some embodiments, the RNA-guided DNA-binding agent is Cas9. In some embodiments, the additional lipid-nucleic acid assembly composition comprises a donor nucleic acid.

[0089] In some embodiments, a method for gene editing in a cell is provided, comprising: a) contacting the cell in vitro with a first lipid-nucleic acid assembly composition comprising a first genome editing tool and a second lipid-nucleic acid assembly composition comprising a second genome editing tool; and b) expanding the cell in vitro, thereby editing the cell, wherein one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRAC, one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRBC, and a further lipid-nucleic acid assembly composition comprises a gRNA targeting B2M. In some embodiments, the further lipid-nucleic acid assembly composition comprises an RNA-guided DNA-binding agent. In some embodiments, the RNA-guided DNA-binding agent is Cas9. In some embodiments, the further lipid-nucleic acid assembly composition comprises a donor nucleic acid.

[0090] In some embodiments, a method for gene editing in a cell is provided, comprising: a) contacting the cell in vitro with a first lipid-nucleic acid assembly composition comprising a first genome editing tool and a second lipid-nucleic acid assembly composition comprising a second genome editing tool; and b) expanding the cell in vitro, thereby editing the cell, wherein one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRAC, one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRBC, and a further lipid-nucleic acid assembly composition comprises a gRNA targeting HLA-A, and optionally the cell is homozygous for HLA-B and homozygous for HLA-C. In some embodiments, the further lipid-nucleic acid assembly composition comprises an RNA-guided DNA-binding agent. In some embodiments, the RNA-guided DNA-binding agent is Cas9. In some embodiments, the further lipid-nucleic acid assembly composition comprises a donor nucleic acid.

[0091] In some embodiments, a method for gene editing in a cell is provided, comprising: a) contacting the cell in vitro with a first lipid-nucleic acid assembly composition comprising a first genome editing tool and a second lipid-nucleic acid assembly composition comprising a second genome editing tool; and b) expanding the cell in vitro, thereby editing the cell, wherein one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRAC, one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRBC, a further lipid-nucleic acid assembly composition comprises a gRNA targeting B2M, and a further lipid-nucleic acid assembly composition comprises a gRNA targeting CIITA. In some embodiments, the further lipid-nucleic acid assembly composition comprises an RNA-guided DNA-binding agent. In some embodiments, the RNA-guided DNA-binding agent is Cas9. In some embodiments, the further lipid-nucleic acid assembly composition comprises a donor nucleic acid.

[0092] In some embodiments, a method for gene editing in a cell is provided, comprising: a) contacting the cell in vitro with a first lipid-nucleic acid assembly composition comprising a first genome editing tool and a second lipid-nucleic acid assembly composition comprising a second genome editing tool; and b) expanding the cell in vitro, thereby editing the cell, wherein one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRAC, one of the lipid-nucleic acid assembly compositions comprises a gRNA targeting TRBC, and a further lipid-nucleic acid assembly composition comprises a gRNA targeting HLA-A; and optionally, the cell is homozygous for HLA-B and homozygous for HLA-C, and the further lipid-nucleic acid assembly composition comprises a gRNA targeting CIITA. In some embodiments, the further lipid-nucleic acid assembly composition comprises an RNA-guided DNA-binding agent. In some embodiments, the RNA-guided DNA-binding agent is Cas9. In some embodiments, the further lipid-nucleic acid assembly composition comprises a donor nucleic acid.

[0093] In some embodiments, T cells are activated by polyclonal activation (or "polyclonal stimulation") (rather than antigen-specific stimulation). In some embodiments, T cells are activated by CD3 stimulation (e.g., providing an anti-CD3 antibody). In some embodiments, T cells are activated by CD3 and CD28 stimulation (e.g., providing an anti-CD3 antibody and an anti-CD28 antibody). In some embodiments, T cells are activated using ready-to-use reagents for activating T cells (e.g., via CD3 / CD28 stimulation). In some embodiments, T cells are activated via CD3 / CD28 stimulation provided by beads. In some embodiments, T cells are activated via CD3 / CD28 stimulation, where one or more components are soluble and / or one or more components are bound to a solid surface (e.g., a plate or beads). In some embodiments, T cells are activated by an antigen-independent mitogen (e.g., a lectin, including, for example, concanavalin A ("ConA"), or PHA).

[0094] In some embodiments, one or more cytokines are used for T cell activation. IL-2 is provided for T cell activation. In some embodiments, the cytokine for T cell activation is a cytokine that binds 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-7 is provided to promote T cell survival. 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 for T cell activation is provided, including, for example, IL-2, IL-7, IL-15, and / or IL-21.

[0095] 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 in a major histocompatibility complex ("MHC") molecule (peptide-MHC complex). Cognate antigen can be presented to 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-presenting cells that have been pulsed with the antigen. In some embodiments, the antigen-presenting cells are pulsed with a peptide of the antigen.

[0096] In some embodiments, T cells may be activated for 12 to 72 hours. In some embodiments, T cells may be activated for 12 to 48 hours. In some embodiments, T cells may be activated for 12 to 24 hours. In some embodiments, T cells may be activated for 24 to 48 hours. In some embodiments, T cells may be activated for 24 to 72 hours. In some embodiments, T cells may be activated for 12 hours. In some embodiments, T cells may be activated for 48 hours. In some embodiments, T cells may be activated for 72 hours.

[0097] In some embodiments, the methods provided herein do not include a selection step. In some embodiments, a selection step is included, optionally a physical sorting step (e.g., FACS or MACS) or a biochemical selection step (e.g., suicide gene, drug resistance selection, or antibody-toxin conjugate selection).

[0098] The lipid nucleic acid assembly composition disclosed herein can be used in in vitro multiple genome editing method.This method overcomes the existing problems associated with such method by reducing the toxicity associated with the transfection process itself.The reduced toxicity of each transfection event allows multiple transactions, thereby allowing multiple genome editing per cell.

[0099] In some embodiments, the genome editing comprises any one or more of an insertion, deletion, or substitution of at least one nucleotide in the target sequence. In some embodiments, the genome editing comprises an insertion of 1, 2, 3, 4, or 5 or more nucleotides in the target sequence. In some embodiments, the genome editing comprises a deletion of 1, 2, 3, 4, or 5 or more nucleotides in the target sequence. In other embodiments, the genome editing comprises an insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In other embodiments, the genome editing comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, the genome editing comprises an indel, which is generally defined in the art as an insertion or deletion of less than 1000 base pairs (bp). In some embodiments, the genome editing comprises an indel that results in a frameshift mutation in the target sequence. In some embodiments, the genome editing comprises the substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, the genome editing comprises one or more of an insertion, deletion, or substitution of nucleotides resulting from integration of a template nucleic acid. In some embodiments, the genome editing comprises the insertion of a donor nucleic acid in the target sequence. In some embodiments, the editing or modification is not transient.

[0100] In some embodiments, one or more donor nucleic acids are provided for insertion at a target sequence. In some embodiments, the target sequence for insertion is a safe harbor locus. A safe harbor locus is a site within a genome that can accommodate integration of an exogenous sequence without causing deleterious changes to the host genome and is known in the art. In some embodiments, the target sequence for insertion is in the beta-2 microglobulin (B2M) gene. In some embodiments, the target sequence for insertion is in the class II major histocompatibility complex transactivator (CIITA) gene. In some embodiments, the target sequence for insertion is in the TRAC gene. In some embodiments, the target sequence for insertion is in AAVS1.

[0101] III. Cell Populations and Methods / Uses A. Cell population In some embodiments, compositions are provided herein comprising a cell population comprising edited cells comprising multiple genome edits per cell. In some embodiments, cell populations comprising edited cells comprising multiple genome edits per cell are provided, wherein at least 50% of the cells in the cell population comprise at least two genome edits, and (i) less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% of the cells in the cell population have target-to-target translocations, or (ii) the cell population has less than two-fold background levels of reciprocal translocations, compound translocations, or off-target translocations. In some embodiments, at least 50% of the cells in the cell population comprise at least two genome edits, and less than 1% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 50% of the cells in the cell population comprise at least two genome edits, and less than 0.5% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 50% of the cells in the cell population comprise at least two genome edits, and less than 0.2% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 50% of the cells in the cell population comprise at least two genome edits, and less than 0.1% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 50% of the cells in the cell population comprise at least two genome edits, and the cell population has less than two-fold background levels of reciprocal, compound, or off-target translocations. In some embodiments, the cell population is capable of 20-fold, 30-fold, 40-fold, or 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 20-fold expansion ex vivo in culture within 14 days after initiation of editing.In some embodiments, the cell population is capable of 30-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 40-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 60-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 70-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 80-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 90-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least one genome edit of the plurality of genome edits is produced by a genome editing tool comprising an RNA-guided DNA binding agent, and the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, at least two genome edits of the plurality of genome edits are produced by a genome editing tool comprising an RNA-guided DNA binding agent, and the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, the plurality of genome edits comprises insertion of a donor nucleic acid, and the insertion is optionally a targeted insertion.

[0102] In some embodiments, a cell population is provided that includes edited cells that contain multiple genome edits per cell, wherein at least 60% of the cells in the cell population contain at least two genome edits, and (i) less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% of the cells in the cell population have target-to-target translocations, or (ii) the cell population has less than two-fold background levels of reciprocal translocations, compound translocations, or off-target translocations. In some embodiments, at least 60% of the cells in the cell population contain at least two genome edits, and less than 1% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 60% of the cells in the cell population contain at least two genome edits, and less than 0.5% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 60% of the cells in the cell population contain at least two genome edits, and less than 0.2% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 60% of the cells in the cell population comprise at least two genome edits, and less than 0.1% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 60% of the cells in the cell population comprise at least two genome edits, and the cell population has less than two-fold background levels of reciprocal, compound, or off-target translocations. In some embodiments, the cell population is capable of 20-fold, 30-fold, 40-fold, or 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 20-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 30-fold expansion ex vivo in culture within 14 days of initiating editing.In some embodiments, the cell population is capable of 40-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 60-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 70-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 80-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 90-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least one genome edit of the plurality of genome edits is produced by a genome editing tool comprising an RNA-guided DNA binding agent, and the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, at least two of the plurality of genome edits are produced by a genome editing tool comprising an RNA-guided DNA binding agent, wherein the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, the plurality of genome edits comprises an insertion of a donor nucleic acid, wherein the insertion is optionally a targeted insertion.

[0103] In some embodiments, a cell population is provided that includes edited cells that contain multiple genome edits per cell, wherein at least 70% of the cells in the cell population contain at least two genome edits, and (i) less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% of the cells in the cell population have target-to-target translocations, or (ii) the cell population has less than two-fold background levels of reciprocal translocations, compound translocations, or off-target translocations. In some embodiments, at least 70% of the cells in the cell population contain at least two genome edits, and less than 1% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 70% of the cells in the cell population contain at least two genome edits, and less than 0.5% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 70% of the cells in the cell population contain at least two genome edits, and less than 0.2% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 70% of the cells in the cell population comprise at least two genome edits, and less than 0.1% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 70% of the cells in the cell population comprise at least two genome edits, and the cell population has less than two-fold background levels of reciprocal, compound, or off-target translocations. In some embodiments, the cell population is capable of 20-fold, 30-fold, 40-fold, or 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 20-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 30-fold expansion ex vivo in culture within 14 days of initiating editing.In some embodiments, the cell population is capable of 40-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 60-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 70-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 80-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 90-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least one genome edit of the plurality of genome edits is produced by a genome editing tool comprising an RNA-guided DNA binding agent, and the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, at least two of the plurality of genome edits are produced by a genome editing tool comprising an RNA-guided DNA binding agent, wherein the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, the plurality of genome edits comprises an insertion of a donor nucleic acid, wherein the insertion is optionally a targeted insertion.

[0104] In some embodiments, a cell population is provided that includes edited cells that contain multiple genome edits per cell, wherein at least 80% of the cells in the cell population contain at least two genome edits, and (i) less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% of the cells in the cell population have target-to-target translocations, or (ii) the cell population has less than two-fold background levels of reciprocal translocations, compound translocations, or off-target translocations. In some embodiments, at least 80% of the cells in the cell population contain at least two genome edits, and less than 1% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 80% of the cells in the cell population contain at least two genome edits, and less than 0.5% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 80% of the cells in the cell population contain at least two genome edits, and less than 0.2% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 80% of the cells in the cell population comprise at least two genome edits, and less than 0.1% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 80% of the cells in the cell population comprise at least two genome edits, and the cell population has less than two-fold background levels of reciprocal, compound, or off-target translocations. In some embodiments, the cell population is capable of 20-fold, 30-fold, 40-fold, or 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 20-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 30-fold expansion ex vivo in culture within 14 days of initiating editing.In some embodiments, the cell population is capable of 40-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 60-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 70-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 80-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 90-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least one genome edit of the plurality of genome edits is produced by a genome editing tool comprising an RNA-guided DNA binding agent, and the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, at least two of the plurality of genome edits are produced by a genome editing tool comprising an RNA-guided DNA binding agent, wherein the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, the plurality of genome edits comprises an insertion of a donor nucleic acid, wherein the insertion is optionally a targeted insertion.

[0105] In some embodiments, a cell population is provided that includes edited cells that contain multiple genome edits per cell, wherein at least 90% of the cells in the cell population contain at least two genome edits, and (i) less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% of the cells in the cell population have target-to-target translocations, or (ii) the cell population has less than two-fold background levels of reciprocal translocations, compound translocations, or off-target translocations. In some embodiments, at least 90% of the cells in the cell population contain at least two genome edits, and less than 1% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 90% of the cells in the cell population contain at least two genome edits, and less than 0.5% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 90% of the cells in the cell population contain at least two genome edits, and less than 0.2% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 90% of the cells in the cell population comprise at least two genome edits, and less than 0.1% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 90% of the cells in the cell population comprise at least two genome edits, and the cell population has less than two-fold background levels of reciprocal, compound, or off-target translocations. In some embodiments, the cell population is capable of 20-fold, 30-fold, 40-fold, or 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 20-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 30-fold expansion ex vivo in culture within 14 days of initiating editing.In some embodiments, the cell population is capable of 40-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 60-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 70-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 80-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 90-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least one genome edit of the plurality of genome edits is produced by a genome editing tool comprising an RNA-guided DNA binding agent, and the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, at least two of the plurality of genome edits are produced by a genome editing tool comprising an RNA-guided DNA binding agent, wherein the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, the plurality of genome edits comprises an insertion of a donor nucleic acid, wherein the insertion is optionally a targeted insertion.

[0106] In some embodiments, a cell population is provided that includes edited cells that contain multiple genome edits per cell, wherein at least 95% of the cells in the cell population contain at least two genome edits, and (i) less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% of the cells in the cell population have target-to-target translocations, or (ii) the cell population has less than two-fold background levels of reciprocal translocations, compound translocations, or off-target translocations. In some embodiments, at least 95% of the cells in the cell population contain at least two genome edits, and less than 1% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 95% of the cells in the cell population contain at least two genome edits, and less than 0.5% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 95% of the cells in the cell population contain at least two genome edits, and less than 0.2% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 95% of the cells in the cell population comprise at least two genome edits, and less than 0.1% of the cells in the cell population have target-to-target translocations. In some embodiments, at least 95% of the cells in the cell population comprise at least two genome edits, and the cell population has less than two-fold background levels of reciprocal, compound, or off-target translocations. In some embodiments, the cell population is capable of 20-fold, 30-fold, 40-fold, or 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 20-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 30-fold expansion ex vivo in culture within 14 days of initiating editing.In some embodiments, the cell population is capable of 40-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 60-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 70-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 80-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 90-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least one genome edit of the plurality of genome edits is produced by a genome editing tool comprising an RNA-guided DNA binding agent, and the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, at least two of the plurality of genome edits are produced by a genome editing tool comprising an RNA-guided DNA binding agent, wherein the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, the plurality of genome edits comprises an insertion of a donor nucleic acid, wherein the insertion is optionally a targeted insertion.

[0107] In some embodiments, a cell population is provided that includes edited cells that comprise multiple genome edits per cell, wherein at least 50% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of a 20-fold, 30-fold, 40-fold, or 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of a 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 50% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 20-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 50% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 30-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 50% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 40-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 50% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 60-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 50% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 70-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 50% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of an 80-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 50% of the cells in the cell population contain at least two genome edits, and the cell population is capable of expanding 90-fold ex vivo in culture within 14 days after initiation of editing.In some embodiments, at least 50% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, fewer than 1% of the cells in the cell population have a target-to-target translocation. In some embodiments, fewer than 0.5% of the cells in the cell population have a target-to-target translocation. In some embodiments, fewer than 0.2% of the cells in the cell population have a target-to-target translocation. In some embodiments, fewer than 0.1% of the cells in the cell population have a target-to-target translocation. In some embodiments, the cell population has less than two-fold background levels of reciprocal translocations, compound translocations, or off-target translocations. In some embodiments, at least one genome edit of the plurality of genome edits is produced by a genome editing tool comprising an RNA-guided DNA binding agent, and the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, at least two genome edits of the plurality of genome edits are produced by a genome editing tool comprising an RNA-guided DNA binding agent, and the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, the plurality of genome edits comprises insertion of a donor nucleic acid, optionally a targeted insertion.

[0108] In some embodiments, a cell population is provided that includes edited cells that comprise multiple genome edits per cell, wherein at least 60% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of a 20-fold, 30-fold, 40-fold, or 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of a 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 60% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 20-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 60% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 30-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 60% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 40-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 60% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 60-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 60% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 70-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 60% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of an 80-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 60% of the cells in the cell population contain at least two genome edits, and the cell population is capable of expanding 90-fold ex vivo in culture within 14 days after initiation of editing.In some embodiments, at least 60% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, fewer than 1% of the cells in the cell population have target-to-target translocations. In some embodiments, fewer than 0.5% of the cells in the cell population have target-to-target translocations. In some embodiments, fewer than 0.2% of the cells in the cell population have target-to-target translocations. In some embodiments, fewer than 0.1% of the cells in the cell population have target-to-target translocations. In some embodiments, the cell population has less than two-fold background levels of reciprocal translocations, compound translocations, or off-target translocations. In some embodiments, at least one genome edit of the plurality of genome edits is produced by a genome editing tool comprising an RNA-guided DNA binding agent, and the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, at least two genome edits of the plurality of genome edits are produced by a genome editing tool comprising an RNA-guided DNA binding agent, and the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, the plurality of genome edits comprises insertion of a donor nucleic acid, optionally a targeted insertion.

[0109] In some embodiments, a cell population is provided that includes edited cells that comprise multiple genome edits per cell, wherein at least 70% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of a 20-fold, 30-fold, 40-fold, or 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of a 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 70% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 20-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 70% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 30-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 70% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 40-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 70% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 60-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 70% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 70-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 70% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of an 80-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 70% of the cells in the cell population contain at least two genome edits, and the cell population is capable of expanding 90-fold ex vivo in culture within 14 days after initiation of editing.In some embodiments, at least 70% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, fewer than 1% of the cells in the cell population have a target-to-target translocation. In some embodiments, fewer than 0.5% of the cells in the cell population have a target-to-target translocation. In some embodiments, fewer than 0.2% of the cells in the cell population have a target-to-target translocation. In some embodiments, fewer than 0.1% of the cells in the cell population have a target-to-target translocation. In some embodiments, the cell population has less than two-fold background levels of reciprocal translocations, compound translocations, or off-target translocations. In some embodiments, at least one genome edit of the plurality of genome edits is produced by a genome editing tool comprising an RNA-guided DNA binding agent, and the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, at least two genome edits of the plurality of genome edits are produced by a genome editing tool comprising an RNA-guided DNA binding agent, and the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, the plurality of genome edits comprises insertion of a donor nucleic acid, optionally a targeted insertion.

[0110] In some embodiments, a cell population is provided that includes edited cells that comprise multiple genome edits per cell, wherein at least 80% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of a 20-fold, 30-fold, 40-fold, or 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of a 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 80% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 20-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 80% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 30-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 80% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 40-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 80% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 60-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 80% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 70-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 80% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of an 80-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 80% of the cells in the cell population contain at least two genome edits, and the cell population is capable of expanding 90-fold ex vivo in culture within 14 days after initiation of editing.In some embodiments, at least 80% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, fewer than 1% of the cells in the cell population have a target-to-target translocation. In some embodiments, fewer than 0.5% of the cells in the cell population have a target-to-target translocation. In some embodiments, fewer than 0.2% of the cells in the cell population have a target-to-target translocation. In some embodiments, fewer than 0.1% of the cells in the cell population have a target-to-target translocation. In some embodiments, the cell population has less than two-fold background levels of reciprocal translocations, compound translocations, or off-target translocations. In some embodiments, at least one genome edit of the plurality of genome edits is produced by a genome editing tool comprising an RNA-guided DNA binding agent, and the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, at least two genome edits of the plurality of genome edits are produced by a genome editing tool comprising an RNA-guided DNA binding agent, and the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, the plurality of genome edits comprises insertion of a donor nucleic acid, optionally a targeted insertion.

[0111] In some embodiments, a cell population is provided that includes edited cells that comprise multiple genome edits per cell, wherein at least 90% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of a 20-fold, 30-fold, 40-fold, or 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of a 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 90% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 20-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 90% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 30-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 90% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 40-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 90% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 60-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 90% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 70-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 90% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of an 80-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 90% of the cells in the cell population contain at least two genome edits, and the cell population is capable of expanding 90-fold ex vivo in culture within 14 days after initiation of editing.In some embodiments, at least 90% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, fewer than 1% of the cells in the cell population have a target-to-target translocation. In some embodiments, fewer than 0.5% of the cells in the cell population have a target-to-target translocation. In some embodiments, fewer than 0.2% of the cells in the cell population have a target-to-target translocation. In some embodiments, fewer than 0.1% of the cells in the cell population have a target-to-target translocation. In some embodiments, the cell population has less than two-fold background levels of reciprocal translocations, compound translocations, or off-target translocations. In some embodiments, at least one genome edit of the plurality of genome edits is produced by a genome editing tool comprising an RNA-guided DNA binding agent, and the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, at least two genome edits of the plurality of genome edits are produced by a genome editing tool comprising an RNA-guided DNA binding agent, and the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, the plurality of genome edits comprises insertion of a donor nucleic acid, optionally a targeted insertion.

[0112] In some embodiments, a cell population is provided that includes edited cells that comprise multiple genome edits per cell, wherein at least 95% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of a 20-fold, 30-fold, 40-fold, or 50-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, the cell population is capable of a 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 95% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 20-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 95% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 30-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 95% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 40-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 95% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 60-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 95% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of a 70-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 95% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of an 80-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, at least 95% of the cells in the cell population contain at least two genome edits, and the cell population is capable of expanding 90-fold ex vivo in culture within 14 days after initiation of editing.In some embodiments, at least 95% of the cells in the cell population comprise at least two genome edits, and the cell population is capable of 100-fold expansion ex vivo in culture within 14 days after initiation of editing. In some embodiments, fewer than 1% of the cells in the cell population have a target-to-target translocation. In some embodiments, fewer than 0.5% of the cells in the cell population have a target-to-target translocation. In some embodiments, fewer than 0.2% of the cells in the cell population have a target-to-target translocation. In some embodiments, fewer than 0.1% of the cells in the cell population have a target-to-target translocation. In some embodiments, the cell population has less than two-fold background levels of reciprocal translocations, compound translocations, or off-target translocations. In some embodiments, at least one genome edit of the plurality of genome edits is produced by a genome editing tool comprising an RNA-guided DNA binding agent, and the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, at least two genome edits of the plurality of genome edits are produced by a genome editing tool comprising an RNA-guided DNA binding agent, and the RNA-guided DNA binding agent is optionally a cleavase. In some embodiments, the plurality of genome edits comprises insertion of a donor nucleic acid, optionally a targeted insertion.

[0113] As used herein, "days in culture" refers to the number of days in culture that begins on the day the cells are thawed and placed in culture if the cells are frozen before culture, before editing, or during the editing step. That is, the number of days in culture may be discrete.

[0114] As used herein, "after initiation of editing" refers to the time from when a cell or population of cells is contacted with a first LNP composition.

[0115] The targeted-to-target translocations described herein can be detected using standard ddPCR assays.

[0116] In some embodiments, the cells of the cell population comprising edited cells are human cells. In some embodiments, the cells of the cell population comprising edited cells are selected from mesenchymal stem cells, hematopoietic stem cells (HSCs), mononuclear cells, endothelial progenitor cells (EPCs), neural stem cells (NSCs), corneal epithelial 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), cells for organ or tissue transplantation, and cells used in ACT therapy.

[0117] In some embodiments, the cells of the cell population comprising edited cells are immune cells. In some embodiments, the cells of the cell population comprising edited cells are immune cells selected from lymphocytes (e.g., T cells, B cells, natural killer cells ("NK cells," and NKT cells, or iNKT cells)), monocytes, macrophages, mast cells, dendritic cells, granulocytes (e.g., neutrophils, eosinophils, and basophils), primary immune cells, CD3+ cells, CD4+ cells, CD8+ T cells, regulatory T cells (Tregs), B cells, NK cells, and dendritic cells (DCs). In some embodiments, the cells of the cell population comprising edited cells are immune cells selected from peripheral blood mononuclear cells (PBMCs), lymphocytes, T cells, optionally CD4+ cells, CD8+ cells, memory T cells, naive T cells, stem cell memory T cells, or B cells, optionally memory B cells, naive B cells, and primary cells. In some embodiments, the cells of the cell population comprising edited cells are T cells, hi some embodiments, the cells of the cell population comprising edited cells are T cells selected from tumor infiltrating lymphocytes (TILs), T cells expressing alpha-beta TCRs, T cells expressing gamma-delta TCRs, regulatory T cells (Tregs), memory T cells, and early stem cell memory T cells (Tscm, CD27+ / CD45+).

[0118] In some embodiments, the cells of the cell population comprising edited cells are immune cells isolated from human donor PBMCs or leukoplakias prior to editing, hi some embodiments, the cells of the cell population comprising edited cells are immune cells derived from progenitor cells.

[0119] In some embodiments, the cells of the cell population comprising edited cells are non-activated immune cells. In some embodiments, the cells of the cell population comprising edited cells are activated immune cells.

[0120] In some embodiments, a cell of the cell population comprising an edited cell comprising multiple genome edits comprises a third genome edit.

[0121] In some embodiments, the cells of the cell population comprising the edited cells are for transplantation into a human subject.

[0122] In some embodiments, at least 95% of the cells in the cell population comprise genomic editing of the endogenous TCR sequence. In some embodiments, at least 96% of the cells in the cell population comprise genomic editing of the endogenous TCR sequence. In some embodiments, at least 97% of the cells in the cell population comprise genomic editing of the endogenous TCR sequence. In some embodiments, at least 98% of the cells in the cell population comprise genomic editing of the endogenous TCR sequence. In some embodiments, at least 99% of the cells in the cell population comprise genomic editing of the endogenous TCR sequence.

[0123] In some embodiments, the cell population comprises edited cells with genome editing comprising insertion of an exogenous nucleic acid sequence encoding a targeting ligand or alternative antigen binding moiety, wherein at least 70% of the cells of the cell population comprise insertion of the exogenous nucleic acid into a target sequence. In some embodiments, the cell population comprises edited cells with genome editing comprising insertion of an exogenous nucleic acid sequence encoding a targeting ligand or alternative antigen binding moiety, wherein at least 80% of the cells of the cell population comprise insertion of the exogenous nucleic acid into a target sequence. In some embodiments, the cell population comprises edited cells with genome editing comprising insertion of an exogenous nucleic acid encoding a targeting ligand or alternative antigen binding moiety, wherein at least 90% of the cells of the cell population comprise insertion of the exogenous nucleic acid into a target sequence. In some embodiments, the cell population comprises edited cells with genome editing comprising insertion of an exogenous nucleic acid encoding a targeting ligand or alternative antigen binding moiety, wherein at least 95% of the cells of the cell population comprise insertion of the exogenous nucleic acid into a target sequence.

[0124] In some embodiments, the cell population comprises edited T cells, and at least 30%, 40%, 50%, 55%, 60%, or 65% of the cells of the cell population have a memory phenotype (CD27+, CD45RA+). In some embodiments, the cell population comprises edited T cells, and at least 30% of the cells of the cell population have a memory phenotype (CD27+, CD45RA+). In some embodiments, the cell population comprises edited T cells, and at least 40% of the cells of the cell population have a memory phenotype (CD27+, CD45RA+). In some embodiments, the cell population comprises edited T cells, and at least 50% of the cells of the cell population have a memory phenotype (CD27+, CD45RA+). In some embodiments, the cell population comprises edited T cells, and at least 55% of the cells of the cell population have a memory phenotype (CD27+, CD45RA+). In some embodiments, the cell population comprises edited T cells, wherein at least 60% of the cells of the cell population have a memory phenotype (CD27+, CD45RA+). In some embodiments, the cell population comprises edited T cells, wherein at least 65% of the cells of the cell population have a memory phenotype (CD27+, CD45RA+).

[0125] In some embodiments, the cell population comprising the edited cells comprises cells with reduced or eliminated surface expression of MHC class I and / or MHC class II. In some embodiments, the cell population comprising the edited cells comprises cells with reduced or eliminated surface expression of both MHC class I and MHC class II. In some embodiments, the cell population comprising the edited cells comprises cells with reduced or eliminated surface expression HLA-A, and the cells are homozygous for HLA-B and homozygous for HLA-C.

[0126] In some embodiments, the cell population comprising edited T cells comprises cells with reduced or eliminated surface expression of MHC class I and / or MHC class II. In some embodiments, the cell population comprising edited T cells comprises cells with reduced or eliminated surface expression of both MHC class I and MHC class II. In some embodiments, the cell population comprising edited T cells comprises cells with reduced or eliminated surface expression HLA-A, and the cells are homozygous for HLA-B and homozygous for HLA-C.

[0127] In some embodiments, a population of cells is produced according to the provided multiplex delivery and genome editing methods. In some embodiments, at least 50% or more of the cells in the population contain more than one genome edit. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells in the population (i.e., all cells as determined by a method of detection) contain more than one genome edit. In some embodiments, the methods disclosed herein result in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, preferably at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cells having at least two genome edits. In other embodiments, the methods disclosed herein result in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, preferably at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cells having two, three, four, five, six, seven, or eight genome edits. In some embodiments, the methods disclosed herein result in about 5% to about 100%, about 10% to about 50%, about 20% to about 100%, about 20% to about 80%, about 40% to about 100%, or about 40% to about 80% of all cells in the population having at least two genome edits. In some embodiments, the cells are not subjected to a selection process, e.g., a FACS or biochemical selection process, upon completion of editing to enrich for the population of edited cells.

[0128] In some embodiments, the delivery and genome editing methods produce in vitro expanded cells with increased survival. In embodiments, the improved survival rate can be compared to cells treated using an electroporation process. In embodiments, the cell survival rate of the expanded cells is at least 70%, 80%, 90%, or 95%.

[0129] In some embodiments, the delivery and genome editing methods produced cells in vitro with low toxicity. For example, in embodiments, cells resulting from the disclosed methods have less than 2%, 1%, 0.5%, 0.2%, or 0.1% translocations, including, for example, target-to-target translocations and / or off-target translocations. In some embodiments, cells resulting from the disclosed methods have less than 1%, 0.5%, 0.2%, or 0.1% target-to-target translocations. In some embodiments, cells resulting from the disclosed methods have no measurable translocations, including, for example, target-to-target translocations and / or off-target translocations. In some embodiments, the resulting cells have no measurable reciprocal translocations, e.g., as determined using the methods provided herein. In some embodiments, the resulting cells have no measurable compound translocations, e.g., as determined using the methods provided herein. In some embodiments, the resulting cells have no measurable off-target translocations, e.g., as determined using the methods provided herein. In some embodiments, the resulting cells have less than two-fold background levels of reciprocal, compound, or off-target translocations, e.g., as determined using the methods provided herein.

[0130] In some embodiments, the genome editing methods produce cells with high editing efficiency. A particular advantage of the disclosed methods is the high editing rate observed in cells with multiple genome edits. For example, in some embodiments, the percent editing efficiency is at least 60%, 70%, 80%, 90%, or 95% at each target site.

[0131] It is understood that the number of cells in a population required for any particular use depends, for example, on the type of cell and the intended use of the cells. The number of cells to be edited also depends on the ability to expand the cells after editing. It is also understood that the level of editing required, or the level of knockdown required, will depend, at least in part, on the particular editing performed and the intended use of the cell population. For example, a population of B cells with, for example, 30% or less, 40% or less, or 50% or less genome editing may be useful in a protein expression system. For example, a higher level of knockdown of endogenous T cell receptors (TCRs) on the surface of T cells for transplantation into a subject is required because low levels of endogenous TCRs on the surface of T cells can result in severe adverse reactions upon transplantation into a subject. Therefore, T cells expressing endogenous TCRs should be present at as low a level as possible in a population of T cells for transplantation purposes. However, editing T cells to produce cytokines or other secreted factors, even for transplantation use, may not require such high levels of editing of endogenous TCRs in a population of T cells for transplantation.

[0132] The size of exemplary edited cell populations is provided below.It is understood that the number of edited cells required for any particular indication may vary, for example, treatment method may vary.In addition, a larger number of cells may be desirable for cell populations used in allogeneic therapy than in autologous therapy.

[0133] In certain embodiments, the population of cells comprising edited cells is a population of T cells. In certain embodiments, the population of T cells comprises 1 x 10e9 edited T cells with multiple edits, i.e., at least two edits. In certain embodiments, the population of T cells comprises 5 x 10e9 edited T cells with at least a single edit. In certain embodiments, the population of T cells comprises 1-10 x 10e9 edited T cells and is useful for TCR-T cell therapy. In certain embodiments, the population of T cells comprises 1 x 10e8 edited T cells and is useful for CAR-T therapy.

[0134] In certain embodiments, the population of cells comprising edited cells is a population of B cells. In certain embodiments, the population of B cells comprises 1-5x10e8 edited B cells with at least a single edit, and preferably edited B cells with multiple edits.

[0135] In certain embodiments, the population of cells comprising edited cells is a population of NK cells. In certain embodiments, the population of NK cells comprises 3x10e9 NK-edited NK cells with at least a single edit. In certain embodiments, the population of NK cells comprises at least 5x10e8 edited NK cells with multiple edits. In certain embodiments, the population of NK cells comprises 1x10e8 to 9x10e9 edited NK cells for use in therapy.

[0136] In certain embodiments, the population of cells comprising edited cells is a population of monocytes or macrophages, hi certain embodiments, the population of monocytes or macrophages comprising edited cells comprises at least 1 x 10e9 monocytes or macrophages with at least a single edit, or at least 2 x 10e8 monocytes or macrophages with multiple edits.

[0137] In certain embodiments, the population of cells comprising edited cells are dendritic cells. In certain embodiments, the population of dendritic cells comprises 5x10e6 to 5x10e7 edited dendritic cells.

[0138] In some embodiments, in vitro genome editing methods for T cells produce high editing efficiency at multiple target sites. In some embodiments, engineered T cells are generated and an endogenous TCR is knocked out. In some embodiments, engineered T cells are generated and expression of an endogenous TCR is reduced. In some embodiments, engineered T cells are generated and three genes are reduced in expression and / or knocked out. In some embodiments, engineered T cells are generated and four genes are reduced in expression and / or knocked out. In some embodiments, engineered T cells are generated and five genes are reduced in expression and / or knocked out. In some embodiments, engineered T cells are generated and six genes are reduced in expression and / or knocked out. In some embodiments, engineered T cells are generated and seven genes are reduced in expression and / or knocked out. In some embodiments, engineered T cells are generated and eight genes are reduced in expression and / or knocked out. In some embodiments, engineered T cells are produced in which 9 genes have reduced expression and / or are knocked out. In some embodiments, engineered T cells are produced in which 10 genes have reduced expression and / or are knocked out. In some embodiments, engineered T cells are produced in which 11 genes have reduced expression and / or are knocked out.

[0139] In some embodiments, engineered T cells are generated, 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 of T cells (e.g., greater than 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) using the disclosed lipid nucleic acid assembly compositions.

[0140] In some embodiments, T cells produced by the disclosed methods have increased cytokine production. In some embodiments, the increased cytokine production can be compared to T cells treated using an electroporation process. For example, in some embodiments, the engineered T cells produced increased levels of IL-2. In some embodiments, the engineered T cells produced increased levels of IFNγ. In some embodiments, the engineered T cells produced increased levels of TNFα. Cytokine levels can be determined by standard methods, including, for example, ELISA, intracellular flow cytometry staining.

[0141] In some embodiments, T cells produced by the disclosed methods exhibit continued proliferation with repeated stimulation. For example, T cells can proliferate after repeated stimulation in in vitro culture with an agent used to stimulate the T cells. In some embodiments, T cells can be stimulated and proliferated in response to repeated stimulation with a cognate antigen for the TCR of the T cells (e.g., a peptide-MHC complex on a cell co-cultured with the T cells). In some embodiments, T cells can be stimulated and proliferated in response to repeated polyclonal stimulation. In some embodiments, the repeated stimulation is at least two, three, four, five, or more times. In some embodiments, the proliferation of cells is expanded to form a population of cells containing the genetic modification.

[0142] In some embodiments, T cells produced by the disclosed methods exhibit increased expansion. In some embodiments, the increased expansion can be compared to T cells treated using an electroporation process. Expansion can be assessed by cell counting, proliferation, or other standard methods for measuring T cell expansion.

[0143] In some embodiments, T cells produced by the disclosed methods exhibit a memory T cell phenotype. In some embodiments, a T cell memory phenotype termed early stem cell memory T cells (or "Tscm") is particularly advantageous and is produced by the disclosed methods. In some embodiments, the engineered T cells have a Tscm phenotype (CD27+, CD45RA+).

[0144] In some embodiments, engineered cells (e.g., T cells) produced by the disclosed methods have reduced or eliminated surface expression of MHC class I and / or MHC class II. In some embodiments, the engineered cells have reduced or eliminated surface expression of both MHC class I and MHC class II. In some embodiments, the engineered cells have reduced or eliminated surface expression of HLA-A, and the cells are homozygous for HLA-B and homozygous for HLA-C.

[0145] In some embodiments, engineered T cells produced by the disclosed methods have reduced or eliminated surface expression of MHC class I and / or MHC class II. In some embodiments, the engineered cells have reduced or eliminated surface expression of both MHC class I and MHC class II. In some embodiments, the engineered cells have reduced or eliminated surface expression of HLA-A, and the cells are homozygous for HLA-B and homozygous for HLA-C.

[0146] In some embodiments, one or more of all of the following advantages of the present methods, reagents used therefor, and products produced thereby are observed compared to products produced by other methods of genome editing known in the art, e.g., electroporation: a. an improved ability to expand edited cells in culture within 14 days of initiating editing, e.g., by 20-fold, 30-fold, 40-fold, or 50-fold expansion, optionally by 60-fold, 70-fold, or 80-fold; b. Insertion rates comparable to alternative methods such as electroporation; c. A reduction in the number / percentage of unedited cells due to higher editing efficiency, including an increase in the percentage of cells with more than one edit, e.g., at least 2, 3, 4, 5, or 6 edits, i.e., preferably without a selection step to remove unedited cells or enrich for edited cells; d. have a memory T cell phenotype (CD27+, CD45RA+), e.g., at least 30%, 40%, preferably at least 50% of a more desirable memory cell phenotype; e. Increased cytokine production (e.g., IL-2, IFNγ, TNFα), or other cytokines depending on the edited cell type; f. Improved cytotoxicity of edited cells; g. Improved growth and / or proliferation capacity of edited cells; h. Enhanced durability of responses, particularly in T cells, following repeated stimulation; and / or i. A reduction in the rate of unwanted side effects and mutations, such as a reduction in the translocation rate, e.g., a translocation rate of less than 2%, 1%, 0.5%, 0.2%, or 0.1% translocations, preferably target-to-target translocations; or less than two-fold the total number of translocations compared to background.

[0147] B. Methods / Uses for Treating Disorders The cells and / or populations of cells provided herein produced by the disclosed multiplex methods can be used in methods of treating a variety of diseases and disorders.

[0148] In some embodiments, the present disclosure provides a method of providing immunotherapy in a subject, the method comprising administering to the subject an effective amount of a cell (e.g., a population of cells) described herein, e.g., a cell of any of the foregoing aspects and embodiments of the cell.

[0149] In some embodiments of the method, the method includes administering to the subject a lymphodepleting agent or an immunosuppressant prior to administering an effective amount of a cell (e.g., a population of cells) described herein, e.g., a cell of any of the foregoing cell aspects and embodiments. In another aspect, the disclosure provides methods of preparing the cells (e.g., population of cells).

[0150] Immunotherapy is the treatment of disease by activating or suppressing the immune system. Immunotherapies designed to induce or amplify an immune response are classified as activated immunotherapies. Cell-based immunotherapy has proven effective in the treatment of several cancers. Immune effector cells, such as lymphocytes, macrophages, dendritic cells, natural killer cells, and cytotoxic T lymphocytes (CTLs), can be programmed to act in response to abnormal antigens expressed on the surface of tumor cells. Cancer immunotherapy thus enables components of the immune system to destroy tumors or other cancer cells. Cell-based immunotherapy has also proven effective in the treatment of autoimmune diseases or transplant rejection. Immune effector cells, such as regulatory T cells (Tregs) or mesenchymal stem cells, can be programmed to act in response to self- or transplant antigens expressed on the surface of normal tissues.

[0151] In some embodiments, the present disclosure provides a population of cells or a method of preparing cells (e.g., a population of cells). The population of cells can be used in immunotherapy.

[0152] The cells of the present disclosure are suitable for further manipulation, for example, by the introduction of further edited or modified genes or alleles. In some embodiments, the polypeptide is a wild-type or variant TCR. The cells of the present disclosure may also be suitable for further manipulation by the introduction of heterologous sequences encoding alternative antigen-binding moieties, for example, by the introduction of heterologous sequences encoding alternative (non-endogenous) TCRs, such as chimeric antigen receptors (CARs) engineered to target specific proteins. CARs are also known as chimeric immune receptors, chimeric T cell receptors, or artificial T cell receptors.

[0153] In some embodiments, the present disclosure provides a method of treating a subject in need of treatment, comprising administering a cell (e.g., a population of cells), e.g., a cell prepared by a method of preparing a cell described herein, e.g., a method of any of the aforementioned aspects and embodiments of the method of preparing a cell.

[0154] In some embodiments, the cells or populations of cells produced by the disclosed methods can be used to treat cancer, an infectious disease, an inflammatory disease, an autoimmune disease, a cardiovascular disease, a neurological disease, an ophthalmological disease, a kidney disease, a liver disease, a musculoskeletal disease, a red blood cell disease, or transplant rejection.

[0155] In some embodiments, the cancer is lymphoma, breast cancer, lung cancer, multiple myeloma, leukemia, liver cancer, urinary tract cancer, kidney cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, carcinoma of the epithelium, mesothelioma, oropharyngeal cancer, cervical cancer, uterine cancer, ovarian cancer, anogenital cancer, or brain cancer. In some embodiments, the lymphoma is diffuse large B-cell lymphoma (DLBCL), aggressive B-cell lymphoma, high-grade B-cell lymphoma, or non-Hodgkin's lymphoma, including mantle cell lymphoma. In some embodiments, the breast cancer is triple-negative breast cancer. In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC). In some embodiments, the leukemia is acute lymphoblastic leukemia or acute myeloid leukemia. In some embodiments, the cancer is a solid tumor.

[0156] In some embodiments, the infectious disease is caused by human immunodeficiency virus (HIV), hepatitis A virus, hepatitis C virus, hepatitis B virus, human cytomegalovirus (CMV), Epstein-Barr virus, human papillomavirus, Mycobacterium tuberculosis, human coronavirus, or invasive Aspergillus fumigatus. In some embodiments, the infectious disease is acquired immunodeficiency syndrome (AIDS), hepatitis A, hepatitis B, hepatitis C, tuberculosis, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), or coronavirus disease 2019 (COVID-19). In some embodiments, the tuberculosis is multidrug-resistant (MDR) tuberculosis or extensively drug-resistant (XDR) tuberculosis. In some embodiments, the human coronavirus is Middle East Respiratory Syndrome coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), or Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV2). In some embodiments, the infectious disease is human papillomavirus-positive cancer, such as uterine cancer, cervical cancer, or oropharyngeal cancer.

[0157] In some embodiments, the inflammatory disease is allergy, asthma, celiac disease, glomerulonephritis, inflammatory bowel disease, gout, rheumatoid arthritis (RA), myositis, scleroderma, ankylosing spondylitis (AS), antiphospholipid syndrome (APS), systemic lupus erythematosus (SLE), Sjogren's syndrome, rheumatic heart disease, chronic obstructive pulmonary disease (COPD), or transplant rejection.

[0158] In some embodiments, the autoimmune disease is type 1 diabetes, multiple sclerosis, Crohn's disease, ulcerative colitis, autoimmune thyroid disease, rheumatoid arthritis (RA), inflammatory bowel disease, antiphospholipid syndrome (APS), Sjogren's syndrome, scleroderma, psoriasis, psoriatic arthritis, Guillain-Barré syndrome, Addison's disease, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, autoimmune uveitis, autoimmune hepatitis, pernicious anemia, celiac disease, or systemic lupus erythematosus (SLE).

[0159] In some embodiments, the cardiovascular disease is ischemic heart disease, coronary heart disease, aortic disease, Marfan syndrome, congenital heart disease, valvular heart disease, pericardial disease, rheumatic heart disease, peripheral arterial disease, or stroke.

[0160] In some embodiments, the neurological disease is Parkinson's disease, amyotrophic lateral sclerosis, stroke, spinal cord injury, Alzheimer's disease, age-related macular degeneration, traumatic brain injury, multiple sclerosis, Huntington's disease, muscular dystrophy, or Guillain-Barré syndrome.

[0161] In some embodiments, the ophthalmological disease is glaucoma, retinopathy, macular degeneration, or cytomegalovirus (CMV) retinitis. In some embodiments, the ophthalmological disease is a retinal disease. In some embodiments, the ophthalmological disease is mediated by VEGF.

[0162] In some embodiments, engineered cells produced by the methods of the present disclosure can be used as cell therapy, including autologous cell therapy. In some embodiments, engineered cells can be used as cell therapy, including allogeneic stem cell therapy. In some embodiments, the cell therapy comprises induced pluripotent stem cells (iPSCs). iPSCs can be induced to differentiate into other cell types, including, for example, beta islet cells, neurons, and blood cells. In some embodiments, the cell therapy comprises hematopoietic stem cells. In some embodiments, the stem cells comprise mesenchymal stem cells, which can develop into bone, cartilage, muscle, and fat cells. In some embodiments, the stem cells comprise ocular stem cells. In some embodiments, the allogeneic stem cell transplant comprises allogeneic bone marrow transplant. In some embodiments, the stem cells comprise pluripotent stem cells (PSCs). In some embodiments, the stem cells comprise induced embryonic stem cells (ESCs).

[0163] In some embodiments, the cell therapy is transgenic T cell therapy. In some embodiments, the cell therapy comprises Wilms' tumor 1 (WT1) targeting transgenic T cells. In some embodiments, the cell therapy comprises a donor nucleic acid encoding a targeting receptor or a targeting receptor for a commercially available T cell therapy, such as CAR T cell therapy. Currently, several targeting receptors are approved for cell therapy. The cells and methods provided herein can be used with these known constructs. Commercially approved cell products containing targeting receptor constructs for use as cell therapies include, for example, Kymriah® (tisagenlecleucel); Yescarta® (axicabtagene ciloleucel); Tecartus™ (brexucabtagene autoleucel); Tabelecleucel (Tab-cel®); Viralym-M (ALVR105); and Viralym-C.

[0164] C. Exemplary Cell Types 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 is a CD3 + , CD4 + , and CD8 + The immune cells may be selected from T cells, regulatory T cells (Treg), B cells, NK cells, and dendritic cells (DCs). In some embodiments, the immune cells are allogeneic.

[0165] 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.

[0166] As used herein, a T cell may be defined as a cell that expresses a T cell receptor ("TCR" or "αβTCR" or "γδTCR"), 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), and thus expression of the protein CD3 may be used as a marker to identify T cells by standard flow cytometry methods. CD3 is a multisubunit 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+ or CD8+ marker.

[0167] In some embodiments, T cells express the glycoprotein CD8 and are therefore CD8+ by standard flow cytometry methods and may be referred to as "cytotoxic" T cells. In some embodiments, T cells express the glycoprotein CD4 and are therefore CD4+ by standard flow cytometry methods and may be referred to as "helper" T cells. CD4+ T cells can be differentiated 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 pro- or anti-inflammatory, survival, or protective functions. T cells can be isolated from a subject by CD4+ or CD8+ selection methods.

[0168] 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 recently infected tissues (effector memory T cells). Memory T cells may be CD8+ T cells. Memory T cells may be CD4+ T cells.

[0169] As used herein, "central memory T cells" may be defined as antigen-experienced T cells that may express, for example, CD62L and CD45RO. Central memory T cells may be detected as CD62L+ and CD45RO+, and because they also express CCR7, may be detected as CCR7+ by standard flow cytometry methods.

[0170] 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 also express CD62L and CCR7 and can therefore 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.

[0171] In some embodiments, the cell is a B cell. As used herein, a "B cell" may be defined as a cell that expresses CD19 and / or CD20, and / or B-cell maturation antigen ("BCMA"), and thus, a B cell is CD19+, and / or CD20+, and / or BCMA+ by standard flow cytometry methods. The B cell is also negative for CD3 and CD56 by standard flow cytometry methods. The B cell may be a plasma cell. The B cell may be a memory B cell. The B cell may be a naive B cell. The B cell may be IgM+ or have a class-switched B cell receptor (e.g., IgG+, or IgA+).

[0172] In some embodiments, the cells are mononuclear cells, such as 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").

[0173] 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 adipose), hematopoietic stem cells (HSCs; e.g., isolated from BM), mononuclear cells (e.g., isolated from BM or PB), endothelial progenitor cells (EPCs; isolated from BM, PB, and UC), neural stem cells (NSCs), corneal epithelial stem cells (LSCs), or tissue-specific primary cells or cells derived therefrom (TSCs). Cells used in ACT therapy further include induced pluripotent stem cells (iPSCs; see, e.g., Mahla, International J. Cell Biol. 2016(Article ID 6940283):1-24(2016)), which can be induced to differentiate into other cell types, including, for example, islet cells, neurons, and blood cells; ocular stem cells; pluripotent stem cells (PSCs); embryonic stem cells (ESCs); and cells for organ or tissue transplantation, such as islet cells, cardiomyocytes, thyroid cells, thymocytes, neurons, skin cells, retinal cells, chondrocytes, muscle cells, and keratinocytes.

[0174] 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. In some embodiments, the cells are isolated from a patient. In some embodiments, the cells are isolated from a donor. In some embodiments, the cells are isolated from human donor PBMCs or leukoplakia. In some embodiments, the cells are from a subject with a condition, disorder, or disease. In some embodiments, the cells are from a human donor with Epstein-Barr virus ("EBV").

[0175] In some embodiments, the cells are homozygous for HLA-B and homozygous for HLA-C. In some embodiments, the cells contain a genetic modification in the HLA-A gene and are homozygous for HLA-B and homozygous for HLA-C.

[0176] In some embodiments, the method is performed ex vivo. As used herein, "ex vivo" refers to an in vitro method in which cells can be introduced into a subject, such as, for example, an ACT therapy. In some embodiments, the ex vivo method is an in vitro method involving a cell or cell population of an ACT therapy.

[0177] 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 readministered 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.

[0178] In some embodiments, the cells are from a cell line. In some embodiments, the cell line is derived from a human subject. In some embodiments, the cell line is a lymphoblastoid cell line ("LCL"). The cells may be cryopreserved and thawed. The cells may not have been previously cryopreserved.

[0179] 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, cells are removed from a subject, genetically modified ex vivo, and transferred to a cell bank. In some embodiments, a population of genetically modified cells is transferred to a cell bank. In some embodiments, a population of genetically modified immune cells is transferred to a cell bank. In some embodiments, a population of genetically modified immune cells comprising first and second subpopulations, 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.

[0180] IV. Exemplary Genome Editing Tools In some embodiments, the lipid-nucleic acid assembly comprises a genome editing tool or a nucleic acid encoding the same.

[0181] As used herein, the term "genome editing tool" (or "gene editing tool") refers to any component of a "genome editing system" (or "gene editing system") that is necessary or useful for producing edits in a cell's genome. In some embodiments, the present disclosure provides methods 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 population of cells). Genome editing tools include, for example, nucleases that can create single- or double-stranded breaks in the DNA or RNA of a cell, e.g., in the genome of a cell. Genome editing tools, e.g., nucleases, can optionally modify the genome of a cell without cleaving the nucleic acid or nickase. 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, for example, fusion proteins comprising a nickase fused to an effector domain, such as an editor domain. Genome editing tools include any items necessary or useful in achieving a genome editing goal, such as, for example, guide RNAs, sgRNAs, dgRNAs, donor nucleic acids, etc.

[0182] Various 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) in target DNA sequences. Cleavage or nicking can occur through the use of specific nucleases, such as engineered ZFNs or TALENs, or by using CRISPR / Cas systems with engineered guide RNAs to induce specific cleavage or nicking of target DNA sequences. Additionally, targeted nucleases have been developed based on the Argonaute system (e.g., from T. thermophilus known as "TtAgo", see Swarts et al (2014) Nature 507(7491):258-261) and may also have potential use in genome editing and gene therapy.

[0183] A. CRISPR / Cas genome editing tools In some embodiments, the genome editing tool is a component of a CRISPR / Cas system.

[0184] 1. Guide RNA (gRNA) In some embodiments, the genome editing tool is a guide RNA (gRNA), which can be a dual guide RNA (dgRNA) or a single guide RNA (sgRNA). The guide RNA directs an RNA-guided DNA-binding agent to a target sequence.

[0185] In some embodiments of the present disclosure, the cargo of the lipid-nucleic acid assembly formulation includes at least one gRNA or a nucleic acid encoding the same. The gRNA may 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, thereby providing specificity for cleavage. In some embodiments, the gRNA and Cas nuclease may form a ribonucleoprotein (RNP), e.g., a CRISPR / Cas complex, such as a CRISPR / Cas9 complex. In some embodiments, the CRISPR / Cas complex may be a Type II CRISPR / Cas9 complex. In some embodiments, the CRISPR / Cas complex may be a Type V CRISPR / Cas complex, such as a Cpf1 / guide RNA complex. The Cas nuclease and cognate gRNA may be paired. The gRNA scaffold structure paired with each Class 2 Cas nuclease varies depending on the particular CRISPR / Cas system.

[0186] In some embodiments, the sgRNA is a "Cas9 sgRNA" capable of mediating RNA-guided DNA cleavage by the Cas9 protein. In some embodiments, the sgRNA is a "Cpf1 sgRNA" capable of mediating RNA-guided DNA cleavage by the Cpf1 protein. In some embodiments, the gRNA comprises sufficient crRNA and tracrRNA to form an active complex with the Cas9 protein and mediate RNA-guided DNA cleavage. In some embodiments, the gRNA comprises sufficient crRNA to form an active complex with the Cpf1 protein and mediate RNA-guided DNA cleavage. See Zetsche 2015.

[0187] Certain embodiments of the present disclosure also provide nucleic acids, e.g., expression cassettes, encoding the gRNAs described herein. "Guide RNA nucleic acid" is used herein to refer to a guide RNA (e.g., sgRNA or dgRNA) and a guide RNA expression cassette, which is a nucleic acid that encodes one or more guide RNAs.

[0188] In some embodiments, the nucleic acid may be a DNA molecule. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding a crRNA. In some embodiments, the nucleotide sequence encoding the crRNA comprises a targeting sequence flanked by all or part of repeat sequences from a naturally occurring CRISPR / Cas system. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding a tracrRNA. In some embodiments, the crRNA and tracrRNA may be encoded by two separate nucleic acids. In other embodiments, the crRNA and tracrRNA may be encoded by a single nucleic acid. In some embodiments, the crRNA and tracrRNA may be encoded by opposite strands of a single nucleic acid. In other embodiments, the crRNA and tracrRNA may be encoded by the same strand of a single nucleic acid. In some embodiments, the gRNA nucleic acid encodes an sgRNA. In some embodiments, the gRNA nucleic acid encodes a Cas9 nuclease sgRNA. In some embodiments, the gRNA nucleic acid encodes a Cpf1 nuclease sgRNA.

[0189] The nucleotide sequence encoding the guide RNA can be operably linked to at least one transcriptional or regulatory control sequence, such as a promoter, a 3'UTR, or a 5'UTR. In one example, the promoter is a tRNA promoter, e.g., a tRNA promoter. Lys3, or tRNA chimeras. 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 U6 and H1 promoters. 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 includes modified nucleosides or nucleotides. In some embodiments, the gRNA nucleic acid includes a 5'-end modification, e.g., a modified nucleoside or nucleotide, to stabilize the nucleic acid and prevent integration. In some embodiments, the gRNA nucleic acid includes double-stranded DNA with a 5'-end modification on each strand. In some embodiments, the gRNA nucleic acid includes an inverted dideoxy-T or an inverted abasic nucleoside or nucleotide as a 5'-end modification. In some embodiments, the gRNA nucleic acid includes a label such as biotin, desthiobiotin-TEG, digoxigenin, and a fluorescent marker, including, for example, FAM, ROX, TAMRA, and AlexaFluor.

[0190] In some embodiments, more than one gRNA nucleic acid, such as a gRNA, can be used with a CRISPR / Cas nuclease system. Each gRNA nucleic acid can contain a different targeting sequence, allowing the CRISPR / Cas system to cleave more than one target sequence. In some embodiments, one or more gRNAs can have the same or different properties, such as activity or stability within a CRISPR / Cas complex. When more than one gRNA is used, each gRNA can be encoded on the same or different gRNA nucleic acid. The promoters used to drive the expression of more than one gRNA can be the same or different.

[0191] Because the nucleic acid substrate of a Cas protein is a double-stranded nucleic acid, the target sequence for the Cas protein includes both the plus and minus strands of genomic DNA (i.e., the given sequence and its reverse complement). Thus, when a guide sequence is said to be "complementary to a target sequence," it should be understood that it can direct the guide RNA 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 certain nucleotides of the target sequence (e.g., the target sequence without the PAM), except for the substitution of U for T in the guide sequence.

[0192] The length of the targeting sequence depends on the CRISPR / Cas system and components used. For example, various Class 2 Cas nucleases derived from various bacterial species have different optimal targeting 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 some embodiments, the Cas nuclease and gRNA scaffold are derived from the same CRISPR / Cas system. In some embodiments, the targeting sequence may comprise or consist of 18 to 24 nucleotides. In some embodiments, the targeting sequence may comprise or consist of 19-21 nucleotides, hi some embodiments, the targeting sequence may comprise or consist of 20 nucleotides.

[0193] 2. RNA-guided DNA binders In some embodiments, the genome editing tool is an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent is a Cas cleavase / nickase and its inactivated form (dCas DNA binding agent). In some embodiments, the RNA-guided DNA binding agent is a Cas nuclease.

[0194] In some embodiments, the genome editing tool is an mRNA encoding an RNA-guided DNA binding agent, hi some embodiments, the genome editing tool is an mRNA encoding a Cas nuclease.

[0195] In some embodiments, the genome editing tool comprises an mRNA, such as a Cas nuclease mRNA, and a gRNA nucleic acid co-encapsulated in a lipid nucleic acid assembly composition. In some embodiments, an mRNA encoding an RNA-guided DNA-binding agent is formulated in a first lipid nucleic acid assembly composition, and a gRNA nucleic acid is formulated in a second lipid nucleic acid assembly composition. In some embodiments, the first and second lipid nucleic acid assembly compositions are administered simultaneously. In other embodiments, the first and second lipid nucleic acid assembly compositions are administered sequentially. In some embodiments, the first and second lipid nucleic acid assembly compositions are combined prior to the pre-incubation step. In some embodiments, the first and second lipid nucleic acid assembly compositions are pre-incubated separately.

[0196] Non-limiting exemplary species from which Cas nucleases can 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。.

[0197] In some embodiments, the Cas nuclease is a Cas9 nuclease from Streptococcus pyogenes. In some embodiments, the Cas nuclease is a Cas9 nuclease from Streptococcus thermophilus. In some 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 some embodiments, the Cas nuclease is a Cpf1 nuclease from Acidaminococcus sp.. In some 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.

[0198] Wild-type Cas9 has two nuclease domains, RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, and the HNH domain cleaves the target strand of DNA. In some embodiments, the Cas9 nuclease comprises more than one RuvC domain and / or more than one HNH domain. In some embodiments, the Cas9 nuclease is wild-type Cas9. In some embodiments, Cas9 is capable of inducing a double-stranded break in the target DNA. In some embodiments, the Cas nuclease may cleave dsDNA, cleave one strand of dsDNA, or may not have DNA cleavage or nickase activity.

[0199] In some embodiments, chimeric Cas nucleases are used, in which one domain or region of a protein is replaced with a portion of a different protein. In some embodiments, a Cas nuclease domain may be replaced with a domain from a different nuclease, such as Fok1. In some embodiments, the Cas nuclease may be a modified nuclease.

[0200] In other embodiments, the Cas nuclease or Cas nickase may be from a type I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a component of the Cascade complex of a type I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a Cas3 protein. In some embodiments, the Cas nuclease may be from a type III CRISPR / Cas system. In some embodiments, the Cas nuclease may have RNA cleavage activity.

[0201] In some embodiments, the RNA-guided DNA binding agent has single-stranded nickase activity, i.e., it can cleave one DNA strand to generate 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 an aspect of a Cas nuclease (e.g., the Cas nucleases discussed above) in which the endonucleolytic active site has been inactivated, e.g., by one or more modifications (e.g., point mutations) in the catalytic domain. See, e.g., U.S. Patent No. 8,889,356 for a discussion of Cas nickases and exemplary catalytic domain modifications. In some embodiments, the Cas nickase, such as the Cas9 nickase, has an inactivated RuvC or HNH domain.

[0202] In some embodiments, the RNA-guided DNA binder is modified to contain only one functional nuclease domain. For example, the drug protein may be modified so 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 that has 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 that has reduced activity is used. In some embodiments, a nickase with an inactive HNH domain is used.

[0203] In some embodiments, conserved amino acids within the Cas protein nuclease domain are substituted to reduce or alter nuclease activity. In some embodiments, the Cas nuclease may comprise an amino acid substitution within the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015) Cell Oct 22:163(3):759-771. In some embodiments, the Cas nuclease may comprise an amino acid substitution within 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))).

[0204] In some embodiments, an mRNA encoding a nickase is provided in combination with a pair of guide RNAs that are complementary to the sense and antisense strands of the target sequence, respectively. In this embodiment, the guide RNAs direct the nickase to the target sequence by generating nicks on opposite strands of the target sequence (i.e., double nicking), thereby introducing a DSB. In some embodiments, the use of double nicking can improve specificity and reduce off-target effects. In some embodiments, a nickase is used with two separate guide RNAs that target opposite strands of DNA, generating a double nick in the target DNA. In some embodiments, a nickase is used with two separate guide RNAs that are selected to be in close proximity to each other, generating a double nick in the target DNA.

[0205] In some embodiments, the RNA-guided DNA binding agent lacks cleavage and nickase activity. In some embodiments, the RNA-guided DNA binding agent comprises a dCas DNA-binding polypeptide. The dCas polypeptide essentially lacks catalytic (cleavage / nickase) activity while possessing DNA-binding activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, an RNA-guided DNA binding agent or dCas DNA-binding polypeptide lacking cleavage and nickase activity is an embodiment of a Cas nuclease (e.g., a Cas nuclease discussed above) whose endonucleolytic 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, US2015 / 0166980A1.

[0206] 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.

[0207] 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-D10A Cas9 nickase by an XTEN linker.

[0208] In some embodiments, the RNA-guided DNA binding agent comprises one or more heterologous functional domains (e.g., is or comprises a fusion polypeptide).

[0209] In some embodiments, the heterologous functional domain may facilitate the transport of the RNA-guided DNA binding agent to the cell nucleus. For example, the heterologous functional domain may be a nuclear localization signal (NLS). In some embodiments, the RNA-guided DNA binding agent may be fused with 1 to 10 NLSs. In some embodiments, the RNA-guided DNA binding agent may be fused with 1 to 5 NLSs. In some embodiments, the RNA-guided DNA binding agent may be fused with one NLS. When one NLS is used, the NLS may be fused at the N-terminal or C-terminal end of the sequence of the RNA-guided DNA binding agent. It may also be inserted within the sequence of the RNA-guided DNA binding agent. In other embodiments, the RNA-guided DNA binding agent may be fused with more than one NLS. In some embodiments, the RNA-guided DNA binding agent may be fused with 2, 3, 4, or 5 NLSs. In some embodiments, the RNA-guided DNA binding agent may be fused with two NLSs. In some situations, the two NLSs may be the same (e.g., two SV40 NLSs) or different. In some embodiments, the RNA-guided DNA binding agent is fused to two NLS sequences (e.g., SV40) fused at the carboxy-terminal end. In some embodiments, the RNA-guided DNA binding agent may be fused with two NLSs, one linked to the N-terminal end and one linked to the C-terminal end. In some embodiments, the RNA-guided DNA binding agent may be fused with three NLSs. In some embodiments, the RNA-guided DNA binding agent may be fused without an NLS. In some embodiments, the NLS may be a single moiety, such as the SV40 NLS, PKKKRKV (SEQ ID NO: 23) or PKKKRRV (SEQ ID NO: 24). In some embodiments, the NLS may be a bimoiety, such as the nucleoplasmin NLS, KRPAATKKAGQAKKKK (SEQ ID NO: 25). In certain embodiments, a single PKKKRKV (SEQ ID NO: 23) NLS may be fused at the C-terminal end of the RNA-guided DNA binding agent. One or more linkers are optionally included at the fusion site.

[0210] In some embodiments, the heterologous functional domain can modify the intracellular half-life of the RNA-guided DNA binding agent. In some embodiments, the half-life of the RNA-guided DNA binding agent can be increased. In some embodiments, the half-life of the RNA-guided DNA binding agent can be decreased. In some embodiments, the heterologous functional domain can increase the stability of the RNA-guided DNA binding agent. In some embodiments, the heterologous functional domain can decrease the stability of the RNA-guided DNA binding agent. In some embodiments, the heterologous functional domain can function as a signal peptide for protein degradation. In some embodiments, the protein degradation can be mediated by proteolytic enzymes such as proteasomes, lysosomal proteases, or calpain proteases. In some embodiments, the heterologous functional domain can comprise a PEST sequence. In some embodiments, the RNA-guided DNA binding agent can be modified by the addition of ubiquitin or polyubiquitin chains. In some embodiments, the ubiquitin can 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), neural progenitor 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).

[0211] 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., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric 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, Softag1, Softag3, 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), β-galactosidase, β-glucuronidase, luciferase, or fluorescent protein.

[0212] In additional embodiments, the heterologous functional domain may target the RNA-guided DNA binding agent to a specific organelle, cell type, tissue, or organ, hi some embodiments, the heterologous functional domain may target the RNA-guided DNA binding agent to mitochondria.

[0213] In further embodiments, the heterologous functional domain can be an effector domain, such as an editor domain. When an RNA-guided DNA-binding agent is directed to its target sequence, for example, when a Cas nuclease is directed to the target sequence by a gRNA, the effector domain, such as an editor domain, can modify or affect the target sequence. In some embodiments, the effector domain, such as 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 transcriptional activation domain, or a transcriptional repression domain. In some embodiments, the heterologous functional domain is a nuclease, such as a FokI nuclease. See, e.g., U.S. Patent No. 9,023,649. In some embodiments, the heterologous functional domain is a transcriptional activator or a transcriptional repressor. See, 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). Thus, RNA-guided DNA binders essentially become transcription factors that can be directed to bind to desired target sequences using guide RNAs.In some embodiments, the DNA-modifying domain is a methylation domain, such as a demethylation domain or a methyltransferase domain. In some embodiments, the effector domain is a DNA-modifying domain, such as a base-editing domain. In certain embodiments, the DNA-modifying domain is a nucleic acid-editing domain that introduces specific modifications into DNA, such as a deaminase domain. See, for example, WO2015 / 089406 and US2016 / 0304846. The nucleic acid-editing domains, deaminase domains, and Cas9 variants described in WO2015 / 089406 and US2016 / 0304846 are incorporated herein by reference.

[0214] Nucleases can contain at least one domain that interacts with a guide RNA ("gRNA"). Additionally, nucleases can be directed to a target sequence by the gRNA. In Class 2 Cas nuclease systems, the gRNA interacts with the nuclease and the target sequence, such that the gRNA directs binding to the target sequence. In some embodiments, the gRNA provides specificity for targeted cleavage, and the nuclease is versatile and can be paired with different gRNAs to cleave different target sequences. Class 2 Cas nucleases can be paired with gRNA scaffolds of the types, orthologs, and exemplary species listed above.

[0215] B. Additional genome editing tools In some embodiments, the genome editing tool is a component of a genome editing system selected from a zinc finger nuclease system, a TALEN system, and a meganuclease system. In some embodiments, the genome editing tool is a nucleic acid encoding one or more components of such a genome editing system. Exemplary components of this system include meganucleases, zinc finger nucleases, TALENS, and fragments thereof.

[0216] 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 in 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 genome editing in a system known as engineered nuclease genome editing. Such methods and compositions for use therein are known in the art. See, for example, WO2019 / 147805, WO2014 / 040370, and WO2018 / 073393, the contents of which are incorporated herein in their entireties.

[0217] 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 a specific desired DNA sequence, allowing the zinc finger nuclease to target a unique sequence within a complex genome. The non-specific cleavage domain from the type IIs restriction endonuclease FokI is typically used as the cleavage domain in ZFN. The cleavage is repaired by endogenous DNA repair mechanisms, allowing ZFN to precisely modify the genome of higher organisms. Such methods and compositions for use therein are known in the art. For example, see WO2011 / 091324, the contents of which are incorporated herein in their entirety.

[0218] V. Exemplary Nucleic Acids for Lipid-Nucleic Acid Assembly Compositions In some embodiments, the lipid-nucleic acid assembly composition delivers nucleic acids (or polynucleotides) to cells. In some embodiments, the nucleic acids comprise nucleosides or nucleoside analogs having nitrogenous heterocyclic bases or base analogs linked together along the backbone, including polymers of traditional RNA, DNA, mixed RNA-DNA, and analogs thereof.

[0219] A. Modified nucleic acids In some embodiments, the lipid nucleic acid assembly composition comprises modified RNA. In some embodiments, the lipid nucleic acid assembly composition comprises modified DNA.

[0220] Modified nucleosides or nucleotides can be present in an RNA, e.g., a gRNA or mRNA. For example, a gRNA or mRNA that includes one or more modified nucleosides or nucleotides is referred to as a "modified" RNA to account for the presence of one or more non-natural and / or naturally occurring components or arrangements used in place of or in addition to the standard A, G, C, and U residues. In some embodiments, the modified RNA is synthesized with non-standard nucleosides or nucleotides and is referred to herein as "modified."

[0221] Modified nucleosides or nucleotides can include one or more of the following: (i) an alteration in the phosphodiester backbone linkage, e.g., replacement of one or both of the non-bridging phosphate oxygens and / or one or more of the bridging phosphate oxygens (exemplary backbone modifications); (ii) an alteration, e.g., replacement, of a component of the ribose sugar, e.g., the 2' hydroxyl of the ribose sugar (exemplary sugar modifications); (iii) extensive replacement of a phosphate moiety with a "dephosphorylated" linker (exemplary backbone modifications); (iv) a modification or replacement of a naturally occurring nucleobase, including with a non-standard nucleobase (exemplary base modifications); (v) a replacement or modification of the ribose phosphate backbone (exemplary backbone modifications); (vi) a modification of the 3' or 5' end of the oligonucleotide, e.g., removal, modification, or replacement of a terminal phosphate group, or conjugation of a moiety, cap, or linker (such 3' or 5' cap modifications can include sugar and / or backbone modifications); and (vii) a modification or replacement of the sugar (exemplary sugar modifications). Certain embodiments include 5'-end modifications to the mRNA, gRNA, or nucleic acid. Certain embodiments include 3'-end modifications to the mRNA, gRNA, or nucleic acid. The modified RNA may include 5'-end and 3'-end modifications. The modified RNA may include one or more modified residues at non-terminal positions. In some embodiments, the gRNA includes at least one modified residue. In some embodiments, the mRNA includes at least one modified residue.

[0222] As used herein, a first sequence is considered to "contain a sequence having at least X% identity" with a second sequence if alignment of the first sequence to the second sequence shows that X% or more of the positions of the second sequence are matched overall by the first sequence. For example, the sequence AAGA contains a sequence having 100% identity with the sequence AAG, because the alignment gives 100% identity in that there is a match at all three positions in the second sequence. Differences between RNA and DNA (generally, the replacement of thymidine with uridine, or vice versa), and the presence of nucleoside analogs such as modified uridines, do not contribute to differences in identity or complementarity between polynucleotides, as long as the related nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all thymidine, uridine, or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as their complement). Thus, for example, the sequence 5'-AXG, where X is any modified uridine, such as pseudouridine, N1-methylpseudouridine, or 5-methoxyuridine, is considered 100% identical to AUG, in that both are perfectly complementary to the same sequence (5'-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well known in the art. Those skilled in the art will understand what algorithm and parameter setting is appropriate for a given pair of sequences to be aligned. Generally, for sequences of similar length and predicted identity greater than 50% for amino acids or 75% for nucleotides, the Needleman-Wunsch algorithm, using the default settings of the Needleman-Wunsch algorithm interface provided by EBI at the www.ebi.ac.uk web server, is generally appropriate.

[0223] In some embodiments, a composition or formulation disclosed herein comprises an mRNA comprising an open reading frame (ORF), e.g., an ORF encoding an RNA-guided DNA binder, such as a Cas nuclease or Class 2 Cas nuclease described herein. In some embodiments, an mRNA comprising an ORF encoding an RNA-guided DNA binder, such as a Cas nuclease or Class 2 Cas nuclease, is provided, used, or administered. In some embodiments, the ORF is codon-optimized. In some embodiments, the ORF encoding the RNA-guided DNA binder is a "modified RNA-guided DNA binder ORF" or simply a "modified ORF," which is used as an abbreviation to indicate that the ORF has been modified in one or more of the following ways: (1) the modified ORF has a uridine content ranging from its minimum uridine content to 150% of the minimum uridine content; (2) the modified ORF has a uridine dinucleotide content ranging from its minimum uridine dinucleotide content to 150% of the minimum uridine dinucleotide content; (3) the modified ORF has at least 90% identity to any one of any of the CasORFs in Table 89; (4) the modified ORF is composed of a set of codons in which at least 75% of the codons are minimal uridine codons for a given amino acid, e.g., codons with the fewest uridines (usually 0 or 1, except for the codon for phenylalanine, which has two uridines); or (5) the modified ORF contains at least one modified uridine. In some embodiments, the modified ORF is modified in at least two, three, or four of the foregoing ways. In some embodiments, the modified ORF contains at least one modified uridine and is modified in at least one, two, three, or all of (1) to (4) above.

[0224] "Modified uridine" is used herein to refer to a nucleoside other than thymidine that has the same hydrogen bond acceptor as uridine and has one or more structural differences from uridine. In some embodiments, the modified uridine is a substituted uridine, i.e., a uridine in which one or more aprotic substituents (e.g., alkoxy, e.g., methoxy) are placed in place of a proton. In some embodiments, the modified uridine is a pseudouridine. In some embodiments, the modified pseudouridine is a substituted pseudouridine, i.e., a pseudouridine in which one or more aprotic substituents (e.g., alkyl, e.g., methyl) are placed in place of a proton. In some embodiments, the modified uridine is any of a substituted uridine, pseudouridine, or substituted pseudouridine.

[0225] As used herein, a "uridine position" refers to a position in a polynucleotide that is occupied by a uridine or a modified uridine. Thus, for example, a polynucleotide in which "100% of the uridine positions are modified uridines" contains a modified uridine at every position that would be a uridine in conventional RNA of the same sequence (where all bases are standard A, U, C, or G bases). Unless otherwise indicated, U in a polynucleotide sequence in this disclosure or in the accompanying sequence tables or sequence listings can be a uridine or a modified uridine.

[0226] Minimal uridine codon: [Table 1]

[0227] In any of the foregoing embodiments, the modified ORF may consist of a set of codons in which at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the codons are codons listed in the above table of minimal uridine codons. In any of the foregoing embodiments, the modified ORF may comprise a sequence having at least 90%, 95%, 98%, 99%, or 100% identity to any one of the Cas ORFs in Table 89.

[0228] In any of the foregoing embodiments, the modified ORF may have a uridine content that ranges from its minimum uridine content to 150%, 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of the minimum uridine content.

[0229] In any of the foregoing embodiments, the modified ORF may have a uridine dinucleotide content that ranges from its minimum uridine dinucleotide content to 150%, 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of the minimum uridine dinucleotide content.

[0230] In any of the foregoing embodiments, the modified ORF can include modified uridines at at least one, more than one, or all uridine positions. In some embodiments, the modified uridine is a uridine modified at the 5-position, e.g., with a halogen, methyl, or ethyl. In some embodiments, the modified uridine is a pseudouridine modified at the 1-position, e.g., with a halogen, methyl, or ethyl. The modified uridine can be, for example, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof. In some embodiments, the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methylpseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5-methoxyuridine.

[0231] In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the uridine positions in an mRNA according to the present disclosure are modified uridines. In some embodiments, 10% to 25%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 90% to 100% of the uridine positions in an mRNA according to the present disclosure are modified uridines, such as 5-methoxyuridine, 5-iodouridine, N1-methylpseudouridine, pseudouridine, or a combination thereof. In some embodiments, 10% to 25%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 90% to 100% of the uridine positions in an mRNA according to the present disclosure are 5-methoxyuridine. In some embodiments, 10% to 25%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 90% to 100% of the uridine positions in an mRNA according to the present disclosure are pseudouridine. In some embodiments, 10% to 25%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 90% to 100% of the uridine positions in an mRNA according to the present disclosure are N1-methylpseudouridine. In some embodiments, 10% to 25%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 90% to 100% of the uridine positions in an mRNA according to the present disclosure are 5-iodouridine. In some embodiments, 10% to 25%, 15 to 25%, 25 to 35%, 35 to 45%, 45 to 55%, 55 to 65%, 65 to 75%, 75 to 85%, 85 to 95%, or 90 to 100% of the uridine positions in an mRNA according to the present disclosure are 5-methoxyuridine, and the remainder are N1-methylpseudouridine.In some embodiments, 10% to 25%, 15 to 25%, 25 to 35%, 35 to 45%, 45 to 55%, 55 to 65%, 65 to 75%, 75 to 85%, 85 to 95%, or 90 to 100% of the uridine positions in an mRNA according to the present disclosure are 5-iodouridine, and the remainder are N1-methylpseudouridine.

[0232] In any of the foregoing embodiments, the modified ORF may contain a reduced uridine dinucleotide (UU) content, such as an ORF that (a) uses minimal uridine codons (as described above) at all positions and (b) encodes the same amino acid sequence as the given ORF. Uridine dinucleotide (UU) content can be expressed in absolute terms based on percentages, such as a count of UU dinucleotides in the ORF or as the percentage of positions occupied by uridines of uridine dinucleotides (e.g., AUUAU has a uridine dinucleotide content of 40% because two of the five positions are occupied by uridines of uridine dinucleotides). Modified uridine residues are considered equivalent to uridine for purposes of assessing minimum uridine dinucleotide content.

[0233] In some embodiments, the mRNA comprises at least one UTR from an expressed mammalian mRNA, for example, a constitutively expressed mRNA. An mRNA is considered to be constitutively expressed in a mammal if the mRNA is continuously transcribed in at least one tissue of a healthy adult mammal. In some embodiments, the mRNA comprises a 5'UTR, a 3'UTR, or 5' and 3'UTR from an expressed mammalian RNA, for example, a constitutively expressed mammalian mRNA. Actin mRNA is an example of a constitutively expressed mRNA.

[0234] In some embodiments, the mRNA comprises at least one UTR from hydroxysteroid 17-beta dehydrogenase 4 (HSD17B4 or HSD), e.g., the 5' UTR from HSD. In some embodiments, the mRNA comprises at least one UTR from a globin mRNA, e.g., human alpha globin (HBA) mRNA, human beta globin (HBB) mRNA, or Xenopus laevis beta globin (XBG) mRNA. In some embodiments, the mRNA comprises the 5' UTR, 3' UTR, or 5' and 3' UTRs from a globin mRNA, e.g., HBA, HBB, or XBG. In some embodiments, the mRNA comprises the 5' UTR from bovine growth hormone, cytomegalovirus (CMV), mouse Hba-a1, HSD, albumin gene, HBA, HBB, or XBG. In some embodiments, the mRNA comprises the 3' UTR from bovine growth hormone, cytomegalovirus, mouse Hba-a1, HSD, albumin gene, HBA, HBB, or XBG. In some embodiments, the mRNA comprises the 5' and 3' UTR from bovine growth hormone, cytomegalovirus, mouse Hba-a1, HSD, albumin gene, HBA, HBB, XBG, heat shock protein 90 (Hsp90), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-actin, alpha-tubulin, tumor protein (p53), or epidermal growth factor receptor (EGFR).

[0235] In some embodiments, the mRNA comprises 5' and 3' UTRs that are from the same source, for example, a constitutively expressed mRNA such as actin, albumin, or a globin such as HBA, HBB, or XBG.

[0236] In some embodiments, the mRNA does not include a 5' UTR, e.g., there are no additional nucleotides between the 5' cap and the start codon. In some embodiments, the mRNA includes a Kozak sequence (described below) between the 5' cap and the start codon, but does not have any additional 5' UTR. In some embodiments, the mRNA does not include a 3' UTR, e.g., there are no additional nucleotides between the stop codon and the polyA tail.

[0237] In some embodiments, the mRNA contains a Kozak sequence. Kozak sequences can affect translation initiation and overall yield of a polypeptide translated from the mRNA. Kozak sequences include a methionine codon that can function as an initiation codon. A minimal Kozak sequence is NNNRUGN, where at least one of the following is true: the first N is A or G and the second N is G. In the context of a nucleotide sequence, R denotes a purine (A or G). In some embodiments, the Kozak sequence is RNNRUGN, NNNRUGG, RNNRUGG, RNNAUGN, NNNAUGG, or RNNAUGG. In some embodiments, the Kozak sequence is rccRUGg with zero, one, or up to two mismatches relative to the lowercase positions. In some embodiments, the Kozak sequence is rccAUGg with zero, one, or up to two mismatches relative to the lowercase positions. In some embodiments, the Kozak sequence is gccRccAUGG (SEQ ID NO:26) with zero, or up to 1, 2, or 3 mismatches relative to the lowercase positions. In some embodiments, the Kozak sequence is gccAccAUG with zero, or up to 1, 2, 3, or 4 mismatches relative to the lowercase positions. In some embodiments, the Kozak sequence is GCCACCAUG. In some embodiments, the Kozak sequence is gccgccRccAUGG (SEQ ID NO:27) with zero, or up to 1, 2, 3, or 4 mismatches relative to the lowercase positions.

[0238] In some embodiments, the mRNA comprising an ORF encoding the RNA-guided DNA binder comprises a sequence having at least 90% identity to any of the CasORFs in Table 89.

[0239] In some embodiments, the mRNA disclosed herein includes a 5' cap, e.g., Cap0, Cap1, or Cap2. The 5' cap is generally a 7-methylguanine ribonucleotide (which may be further modified as discussed below, e.g., with respect to ARCA) linked via a 5'-triphosphate to the 5' position of the first nucleotide of the 5'-3' strand of the mRNA (i.e., the first cap-proximal nucleotide). In Cap0, the riboses of the first and second cap-proximal nucleotides of the mRNA both include a 2'-hydroxyl. In Cap1, the riboses of the first and second transcribed nucleotides of the mRNA include a 2'-methoxy and a 2'-hydroxyl, respectively. In Cap2, the riboses of the first and second cap-proximal nucleotides of the mRNA both include a 2'-methoxy. See, for example, Katibah et al. (2014) Proc Natl Acad Sci USA 111(33):12025-30 and Abbas et al. (2017) Proc Natl Acad Sci USA 114(11):E2106-E2115. Most endogenous higher eukaryotic mRNAs, including mammalian mRNAs such as human mRNAs, contain Cap1 or Cap2. Cap0 and other cap structures distinct from Cap1 and Cap2 are recognized as "non-self" by components of the innate immune system, such as IFIT-1 and IFIT-5, and can be immunogenic in mammals, including humans, and can cause elevated levels of cytokines, including type I interferons. Components of the innate immune system, such as IFIT-1 and IFIT-5, can also compete with eIF4E for binding to mRNAs with caps other than Cap1 or Cap2, potentially inhibiting mRNA translation.

[0240] A cap can be included co-transcriptionally. For example, ARCA (anti-reverse cap analog, Thermo Fisher Scientific catalog number AM8045) is a cap analog containing 7-methylguanine 3'-methoxy-5'-triphosphate linked to the 5' position of guanine ribonucleotides and can be incorporated into transcripts in vitro during initiation. ARCA results in a Cap0 cap in which the 2' position of the first cap-proximal nucleotide is hydroxyl. See, for example, Stepinski et al. (2001) "Synthesis and properties of mRNAs containing the novel 'anti-reverse' cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl(3'deoxy)GpppG," RNA 7:1486-1495. The structure of ARCA is shown below. [ka]

[0241] CleanCap™ AG (m7G(5')ppp(5')(2'OMeA)pG, TriLink Biotechnologies catalog number N-7113) or CleanCap™ GG (m7G(5')ppp(5')(2'OMeG)pG, TriLink Biotechnologies catalog number N-7133) can be used to co-transcriptionally provide the Cap1 structure. 3'-O-methylated versions of CleanCap™ AG and CleanCap™ GG are also available from TriLink Biotechnologies as catalog numbers N-7413 and N-7433, respectively. The structure of CleanCap™ AG is shown below. [ka]

[0242] Alternatively, a cap can be added to RNA post-transcriptionally. For example, Vaccinia capping enzyme is commercially available (New England Biolabs, catalog number M2080S), which possesses RNA triphosphatase and guanylyltransferase activities provided by its D1 subunit and guanine methyltransferase activity provided by its D12 subunit. Thus, in the presence of S-adenosylmethionine and GTP, 7-methylguanine can be added to RNA to give Cap0. See, e.g., Guo, P. and Moss, B. (1990) Proc. Natl. Acad. Sci. USA 87, 4023-4027; Mao, X. and Shuman, S. (1994) J. Biol. Chem. 269, 24472-24479.

[0243] In some embodiments, the mRNA further comprises a polyadenylation (poly-A) tail. In some embodiments, the poly-A tail comprises at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, and optionally up to 300 adenines. In some embodiments, the poly-A tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides. In some cases, the poly-A tail is "interrupted" with one or more non-adenine nucleotide "anchors" at one or more positions within the poly-A tail. The poly-A tail may comprise at least eight consecutive adenine nucleotides, but may also include one or more non-adenine nucleotides. As used herein, "non-adenine nucleotide" refers to any natural or non-natural nucleotide that does not contain adenine. Guanine, thymine, and cytosine nucleotides are exemplary non-adenine nucleotides. Thus, the poly-A tail on the mRNA described herein may comprise consecutive adenine nucleotides located 3' to the nucleotides encoding the RNA-guided DNA-binding agent or sequence of interest. In some cases, the poly-A tail on the mRNA comprises non-consecutive adenine nucleotides located 3' to the nucleotides encoding the RNA-guided DNA-binding agent or sequence of interest, where the non-adenine nucleotides interrupt the adenine nucleotides at regular or irregular intervals.

[0244] As used herein, "non-adenine nucleotide" refers to any natural or non-natural nucleotide that does not contain adenine. Guanine, thymine, and cytosine nucleotides are exemplary non-adenine nucleotides. Thus, the poly-A tail on the mRNA described herein can include consecutive adenine nucleotides located 3' to the nucleotides encoding the RNA-guided DNA binding agent or sequence of interest. In some cases, the poly-A tail on the mRNA includes non-consecutive adenine nucleotides located 3' to the nucleotides encoding the RNA-guided DNA binding agent or sequence of interest, and the non-adenine nucleotides interrupt the adenine nucleotides at regular or irregular intervals.

[0245] In some embodiments, mRNA is purified. In some embodiments, mRNA is purified using a precipitation method (e.g., LiCl precipitation, alcohol precipitation, or equivalent method, e.g., those described herein). In some embodiments, mRNA is purified using a chromatography-based method, such as an HPLC-based method or equivalent method (e.g., those described herein). In some embodiments, mRNA is purified using both a precipitation method (e.g., LiCl precipitation) and an HPLC-based method.

[0246] In some embodiments, at least one gRNA is provided in combination with the mRNA disclosed herein.In some embodiments, the gRNA is provided as a molecule separate from the mRNA.In some embodiments, the gRNA is provided as a part of the mRNA disclosed herein, for example, as a part of the UTR.

[0247] B. Chemically modified nucleic acids In some embodiments, the nucleic acid is RNA, such as chemically modified RNA. In some embodiments, the nucleic acid is or comprises DNA, such as chemically modified DNA.

[0248] RNA containing one or more modified nucleosides or nucleotides is referred to as "modified" or "chemically modified" RNA to describe the presence of one or more non-natural and / or naturally occurring components or structures used in place of, or in addition to, the standard A, G, C, and U residues. In some embodiments, modified RNA is synthesized using non-standard nucleosides or nucleotides and is referred to herein as "modified." Modified nucleosides or nucleotides can include one or more of the following: (i) an alteration in the phosphodiester backbone linkage, e.g., replacement of one or both of the non-bridging phosphate oxygens and / or one or more of the bridging phosphate oxygens (exemplary backbone modifications); (ii) an alteration, e.g., replacement, of a component of the ribose sugar, e.g., the 2' hydroxyl of the ribose sugar (exemplary sugar modifications); (iii) extensive replacement of a phosphate moiety with a "dephosphorylated" linker (exemplary backbone modifications); (iv) a modification or replacement of a naturally occurring nucleobase, including with a non-standard nucleobase (exemplary base modifications); (v) a replacement or modification of the ribose phosphate backbone (exemplary backbone modifications); (vi) a modification of the 3' or 5' end of the oligonucleotide, e.g., removal, modification, or replacement of a terminal phosphate group, or conjugation of a moiety, cap, or linker (such 3' or 5' cap modifications can include sugar and / or backbone modifications); and (vii) a modification or replacement of the sugar (exemplary sugar modifications).

[0249] gRNAs that include one or more modified nucleosides or nucleotides are referred to as "modified" gRNAs or "chemically modified" RNAs to describe the presence of one or more non-natural and / or naturally occurring components or structures used in place of, or in addition to, the standard A, G, C, and U residues. In some embodiments, modified gRNAs are synthesized using non-standard nucleosides or nucleotides and are referred to herein as "modified."

[0250] Chemical modifications such as those listed above can be combined to provide modified nucleic acids, DNA, RNA, or gRNAs comprising nucleosides and nucleotides (collectively "residues") that may have two, three, four, or more modifications. For example, modified residues may have a modified sugar and a modified nucleobase. In some embodiments, each base of a gRNA is modified, e.g., all bases have a modified phosphate group, such as phosphorothioate. In some embodiments, all, or substantially all, of the phosphate groups of a gRNA molecule are replaced with phosphorothioate groups. In some embodiments, a modified gRNA comprises at least one modified residue at or near the 5' end of the RNA. In some embodiments, a modified gRNA comprises at least one modified residue at or near the 3' end of the RNA.

[0251] In some embodiments, a nucleic acid, such as a gRNA, comprises one, two, three, or more modified residues. In some embodiments, at least 5% (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) of the positions in the modified gRNA are modified nucleosides or nucleotides.

[0252] Unmodified nucleic acids may be susceptible to degradation, for example, by intracellular nucleases or those found in serum. For example, nucleases can hydrolyze phosphodiester bonds of nucleic acids. Thus, in one aspect, modified nucleic acids, such as gRNAs described herein, can contain one or more modified nucleosides or nucleotides, for example, to introduce stability against intracellular or serum-based nucleases. In some embodiments, modified gRNA molecules described herein can exhibit a reduced innate immune response when introduced into cell populations both in vivo and ex vivo. The term "innate immune response" includes cellular responses to exogenous nucleic acids, including single-stranded nucleic acids, including induction of cytokine (particularly interferon) expression and release and cell death.

[0253] In some embodiments of backbone modifications, the phosphate group of the modified residue may be modified by replacing one or more oxygens with different substituents. Furthermore, modified residues, such as those present in modified nucleic acids, may include extensive replacement of unmodified phosphate moieties with modified phosphate groups, as described herein. In some embodiments, backbone modifications of the phosphate backbone may include alterations that result in either uncharged linkers or charged linkers with asymmetric charge distributions.

[0254] Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. The phosphate atom in an unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygens with one of the atoms or groups of atoms described above can make the phosphorus atom chiral. The stereogenic 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 oxygen (i.e., the oxygen connecting the phosphate to the nucleoside) with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates). Replacement can occur at either or both bridging oxygens.

[0255] The phosphate group can be replaced by a non-phosphorus-containing linking group 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 can include, but are not limited to, methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, and methyleneoxymethylimino.

[0256] In some embodiments, the present disclosure includes sgRNAs comprising one or more modifications in one or more of the following regions: nucleotides at the 5' end, the lower stem region, the bulge region, the upper stem region, the nexus region, the hairpin 1 region, the hairpin 2 region, and the nucleotides at the 3' end. In some embodiments, the modification comprises 2'-O-methyl (2'-O-Me) modified nucleotides. In some embodiments, the modification comprises 2'-fluoro (2'-F) modified nucleotides. In some embodiments, the modification comprises an internucleotide phosphorothioate (PS) linkage.

[0257] In some embodiments, the first 3 or 4 nucleotides at the 5' end and the last 3 or 4 nucleotides at the 3' end are modified. In some embodiments, the first 4 nucleotides at the 5' end and the last 4 nucleotides at the 3' end are linked using phosphorothioate (PS) linkages. In some embodiments, the modification comprises 2'-O-Me. In some embodiments, the modification comprises 2'-F.

[0258] In some embodiments, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked by PS bonds, and the first three nucleotides at the 5' end and the last three nucleotides at the 3' end contain 2'-O-Me modifications.

[0259] In some embodiments, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked with PS linkages, and the first three nucleotides at the 5' end and the last three nucleotides at the 3' end contain 2'-F modifications.

[0260] In some embodiments, the sgRNA comprises the modification pattern (mN*mN*mN*NNNNNNNNNNNNNNNNNGUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 28), where N is any natural or unnatural nucleotide. A, C, G, and U are adenine nucleotides, cytidine nucleotides, A, C, G, and U are guanine nucleotides and uridine nucleotides. In certain embodiments, A, C, G, and U are each independently a natural or non-naturally occurring nucleotide having the indicated base. In certain embodiments, A, C, G, and U are RNA nucleotides. In some embodiments, the sgRNA comprises a sequence disclosed in the preceding sentence about this sgRNA. In some embodiments, the sgRNA comprises 2'O-methyl modifications of the first three residues at its 5' end, with phosphorothioate linkages between residues 1-2, 2-3, and 3-4 of the RNA.

[0261] C. Template Nucleic Acid The compositions and methods disclosed herein may include a donor nucleic acid, i.e., a template nucleic acid. The template may be used to modify or insert a nucleic acid sequence at or near a target site for a Cas nuclease. In some embodiments, the method includes introducing the template into a cell. In some embodiments, a single template may be provided. In other embodiments, two or more templates may be provided so that editing can occur at two or more target sites. For example, different templates may be provided to edit a single gene in a cell or two different genes in a cell.

[0262] In some embodiments, the template may be used in homologous recombination. In some embodiments, homologous recombination may result in the integration of the template sequence or a portion of the template sequence into a target nucleic acid molecule. In other embodiments, the template may be used in homology-directed repair, which involves DNA strand invasion at the nucleic acid cleavage site. In some embodiments, homology-directed repair may result in the inclusion of the template sequence in an edited target nucleic acid molecule. In still other embodiments, the template may be used in gene editing mediated by non-homologous end joining. In some embodiments, the template sequence is dissimilar to the nucleic acid sequence near the cleavage site. In some embodiments, the template or a portion of the template sequence is integrated. In some embodiments, the template includes adjacent inverted terminal repeat (ITR) sequences.

[0263] In some embodiments, the template can include a first homology arm and a second homology arm (also referred to as a first and second nucleotide sequence) that are complementary to sequences located upstream and downstream of the cleavage site, respectively. When the template includes two homology arms, each arm can be the same length or different lengths, and the sequence between the homology arms can be substantially similar or identical to the target sequence between the homology arms, or can be entirely unrelated. In some embodiments, the degree of complementarity or percent identity between the first nucleotide sequence on the template and the sequence upstream of the cleavage site, and between the second nucleotide sequence on the template and the sequence downstream of the cleavage site, can enable homologous recombination, e.g., high-fidelity homologous recombination, between the template and the target nucleic acid molecule. In some embodiments, the degree of complementarity can be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the degree of complementarity may be about 95%, 97%, 98%, 99%, or 100%. In some embodiments, the degree of complementarity may be at least 98%, 99%, or 100%. In some embodiments, the degree of complementarity may be 100%. In some embodiments, the percent identity may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the percent identity may be about 95%, 97%, 98%, 99%, or 100%. In some embodiments, the percent identity may be at least 98%, 99%, or 100%. In some embodiments, the percent identity may be 100%.

[0264] In some embodiments, the template sequence may correspond to, comprise, or consist of an endogenous sequence of the target cell. The template sequence may also, or alternatively, correspond to, comprise, or consist of an exogenous sequence of the target cell. As used herein, the term "endogenous sequence" refers to a sequence native to the cell. The term "exogenous sequence" refers to a sequence not native to the cell or a sequence at a different location than its native location in the genome of the cell. In some embodiments, the endogenous sequence may be a genomic sequence of the cell. In some embodiments, the endogenous sequence may be a chromosomal or extrachromosomal sequence. In some embodiments, the endogenous sequence may be a plasmid sequence of the cell. In some embodiments, the template sequence may be substantially identical to a portion of the endogenous sequence in the cell at or near the cleavage site, but contains at least one nucleotide change. In some embodiments, editing the cleaved target nucleic acid molecule using the template may result in a mutation including an insertion, deletion, or substitution of one or more nucleotides in the target nucleic acid molecule. In some embodiments, the mutation may result in one or more amino acid changes in the protein expressed from the gene comprising the target sequence.

[0265] In some embodiments, the mutation may result in one or more nucleotide changes in the RNA expressed from the target insertion site. In some embodiments, the mutation may alter the expression level of the target gene. In some embodiments, the mutation may result in increased or decreased expression of the target gene. In some embodiments, the mutation may result in gene knockdown. In some embodiments, the mutation may result in gene knockout. In some embodiments, the mutation may result in restoration of gene function. In some embodiments, editing the cleaved target nucleic acid molecule with a template may result in changes to exon sequences, intron sequences, regulatory sequences, transcriptional regulatory sequences, translational regulatory sequences, splice sites, or non-coding sequences of the target nucleic acid molecule, such as DNA.

[0266] In other embodiments, the template sequence may include an exogenous sequence. In some embodiments, the exogenous sequence may include a coding sequence. In some embodiments, the exogenous sequence may include a protein or RNA coding sequence (e.g., ORF) operably linked to an exogenous promoter sequence such that, upon integration of the exogenous sequence into a target nucleic acid molecule, the cell can express the protein or RNA encoded by the integrated sequence. In other embodiments, upon integration of the exogenous sequence into a target nucleic acid molecule, expression of the integrated sequence may be regulated by an endogenous promoter sequence. In some embodiments, the exogenous sequence may provide a cDNA sequence encoding a protein or a portion of a protein. In yet other embodiments, the exogenous sequence may comprise or consist of exon sequences, intron sequences, regulatory sequences, transcriptional control sequences, translational control sequences, splice sites, or non-coding sequences. In some embodiments, integration of the exogenous sequence may result in restoration of gene function. In some embodiments, integration of the exogenous sequence may result in gene knock-in. In some embodiments, integration of the exogenous sequence may result in gene knock-out.

[0267] The template can be of any suitable length. In some embodiments, the template can comprise 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, or more nucleotides in length. The template can be a single-stranded nucleic acid. The template can be a double-stranded or partially double-stranded nucleic acid. In some embodiments, a single-stranded template is 20, 30, 40, 50, 75, 100, 125, 150, 175, or 200 nucleotides in length. In some embodiments, the template can comprise a nucleic acid sequence that is complementary to a portion of a target nucleic acid molecule that comprises the target sequence (i.e., a "homology arm"). In some embodiments, the template can comprise a homology arm that is complementary to a sequence located upstream or downstream of the cleavage site on the target nucleic acid molecule.

[0268] In some embodiments, the template comprises ssDNA or dsDNA containing 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.

[0269] D. Nucleic Acid Purification In some embodiments, nucleic acids are purified. In some embodiments, nucleic acids are purified using a precipitation method (e.g., LiCl precipitation, alcohol precipitation, or equivalent method, e.g., those described herein). In some embodiments, nucleic acids are purified using a chromatography-based method, such as an HPLC-based method or equivalent method (e.g., those described herein). In some embodiments, nuclei are purified using both a precipitation method (e.g., LiCl precipitation) and an HPLC-based method.

[0270] E. Target Sequence In some embodiments, the CRISPR / Cas system of the present disclosure can be directed to and cleave a target sequence on a target nucleic acid molecule. For example, the target sequence can be recognized and cleaved by a Cas nuclease. In some embodiments, the target sequence of the Cas nuclease is located near the nuclease's cognate PAM sequence. In some embodiments, a Class 2 Cas nuclease can be directed to a target sequence on a target nucleic acid molecule by a gRNA, where the gRNA hybridizes to the target sequence and a Class 2 Cas protein cleaves the target sequence. In some embodiments, a guide RNA hybridizes to the target sequence adjacent to or including its cognate PAM, and the Class 2 Cas nuclease cleaves the target sequence adjacent to or including its cognate PAM. In some embodiments, the target sequence can be complementary to the targeting sequence of the guide RNA. In some embodiments, the degree of complementarity between the targeting sequence of the guide RNA and the portion of the corresponding target sequence that hybridizes to the guide RNA can be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the percent identity between the targeting sequence of the guide RNA and the portion of the corresponding target sequence that hybridizes to the guide RNA can be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the target homology region is adjacent to the cognate PAM sequence. In some embodiments, the target sequence can comprise a sequence that is 100% complementary to the targeting sequence of the guide RNA. In other embodiments, the target sequence may contain at least one mismatch, deletion, or insertion compared to the targeting sequence of the guide RNA.

[0271] The length of the target sequence can depend on the nuclease system used. For example, the targeting sequence of a guide RNA for a CRISPR / Cas system can 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, with the target sequence of the corresponding length optionally flanked by a PAM sequence. In some embodiments, the target sequence can comprise 15-24 nucleotides in length. In some embodiments, the target sequence can comprise 17-21 nucleotides in length. In some embodiments, the target sequence can comprise 20 nucleotides in length. When a nickase is used, the target sequence can comprise a pair of target sequences recognized by a pair of nickases that cleave opposing strands of a DNA molecule. In some embodiments, a target sequence may comprise a pair of target sequences recognized by a pair of nickases that cleave the same strand of a DNA molecule, hi some embodiments, a target sequence may comprise a portion of a target sequence recognized by one or more Cas nucleases.

[0272] A target nucleic acid molecule can be any DNA or RNA molecule that is endogenous or exogenous to a cell. In some embodiments, a target nucleic acid molecule can be episomal DNA, a plasmid, genomic DNA, a viral genome, mitochondrial DNA, or chromosomal DNA from or within a cell. In some embodiments, the target sequence of a target nucleic acid molecule can be a genomic sequence from or within a cell, including a human cell.

[0273] In further embodiments, the target sequence may be a viral sequence. In further embodiments, the target sequence may be a pathogen sequence. In yet other embodiments, the target sequence may be a synthetic sequence. In further embodiments, the target sequence may be a chromosomal sequence. In certain embodiments, the target sequence may comprise a translocation junction, e.g., a translocation associated with cancer. In some embodiments, the target sequence may be on a eukaryotic chromosome, e.g., a human chromosome.

[0274] In some embodiments, the target sequence may be located within a gene's coding sequence, a gene's intron sequence, a regulatory sequence, a gene's transcriptional regulatory sequence, a gene's translational regulatory sequence, a splice site, or an intergenic non-coding sequence. In some embodiments, the gene may be a protein-coding gene. In other embodiments, the gene may be a non-coding RNA gene. In some embodiments, the target sequence may include all or a portion of a disease-associated gene. In some embodiments, the target sequence may be located at a non-gene functional site in the genome, for example, a site that controls aspects of chromatin organization, such as a scaffolding site or locus control region.

[0275] In some embodiments involving Cas nucleases, such as Class 2 Cas nucleases, the target sequence may be adjacent to a protospacer adjacent motif ("PAM"). In some embodiments, the PAM may be adjacent to the 3' end of the target sequence or within 1, 2, 3, or 4 nucleotides of it. The length and sequence of the PAM may depend on the Cas protein used. For example, the PAM may be selected from consensus sequences or specific PAM sequences for particular Cas9 proteins or Cas9 orthologs, including those disclosed in Figure 1 of Ran et al., Nature, 520:186-191 (2015) and Figure S5 of Zetsche 2015, the relevant disclosures of each of which are incorporated herein by reference. In some embodiments, the PAM may be 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Non-limiting exemplary PAM sequences include NGG, NGGNG, NG, NAAAAN, NNAAAAW, NNNNACA, GNNNCNNA, TTN, and NNNNGATT (N is defined as any nucleotide and W is defined as either A or T). In some embodiments, the PAM sequence can be NGG. In some embodiments, the PAM sequence can be NGGNG. In some embodiments, the PAM sequence can be TTN. In some embodiments, the PAM sequence can be NNAAAAW.

[0276] VI. Exemplary Lipid-Nucleic Acid Assemblies Disclosed herein are various embodiments that use lipid-nucleic acid assemblies containing genome editing tools such as RNA, including CRISPR / Cas components and RNAs that express them.

[0277] As used herein, "lipid-nucleic acid assembly compositions" refer to lipid-based delivery compositions, including lipid nanoparticles (LNPs) and lipoplexes. In some embodiments, "LNP compositions" are used interchangeably with "LNPs" or "LNPs."

[0278] In some embodiments, LNP refers to lipid nanoparticles having a diameter of less than 100 nM, or a population of LNPs having an average diameter of less than 100 nM. In certain embodiments, LNPs have a diameter of about 1-250 nm, 10-200 nm, about 20-150 nm, about 50-150 nm, about 50-100 nm, about 50-120 nm, about 60-100 nm, about 75-150 nm, about 75-120 nm, or about 75-100 nm, or a population of LNPs having an average diameter of about 10-200 nm, about 20-150 nm, about 50-150 nm, about 50-100 nm, about 50-120 nm, about 60-100 nm, about 75-150 nm, about 75-120 nm, or about 75-100 nm. In a preferred embodiment, the LNP composition has a diameter of 75-150 nm.

[0279] LNPs are formed by precisely mixing a lipid component (e.g., in ethanol) with an aqueous nucleic acid component, and LNPs are uniform in size. Lipoplexes are particles formed by bulk mixing of lipid and nucleic acid components, and are approximately 100 nm to 1 micron in size. In certain embodiments, the lipid-nucleic acid assembly is an LNP. As used herein, a "lipid-nucleic acid assembly" comprises multiple (i.e., more than one) lipid molecules physically associated with each other through intermolecular forces. The lipid-nucleic acid assembly may comprise bioavailable lipids with pKa values ​​less than 7.5 or less than 7. The lipid-nucleic acid assembly is formed by mixing a nucleic acid-containing aqueous solution with an organic solvent-based lipid solution, e.g., 100% ethanol. Suitable solutions or solvents may include or contain water, PBS, Tris buffer, NaCl, citrate buffer, ethanol, chloroform, diethyl ether, cyclohexane, tetrahydrofuran, methanol, or isopropanol. A pharmaceutically acceptable buffer may optionally be included in a pharmaceutical formulation containing the lipid-nucleic acid assembly, e.g., for ex vivo ACT therapy. In some embodiments, the aqueous solution comprises RNA, such as mRNA or gRNA, hi some embodiments, the aqueous solution comprises mRNA encoding an RNA-guided DNA-binding agent, such as Cas9.

[0280] In some embodiments, lipid nucleic acid assembly formulations include "amine lipids" (sometimes referred to herein or elsewhere as "ionizable lipids" or "biodegradable lipids"), along with optional "helper lipids," "neutral lipids," and stealth lipids such as PEG lipids. In some embodiments, the amine lipids or ionizable lipids are cationic depending on the pH.

[0281] A. Amine lipids In some embodiments, lipid nucleic acid assembly compositions comprise "amine lipids," which are ionizable lipids such as, for example, lipid A, or lipid D, or their equivalents, including acetal analogs of lipid A or lipid D.

[0282] In some embodiments, the amine lipid is lipid A, which is (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. Lipid A can be depicted as follows: [ka]

[0283] Lipid A can be synthesized according to WO2015 / 095340 (e.g., pages 84-86). In some embodiments, the amine lipid is lipid A or an amine lipid provided in WO2020 / 219876, which is incorporated herein by reference.

[0284] In some embodiments, the amine lipid is an analog of lipid A. In some embodiments, the lipid A analog is an acetal analog of lipid A. In certain lipid nucleic acid assembly compositions, the acetal analog is a C4-C12 acetal analog. In some embodiments, the acetal analog is a C5-C12 acetal analog. In additional embodiments, the acetal analog is a C5-C10 acetal analog. In further embodiments, the acetal analog is selected from C4, C5, C6, C7, C9, C10, C11, and C12 acetal analogs.

[0285] In some embodiments, the amine lipid has the formula IA [ka] A compound having the structure During the ceremony, X1A is O, NH or a direct bond; X2A is a C2-3 alkylene; R3A is C1-3 alkyl, R2A is C1-3 alkyl, or R2A together with the nitrogen atom to which it is attached and 2 to 3 carbon atoms of X2A form a 5- or 6-membered ring, or R2A together with R3A and the nitrogen atom to which they are attached form a 5-membered ring; Y1A is a C6-10 alkylene; Y2A is [ka] is selected from R4A is C4-11 alkyl, Z1A is a C2-5 alkylene; The Z2A is [ka] or not present, R5A is C6-8 alkyl or C6-8 alkoxy; R6A is C6-8 alkyl or C6-8 alkoxy, or a salt thereof.

[0286] In some embodiments, the amine lipid has the formula (IIA): [ka] A compound of the formula: During the ceremony, X1A is O, NH or a direct bond; X2A is a C2-3 alkylene; Z1A is a C3 alkylene and R5A and R6A are each a C6 alkyl, or Z1A is a direct bond and R5A and R6A are each a C8 alkoxy; The R8A is [ka] or a salt thereof.

[0287] In certain embodiments, X 1A is O. In other embodiments, X 1A is NH. In yet other embodiments, X 1A is a direct bond.

[0288] In certain embodiments, X2A is C3 alkylene. In certain embodiments, X2A is C2 alkylene.

[0289] In certain embodiments, Z1A is a direct bond, and R5A and R6A are each C8 alkoxy. In some embodiments, Z1A is a C3 alkylene, and R5A and R6A are each C6 alkyl.

[0290] In certain embodiments, R8A is [ka] In other embodiments, R8A is [ka] is.

[0291] In certain embodiments, the amine lipid is a salt.

[0292] Representative compounds of formula (IA) include: [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] or a salt thereof, for example, a pharmaceutically acceptable salt thereof.

[0293] In some embodiments, the amine lipid is nonyl 8-((7,7-bis(octyloxy)heptyl)(2-hydroxyethyl)amino)octanoate: [ka] or a salt thereof.

[0294] Lipid D can be synthesized according to WO2020072605, and Mol. Ther. 2018, 26(6), 1509-1519 ("Sabnis"), which are incorporated by reference in their entireties. In some embodiments, lipid D, or an amine lipid, is an amine lipid provided in WO2020072605, which is incorporated by reference herein.

[0295] In some embodiments, the amine lipid has formula IB: [ka] A compound having the structure During the ceremony, X 1B is C 6~7 is alkylene, X 2B is or does not exist, except for X 2B but [ka] If R 2B is not an alkoxy; Z 1B is C 2~3 is alkylene, Z 2B is selected from -OH, -NHC(=O)OCH3, and -NHS(=O)2CH3; R 1B is C 7~9 is an unbranched alkyl; Each R 2B are independently C8 alkyl or C8 alkoxy, or a salt thereof

[0296] In some embodiments, the amine lipid has the formula (IIB): [ka] A compound of the formula: During the ceremony, X 1B is C 6~7 is alkylene, Z 1B is C 2~3 is alkylene, R 1B is C 7~9 is an unbranched alkyl; Each R 2B is a C8 alkyl, or a salt thereof.

[0297] In certain embodiments, X 1B is C6 alkylene. 1B is a C7 alkylene.

[0298] In certain embodiments, Z 1B is a direct bond, and R 5B and R 6B are each C alkoxy. 1B is a C3 alkylene, and R 5B and R 6B are each C6 alkyl.

[0299] In certain embodiments, X 2B teeth, [ka] and R 2B is not alkoxy. 2B does not exist.

[0300] In certain embodiments, Z 1B is C2 alkylene, and in other embodiments, Z1B is a C3 alkylene.

[0301] In certain embodiments, Z 2B is —OH. In another embodiment, Z 2B is —NHC(═O)OCH 3 . In another embodiment, Z 2B is -NHS(=O)2CH3.

[0302] In certain embodiments, R 1B is a C7 unbranched alkylene. 1B is a C8 branched or unbranched alkylene. 1B is a C9 branched or unbranched alkylene.

[0303] In certain embodiments, the amine lipid is a salt.

[0304] Representative compounds of formula (IB) include: [Table 3-1] [Table 3-2] or a salt thereof, for example, a pharmaceutically acceptable salt thereof.

[0305] Amine lipids and other "biodegradable lipids" suitable for use in the lipid nucleic acid assemblies described herein are biodegradable in vivo or ex vivo. Amine lipids have low toxicity (e.g., are tolerated in animal models without side effects at doses of 10 mg / kg or greater). In some embodiments, lipid nucleic acid assemblies comprising amine lipids include those in which at least 75% of the amine lipids are cleared from plasma or engineered cells within 8, 10, 12, 24, or 48 hours, or within 3, 4, 5, 6, 7, or 10 days. In some embodiments, lipid nucleic acid assemblies comprising amine lipids include those in which at least 50% of the nucleic acid, e.g., mRNA or gRNA, is cleared from plasma within 8, 10, 12, 24, or 48 hours, or within 3, 4, 5, 6, 7, or 10 days. In some embodiments, lipid nucleic acid assemblies comprising amine lipids include those in which at least 50% of the lipid nucleic acid assemblies are cleared from plasma within 8, 10, 12, 24, or 48 hours, or within 3, 4, 5, 6, 7, or 10 days, e.g., by measuring lipid (e.g., amine lipid), nucleic acid, e.g., RNA / mRNA, or other components. In some embodiments, lipid-encapsulated versus free lipid, RNA, or nucleic acid components of the lipid nucleic acid assemblies are measured.

[0306] Biodegradable lipids include, for example, those described in WO / 2020 / 219876 (e.g., at pages 13-33, 66-87), WO / 2020 / 118041, WO / 2020 / 072605 (e.g., at pages 5-12, 21-29, 61-68), WO / 2019 / 067992, WO / 2017 / 173054, WO2015 / 095340, and WO2014 / 136086, and LNPs include the LNP compositions described therein, the lipids and compositions of which are incorporated herein by reference.

[0307] Lipid clearance may be measured as described in the literature. See Maier, MA, et al. Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics. Mol. Ther. 2013, 21(8), 1570-78 ("Maier"). For example, in Maier, an LNP-siRNA system containing siRNA targeting luciferase was administered at 0.3 mg / kg via intravenous bolus injection via the lateral tail vein to 6-8 week-old male C57Bl / 6 mice. Blood, liver, and spleen samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96, and 168 hours post-administration. Mice were perfused with saline prior to tissue collection, and blood samples were processed to obtain plasma. All samples were processed and analyzed by LC-MS. Furthermore, Maier describes a procedure for assessing the toxicity of LNP-siRNA formulations after administration. For example, siRNA targeting luciferase was administered to male Sprague-Dawley rats at 0, 1, 3, 5, and 10 mg / kg (5 animals / group) via a single intravenous bolus injection at a dose volume of 5 mL / kg. After 24 hours, approximately 1 mL of blood was obtained from the jugular vein of conscious animals, and serum was isolated. Seventy-two hours after administration, all animals were euthanized for necropsy. Clinical signs, body weight, serum chemistry, organ weight, and histopathology were assessed. While Maier describes methods for assessing siRNA-LNP formulations, these methods can be applied to assessing clearance, pharmacokinetics, and toxicity of the lipid-nucleic acid assembly compositions of the present disclosure.

[0308] Ionized lipid and bioavailable lipid are suitable for LNP delivery of nucleic acid known in the art.Lipid may be ionized according to the pH of the medium in which it is contained.For example, in a slightly acidic medium, lipid such as amine lipid may be protonated and therefore carry positive charge.On the other hand, in a slightly basic medium such as blood, whose pH is about 7.35, lipid such as amine lipid may not be protonated and therefore carry no charge.

[0309] The ability of a lipid to carry a charge is related to its inherent pKa. In some embodiments, the amine lipids of the present disclosure may each independently have a pKa in the range of about 5.1 to about 7.4. In some embodiments, the bioavailable lipids of the present disclosure may each independently have a pKa in the range of about 5.1 to about 7.4, e.g., about 5.5 to about 6.6, about 5.6 to about 6.4, about 5.8 to about 6.2, or about 5.8 to about 6.5. For example, the amine lipids of the present disclosure may each independently have a pKa in the range of about 5.8 to about 6.5. Lipids with a pKa in the range of about 5.1 to about 7.4 are effective in delivering cargo in vivo, for example, to the liver. Furthermore, lipids with a pKa in the range of about 5.3 to about 6.4 have been found to be effective in delivering cargo in vivo, for example, to tumors. See, e.g., WO2014 / 136086.

[0310] B. 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 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), pohsphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearo ... 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 one embodiment, the neutral phospholipid may be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidylethanolamine (DMPE). In another embodiment, the neutral phospholipid may be distearoylphosphatidylcholine (DSPC).

[0311] "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 one embodiment, the helper lipid may be cholesterol. In one embodiment, the helper lipid may be cholesterol hemisuccinate.

[0312] A "stealth lipid" is a lipid that alters the length of time that nanoparticles can exist in vivo (e.g., in blood vessels). Stealth lipids are useful in formulation processes, for example, by reducing particle aggregation and controlling particle size. Stealth lipids used herein can modulate the pharmacokinetic properties of lipid-nucleic acid assemblies or aid in the stability of nanoparticles ex vivo. Stealth lipids suitable for use in the lipid compositions of the present disclosure include, but are not limited to, stealth lipids having a hydrophilic head group linked to the lipid moiety. Information regarding stealth lipids suitable for use in the lipid compositions of the present disclosure and the biochemistry of such lipids can be found in Romberg et al., Pharmaceutical Research, Vol. 25, No. 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 WO 2006 / 007712.

[0313] In one embodiment, the hydrophilic head group of the stealth lipid comprises a polymer moiety selected from PEG-based polymers. The stealth lipid may comprise a lipid moiety. In some embodiments, the stealth lipid is a PEG lipid.

[0314] In one embodiment, the stealth lipid comprises a polymer moiety selected from PEG-based polymers (sometimes referred to as poly(ethylene oxide)), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), polyamino acids, and poly[N-(2-hydroxypropyl)methacrylamide].

[0315] In one embodiment, the PEG lipid comprises a PEG-based polymer moiety (sometimes referred to as poly(ethylene oxide)).

[0316] The PEG-lipid further comprises a lipid moiety. In some embodiments...

Claims

1. A cell population comprising edited cells comprising multiple genome edits per cell, wherein at least 50% of the cells in the cell population comprise at least two genome edits, and wherein (i) less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% of the cells in the cell population have target-to-target translocations, or (ii) the cell population has less than two-fold background levels of reciprocal, compound, or off-target translocations.

2. The cell population described in claim 1, wherein the cell population is capable of expanding 20-fold, 30-fold, 40-fold, or 50-fold ex vivo in culture within 14 days after initiation of editing.

3. A cell population comprising edited cells containing multiple genome edits per cell, wherein at least 50% of the cells in the cell population contain at least two genome edits, and wherein the cell population is capable of a 50-fold expansion ex vivo in culture within 14 days after initiation of editing.

4. The cell population of claim 3, wherein less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% of the cells have target-to-target translocations or have less than two-fold background levels of reciprocal, complex, or off-target translocations.

5. The cell population described in any one of claims 1 to 4, wherein at least one of the plurality of genome edits is produced by a genome editing tool comprising an RNA-guided DNA binding agent, and the RNA-guided DNA binding agent is optionally a cleavase.

6. The cells are selected from mesenchymal stem cells, hematopoietic stem cells (HSCs), mononuclear cells, endothelial progenitor cells (EPCs), neural stem cells (NSCs), corneal epithelial 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), cells for organ or tissue transplantation, and optionally cells for use in ACT therapy; or the cell is an immune cell, and optionally the immune cells are selected from lymphocytes (e.g., T cells, B cells, natural killer cells ("NK cells", and NKT cells, or iNKT cells)), monocytes, macrophages, mast cells, dendritic cells, granulocytes (e.g., neutrophils, eosinophils, and basophils), primary immune cells, CD3+ cells, CD4+ cells, CD8+ T cells, regulatory T cells (Treg), B cells, NK cells, and dendritic cells (DCs)); and optionally the immune cells are selected from peripheral blood mononuclear cells (PBMCs), lymphocytes, T cells, optionally CD4+ cells, CD8+ cells, memory T cells, naive T cells, stem cell memory T cells, or B cells, optionally memory B cells, naive B cells, and primary cells; The cell population according to any one of claims 1 to 5.

7. The cell population of claim 6, wherein the cells are T cells, and optionally the T cells are selected from tumor infiltrating lymphocytes (TILs), T cells expressing alpha-beta TCR, T cells expressing gamma-delta TCR, regulatory T cells (Tregs), memory T cells, and early stem cell memory T cells (Tscm, CD27+ / CD45+).

8. The cell population of claim 7, wherein at least 95% of the cells in the cell population contain genomic editing of an endogenous T cell receptor (TCR) sequence.

9. The cell population of claim 7 or 8, wherein the genome editing comprises insertion of an exogenous nucleic acid encoding a targeting ligand or an alternative antigen binding moiety, and wherein at least 70% of the cells in the cell population comprise insertion of an exogenous nucleic acid into a target sequence.

10. The cell population of any one of claims 1 to 9, wherein the cells are non-activated immune cells, or the cells are activated immune cells.

11. A method for producing multiple genome edits in an in vitro cultured cell, comprising: contacting the cell in vitro with at least a first lipid nanoparticle (LNP) composition and a second LNP composition, wherein the first LNP composition comprises a first guide RNA (gRNA) directed to a first target sequence and optionally a first nucleic acid genome editing tool, and the second LNP composition comprises a second gRNA directed to a second target sequence different from the first target sequence and optionally a second nucleic acid genome editing tool; b. Expanding the cells in vitro; thereby producing multiple genome edits in the cell.

12. A method for producing multiple genome edits in ex vivo cultured cells, comprising: contacting the cell in vitro with at least a first lipid nanoparticle (LNP) composition and a second LNP composition, wherein the first lipid LNP composition comprises a first guide RNA (gRNA) directed to a first target sequence and optionally a first nucleic acid genome editing tool, and the second LNP composition comprises a second gRNA directed to a second target sequence different from the first target sequence and optionally a second nucleic acid genome editing tool; b. culturing the cells ex vivo; thereby producing multiple genome edits in the cell.

13. The method described in claim 11 or 12, wherein the first genome editing tool or the second genome editing tool comprises a nucleic acid encoding an RNA-guided DNA binding agent.

14. The method of any one of claims 11 to 13, wherein the cell is further contacted with a donor nucleic acid for insertion into a target sequence.

15. A method for delivering a lipid nanoparticle (LNP) composition to a population of in vitro cultured cells, comprising: a. contacting said population of cells in vitro with at least a first LNP composition comprising a first nucleic acid, thereby producing a population of contacted cells; b. culturing said population of contacted cells in vitro, thereby producing a cultured population of contacted cells; c. contacting the contacted population of cells or the cultured contacted population of cells in vitro with at least a second LNP composition comprising a second nucleic acid, wherein the second nucleic acid is different from the first nucleic acid; d. Expanding the population of cells in vitro; wherein the expanded population of cells exhibits a viability of at least 70%, 80%, 90%, or 95% upon 24 hours of expansion.

16. A method for delivering a lipid nanoparticle (LNP) composition to a population of in vitro cultured cells, comprising: a. contacting said population of cells in vitro with at least a first LNP composition comprising a first nucleic acid, thereby producing a population of contacted cells; b. culturing said population of contacted cells in vitro, thereby producing a cultured population of contacted cells; c. contacting the contacted population of cells or the cultured contacted population of cells in vitro with at least a second LNP composition comprising a second nucleic acid, wherein the second nucleic acid is different from the first nucleic acid; wherein at least 70%, 80%, 90%, or 95% of the cells in the population of cells are viable 24 hours after last contact with an LNP composition.

17. Contacting the contacted population of cells or the cultured contacted population of cells with a total of 2 to 12 LNP compositions, 2 to 8 LNP compositions, 2 to 6 LNP compositions, 3 to 8 LNP compositions, 3 to 6 LNP compositions, 4 to 6 LNP compositions, 6 to 12 LNP compositions, or 3, 4, 5, or 6 LNP compositions; or The contacted population of cells or the cultured contacted population of cells are simultaneously contacted with the LNP composition; or The contacted population of cells or the cultured contacted population of cells is simultaneously contacted with six or fewer LNP compositions; or contacting the population of contacted cells or the cultured population of contacted cells with no more than two LNP compositions simultaneously; 17. The method according to claim 15 or 16.

18. A method of gene editing in a population of cells, comprising: a. contacting the population of cells in vitro with a first lipid nanoparticle (LNP) composition comprising a first genome editing tool and a second LNP composition comprising a second genome editing tool; b. expanding the population of cells in vitro; thereby editing the population of cells.

19. A method of gene editing in a population of cells, comprising: a. contacting the population of cells in vitro with a first lipid nanoparticle (LNP) composition comprising a first genome editing tool and a second LNP composition comprising a second genome editing tool; b. culturing the population of cells in vitro, wherein at least 70%, 80%, 90%, or 95% of the cells in the population of cells are viable 24 hours after last contact with an LNP composition; thereby editing the population of cells.

20. The method described in claim 18 or 19, wherein the first genome editing tool or the second genome editing tool comprises a guide RNA.

21. The method of any one of claims 18 to 20, further comprising contacting the cell population in vitro with a third LNP composition comprising a genome editing tool, wherein at least two of the LNP compositions comprise a gRNA.

22. The method of any one of claims 18 to 21, wherein at least one LNP composition comprises an RNA-guided DNA binding agent, and optionally, the RNA-guided DNA binding agent is Cas9.

23. The method of any one of claims 18 to 22, further comprising contacting the cell population with donor nucleic acid.

24. The cells are immune cells, optionally mesenchymal stem cells, hematopoietic stem cells (HSC), mononuclear cells, endothelial progenitor cells (EPC), neural stem cells (NSC), corneal epithelial stem cells (LSC), tissue-specific primary cells or cells derived therefrom (TSC), induced pluripotent stem cells (iPSC), ocular stem cells, pluripotent stem cells (PSC), embryonic stem cells (ESC), cells for organ or tissue transplantation, and optionally cells for use in ACT therapy; or the cell population is immune cells, optionally mesenchymal stem cells, hematopoietic stem cells (HSC), mononuclear cells, endothelial progenitor cells (EPC), neural stem cells (NSC), corneal epithelial stem cells (LSC), tissue-specific primary cells or cells derived therefrom (TSC), induced pluripotent stem cells (iPSC), ocular stem cells, pluripotent stem cells (PSC), embryonic stem cells (ESC), cells for organ or tissue transplantation, and optionally cells for use in ACT therapy; or the cells are lymphocytes, optionally T cells, B cells, natural killer cells ("NK cells" and NKT cells, or iNKT cells), monocytes, macrophages, mast cells, dendritic cells, granulocytes (e.g., neutrophils, eosinophils, and basophils), primary immune cells, CD3+ cells, CD4+ cells, CD8+ T cells, regulatory T cells (Treg), or dendritic cells (DC); or the cell population is lymphocytes, optionally T cells, B cells, natural killer cells ("NK cells" and NKT cells, or iNKT cells), monocytes, macrophages, mast cells, dendritic cells, granulocytes (e.g., neutrophils, eosinophils, and basophils), primary immune cells, CD3+ cells, CD4+ cells, CD8+ T cells, regulatory T cells (Treg), or dendritic cells (DC); The method according to any one of claims 11 to 23.

25. The method of any one of claims 11 to 24, wherein the cell is a T cell or the cell population is a T cell.

26. The method of any one of claims 11 to 25, wherein the cells are non-activated cells or the cell population is non-activated cells.

27. The method of any one of claims 11 to 25, wherein the cells are activated cells or the cell population is activated cells.

28. The method of any one of claims 11 to 25, wherein the cells or cell population of (a) are activated following contact with at least one LNP composition.

29. A method for producing multiple genome edits in in vitro cultured T cells, comprising: contacting the T cells in vitro with (i) a first lipid nanoparticle (LNP) composition comprising a guide RNA (gRNA) directed to a first target sequence, and optionally (ii) one or two additional LNP compositions, each additional LNP composition comprising a gRNA and / or a genome editing tool directed to a target sequence different from the first target sequence; b. activating the T cells in vitro; c. Contacting the activated T cells in vitro with (i) an additional LNP composition comprising an additional guide RNA directed to a target sequence different from the target sequence of (a), and optionally (ii) one or more LNP compositions, each LNP composition comprising a guide RNA directed to a target sequence different from the target sequence of (a) and different from each other, and / or a genome editing tool; d. Expanding the cells in vitro; thereby producing multiple genome edits in the T cell.

30. The method of any one of claims 11 to 29, wherein the cells are contacted simultaneously with 2 to 8, or 2 to 6, optionally 2 to 5, 3 to 5, or 3 to 6 LNP compositions.

31. A method for genetically modifying a primary cell, comprising the steps of: a. culturing primary cells in a cell culture medium; b. providing a first lipid nanoparticle (LNP) composition comprising a nucleic acid; c. combining the primary cells of (a) in vitro with the LNP composition of (b); d. Optionally, confirming that the primary cells are genetically modified; and e. Optionally, expanding the primary cells. A method comprising:

32. The method of claim 31, wherein the primary cells are primary immune cells.

33. The method of claim 32, comprising performing the combining step (c) on non-activated immune cells, and optionally further comprising activating the immune cells after step (c).

34. The method of claim 31 or 32, comprising performing the combining step (c) on activated immune cells. (b2) providing a second LNP composition comprising a second nucleic acid; and (c2) combining the genetically modified cells of step (c) in vitro with the second LNP composition; (d2) optionally confirming that the cell has been genetically modified using the second nucleic acid for genetic modification; 33. The method of claim 31 or 32, optionally further comprising expanding the cells. (b3) providing a third LNP composition comprising a third nucleic acid; and (c3) combining the genetically modified cells of step (c2) with the third LNP composition in vitro; (d2) optionally confirming that the cell has been genetically modified using the third nucleic acid for genetic modification; 36. The method of claim 35, further comprising (e) optionally expanding the cells.

37. A method of administering at least two LNP compositions sequentially, the sequential administration comprising culturing the cells for a period including 10 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours from administration, or providing an LNP composition to the cells and providing a subsequent LNP composition to the cells; or The cells are expanded or grown at least 20, 30, 40, or 50 fold, and optionally the expansion or growth is in culture for no more than 14 days after initiation of editing.

37. The method according to any one of claims 11 to 36.

38. The nucleic acid or nucleic acid genome editing tool or gRNA comprises RNA, or The nucleic acid or nucleic acid genome editing tool comprises a guide RNA (gRNA); or The nucleic acid or nucleic acid genome editing tool or gRNA comprises an sgRNA; or The nucleic acid or nucleic acid genome editing tool or gRNA comprises a dgRNA; or The nucleic acid or nucleic acid genome editing tool comprises mRNA; or The nucleic acid or nucleic acid genome editing tool comprises an mRNA encoding a genome editing tool; 38. The method according to any one of claims 11 to 37.

39. A method described in any one of claims 11 to 38, wherein the first nucleic acid genome editing tool or the second nucleic acid genome editing tool comprises a donor nucleic acid.

40. A method described in any one of claims 11 to 39, wherein the first nucleic acid genome editing tool or the second nucleic acid genome editing tool comprises an RNA-guided DNA binding agent.

41. The method of any one of claims 11 to 40, further comprising contacting the cell or cell population with a DNA-dependent protein kinase inhibitor (DNA-PKi), optionally wherein the DNA-PKi is selected from compound 1 and compound 4.

42. The method of any one of claims 11 to 41, further comprising contacting the cell or cell population with one or more donor nucleic acids, at least one of the one or more donor nucleic acids comprising a region having homology to a corresponding region of the TRAC locus, the B2M locus, the AAVS1 locus, the CIITA locus, or the TRBC locus.

43. The method of any one of claims 11 to 42, wherein one of the LNP compositions comprises a gRNA targeting TRAC, one of the LNP compositions comprises a gRNA targeting TRBC, a further LNP composition comprises a gRNA targeting HLA-A, and a further LNP composition comprises a gRNA targeting CIITA.

44. The method of any one of claims 11 to 43, wherein the cells are T cells and at least 95% of the cells in the edited population comprise a genomic edit of an endogenous T cell receptor (TCR) sequence.

45. The method of any one of claims 11 to 44, wherein genome editing comprises insertion of a heterologous sequence encoding a targeting ligand or an alternative antigen binding moiety in 70%, 75%, 80%, or 85% of the cells of the population.

46. ​​The method of any one of claims 11 to 45, wherein the N / P ratio of the first LNP composition or the second LNP composition is about 6.

47. The method of any one of claims 11 to 46, wherein the first LNP composition or the second LNP composition comprises an amine lipid, a helper lipid, and a PEG lipid.

48. The LNP composition comprises a lipid component, the lipid component comprising about 25-45 mol% of an amine lipid such as lipid A, about 10-30 mol% of a neutral lipid, about 25-65 mol% of a helper lipid, and about 1.5-3.5 mol% of a stealth lipid (e.g., a PEG lipid), the N / P ratio of the LNP composition being about 3-7, and optionally a. the amount of the amine lipid is about 29-44 mol% of the lipid building block, the amount of the neutral lipid is about 11-28 mol% of the lipid building block, the amount of the helper lipid is about 28-55 mol% of the lipid building block, and the amount of the PEG lipid is about 2.3-3.5 mol% of the lipid building block; b. the amount of the amine lipid is about 29-38 mol% of the lipid building block, the amount of the neutral lipid is about 11-20 mol% of the lipid building block, the amount of the helper lipid is about 43-55 mol% of the lipid building block, and the amount of the PEG lipid is about 2.3-2.7 mol% of the lipid building block; c. the amount of the amine lipid is about 25-34 mol% of the lipid building block, the amount of the neutral lipid is about 10-20 mol% of the lipid building block, the amount of the helper lipid is about 45-65 mol% of the lipid building block, and the amount of the PEG lipid is about 2.5-3.5 mol% of the lipid building block, or d. the amount of the amine lipid is about 30-43 mol% of the lipid building block, the amount of the neutral lipid is about 10-17 mol% of the lipid building block, the amount of the helper lipid is about 43.5-56 mol% of the lipid building block, and the amount of the PEG lipid is about 1.5-3 mol% of the lipid building block; or, The LNP composition comprises a lipid component, the lipid component comprising about 25-50 mol% of an amine lipid, such as lipid D, about 7-25 mol% of a neutral lipid, about 39-65 mol% of a helper lipid, and about 0.5-1.8 mol% of a stealth lipid (e.g., a PEG lipid), the N / P ratio of the LNP composition is about 3-7, and optionally a. the amount of the ionizable lipid is about 27-40 mol% of the lipid building block, the amount of the neutral lipid is about 10-20 mol% of the lipid building block, the amount of the helper lipid is about 50-60 mol% of the lipid building block, and the amount of the PEG lipid is about 0.9-1.6 mol% of the lipid building block; b. the amount of the ionizable lipid is about 30-45 mol% of the lipid component, the amount of the neutral lipid is about 10-15 mol% of the lipid component, the amount of the helper lipid is about 39-59 mol% of the lipid component, and the amount of the PEG lipid is about 1-1.5 mol% of the lipid component; c. The amount of the ionizable lipid is about 30 mol% of the lipid component, the amount of the neutral lipid is about 10 mol% of the lipid component, the amount of the helper lipid is about 59 mol% of the lipid component, and the amount of the PEG lipid is about 1 mol% of the lipid component; d. the amount of the ionizable lipid is about 40 mol% of the lipid building block, the amount of the neutral lipid is about 15 mol% of the lipid building block, the amount of the helper lipid is about 43.5 mol% of the lipid building block, and the amount of the PEG lipid is about 1.5 mol% of the lipid building block, or e. the amount of the ionizable lipid is about 50 mol% of the lipid building block, the amount of the neutral lipid is about 10 mol% of the lipid building block, the amount of the helper lipid is about 39 mol% of the lipid building block, and the amount of the PEG lipid is about 1 mol% of the lipid building block; and optionally the amine lipid is lipid A; or the amine lipid is lipid D; or The neutral lipid is DSPC; or the stealth lipid is PEG2k-DMG; or The helper lipid is cholesterol.

48. The method according to any one of claims 11 to 47.

49. The method of any one of claims 11 to 48, wherein the LNP composition is pretreated with recombinant ApoE3 or ApoE4 prior to contacting the cells, and optionally the ApoE3 or ApoE4 is human ApoE3 or ApoE4.

50. A cell population produced or obtainable by a method according to any one of claims 11 to 49.

51. A cell population described in any one of claims 1 to 10 and 50 for use in a therapeutic method or pharmaceutical composition.

52. The cell population described in claim 51, wherein the therapeutic method or pharmaceutical composition is for the treatment of cancer or autoimmune therapy, or for adoptive cell transfer therapy.

53. A method for producing a cell bank, comprising genetically modifying a cell or population of cells, optionally an immune cell or population of immune cells, using a method according to any one of claims 11 to 49 to obtain a population of genetically modified cells, and transferring said genetically modified cells into a cell bank.

Images